Astyanax transgenesis and husbandry: how cavefish enters the laboratory.
Yannick, Y. Elipot ; Laurent, L. Legendre ; Stéphane, S. Père ; Frédéric, F. Sohm ; Sylvie, S. Rétaux.
1 CNRS UPR3294, DECA Group, Institut Alfred Fessard , Gif-sur-Yvette, France .
Astyanax mexicanus, a teleost fish comprising both sighted river-dwelling and blind cave-dwelling morphs, is becoming increasingly used in the field of developmental and evolutionary biology. Thus, new experimental and technological tools are needed on this emerging fish model by the expanding scientific community. Here, we describe Astyanax husbandry and egg spawning habits, a prerequisite to the successful establishment of Astyanax transgenic lines. We then compare two different transgenesis methods on both surface and cave Astyanax. Both meganuclease (I-SceI)- and transposase (Tol2)-mediated transgenesis are equivalently efficient, resulting in ∼40% mosaic transgenic fish in F0. Furthermore, the transmission rate was analyzed in F1 in the case of the I-SceI method and was found to be 16%. Finally, the transgene was found stable up the F3 generation, demonstrating the feasibility of generating stable transgenic lines in Astyanax and opening a wide range of possibilities for this fish model.The proximal promoter region of the zebrafish gsdf gene is sufficient to mimic the spatio-temporal expression pattern of the endogenous gene in Sertoli and granulosa cells.
Aude, A. Gautier ; Frédéric, F. Sohm ; Jean-Stéphane, JS. Joly ; Florence, F. Le Gac ; Jean-Jacques, JJ. Lareyre.
INRA, UR1037 SCRIBE, Testicular Physiology and Puberty Research Team, IFR140, Ouest-BioGenOuest, Rennes, France.
The gonadal soma-derived factor (GSDF) is a new member of the transforming growth factor beta (TGF-beta) superfamily that regulates the proliferation of the primordial germ cells (PGC) in developing embryos and spermatogonia in juvenile male trout. The gsdf transcripts are expressed in the somatic cells supporting germ cell development. In zebrafish, we show that GSDF is encoded by a single copy gene that generates polymorphic transcripts and proteins. We determined that gsdf gene expression occurs before gonadal differentiation and is restricted to the gonads. Gene expression is maintained in adult granulosa cells and Sertoli cells but decreases in the cells that are in contact with meiotic and postmeiotic germ cells. Using zebrafish transgenic lines, we demonstrate that the 2-kb proximal promoter region of the gsdf gene targets high levels of transgene expression in the Sertoli and granulosa cells, and is sufficient to mimic the temporal expression pattern of the endogenous gsdf gene from 16 days postfertilization onward. We identified within the first 500 bp evolutionarily conserved DNA motifs that may be involved in Sertoli and granulosa cell-specific expression. However, the 2-kb proximal promoter region failed to drive efficient expression of the transgene in the gonads in four transgenic medaka lines. We propose that the proximal promoter region can be used to target candidate gene deregulation in zebrafish granulosa and Sertoli cells. Furthermore, the green fluorescent protein-expressing zebrafish lines produced in the present study are new valuable models for cell lineage tracing during sex differentiation and gametogenesis.Isolation and expression of tilapia (Oreochromis niloticus) serine 8-type GnRH coding and regulatory sequences.
Hamid, H. Farahmand ; M Azizur, MA. Rahman ; Frédéric, F. Sohm ; Gyu-Lin, GL. Hwang ; Norman, N. Maclean.
University of Southampton, School of Biological Sciences, Division of Cell Sciences, Biomedical Sciences Building, Bassett Crescent East, Southampton SO16 7PX, UK.
The complete Serine 8-type gonadotropin releasing hormone (GnRH) coding sequence with a substantial 5-prime regulatory sequence (5 kb) has been isolated and characterised in Nile Tilapia (Oreochromis niloticus) from a relevant genomic library. The primary structure of the protein precursor was identified for this gene. The promoter efficacy has been tested using 0.6 kb of the GnRH promoter driving a lacZ reporter gene in both cultured spleen cells and transiently expressing zebrafish. In the cell transfection experiments, the average level of beta-galactosidase activity in transfected cells was more than 2.1 (P<0.05) times higher than the control promoter-less vector in five independent cultures indicating that the 0.6 GnRH/lacZ construct is able to express in spleen cells. In addition, the transient expression of the lacZ gene was detected in the brain of G0 zebrafish embryos (Danio rerio) 4 days after fertilisation following egg injection with the construct, which demonstrated the efficacy of the tilapia GnRH promoter.Isolation and characterisation of tilapia beta-actin promoter and comparison of its activity with carp beta-actin promoter.
Gyu-Lin, GL. Hwang ; M, M. Azizur Rahman ; Shaharudin, S. Abdul Razak ; Frédéric, F. Sohm ; Hamid, H. Farahmand ; Alan, A. Smith ; Carly, C. Brooks ; Norman, N. Maclean.
Division of Cell Sciences, School of Biological Sciences, University of Southampton, UK.
The regulatory sequence including proximal promoter, untranslated exon 1 and intron 1 of the beta-actin gene from tilapia (Oreochromis niloticus) has been isolated and spliced to a beta-galactosidase reporter gene to test its activity. Comparisons of promoter activity have been carried out with three different constructs: (1) 1.6 kb tilapia beta-actin regulatory sequence, (2) 1.5 kb carp beta-actin regulatory sequence, and (3) 4.7 kb carp beta-actin regulatory sequence. Although the 1.6 kb tilapia beta-actin regulatory sequence gave slightly different expression patterns in tilapia embryos assayed by in situ X-gal staining, no difference was observed in expression level when the tilapia sequence was compared with the 4.7 kb carp beta-actin regulatory sequence by quantitative assay. In comparison with the 1.5 kb carp beta-actin regulatory sequence, the 1.6 kb tilapia beta-actin regulatory sequence gave higher expression levels in tilapia embryos, while a reverse result was observed in zebrafish embryos. In cell transfection experiments, the 1.6 kb tilapia beta-actin regulatory sequence showed three to four times better activity in blue gill cells than either the 4.7 kb carp beta-actin or the 1.5 kb carp beta-actin regulatory sequences. The 1.6 kb tilapia beta-actin regulatory sequence also drove higher reporter gene activity in somatic cells of tilapia than did the 4.7 kb carp beta-actin regulatory sequence following direct injection of constructs into muscle. Therefore, taken together, the data demonstrate that the tilapia beta-actin promoter can be used as an efficient regulatory sequence to produce autotransgenic tilapia.
The proto-MHC of placozoans, a region specialized in cellular stress and ubiquitination/proteasome pathways.
Jaanus, J. Suurväli ; Luc, L. Jouneau ; Dominique, D. Thépot ; Simona, S. Grusea ; Pierre, P. Pontarotti ; Louis, L. Du Pasquier ; Sirje, S. Rüütel Boudinot ; Pierre, P. Boudinot.
Department of Gene Technology, Tallinn University of Technology, Tallinn 12618, Estonia;
The MHC is a large genetic region controlling Ag processing and recognition by T lymphocytes in vertebrates. Approximately 40% of its genes are implicated in innate or adaptive immunity. A putative proto-MHC exists in the chordate amphioxus and in the fruit fly, indicating that a core MHC region predated the emergence of the adaptive immune system in vertebrates. In this study, we identify a putative proto-MHC with archetypal markers in the most basal branch of Metazoans--the placozoan Trichoplax adhaerens, indicating that the proto-MHC is much older than previously believed--and present in the common ancestor of bilaterians (contains vertebrates) and placozoans. Our evidence for a T. adhaerens proto-MHC was based on macrosynteny and phylogenetic analyses revealing approximately one third of the multiple marker sets within the human MHC-related paralogy groups have unique counterparts in T. adhaerens, consistent with two successive whole genome duplications during early vertebrate evolution. A genetic ontologic analysis of the proto-MHC markers in T. adhaerens was consistent with its involvement in defense, showing proteins implicated in antiviral immunity, stress response, and ubiquitination/proteasome pathway. Proteasome genes psma, psmb, and psmd are present, whereas the typical markers of adaptive immunity, such as MHC class I and II, are absent. Our results suggest that the proto-MHC was involved in intracellular intrinsic immunity and provide insight into the primordial architecture and functional landscape of this region that later in evolution became associated with numerous genes critical for adaptive immunity in vertebrates.Salmonids have an extraordinary complex type I IFN system: characterization of the IFN locus in rainbow trout oncorhynchus mykiss reveals two novel IFN subgroups.
Jun, J. Zou ; Bartolomeo, B. Gorgoglione ; Nicholas G H, NG. Taylor ; Thitiya, T. Summathed ; Po-Tsang, PT. Lee ; Akshaya, A. Panigrahi ; Carine, C. Genet ; Young-Mao, YM. Chen ; Tzong-Yueh, TY. Chen ; Mahmood, M. Ul Hassan ; Sharif M, SM. Mughal ; Pierre, P. Boudinot ; Christopher J, CJ. Secombes.
Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Aberdeen AB24 2TZ, United Kingdom; email@example.com firstname.lastname@example.org.
Fish type I IFNs are classified into two groups with two (group I) or four (group II) cysteines in the mature peptide and can be further divided into four subgroups, termed IFN-a, -b, -c, and -d. Salmonids possess all four subgroups, whereas other teleost species have one or more but not all groups. In this study, we have discovered two further subgroups (IFN-e and -f) in rainbow trout Oncorhynchus mykiss and analyzed the expression of all six subgroups in rainbow trout and brown trout Salmo trutta. In rainbow trout RTG-2 and RTS-11 cells, polyinosinic-polycytidylic acid stimulation resulted in early activation of IFN-d, whereas the IFN-e subgroup containing the highest number of members showed weak induction. In contrast with the cell lines, remarkable induction of IFN-a, -b, and -c was detected in primary head kidney leukocytes after polyinosinic-polycytidylic acid treatment, whereas a moderate increase of IFNs was observed after stimulation with resiquimod. Infection of brown trout with hemorrhagic septicemia virus resulted in early induction of IFN-d, -e, and -f and a marked increase of IFN-b and IFN-c expression in kidney and spleen. IFN transcripts were found to be strongly correlated with the viral burden and with marker genes of the IFN antiviral cascade. The results demonstrate that the IFN system of salmonids is far more complex than previously realized, and in-depth research is required to fully understand its regulation and function.A tetrapod-like repertoire of innate immune receptors and effectors for coelacanths.
Pierre, P. Boudinot ; Jun, J. Zou ; Tatsuya, T. Ota ; Francesco, F. Buonocore ; Giuseppe, G. Scapigliati ; Adriana, A. Canapa ; John, J. Cannon ; Gary, G. Litman ; John D, JD. Hansen.
Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, Jouy-en-Josas, France.
The recent availability of both robust transcriptome and genome resources for coelacanth (Latimeria chalumnae) has led to unique discoveries for coelacanth immunity such as the lack of IgM, a central component of adaptive immunity. This study was designed to more precisely address the origins and evolution of gene families involved in the initial recognition and response to microbial pathogens, which effect innate immunity. Several multigene families involved in innate immunity are addressed, including: Toll-like receptors (TLRs), retinoic acid inducible gene 1 (RIG1)-like receptors (RLRs), the nucleotide-binding domain and leucine-rich repeat containing proteins (NLRs), diverse immunoglobulin domain-containing proteins (DICP) and modular domain immune-type receptors (MDIRs). Our analyses also include the tripartite motif-containing proteins (TRIM), which are involved in pathogen recognition as well as the positive regulation of antiviral immunity. Finally, this study addressed some of the downstream effectors of the antimicrobial response including IL-1 family members, type I and II interferons (IFN) and IFN-stimulated effectors (ISGs). Collectively, the genes and gene families in coelacanth that effect innate immune functions share characteristics both in content, structure and arrangement with those found in tetrapods but not in teleosts. The findings support the sister group relationship of coelacanth fish with tetrapods.The past, present, and future of immune repertoire biology - the rise of next-generation repertoire analysis.
Adrien, A. Six ; Maria Encarnita, ME. Mariotti-Ferrandiz ; Wahiba, W. Chaara ; Susana, S. Magadan ; Hang-Phuong, HP. Pham ; Marie-Paule, MP. Lefranc ; Thierry, T. Mora ; Véronique, V. Thomas-Vaslin ; Aleksandra M, AM. Walczak ; Pierre, P. Boudinot.
UPMC University Paris 06, UMR 7211, Immunology-Immunopathology-Immunotherapy (I3) , Paris , France ; CNRS, UMR 7211, Immunology-Immunopathology-Immunotherapy (I3) , Paris , France ; INSERM, UMR_S 959, Immunology-Immunopathology-Immunotherapy (I3) , Paris
T and B cell repertoires are collections of lymphocytes, each characterized by its antigen-specific receptor. We review here classical technologies and analysis strategies developed to assess immunoglobulin (IG) and T cell receptor (TR) repertoire diversity, and describe recent advances in the field. First, we describe the broad range of available methodological tools developed in the past decades, each of which answering different questions and showing complementarity for progressive identification of the level of repertoire alterations: global overview of the diversity by flow cytometry, IG repertoire descriptions at the protein level for the identification of IG reactivities, IG/TR CDR3 spectratyping strategies, and related molecular quantification or dynamics of T/B cell differentiation. Additionally, we introduce the recent technological advances in molecular biology tools allowing deeper analysis of IG/TR diversity by next-generation sequencing (NGS), offering systematic and comprehensive sequencing of IG/TR transcripts in a short amount of time. NGS provides several angles of analysis such as clonotype frequency, CDR3 diversity, CDR3 sequence analysis, V allele identification with a quantitative dimension, therefore requiring high-throughput analysis tools development. In this line, we discuss the recent efforts made for nomenclature standardization and ontology development. We then present the variety of available statistical analysis and modeling approaches developed with regards to the various levels of diversity analysis, and reveal the increasing sophistication of those modeling approaches. To conclude, we provide some examples of recent mathematical modeling strategies and perspectives that illustrate the active rise of a "next-generation" of repertoire analysis.The antiviral innate immune response in fish: evolution and conservation of the IFN system.
Christelle, C. Langevin ; Elina, E. Aleksejeva ; Gabriella, G. Passoni ; Nuno, N. Palha ; Jean-Pierre, JP. Levraud ; Pierre, P. Boudinot.
Virologie et Immunologie Moléculaire, INRA, F-78352, Jouy-en-Josas France.
Innate immunity constitutes the first line of the host defense after pathogen invasion. Viruses trigger the expression of interferons (IFNs). These master antiviral cytokines induce in turn a large number of interferon-stimulated genes, which possess diverse effector and regulatory functions. The IFN system is conserved in all tetrapods as well as in fishes, but not in tunicates or in the lancelet, suggesting that it originated in early vertebrates. Viral diseases are an important concern of fish aquaculture, which is why fish viruses and antiviral responses have been studied mostly in species of commercial value, such as salmonids. More recently, there has been an interest in the use of more tractable model fish species, notably the zebrafish. Progress in genomics now makes it possible to get a relatively complete image of the genes involved in innate antiviral responses in fish. In this review, by comparing the IFN system between teleosts and mammals, we will focus on its evolution in vertebrates.The zebrafish as a new model for the in vivo study of Shigella flexneri interaction with phagocytes and bacterial autophagy.
Serge, S. Mostowy ; Laurent, L. Boucontet ; Maria J, MJ. Mazon Moya ; Andrea, A. Sirianni ; Pierre, P. Boudinot ; Michael, M. Hollinshead ; Pascale, P. Cossart ; Philippe, P. Herbomel ; Jean-Pierre, JP. Levraud ; Emma, E. Colucci-Guyon.
Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom ; Institut Pasteur, Unité des Interactions Bactéries-Cellules, Département de Biologie Cellulaire et Infection, Paris, France ; I
Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, the role of bacterial autophagy in vivo remains poorly understood. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional. Here, we have characterized the susceptibility of zebrafish larvae to Shigella flexneri, a paradigm for bacterial autophagy, and have used this model to study Shigella-phagocyte interactions in vivo. Depending on the dose, S. flexneri injected in zebrafish larvae were either cleared in a few days or resulted in a progressive and ultimately fatal infection. Using high resolution live imaging, we found that S. flexneri were rapidly engulfed by macrophages and neutrophils; moreover we discovered a scavenger role for neutrophils in eliminating infected dead macrophages and non-immune cell types that failed to control Shigella infection. We observed that intracellular S. flexneri could escape to the cytosol, induce septin caging and be targeted to autophagy in vivo. Depletion of p62 (sequestosome 1 or SQSTM1), an adaptor protein critical for bacterial autophagy in vitro, significantly increased bacterial burden and host susceptibility to infection. These results show the zebrafish larva as a new model for the study of S. flexneri interaction with phagocytes, and the manipulation of autophagy for anti-bacterial therapy in vivo.Comprehensive survey and genomic characterization of Toll-like receptors (TLRs) in channel catfish, Ictalurus punctatus: identification of novel fish TLRs.
Catfish Genetics Research Unit, USDA-ARS, 141 Experiment Station Rd, Stoneville, MS 38776, USA. email@example.com
A comprehensive survey of channel catfish Toll-like receptors (TLRs) was undertaken following a genomic PCR approach based on degenerate primers. Twenty different TLRs were identified in channel catfish. Channel catfish TLR sequences were characterized by phylogenetic analysis based on their conserved Toll/interleukin-1 receptor domain and by in-depth analysis of leucine-rich repeat (LRR) motifs of the ligand binding extracellular domain (ECD). The catfish have representatives of all the TLR types defined in vertebrates with the exception of TLR6, TLR10, TLR11, TLR12, TLR13, TLR15, TLR23, and TLR24. Additionally, two new types were discovered: TLR25 and TLR26. TLR25 is also present in cyprinids, cichlids, plecoglossids, and adrianichthyids, suggesting its presence early in fish evolution. To date, TLR26 was found only in channel catfish. Like TLR18-23, TLR25 and TLR26 were not found in any other vertebrate classes and appear to be fish specific. Data mining using the catfish TLR sequences revealed that in addition to ictalurids and cyprinids, TLR4 is also present in salmonids. TLR19 and TLR20 were both found in ictalurids, cyprinids, and salmonids, demonstrating a wider range than previously known. The LRR structure within ECDs appeared generally well conserved. TLR7 demonstrated a very high identity to human TLR7 strongly suggesting that ligand specificity maybe conserved. Finally, expression profiling confirmed that most TLRs are widely expressed in a diversity of tissues and revealed marked differences of expression level.Lack of correlation between the resistances to two rhabdovirus infections in rainbow trout.
Eloi R, ER. Verrier ; Aude, A. Ehanno ; Stéphane, S. Biacchesi ; Sandrine, S. Le Guillou ; Nicolas, N. Dechamp ; Pierre, P. Boudinot ; Michel, M. Bremont ; Edwige, E. Quillet.
INRA, UMR 1313 Génétique Animale et Biologie Intégrative, Jouy-en-Josas, France.
The Viral Hemorrhagic Septicemia Virus (VHSV) and the Infectious Hematopoietic Necrosis Virus (IHNV) are two rhabdoviruses responsible for serious outbreaks in salmonid farms. To date, little is known about the variability of host response to these viruses. Using gynogenetic clonal lines of rainbow trout exhibiting a wide range of resistance to viral infections, we showed that there was no correlation between the resistance to VHSV and IHNV. We also confirmed the importance of fish weight for its susceptibility to IHNV infection. Finally, using a chimeric recombinant IHNV expressing the VHSV glycoprotein, we showed that the glycoprotein plays a key role in the virulence and in the level of resistance observed in different genetic backgrounds. Taken together, our results provide new prospects for a better understanding of host responses to rhabdovirus infections in salmonids.Contrasted TCRβ diversity of CD8+ and CD8- T cells in rainbow trout.
Rosario, R. Castro ; Fumio, F. Takizawa ; Wahiba, W. Chaara ; Aurélie, A. Lunazzi ; Thi Huong, TH. Dang ; Bernd, B. Koellner ; Edwige, E. Quillet ; Adrien, A. Six ; Uwe, U. Fischer ; Pierre, P. Boudinot.
Institut National de la Recherche Agronomique, Virologie et Immunologie Moléculaires, Jouy-en-Josas, France.
Teleost fish express highly diverse naive TCRβ (TRB) repertoires and mount strong public and private clonal responses upon infection with pathogens. Fish T cells express typical markers such as CD8, CD4-1 and CD4-2, CD3, CD28 and CTLA4. Fish CD8(+) T cells have been shown to be responsible for antigen-specific cell-mediated cytotoxicity in in vitro systems using histo-compatible effector and target cells. We compare here the complexity of TRB repertoires between FACS sorted CD8(+) and CD8(-) T cells from spleen and pronephros of rainbow trout. In contrast to human, while the TRB repertoire is highly diverse and polyclonal in CD8(+) T cells of naïve fish, it appeared very different in CD8(-) lymphocytes with irregular CDR3 length distributions suggesting a dominance of activated clones already in naïve fish or the presence of non conventional T cells. After infection with a systemic virus, CD8(+) T cells mount a typical response with significant skewing of CDR3 length profiles. The infection also induces significant modifications of the TRB repertoire expressed by the CD8(-) fraction, but for a different set of V/J combinations. In this fraction, the antiviral response results in an increase of the peak diversity of spectratypes. This unusual observation reflects the presence of a number of T cell expansions that rise the relative importance of minor peaks of the highly skewed distributions observed in unchallenged animals. These results suggest that the diversity of TRB expressed by CD8(+) and CD8(-) αβ T cells may be subjected to different regulatory patterns in fish and in mammals.The astonishing diversity of Ig classes and B cell repertoires in teleost fish.
Simon, S. Fillatreau ; Adrien, A. Six ; Susanna, S. Magadan ; Rosario, R. Castro ; J Oriol, JO. Sunyer ; Pierre, P. Boudinot.
Deutsches Rheuma-Forschungszentrum, Leibniz Institute Berlin, Germany.
With lymphoid tissue anatomy different than mammals, and diverse adaptations to all aquatic environments, fish constitute a fascinating group of vertebrate to study the biology of B cell repertoires in a comparative perspective. Fish B lymphocytes express immunoglobulin (Ig) on their surface and secrete antigen-specific antibodies in response to immune challenges. Three antibody classes have been identified in fish, namely IgM, IgD, and IgT, while IgG, IgA, and IgE are absent. IgM and IgD have been found in all fish species analyzed, and thus seem to be primordial antibody classes. IgM and IgD are normally co-expressed from the same mRNA through alternative splicing, as in mammals. Tetrameric IgM is the main antibody class found in serum. Some species of fish also have IgT, which seems to exist only in fish and is specialized in mucosal immunity. IgM/IgD and IgT are expressed by two different sub-populations of B cells. The tools available to investigate B cell responses at the cellular level in fish are limited, but the progress of fish genomics has started to unravel a rich diversity of IgH and immunoglobulin light chain locus organization, which might be related to the succession of genome remodelings that occurred during fish evolution. Moreover, the development of deep sequencing techniques has allowed the investigation of the global features of the expressed fish B cell repertoires in zebrafish and rainbow trout, in steady state or after infection. This review provides a description of the organization of fish Ig loci, with a particular emphasis on their heterogeneity between species, and presents recent data on the structure of the expressed Ig repertoire in healthy and infected fish.Resistance to a rhabdovirus (VHSV) in rainbow trout: identification of a major QTL related to innate mechanisms.
Eloi R, ER. Verrier ; Michel, M. Dorson ; Stéphane, S. Mauger ; Corinne, C. Torhy ; Céline, C. Ciobotaru ; Caroline, C. Hervet ; Nicolas, N. Dechamp ; Carine, C. Genet ; Pierre, P. Boudinot ; Edwige, E. Quillet.
INRA, UMR 1313 Génétique Animale et Biologie Intégrative, Jouy-en-Josas, France.
Health control is a major issue in animal breeding and a better knowledge of the genetic bases of resistance to diseases is needed in farm animals including fish. The detection of quantitative trait loci (QTL) will help uncovering the genetic architecture of important traits and understanding the mechanisms involved in resistance to pathogens. We report here the detection of QTL for resistance to Viral Haemorrhagic Septicaemia Virus (VHSV), a major threat for European aquaculture industry. Two induced mitogynogenetic doubled haploid F2 rainbow trout (Oncorhynchus mykiss) families were used. These families combined the genome of susceptible and resistant F0 breeders and contained only fully homozygous individuals. For phenotyping, fish survival after an immersion challenge with the virus was recorded, as well as in vitro virus replication on fin explants. A bidirectional selective genotyping strategy identified seven QTL associated to survival. One of those QTL was significant at the genome-wide level and largely explained both survival and viral replication in fin explants in the different families of the design (up to 65% and 49% of phenotypic variance explained respectively). These results evidence the key role of innate defence in resistance to the virus and pave the way for the identification of the gene(s) responsible for resistance. The identification of a major QTL also opens appealing perspectives for selective breeding of fish with improved resistance.Teleost fish mount complex clonal IgM and IgT responses in spleen upon systemic viral infection.
Rosario, R. Castro ; Luc, L. Jouneau ; Hang-Phuong, HP. Pham ; Olivier, O. Bouchez ; Véronique, V. Giudicelli ; Marie-Paule, MP. Lefranc ; Edwige, E. Quillet ; Abdenour, A. Benmansour ; Frédéric, F. Cazals ; Adrien, A. Six ; Simon, S. Fillatreau ; Oriol, O. Sunyer ; Pierre, P. Boudinot.
Virologie et Immunologie Moléculaires, INRA, Jouy-en-Josas, France.
Upon infection, B-lymphocytes expressing antibodies specific for the intruding pathogen develop clonal responses triggered by pathogen recognition via the B-cell receptor. The constant region of antibodies produced by such responding clones dictates their functional properties. In teleost fish, the clonal structure of B-cell responses and the respective contribution of the three isotypes IgM, IgD and IgT remain unknown. The expression of IgM and IgT are mutually exclusive, leading to the existence of two B-cell subsets expressing either both IgM and IgD or only IgT. Here, we undertook a comprehensive analysis of the variable heavy chain (VH) domain repertoires of the IgM, IgD and IgT in spleen of homozygous isogenic rainbow trout (Onchorhynchus mykiss) before, and after challenge with a rhabdovirus, the Viral Hemorrhagic Septicemia Virus (VHSV), using CDR3-length spectratyping and pyrosequencing of immunoglobulin (Ig) transcripts. In healthy fish, we observed distinct repertoires for IgM, IgD and IgT, respectively, with a few amplified μ and τ junctions, suggesting the presence of IgM- and IgT-secreting cells in the spleen. In infected animals, we detected complex and highly diverse IgM responses involving all VH subgroups, and dominated by a few large public and private clones. A lower number of robust clonal responses involving only a few VH were detected for the mucosal IgT, indicating that both IgM(+) and IgT(+) spleen B cells responded to systemic infection but at different degrees. In contrast, the IgD response to the infection was faint. Although fish IgD and IgT present different structural features and evolutionary origin compared to mammalian IgD and IgA, respectively, their implication in the B-cell response evokes these mouse and human counterparts. Thus, it appears that the general properties of antibody responses were already in place in common ancestors of fish and mammals, and were globally conserved during evolution with possible functional convergences.R4 regulators of G protein signaling (RGS) identify an ancient MHC-linked synteny group.
Department of Gene Technology, Tallinn University of Technology, Tallinn, Estonia. firstname.lastname@example.org
Regulators of G protein signaling (RGS) are key regulators of G protein signaling. RGS proteins of the R4 RGS group are composed of a mere RGS domain and are mainly involved in immune response modulation. In both human and mouse, most genes encoding the R4 RGS proteins are located in the same region of chromosome 1. We show here that the RGS1/RGS16 neighborhood constitutes a synteny group well conserved across tetrapods and closely linked to the MHC paralogon of chromosome 1. Genes located in the RGS1/RGS16 region have paralogs close to the MHC on chromosome 6 or close to the other MHC paralogons. In amphioxus, a cephalochordate, these genes possess orthologs that are located in the same scaffolds as a number of markers defining the proto-MHC in this species (Abi-Rached et al., Nat Genet 31:100-115, 2002). We therefore propose that the RGS1/RGS16 region provides useful markers to investigate the origins and the evolution of the MHC. In addition, we show that some genes of the region appear to have immune functions not only in human, but also in Xenopus.Inflammatory chemokines direct and restrict leukocyte migration within live tissues as glycan-bound gradients.
Milka, M. Sarris ; Jean-Baptiste, JB. Masson ; Damien, D. Maurin ; Lieke M, LM. Van der Aa ; Pierre, P. Boudinot ; Hugues, H. Lortat-Jacob ; Philippe, P. Herbomel.
Macrophages and Development of Immunity Unit, Department of Developmental and Stem Cell Biology, Institut Pasteur, Paris, France. email@example.com
Chemokines are essential in many cell migration processes, including the recruitment of leukocytes to sites of infection. In the latter context, chemokines promote leukocyte extravasation into the relevant tissue through a well-studied cascade of events. It is widely believed that chemokines further guide leukocytes within tissues via chemotaxis, the directed migration along gradients of soluble ligands. However, the basic mechanism of chemokine action within tissues has yet to be formally addressed in vivo. We identified a chemokine (zCxcl8) that recruits zebrafish neutrophils to infection loci and analyzed its function directly within interstitial tissues of living larvae. Using noninvasive imaging and a controlled cellular source of zCxcl8, we found that zCxcl8 guides neutrophils in a 2-fold manner: by biasing cell speed according to direction (orthotaxis) and by restricting cell motility near the source. We further show that zCxcl8 establishes tissue-bound gradients in vivo by binding to heparan sulfate proteoglycans (HSPGs). Inhibition of this interaction compromised both directional guidance and restriction of neutrophil motility. Thus, by interacting with extracellular HSPGs, chemokines establish robust surface-bound (haptotactic) gradients that mediate both recruitment and retention of leukocytes at sites of infection.Diversification of IFNγ-inducible CXCb chemokines in cyprinid fish.
Lieke M, LM. van der Aa ; Magdalena, M. Chadzinska ; Wouter, W. Derks ; Marleen, M. Scheer ; Jean-Pierre, JP. Levraud ; Pierre, P. Boudinot ; B M, BM. Lidy Verburg-van Kemenade.
Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University, P.O. Box 338, 6700 AH, Wageningen, The Netherlands.
We earlier identified two CXCL8-like lineages in cyprinid fish, which are functional homologues of the mammalian CXCL8, but with diverged functions. We here investigated whether the carp IFN-γ-inducible CXCb gene, related to the mammalian CXCL9, -10 and -11 chemokines, was subject to a similar diversification. On the zebrafish genome, a cluster of seven CXCb genes was found on chromosome five. Analysis of the promoter of the zebrafish CXCb genes suggests a partially shared, but differential induction. A second CXCb gene, CXCb2, was identified in common carp by homology cloning. CXCb2 is constitutively expressed in immune-related tissues, predominantly in head kidney lymphocytes/monocytes. Interestingly, an induction of CXCb2 gene expression with recombinant carp IFN-γ2 and LPS was observed in macrophages and granulocytes. Finally, difference in sensitivity to LPS, and kinetics of CXCb1 and CXCb2 gene expression during zymosan-induced peritonitis, was observed. These results indicate a functional diversification for cyprinid CXCb chemokines, with functional homology to mammalian CXCL9-11.Transcriptional responses of resistant and susceptible fish clones to the bacterial pathogen Flavobacterium psychrophilum.
Christelle, C. Langevin ; Mar, M. Blanco ; Samuel A M, SA. Martin ; Luc, L. Jouneau ; Jean-Francois, JF. Bernardet ; Armel, A. Houel ; Aurélie, A. Lunazzi ; Eric, E. Duchaud ; Christian, C. Michel ; Edwige, E. Quillet ; Pierre, P. Boudinot.
INRA, Molecular Virology and Immunology, Domaine de Vilvert, Jouy en Josas, France.
Flavobacterium psychrophilum is a bacterial species that represents one of the most important pathogens for aquaculture worldwide, especially for salmonids. To gain insights into the genetic basis of the natural resistance to F. psychrophilum, we selected homozygous clones of rainbow trout with contrasted susceptibility to the infection. We compared the transcriptional response to the bacteria in the pronephros of a susceptible and a resistant line by micro-array analysis five days after infection. While the basal transcriptome of healthy fish was significantly different in the resistant and susceptible lines, the transcriptome modifications induced by the bacteria involved essentially the same genes and pathways. The response to F. psychrophilum involved antimicrobial peptides, complement, and a number of enzymes and chemokines. The matrix metalloproteases mmp9 and mmp13 were among the most highly induced genes in both genetic backgrounds. Key genes of both pro- and anti-inflammatory response such as IL1 and IL10, were up-regulated with a greater magnitude in susceptible animals where the bacterial load was also much higher. While higher resistance to F. psychrophilum does not seem to be based on extensive differences in the orientation of the immune response, several genes including complement C3 showed stronger induction in the resistant fish. They may be important for the variation of susceptibility to the infection.Genetic resistance to rhabdovirus infection in teleost fish is paralleled to the derived cell resistance status.
Eloi R, ER. Verrier ; Christelle, C. Langevin ; Corinne, C. Tohry ; Armel, A. Houel ; Vincent, V. Ducrocq ; Abdenour, A. Benmansour ; Edwige, E. Quillet ; Pierre, P. Boudinot.
INRA, Molecular Virology and Immunology, Jouy en Josas, France.
Genetic factors of resistance and predisposition to viral diseases explain a significant part of the clinical variability observed within host populations. Predisposition to viral diseases has been associated to MHC haplotypes and T cell immunity, but a growing repertoire of innate/intrinsic factors are implicated in the genetic determinism of the host susceptibility to viruses. In a long-term study of the genetics of host resistance to fish rhabdoviruses, we produced a collection of double-haploid rainbow trout clones showing a wide range of susceptibility to Viral Hemorrhagic Septicemia Virus (VHSV) waterborne infection. The susceptibility of fibroblastic cell lines derived from these clonal fish was fully consistent with the susceptibility of the parental fish clones. The mechanisms determining the host resistance therefore did not associate with specific host immunity, but rather with innate or intrinsic factors. One cell line was resistant to rhabdovirus infection due to the combination of an early interferon IFN induction--that was not observed in the susceptible cells--and of yet unknown factors that hamper the first steps of the viral cycle. The implication of IFN was well consistent with the wide range of resistance of this genetic background to VSHV and IHNV, to the birnavirus IPNV and the orthomyxovirus ISAV. Another cell line was even more refractory to the VHSV infection through different antiviral mechanisms. This collection of clonal fish and isogenic cell lines provides an interesting model to analyze the relative contribution of antiviral pathways to the resistance to different viruses.Early antiviral response and virus-induced genes in fish.
INRA, Fish Infection and Immunity, Molecular Virology and Immunology, Domaine de Vilvert, 78352 Jouy en Josas, France.
In fish as in mammals, virus infections induce changes in the expression of many host genes. Studies conducted during the last fifteen years revealed a major contribution of the interferon system in fish antiviral response. This review describes the screening methods applied to compare the impact of virus infections on the transcriptome in different fish species. These approaches identified a "core" set of genes that are strongly induced in most viral infections. The "core" interferon-induced genes (ISGs) are generally conserved in vertebrates, some of them inhibiting a wide range of viruses in mammals. A selection of ISGs -PKR, vig-1/viperin, Mx, ISG15 and finTRIMs - is further analyzed here to illustrate the diversity and complexity of the mechanisms involved in establishing an antiviral state. Most of the ISG-based pathways remain to be directly determined in fish. Fish ISGs are often duplicated and the functional specialization of multigenic families will be of particular interest for future studies.FinTRIMs, fish virus-inducible proteins with E3 ubiquitin ligase activity.
Lieke M, LM. van der Aa ; Luc, L. Jouneau ; Emmanuel, E. Laplantine ; Olivier, O. Bouchez ; Lidy, L. Van Kemenade ; Pierre, P. Boudinot.
Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, Jouy-en-Josas, France.
TRIM proteins have recently emerged as novel players in antiviral defense. TRIM proteins contain a tri-partite motif, composed of a RING zinc finger, one or two B-boxes and a coiled-coil domain. Many members of this large protein family of E3 ubiquitin ligases catalyze the attachment of ubiquitin to a substrate protein, an activity dependent on the RING domain. We earlier made a full description of the TRIM gene family in zebrafish and pufferfish and identified three multigene TRIM subsets, a feature unique to fish. To determine their biological role, we further characterized members of the finTRIM subset. FinTRIM gene expression was studied during development and in multiple tissues in adult rainbow trout. Upregulation of a large number of finTRIM upon viral stimulation suggests they are involved in antiviral immunity. We also demonstrate that two finTRIM members display E3 ubiquitin ligase activity, indicating that finTRIMs could regulate antiviral signaling through ubiquitination.Origin and evolution of TRIM proteins: new insights from the complete TRIM repertoire of zebrafish and pufferfish.
Pierre, P. Boudinot ; Lieke M, LM. van der Aa ; Luc, L. Jouneau ; Louis, L. Du Pasquier ; Pierre, P. Pontarotti ; Valérie, V. Briolat ; Abdenour, A. Benmansour ; Jean-Pierre, JP. Levraud.
Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, Jouy-en-Josas, France. firstname.lastname@example.org
Tripartite motif proteins (TRIM) constitute a large family of proteins containing a RING-Bbox-Coiled Coil motif followed by different C-terminal domains. Involved in ubiquitination, TRIM proteins participate in many cellular processes including antiviral immunity. The TRIM family is ancient and has been greatly diversified in vertebrates and especially in fish. We analyzed the complete sets of trim genes of the large zebrafish genome and of the compact pufferfish genome. Both contain three large multigene subsets--adding the hsl5/trim35-like genes (hltr) to the ftr and the btr that we previously described--all containing a B30.2 domain that evolved under positive selection. These subsets are conserved among teleosts. By contrast, most human trim genes of the other classes have only one or two orthologues in fish. Loss or gain of C-terminal exons generated proteins with different domain organizations; either by the deletion of the ancestral domain or, remarkably, by the acquisition of a new C-terminal domain. Our survey of fish trim genes in fish identifies subsets with different evolutionary dynamics. trims encoding RBCC-B30.2 proteins show the same evolutionary trends in fish and tetrapods: they evolve fast, often under positive selection, and they duplicate to create multigenic families. We could identify new combinations of domains, which epitomize how new trim classes appear by domain insertion or exon shuffling. Notably, we found that a cyclophilin-A domain replaces the B30.2 domain of a zebrafish fintrim gene, as reported in the macaque and owl monkey antiretroviral TRIM5α. Finally, trim genes encoding RBCC-B30.2 proteins are preferentially located in the vicinity of MHC or MHC gene paralogues, which suggests that such trim genes may have been part of the ancestral MHC.Processing of fish Ig heavy chain transcripts: diverse splicing patterns and unusual nonsense mediated decay.
USDA-ARS, Catfish Genetics Research Unit, Stoneville, MS 38776, USA.
While the diversification of the antigen-binding sites is realized by genomic VDJ rearrangements during B cell differentiation, different forms of immunoglobulin (Ig) heavy (H) chains can be produced through multiple splicing pathways. In most vertebrates, the secreted (S) and membrane (Mb) forms of IgM chain are created by alternative splicing through usage of a cryptic splice site in Cμ4 allowing the junction to the TM exon. The processing pattern for Igμ is different in teleosts, which generally use the Cμ3 donor site instead. In ancient fish lineages, multiple unusual splicing patterns were found for Ig H chain, involving donor sites that do not always follow the classical consensus. The production of IgD versus IgM H chains seems to be generally realized by alternative splicing in all vertebrates, but typical teleost IgD H chains are chimeric and contains a Cμ1 domain. Together, these observations raise questions on how different fish regulate RNA splicing and if their splicing machinery is especially complex. A preliminary scan of the zebrafish and stickleback genomes provides evidence that gene orthologs to the mammalian main splice factors are highly conserved as single copy genes, while the snRNPs U repertoire may be different and may explain other particular features of RNA processing in fish.Identification of two FoxP3 genes in rainbow trout (Oncorhynchus mykiss) with differential induction patterns.
Tiehui, T. Wang ; Milena M, MM. Monte ; Wenshu, W. Huang ; Pierre, P. Boudinot ; Samuel A M, SA. Martin ; Christopher J, CJ. Secombes.
Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Aberdeen AB24 2TZ, UK.
FoxP3 is a master transcription factor for the development and function of regulatory T cells in mammals, but little is known about this molecule in fish. Two paralogues of mammalian FoxP3 that share 83.9% identity at the amino acid level have been identified in rainbow trout (Oncorhynchus mykiss). The C-terminal region containing a Zn_C2H2 domain, a leucine zipper-like domain and a forkhead (FH) domain important for dimerization, nuclear translocation, and DNA binding, is well conserved between fish and other vertebrate FoxP3. However, the N-terminal of FoxP3 that is required for FoxP3-mediated repression of transcription is greatly diverged between fish, amphibians and monotreme mammals compared to eutherian mammals, suggesting that FoxP3 in fish, frog and platypus may have a different role to the human and mouse counterpart that defines the Treg cellular lineage and mediates the immune regulatory function. The expression of both trout (t) FoxP3a and tFoxP3b are detectable in all the 14 tissues examined without any significant difference except in muscle in which the expression of tFoxP3a was higher. Both tFoxP3a and tFoxP3b are highly expressed in thymus and in immune related organs including the spleen, kidney, gills and intestine, and are up-regulated by phytohaemagglutinin (PHA) in splenocytes and thymocytes. Whilst the up-regulated tFoxP3b expression induced by PHA was dose-dependent it required a higher PHA concentration to achieve maximal expression relative to tFoxP3a where the highest expression level was seen using 1 μg/ml PHA with higher concentrations having no further effects. In addition, the tFoxP3b expression increased during development from eyed eggs to fry, when it reached a comparable level to that of tFoxP3a. In contrast, tFoxP3a expression was at a high and almost constant level over all of the developmental stages examined. The high level of tFoxP3a expression in early development may be related to the relatively high constitutive level of tFoxP3a expression seen in muscle, perhaps suggesting novel roles of tFoxP3 in fish muscle. The structural and expression analysis suggests that the tFoxP3a and tFoxP3b are subject to differential modulation of expression and may have evolved novel functions. The identification of the two trout FoxP3 paralogues will help to clarify the existence of Treg cells and to dissect the T cell differentiation pathways in fish.In vivo analysis of Ifn-γ1 and Ifn-γ2 signaling in zebrafish.
Dina, D. Aggad ; Cornelia, C. Stein ; Dirk, D. Sieger ; Martine, M. Mazel ; Pierre, P. Boudinot ; Philippe, P. Herbomel ; Jean-Pierre, JP. Levraud ; Georges, G. Lutfalla ; Maria, M. Leptin.
Dynamique des Intéractions Membranaires Normales et Pathologiques, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5235, Montpellier, France.
The zebrafish genome contains a large number of genes encoding potential cytokine receptor genes as judged by homology to mammalian receptors. The sequences are too divergent to allow unambiguous assignments of all receptors to specific cytokines, and only a few have been assigned functions by functional studies. Among receptors for class II helical cytokines-i.e., IFNs that include virus-induced Ifns (Ifn-) and type II Ifns (Ifn-γ), together with Il-10 and its related cytokines (Il-20, Il-22, and Il-26)-only the Ifn--specific complexes have been functionally identified, whereas the receptors for the two Ifn-γ (Ifn-γ1 and Ifn-γ2) are unknown. In this work, we identify conditions in which Ifn-γ1 and Ifn-γ2 (also called IFNG or IFN-γ and IFN-gammarel) are induced in fish larvae and adults. We use morpholino-mediated loss-of-function analysis to screen candidate receptors and identify the components of their receptor complexes. We find that Ifn-γ1 and Ifn-γ2 bind to different receptor complexes. The receptor complex for Ifn-γ2 includes cytokine receptor family B (Crfb)6 together with Crfb13 and Crfb17, whereas the receptor complex for Ifn-γ1 does not include Crfb6 or Crfb13 but includes Crfb17. We also show that of the two Jak2 paralogues present in the zebrafish Jak2a but not Jak2b is involved in the intracellular transmission of the Ifn-γ signal. These results shed new light on the evolution of the Ifn-γ signaling in fish and tetrapods and contribute toward an integrated view of the innate immune regulation in vertebrates.Suppressive functions of activated B cells in autoimmune diseases reveal the dual roles of Toll-like receptors in immunity.
Vicky, V. Lampropoulou ; Elisabeth, E. Calderon-Gomez ; Toralf, T. Roch ; Patricia, P. Neves ; Ping, P. Shen ; Ulrik, U. Stervbo ; Pierre, P. Boudinot ; Stephen M, SM. Anderton ; Simon, S. Fillatreau.
Laboratory of immune regulation, Deutsches Rheuma-Forschungszentrum, Berlin, Germany.
B lymphocytes contribute to immunity through production of antibodies, antigen presentation to T cells, and secretion of cytokines. B cells are generally considered in autoimmune diseases as drivers of pathogenesis. This view is certainly justified, given the successful utilization of the B cell-depleting reagent rituximab in patients with rheumatoid arthritis or other autoimmune pathologies. In a number of cases, however, the depletion of B cells led to an exacerbation of symptoms in patients with autoimmune disorders. In a similar manner, mice lacking B cells can develop an aggravated course of disease in several autoimmune models. These paradoxical observations are now explained by the concept that activated B cells can suppress immune responses through the production of cytokines, especially interleukin-10. Here, we review the stimulatory signals that induce interleukin-10 secretion and suppressive functions in B cells and the phenotype of the B cells with such characteristics. Finally, we formulate a model explaining how this process of immune regulation by activated B cells can confer advantageous properties to the immune system in its combat with pathogens. Altogether, this review proposes that B-cell-mediated regulation is a fundamental property of the immune system, with features of great interest for the development of new cell-based therapies for autoimmune diseases.The two groups of zebrafish virus-induced interferons signal via distinct receptors with specific and shared chains.
Dina, D. Aggad ; Martine, M. Mazel ; Pierre, P. Boudinot ; Knud Erik, KE. Mogensen ; Ole Jensen, OJ. Hamming ; Rune, R. Hartmann ; Sergei, S. Kotenko ; Philippe, P. Herbomel ; Georges, G. Lutfalla ; Jean-Pierre, JP. Levraud.
Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Montpellier, France.
Because the availability of fish genomic data, the number of reported sequences for fish type II helical cytokines is rapidly growing, featuring different IFNs including virus-induced IFNs (IFNphi) and IFN-gamma, and IL-10 with its related cytokines (IL-20, IL-22, and IL-26). Many candidate receptors exist for these cytokines and various authors have postulated which receptor chain would be involved in which functional receptor in fish. To date, only the receptor for zebrafish IFNphi1 has been identified functionally. Three genes encoding virus-induced IFNphis have been reported in zebrafish. In addition to these genes clustered on chromosome 3, we have identified a fourth IFNphi gene on chromosome 12. All these genes possess the intron-exon organization of mammalian lambda IFNs. In the zebrafish larva, all induce the expression of reporter antiviral genes; protection in a viral challenge assay was observed for IFNphi1 and IFNphi2. Using a combination of gain- and loss-of-function experiments, we also show that all zebrafish IFNphis do not bind to the same receptor. Two subgroups of fish virus-induced IFNs have been defined based on conserved cysteines, and we find that this subdivision correlates with receptor usage. Both receptor complexes include a common short chain receptor (CRFB5) and a specific long chain receptor (CRFB1 or CRFB2).Mitochondrial antiviral signaling protein plays a major role in induction of the fish innate immune response against RNA and DNA viruses.
Stéphane, S. Biacchesi ; Monique, M. LeBerre ; Annie, A. Lamoureux ; Yoann, Y. Louise ; Emilie, E. Lauret ; Pierre, P. Boudinot ; Michel, M. Brémont.
INRA, CRJ, Jouy-en-Josas, France. email@example.com
Viral infection triggers host innate immune responses through cellular sensor molecules which activate multiple signaling cascades that induce the production of interferons (IFN) and other cytokines. The recent identification of mammalian cytoplasmic viral RNA sensors, such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and their mitochondrial adaptor, the mitochondrial antiviral signaling protein (MAVS), also called IPS-1, VISA, and Cardif, highlights the significance of these molecules in the induction of IFN. Teleost fish also possess a strong IFN system, but nothing is known concerning the RLRs and their downstream adaptor. In this study, we cloned MAVS cDNAs from several fish species (including salmon and zebrafish) and showed that they were orthologs of mammalian MAVS. We demonstrated that overexpression of these mitochondrial proteins in fish cells led to a constitutive induction of IFN and IFN-stimulated genes (ISGs). MAVS-overexpressing cells were almost fully protected against RNA virus infection, with a strong inhibition of both DNA and RNA virus replication (1,000- and 10,000-fold decreases, respectively). Analyses of MAVS deletion mutants showed that both the N-terminal CARD-like and C-terminal transmembrane domains, but not the central proline-rich region, were indispensable for MAVS signaling function. In addition, we cloned the cDNAs encoding a RIG-I-like molecule from salmonid and cyprinid cell lines. Like the case with MAVS, overexpression of RIG-I CARDs in fish cells led to a strong induction of both IFN and ISGs, conferring on fish cells full protection against RNA virus infection. This report provides the first demonstration that teleost fish possess a functional RLR pathway in which MAVS may play a central role in the induction of the innate immune response.The B7 family of immunoregulatory receptors: a comparative and evolutionary perspective.
John D, JD. Hansen ; Louis, L. Du Pasquier ; Marie-Paule, MP. Lefranc ; Virginie, V. Lopez ; Abdenour, A. Benmansour ; Pierre, P. Boudinot.
US Geological Survey-Western Fisheries Research Center, Seattle, WA 98115, USA. firstname.lastname@example.org
In mammals, T cell activation requires specific recognition of the peptide-MHC complex by the TcR and co-stimulatory signals. Important co-stimulatory receptors expressed by T cells are the molecules of the CD28 family, that regulate T cell activation, proliferation and tolerance. These receptors recognize B7s and B7-homologous (B7H) molecules that are typically expressed by the antigen presenting cells. In teleost fish, typical T cell responses have been described and the TcR, MHC and CD28/CTLA4 genes have been characterized. In contrast, the members of the B7 gene family have only been described in mammals and birds and have yet to be addressed in lower vertebrates. To learn more about the evolution of components guiding T cell activation in vertebrates, we performed a systematic genomic survey for the B7 co-stimulatory and co-inhibitory IgSF receptors in lower vertebrates with an emphasis on teleost fish. Our search identified fish sequences that are orthologous to B7, B7-H1/B7-DC, B7-H3 and B7-H4 as defined by sequence identity, phylogeny and combinations of short or long-range syntenic relationships. However, we were unable to identify clear orthologs for B7-H2 (CD275, ICOS ligand) in bony fish, which correlates with our prior inability to find ICOS in fish. Interestingly, our results indicate that teleost fish possess a single B7.1/B7.2 (CD80/86) molecule that likely interacts with CD28/CTLA4 as the ligand-binding regions seem to be conserved in both partners. Overall, our analyses implies that gene duplication (and loss) have shaped a molecular repertoire of B7-like molecules that was recruited for the refinement of T cell activation during the evolution of the vertebrates.New perspectives for large-scale repertoire analysis of immune receptors.
Pierre, P. Boudinot ; Maria Encarnita, ME. Marriotti-Ferrandiz ; Louis Du, LD. Pasquier ; Abdenour, A. Benmansour ; Pierre-André, PA. Cazenave ; Adrien, A. Six.
Institut National de la Recherche Agronomique Unité de Virologie et Immunologie Moléculaires 78352, Jouy-en-Josas Cedex, France. email@example.com
In vertebrates, the world of antigenic motifs is matched to large populations of lymphocytes through specific recognition of an epitope by a given receptor unique to a lymphocyte clone. The concept of immune repertoire was proposed to describe this diversity of lymphocyte receptors - Ig and TCR - required by the network of interactions. The immune repertoires became useful tools to describe lymphocyte and receptor populations through the development of the immune system and in pathological situations. Recently, the development of mass technologies made possible a comprehensive survey of immune repertoires at the genome, transcript and protein levels, and some of these techniques have been already adapted to TCR and Ig repertoire analyses. Such approaches generate very big datasets, which necessitates complex and multi-parametric annotations in dedicated databases. They also require new analysis methods, leading to the integration of structure and dynamics of the immune repertoires, at different time scales (immune response, development of the individual, evolution of the species). Such methods may be extended to the analysis of new classes of adaptive-like receptors, which were recently discovered in different invertebrates and in agnathans. Ultimately, they may allow a parallel monitoring of pathogen and immune repertoires addressing the reciprocal influences that decide for the host survival or death. In this review, we first study the characteristics of Ig and TCR repertoires, and we examine several systematic approaches developed for the analysis of these "classical" immune repertoires at different levels. We then consider examples of the recent developments of modeling and statistical analysis, and we discuss their relevance and their importance for the study of the immune diversity. An extended view of immune repertoires is proposed, integrating the diversity of other receptors involved in immune recognition. Also, we discuss how repertoire studies could link pathogen variation and immune diversity to reveal regulatory patterns and rules driving their co-diversification race.Genome and polypeptides characterization of Tellina virus 1 reveals a fifth genetic cluster in the Birnaviridae family.
Isabelle, I. Nobiron ; Marie, M. Galloux ; Celine, C. Henry ; Corinne, C. Torhy ; Pierre, P. Boudinot ; Nathalie, N. Lejal ; Bruno, B. Da Costa ; Bernard, B. Delmas.
INRA, Unité de Virologie et Immunologie moléculaires UR892, Domaine de Vilvert, 78350 Jouy-en-Josas, France.
We characterized tellina virus 1 (TV-1), a birnavirus isolated from the marine bivalve mollusk Tellina tenuis. Genome sequence analysis established that TV-1 is representative of a viral cluster distant from other birnaviruses. The maturation process of the polyprotein encoded by the genomic segment A was delineated with the identification of the N-termini of the viral protease VP4 and the ribonucleoprotein VP3, and the characterization of peptides deriving from the processing of pVP2, the VP2 capsid protein precursor. One of these peptides was shown to possess a membrane-disrupting domain. Like the blotched snakehead virus, the polyprotein exhibits a non-structural polypeptide (named [X]) located between pVP2 and VP4. Mutagenesis analysis allowed the identification in VP4 of a catalytic Ser-Lys dyad that does not possess the common Gly-X-Ser signature of the serine hydrolases. The genomic segment B encodes the viral RNA-dependent RNA-polymerase VP1 with the unique sequence motif arrangement identified in other birnavirus VP1s.Wide range of susceptibility to rhabdoviruses in homozygous clones of rainbow trout.
Edwige, E. Quillet ; Michel, M. Dorson ; Sandrine, S. Le Guillou ; Abdenour, A. Benmansour ; Pierre, P. Boudinot.
Unité de Génétique des Poissons, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas, France. firstname.lastname@example.org
Inbred lines differentially susceptible to diseases are a powerful tool to get insights into the mechanisms of genetic resistance to pathogens. In fish, chromosome manipulation techniques allow a quick production of such homozygous lines. Using gynogenesis, we produced nine homozygous clones of rainbow trout from a domestic population (INRA Sy strain). We examined the variability between clones for resistance to two rhabdoviruses, the viral haemorrhagic septicaemia virus (VHSV) and the infectious haematopoietic necrosis virus (IHNV). Intraperitoneal injections and waterborne infections were performed in parallel for both viruses. No survival was recorded after intraperitoneal injection of VHSV or IHNV, indicating that fish from all clones were fully susceptible to both viruses by this route of infection. In contrast, the different clones showed a wide range of survival frequency after waterborne infection. The resistance levels to VHSV ranged from 0 to 99% and resistance was not abrogated when resistant and sensitive animals were mixed and subjected to waterborne infection. VHSV was recovered from 10% of resistant fish after waterborne infection, confirming that virus replication was possible in this context but effective only in a low proportion of the population. The different clones also exhibited a wide range of survival (0-68%) after a waterborne infection with IHNV. Although VHSV-resistant clones were not fully resistant to IHNV, the susceptibility to IHNV and VHSV tended to be correlated, suggesting that non-specific mechanisms common to both viruses were involved.Costimulatory receptors in jawed vertebrates: conserved CD28, odd CTLA4 and multiple BTLAs.
David, D. Bernard ; John D, JD. Hansen ; Louis, L. Du Pasquier ; Marie-Paule, MP. Lefranc ; Abdenour, A. Benmansour ; Pierre, P. Boudinot.
Institut National de la Recherche Agronomique, Unité de Virologie et Immunologie Moléculaires, 78352 Jouy-en-Josas cedex, France.
CD28 family of costimulatory receptors is comprised of molecules with a single V-type extracellular Ig domain, a transmembrane and an intracytoplasmic region with signaling motifs. CD28 and cytotoxic T lymphocyte antigen-4 (CTLA4) homologs have been recently identified in rainbow trout. Other sequences similar to mammalian CD28 family members have now been identified using teleost, Xenopus and chicken databases. CD28- and CTLA4 homologs were found in all vertebrate classes whereas inducible costimulatory signal (ICOS) was restricted to tetrapods, and programmed cell death-1 (PD1) was limited to mammals and chicken. Multiple B and T Lymphocyte Attenuator (BTLA) sequences were found in teleosts, but not in Xenopus or in avian genomes. The intron/exon structure of btlas was different from that of cd28 and other members of the family. The Ig domain encoded in all the btla genes has features of the C-type structure, which suggests that BTLA does not belong to the CD28 family. The genomic localization of these genes in vertebrate genomes supports the split between the BTLA and CD28 families.Costimulatory receptors in a teleost fish: typical CD28, elusive CTLA4.
David, D. Bernard ; Béatrice, B. Riteau ; John D, JD. Hansen ; Ruth B, RB. Phillips ; Frédérique, F. Michel ; Pierre, P. Boudinot ; Abdenour, A. Benmansour.
Institut National de la Recherche Agronomique, Unité de Virologie et Immunologie Moléculaires, Jouy-en-Josas, France.
T cell activation requires both specific recognition of the peptide-MHC complex by the TCR and additional signals delivered by costimulatory receptors. We have identified rainbow trout sequences similar to CD28 (rbtCD28) and CTLA4 (rbtCTLA4). rbtCD28 and rbtCTLA4 are composed of an extracellular Ig-superfamily V domain, a transmembrane region, and a cytoplasmic tail. The presence of a conserved ligand binding site within the V domain of both molecules suggests that these receptors likely recognize the fish homologues of the B7 family. The mRNA expression pattern of rbtCD28 and rbtCTLA4 in naive trout is reminiscent to that reported in humans and mice, because rbtCTLA4 expression within trout leukocytes was quickly up-regulated following PHA stimulation and virus infection. The cytoplasmic tail of rbtCD28 possesses a typical motif that is conserved in mammalian costimulatory receptors for signaling purposes. A chimeric receptor made of the extracellular domain of human CD28 fused to the cytoplasmic tail of rbtCD28 promoted TCR-induced IL-2 production in a human T cell line, indicating that rbtCD28 is indeed a positive costimulator. The cytoplasmic tail of rbtCTLA4 lacked obvious signaling motifs and accordingly failed to signal when fused to the huCD28 extracellular domain. Interestingly, rbtCTLA4 and rbtCD28 are not positioned on the same chromosome and thus do not belong to a unique costimulatory cluster as in mammals. Finally, our results raise questions about the origin and evolution of positive and negative costimulation in vertebrate immune systems.The glycoprotein of a fish rhabdovirus profiles the virus-specific T-cell repertoire in rainbow trout.
Pierre, P. Boudinot ; David, D. Bernard ; Samira, S. Boubekeur ; Maria-Isabel, MI. Thoulouze ; Michel, M. Bremont ; Abdenour, A. Benmansour.
Institut National de la Recherche Agronomique, Unité de Virologie et Immunologie Moléculaires, 78352 Jouy-en-Josas, France.
T-cell responses to viruses are still poorly investigated in lower vertebrates. In rainbow trout, a specific clonal expansion of T cells in response to infection with viral haemorrhagic septicaemia virus (VHSV) was recently identified. Expanded T-cell clones expressed a unique 8 aa Vbeta4-Jbeta1 junction (SSGDSYSE) in different individuals, reminiscent of a typical public response. To get further insight into the nature of this response the modifications of the T-cell repertoire following immunization with plasmid expressing the VHSV external glycoprotein (G), which is the only protein involved in protective immunity, was analysed. After G-based DNA immunization, CDR3-length spectratypes were skewed for several Vbeta-Jbeta combinations, including Vbeta4-Jbeta1. In Vbeta4-Jbeta1, biases consisted of 6 and 8 aa junctions that were detected from day 52, and were still present 3 months after DNA immunization. Sequence analysis of the Vbeta4-Jbeta1 junctions showed that the 8 aa junction (SSGDSYSE) was clearly expanded, indicating that viral G protein was probably the target of the anti-VHSV public response. Additional 6 and 8 aa Vbeta4-Jbeta1 junctions were also expanded in G-DNA-vaccinated fish, showing that significant clonotypic diversity was selected in response to the plasmid-delivered G protein. This higher clonotypic diversity may be related to the demonstrated higher efficiency of G-based DNA vaccines over whole virus immunization. The use of infectious hematopietic necrosis virus (IHNV) recombinant viruses, expressing the VHSV G protein, further substantiated the VHSV G-protein specificity of the 8 aa Vbeta4-Jbeta1 response and designated the 6 aa Vbeta4-Jbeta1 response as potentially directed to a T-cell epitope common to VHSV and IHNV.An Mx1 promoter-reporter system to study interferon pathways in rainbow trout.
Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen AB24 2TZ, Scotland, UK.
A rainbow trout interferon (IFN) reporter system has been established by selection of a stable cell line, RTG-P1, transfected with a plasmid expressing the firefly luciferase gene under the control of the promoter for the IFN-induced gene Mx1. After 148 passages, the luciferase expression was still highly induced by polyinosinic:polycytidylic acid (poly I:C) in RTG-P1 cells. Different IFN inducers (dsRNA, viral hemorrhagic septicaemia virus or conditioned medium containing rainbow trout antiviral activity) were able to stimulate the IFN-reporter system in RTG-P1, showing that this cell line can be used to study the activation of the IFN pathway in various contexts. Pyrrolidine dithiocarbamate (PDTC), an NF-kappaB inhibitor, significantly blocked poly I:C induced luciferase accumulation in RTG-P1 at intermediate doses (1-10 microM), suggesting that Mx1 induction through the IFN signalling pathway is NF-kappaB-dependent in fish. This inhibition was not observed for doses of 50 microM or higher. The RTG-P1 reporter system constitutes an interesting tool to study the induction and regulation of IFN signalling in teleost fish.
Mechanical and non-mechanical functions of Dystrophin can prevent cardiac abnormalities in Drosophila.
Development and Aging Program, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Rd, Building 7 Room 7125, La Jolla, CA 92037, USA; GReD, INSERM U1103, CNRS UMR6293-Clermont University, Faculty of Medicine 28, Place Henri Dunant, 63000
Dystrophin-deficiency causes cardiomyopathies and shortens the life expectancy of Duchenne and Becker muscular dystrophy patients. Restoring Dystrophin expression in the heart by gene transfer is a promising avenue to explore as a therapy. Truncated Dystrophin gene constructs have been engineered and shown to alleviate dystrophic skeletal muscle disease, but their potential in preventing the development of cardiomyopathy is not fully understood. In the present study, we found that either the mechanical or the signaling functions of Dystrophin were able to reduce the dilated heart phenotype of Dystrophin mutants in a Drosophila model. Our data suggest that Dystrophin retains some function in fly cardiomyocytes in the absence of a predicted mechanical link to the cytoskeleton. Interestingly, cardiac-specific manipulation of nitric oxide synthase expression also modulates cardiac function, which can in part be reversed by loss of Dystrophin function, further implying a signaling role of Dystrophin in the heart. These findings suggest that the signaling functions of Dystrophin protein are able to ameliorate the dilated cardiomyopathy, and thus might help to improve heart muscle function in micro-Dystrophin-based gene therapy approaches.Tailup plays multiple roles during cardiac outflow assembly in Drosophila.
The Drosophila LIM-homeodomain transcription factor Tailup and its vertebrate counterpart Islet1 are expressed in cardiac progenitor cells where they play a specification role. Loss of function of Islet1 leads to a complete absence of the right ventricle and affects the development of the cardiac outflow tract in mouse embryos. Similarly, tailup mutant embryos display a reduced number of cardiac cells but the role of tailup in cardiac outflow formation in Drosophila remains unknown. Here, we show that tailup is expressed in the main Drosophila cardiac outflow components, i.e., heart anchoring cells (HANC) and cardiac outflow muscles (COM) and that loss of its function and/or tissue-specific knockdowns dramatically affect cardiac outflow morphogenesis. Our data demonstrate that tailup plays many roles and is required for the acquisition of HANC and COM properties. We also show that tailup regulates HANC motility, COM shapes and their attachment to the heart tip and genetically interacts with ladybird, shotgun and slit, which are known to be involved in cardiac outflow assembly. Furthemore, using tissue-specific overexpression of dominant negative tailup constructs lacking sequences encoding either the homeodomain or the LIM domain, we demonstrate that tailup can exert its function not only in transcription factor mode but also via its protein-protein interaction domain. We identify Tailup as an evolutionarily-conserved regulator of cardiac outflow formation and provide further evidence for its conserved role in heart development.Glycolysis supports embryonic muscle growth by promoting myoblast fusion.
Vanessa, V. Tixier ; Laetitia, L. Bataillé ; Christelle, C. Etard ; Teresa, T. Jagla ; Meltem, M. Weger ; Jean Philippe, JP. Daponte ; Uwe, U. Strähle ; Thomas, T. Dickmeis ; Krzysztof, K. Jagla.
Unité de Génétique, Reproduction et Développement, Institut National de la Santé et de la Recherche Médicale U1103, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6293, University of Clermont-Ferrand, 63001 Clermont-Ferrand, France.
Muscles ensure locomotion behavior of invertebrate and vertebrate organisms. They are highly specialized and form using conserved developmental programs. To identify new players in muscle development we screened Drosophila and zebrafish gene expression databases for orthologous genes expressed in embryonic muscles. We selected more than 100 candidates. Among them is the glycolysis gene Pglym78/pgam2, the attenuated expression of which results in the formation of thinner muscles in Drosophila embryos. This phenotype is also observed in fast muscle fibers of pgam2 zebrafish morphants, suggesting affected myoblast fusion. Indeed, a detailed analysis of developing muscles in Pglym78 RNAi embryos reveals loss of fusion-associated actin foci and an inefficient Notch decay in fusion competent myoblasts, both known to be required for fusion. In addition to Pglym78, our screen identifies six other genes involved in glycolysis or in pyruvate metabolism (Pfk, Tpi, Gapdh, Pgk, Pyk, and Impl3). They are synchronously activated in embryonic muscles and attenuation of their expression leads to similar muscle phenotypes, which are characterized by fibers with reduced size and the presence of unfused myoblasts. Our data also show that the cell size triggering insulin pathway positively regulates glycolysis in developing muscles and that blocking the insulin or target of rapamycin pathways phenocopies the loss of function phenotypes of glycolytic genes, leading to myoblast fusion arrest and reduced muscle size. Collectively, these data suggest that setting metabolism to glycolysis-stimulated biomass production is part of a core myogenic program that operates in both invertebrate and vertebrate embryos and promotes formation of syncytial muscles.Novel Drosophila model of myotonic dystrophy type 1: phenotypic characterization and genome-wide view of altered gene expression.
Lucie, L. Picchio ; Emilie, E. Plantie ; Yoan, Y. Renaud ; Preethi, P. Poovthumkadavil ; Krzysztof, K. Jagla.
GReD Genetics, Reproduction and Development laboratory, INSERM U1103, CNRS UMR6293, University of Clermont-Ferrand, 28 place Henri Dunant, 63000 Clermont-Ferrand, France.
Myotonic dystrophy type 1 (DM1) is a multisystemic RNA-dominant disorder characterized by myotonia and muscle degeneration. In DM1 patients, the mutant DMPK transcripts containing expanded CUG repeats form nuclear foci and sequester the Muscleblind-like 1 splicing factor, resulting in mis-splicing of its targets. However, several pathological defects observed in DM1 and their link with disease progression remain poorly understood. In an attempt to fill this gap, we generated inducible transgenic Drosophila lines with increasing number of CTG repeats. Targeting the expression of these repeats to the larval muscles recapitulated in a repeat-size-dependent manner the major DM1 symptoms such as muscle hypercontraction, splitting of muscle fibers, reduced fiber size or myoblast fusion defects. Comparative transcriptional profiling performed on the generated DM1 lines and on the muscleblind (mbl)-RNAi line revealed that nuclear accumulation of toxic CUG repeats can affect gene expression independently of splicing or Mbl sequestration. Also, in mblRNAi contexts, the largest portion of deregulated genes corresponded to single-transcript genes, revealing an unexpected impact of the indirect influence of mbl on gene expression. Among the single-transcript Mbl targets is Muscle protein 20 involved in myoblast fusion and causing the reduced number of nuclei in muscles of mblRNAi larvae. Finally, by combining in silico prediction of Mbl targets with mblRNAi microarray data, we found the calcium pump dSERCA as a Mbl splice target and show that the membrane dSERCA isoform is sufficient to rescue a DM1-induced hypercontraction phenotype in a Drosophila model.Diversification of muscle types in Drosophila: upstream and downstream of identity genes.
GReD INSERM UMR1103, CNRS UMR6293, University of Clermont-Ferrand, Clermont-Ferrand, France.
Understanding gene regulatory pathways underlying diversification of cell types during development is one of the major challenges in developmental biology. Progressive specification of mesodermal lineages that are at the origin of body wall muscles in Drosophila embryos has been extensively studied during past years, providing an attractive framework for dissecting cell type diversification processes. In particular, it has been found that muscle founder cells that are at the origin of individual muscles display specific expression of transcription factors that control diversification of muscle types. These factors, encoded by genes collectively called muscle identity genes, are activated in discrete subsets of muscle founders. As a result, each founder cell is thought to carry a unique combinatorial code of identity gene expression. Considering this, to define temporally and spatially restricted expression of identity genes, a set of coordinated upstream regulatory inputs is required. But also, to realize the identity program and to form specific muscle types with distinct properties, an efficient battery of downstream identity gene targets needs to be activated. Here we review how the specificity of expression and action of muscle identity genes is acquired.ChIP-enriched in silico targets (ChEST), a ChIP-on-chip approach applied to analyzing skeletal muscle genes.
GReD, INSERM U931, CNRS UMR6247, Faculté de Medecine, Clermont University, Clermont-Ferrand, France.
Mapping the cis-regulatory modules (CRMs) to which bind myogenic transcription factors is an -obligatory step towards understanding gene regulatory networks governing muscle development and function. This can be achieved in silico or by chromatin immunoprecipitation (ChIP) approaches. We have developed a ChIP-enriched in silico targets (ChEST) strategy designed for mapping the CRMs by combining in silico and ChIP methods. ChEST involves a software-assisted prediction of transcription factor (TF) - specific CRMs, which are spotted to produce a computed genomic CRM microarray. In parallel, the in vivo pool of targets of a given TF is isolated by ChIP and used as a probe for hybridization with the array generated. Here we describe ChEST strategy applied to identify direct targets of Myogenic Enhancer Factor, Dmef2 in Drosophila embryos.Specification and behavior of AMPs, muscle-committed transient Drosophila stem cells.
Nicolas, N. Figeac ; Teresa, T. Jagla ; Rajaguru, R. Aradhya ; Jean Philippe, JP. Da Ponte ; Krzysztof, K. Jagla.
GReD, INSERM U931, CNRS UMR6247, Clermont University, Clermont-Ferrand, France.
During development, transient stem cells play critical roles in the formation of specific tissues. Adult Muscle Precursors (AMPs) are at the origin of all adult Drosophila muscles and as we report here represent a novel population of muscle-committed transient stem cells. Similar to vertebrate muscle stem cells, AMPs keep Notch signaling active and express Enhancer of split m6 (E(spl)m6) gene, a read-out of Notch pathway. To get insights into AMP cell specification we performed a gain-of-function screen and found that the rhomboid-triggered Epidermal Growth Factor (EGF) signaling pathway controls both the specification and the subsequent maintenance of AMPs. Our findings are supported by the identification of EGF-secreting cells in the lateral domain and the EGF-dependent regulatory modules that drive expression of the ladybird gene in lateral AMPs. Interestingly, by targeting GFP to the AMP cell membranes we also demonstrated that AMPs send long cellular processes and form a network of interconnected cells. As revealed by laser ablation experiments, the main role of AMP cell connections is to maintain their correct spatial positioning.Regulation and functions of the lms homeobox gene during development of embryonic lateral transverse muscles and direct flight muscles in Drosophila.
Dominik, D. Müller ; Teresa, T. Jagla ; Ludivine Mihaila, LM. Bodart ; Nina, N. Jährling ; Hans-Ulrich, HU. Dodt ; Krzysztof, K. Jagla ; Manfred, M. Frasch.
Division of Developmental Biology, Department of Biology, University of Erlangen-Nuremberg, Erlangen, Germany.
Diversification of muscle types: recent insights from Drosophila.
BACKGROUND:Patterning and differentiation of developing musculatures require elaborate networks of transcriptional regulation. In Drosophila, significant progress has been made into identifying the regulators of muscle development and defining their interactive networks. One major family of transcription factors involved in these processes consists of homeodomain proteins. In flies, several members of this family serve as muscle identity genes to specify the fates of individual muscles, or groups thereof, during embryonic and/or adult muscle development. Herein, we report on the expression and function of a new Drosophila homeobox gene during both embryonic and adult muscle development.
GReD, INSERM U931, CNRS UMR6247, Clermont University, Faculty of Medicine, 28 place Henri Dunant, Clermont-Ferrand, France.
Myogenesis is a highly conserved process ending up by the formation of contracting muscles. In Drosophila embryos, myogenesis gives rise to a segmentally repeated array of thirty distinct fibres, each of which represents an individual muscle. Since Drosophila offers a large range of genetic tools for easily testing gene functions, it has become one of the most studied and consequently best-described model organisms for muscle development. Over the last two decades, the Drosophila model system has enabled major advances in our understanding of how the initially equivalent mesodermal cells become competent for entering myogenic differentiation and how each distinct type of muscle is specified. Here we present an overview of Drosophila muscle development with a special focus on the diversification of muscle types and the genes that control acquisition of distinct muscle properties.Downstream of identity genes: muscle-type-specific regulation of the fusion process.
Laetitia, L. Bataillé ; Isabelle, I. Delon ; Jean Philippe, JP. Da Ponte ; Nicholas H, NH. Brown ; Krzysztof, K. Jagla.
GReD, INSERM U, CNRS UMR, University of Clermont-Ferrand, France.
In all metazoan organisms, the diversification of cell types involves determination of cell fates and subsequent execution of specific differentiation programs. During Drosophila myogenesis, identity genes specify the fates of founder myoblasts, from which derive all individual larval muscles. Here, to understand how cell fate information residing within founders is translated during differentiation, we focus on three identity genes, eve, lb, and slou, and how they control the size of individual muscles by regulating the number of fusion events. They achieve this by setting expression levels of Mp20, Pax, and mspo, three genes that regulate actin dynamics and cell adhesion and, as we show here, modulate the fusion process in a muscle-specific manner. Thus, these data show how the identity information implemented by transcription factors is translated via target genes into cell-type-specific programs of differentiation.Drosophila adult muscle precursors form a network of interconnected cells and are specified by the rhomboid-triggered EGF pathway.
Nicolas, N. Figeac ; Teresa, T. Jagla ; Rajaguru, R. Aradhya ; Jean Philippe, JP. Da Ponte ; Krzysztof, K. Jagla.
GReD, INSERM U931, CNRS UMR6247, Clermont University, Faculté de Médecine, 28 Place Henri Dunant, Clermont-Ferrand, 63000, France.
In Drosophila, a population of muscle-committed stem-like cells called adult muscle precursors (AMPs) keeps an undifferentiated and quiescent state during embryonic life. The embryonic AMPs are at the origin of all adult fly muscles and, as we demonstrate here, they express repressors of myogenic differentiation and targets of the Notch pathway known to be involved in muscle cell stemness. By targeting GFP to the AMP cell membranes, we show that AMPs are tightly associated with the peripheral nervous system and with a subset of differentiated muscles. They send long cellular processes running along the peripheral nerves and, by the end of embryogenesis, form a network of interconnected cells. Based on evidence from laser ablation experiments, the main role of these cellular extensions is to maintain correct spatial positioning of AMPs. To gain insights into mechanisms that lead to AMP cell specification, we performed a gain-of-function screen with a special focus on lateral AMPs expressing the homeobox gene ladybird. Our data show that the rhomboid-triggered EGF signalling pathway controls both the specification and the subsequent maintenance of AMP cells. This finding is supported by the identification of EGF-secreting cells in the lateral domain and the EGF-dependent regulatory modules that drive expression of the ladybird gene in lateral AMPs. Taken together, our results reveal an unsuspected capacity of embryonic AMPs to form a cell network, and shed light on the mechanisms governing their specification and maintenance.Muscle development and regeneration in normal and pathological conditions: learning from Drosophila.
Malgorzata, M. Daczewska ; Lucie, L. Picchio ; Teresa, T. Jagla ; Nicolas, N. Figeac ; Krzysztof, K. Jagla.
Zoology Dept, University of Wroclaw, 21, Sienkiewicza Street, 50-335 Wroclaw, Poland.
The recent demonstration that, throughout evolution, many molecular mechanisms have been highly conserved is fundamental to the advancement of our knowledge on muscle development and regeneration. Research has provided new insights into genetic cascades governing early steps of embryonic myogenesis and the regeneration of adult muscle in normal and pathological conditions, thus revealing significant similarity of both processes. Here we provide a current view on genetic mechanisms underlying muscle regeneration with a special focus on regeneration processes that take place in diseased and aging human muscle. Through examples of Drosophila models of human muscular diseases, we discuss potential impact they might have on uncovering molecular bases and identifying new treatments of muscle disorders. Taking advantage of evolutionarily conserved aspects of muscle development and the relative ease by which molecular pathways can be uncovered and dissected in a simple animal model, the fruit fly, we provide a comprehensive analysis of muscle development in Drosophila. Importantly, identification of muscle stem cell like adult muscle precursors in Drosophila makes fruit fly an attractive model system for studying muscle stem cell biology and muscle regeneration. In support of this assumption, recent studies in our laboratory provide arguments that important insights into the biology of vertebrate muscle stem cells can be gained from genetic analysis in Drosophila.Neprilysin 4, a novel endopeptidase from Drosophila melanogaster, displays distinct substrate specificities and exceptional solubility states.
Heiko, H. Meyer ; Mareike, M. Panz ; Monika, M. Zmojdzian ; Krzysztof, K. Jagla ; Achim, A. Paululat.
Department of Zoology/Developmental Biology, University of Osnabrück, Germany.
Proteins belonging to the family of neprilysins are typically membrane bound M13 endopeptidases responsible for the inactivation and/or activation of peptide signaling events on cell surfaces. Mammalian neprilysins are known to be involved in the metabolism of various regulatory peptides especially in the nervous, immune, cardiovascular and inflammatory systems. Although there is still much to learn about their participation in various diseases, they are potential therapeutic targets. Here we report on the identification and first characterization of neprilysin 4 (NEP4) from Drosophila melanogaster. Reporter lines as well as in situ hybridization combined with immunolocalization demonstrated NEP4 expression during embryogenesis in pericardial cells, muscle founder cells, glia cells and male gonads. Western blot analysis confirmed the prediction of one membrane bound and one soluble isoform, a finding quite unusual among neprilysins with presumably strong physiological relevance. At least one NEP4 isoform was found in every developmental stage indicating protein activities required throughout the whole life cycle of Drosophila. Heterologously expressed NEP4 exhibited substrate preferences comparable to human neprilysin 2 with distinct cleavage of substance P and angiotensin I.Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation.
Caroline, C. Medioni ; Martine, M. Astier ; Monika, M. Zmojdzian ; Krzysztof, K. Jagla ; Michel, M. Sémériva.
Institut de Biologie du Développement de Marseille-Luminy, Centre National de la Recherche Scientifique UMR 6216, Université de la Méditerranée, 13288 Marseille, Cedex 9, France.
Tubulogenesis is an essential component of organ development, yet the underlying cellular mechanisms are poorly understood. We analyze here the formation of the Drosophila melanogaster cardiac lumen that arises from the migration and subsequent coalescence of bilateral rows of cardioblasts. Our study of cell behavior using three-dimensional and time-lapse imaging and the distribution of cell polarity markers reveals a new mechanism of tubulogenesis in which repulsion of prepatterned luminal domains with basal membrane properties and cell shape remodeling constitute the main driving forces. Furthermore, we identify a genetic pathway in which roundabout, slit, held out wings, and dystroglycan control cardiac lumen formation by establishing nonadherent luminal membranes and regulating cell shape changes. From these data we propose a model for D. melanogaster cardiac lumen formation, which differs, both at a cellular and molecular level, from current models of epithelial tubulogenesis. We suggest that this new example of tube formation may be helpful in studying vertebrate heart tube formation and primary vasculogenesis.Genetic control of muscle development: learning from Drosophila.
INSERM U384, 28, place Henri Dunant, 63000 Clermont-Ferrand, France.
Muscle development involves a complex sequence of time and spatially regulated cellular events leading to the formation of highly specialised syncytial muscle cells displaying a common feature, the capacity of contraction. Analyses of mechanisms controlling muscle development reveals that the main steps of muscle formation including myogenic determination, diversification of muscle precursors, myoblast fusion and terminal differentiation involve the actions of evolutionarily conserved genes. Thus dissecting the genetic control of muscle development in simple model organisms appears to be an attractive way to get insights into core genetic cascade that orchestrate myogenesis. In this respect, particularly insightful have been data generated using Drosophila as a model system. Notably, the interplay between intrinsic and extrinsic cues that determine the early myogenic decisions leading to the specification of muscle progenitors and those controlling myoblasts fusion are much better characterised in Drosophila than in vertebrate species. Also, adult Drosophila myogenesis, which leads to the formation of vertebrate-like multi-fibre muscles, emerges as a particularly well-adapted system to study normal and aberrant muscle development.Cellular components and signals required for the cardiac outflow tract assembly in Drosophila.
Unité Mixte de Recherche, Centre National de la Recherche Scientifique 6247-GreD, Clermont-Ferrand University, Institut National de la Santé et de la Recherche Médicale Clermont-Ferrand, Clermont-Ferrand, France.
Specification of cardiac primordia and formation of the Drosophila heart tube is highly reminiscent of the early steps of vertebrate heart development. We previously reported that the final morphogenesis of the Drosophila heart involves a group of nonmesodermal cells called heart-anchoring cells and a pair of derived from the pharyngeal mesoderm cardiac outflow muscles. Like the vertebrate cardiac neural crest cells, heart-anchoring cells migrate, interact with the tip of the heart, and participate in shaping the cardiac outflow tract. To better understand this process, we performed an in-depth analysis of how the Drosophila outflow tract is formed. We found that the most anterior cardioblasts that form a central outflow tract component, the funnel-shaped heart tip, do not originate from the cardiac primordium. They are initially associated with the pharyngeal cardiac outflow muscles and join the anterior aorta during outflow tract assembly. The particular morphology of the heart tip is disrupted in embryos in which heart-anchoring cells were ablated, revealing their critical role in outflow tract morphogenesis. We also demonstrate that Slit and Robo are required for directed movements of heart-anchoring cells toward the heart tip and that the cell-cell contact between the heart-anchoring cells and the ladybird-expressing cardioblasts is critically dependent on DE-cadherin Shotgun. Our observations suggest that the similarities between Drosophila and vertebrate cardiogenesis extend beyond the early developmental events.Genome-wide view of cell fate specification: ladybird acts at multiple levels during diversification of muscle and heart precursors.
Guillaume, G. Junion ; Laetitia, L. Bataillé ; Teresa, T. Jagla ; Jean Philippe, JP. Da Ponte ; Romain, R. Tapin ; Krzysztof, K. Jagla.
Institut National de la Santé et de la Recherche Médicale U384, 63000 Clermont-Ferrand, France.
Correct diversification of cell types during development ensures the formation of functional organs. The evolutionarily conserved homeobox genes from ladybird/Lbx family were found to act as cell identity genes in a number of embryonic tissues. A prior genetic analysis showed that during Drosophila muscle and heart development ladybird is required for the specification of a subset of muscular and cardiac precursors. To learn how ladybird genes exert their cell identity functions we performed muscle and heart-targeted genome-wide transcriptional profiling and a chromatin immunoprecipitation (ChIP)-on-chip search for direct Ladybird targets. Our data reveal that ladybird not only contributes to the combinatorial code of transcription factors specifying the identity of muscle and cardiac precursors, but also regulates a large number of genes involved in setting cell shape, adhesion, and motility. Among direct ladybird targets, we identified bric-a-brac 2 gene as a new component of identity code and inflated encoding alphaPS2-integrin playing a pivotal role in cell-cell interactions. Unexpectedly, ladybird also contributes to the regulation of terminal differentiation genes encoding structural muscle proteins or contributing to muscle contractility. Thus, the identity gene-governed diversification of cell types is a multistep process involving the transcriptional control of genes determining both morphological and functional properties of cells.Muscle stem cells and model systems for their investigation.
INSERM U384, Clermont-Ferrand, France.
Stem cells are characterized by their clonal ability both to generate differentiated progeny and to undergo self-renewal. Studies of adult mammalian organs have revealed stem cells in practically every tissue. In the adult skeletal muscle, satellite cells are the primary muscle stem cells, responsible for postnatal muscle growth, hypertrophy, and regeneration. In the past decade, several molecular markers have been found that identify satellite cells in quiescent and activated states. However, despite their prime importance, surprisingly little is known about the biology of satellite cells, as their analysis was for a long time hampered by a lack of genetically amenable experimental models where their properties can be dissected. Here, we review how the embryonic origin of satellite cells was discovered using chick and mouse model systems and discuss how cells from other sources can contribute to muscle regeneration. We present evidence for evolutionarily conserved properties of muscle stem cells and their identification in lower vertebrates and in the fruit fly. In Drosophila, muscle stem cells called adult muscle precursors (AMP) can be identified in embryos and in larvae by persistent expression of a myogenic basic helix-loop-helix factor Twist. AMP cells play a crucial role in the Drosophila life cycle, allowing de novo formation and regeneration of adult musculature during metamorphosis. Based on the premise that AMPs represent satellite-like cells of the fruit fly, important insight into the biology of vertebrate muscle stem cells can be gained from genetic analysis in Drosophila.Shaping leg muscles in Drosophila: role of ladybird, a conserved regulator of appendicular myogenesis.
Tariq, T. Maqbool ; Cedric, C. Soler ; Teresa, T. Jagla ; Malgorzata, M. Daczewska ; Neha, N. Lodha ; Sudhir, S. Palliyil ; K, K. VijayRaghavan ; Krzysztof, K. Jagla.
Institut National de la Santé et de la Recherche Médicale U384, Faculté de Medecine, Clermont-Ferrand, France.
Legs are locomotor appendages used by a variety of evolutionarily distant vertebrates and invertebrates. The primary biological leg function, locomotion, requires the formation of a specialised appendicular musculature. Here we report evidence that ladybird, an orthologue of the Lbx1 gene recognised as a hallmark of appendicular myogenesis in vertebrates, is expressed in leg myoblasts, and regulates the shape, ultrastructure and functional properties of leg muscles in Drosophila. Ladybird expression is progressively activated in myoblasts associated with the imaginal leg disc and precedes that of the founder cell marker dumbfounded. The RNAi-mediated attenuation of ladybird expression alters properties of developing myotubes, impairing their ability to grow and interact with the internal tendons and epithelial attachment sites. It also affects sarcomeric ultrastructure, resulting in reduced leg muscle performance and impaired mobility in surviving flies. The over-expression of ladybird also results in an abnormal pattern of dorsally located leg muscles, indicating different requirements for ladybird in dorsal versus ventral muscles. This differential effect is consistent with the higher level of Ladybird in ventrally located myoblasts and with positive ladybird regulation by extrinsic Wingless signalling from the ventral epithelium. In addition, ladybird expression correlates with that of FGF receptor Heartless and the read-out of FGF signalling downstream of FGF. FGF signals regulate the number of leg disc associated myoblasts and are able to accelerate myogenic differentiation by activating ladybird, leading to ectopic muscle fibre formation. A key role for ladybird in leg myogenesis is further supported by its capacity to repress vestigial and to down-regulate the vestigial-governed flight muscle developmental programme. Thus in Drosophila like in vertebrates, appendicular muscles develop from a specialised pool of myoblasts expressing ladybird/Lbx1. The ladybird/Lbx1 gene family appears as a part of an ancient genetic circuitry determining leg-specific properties of myoblasts and making an appendage adapted for locomotion.Hedgehog and RAS pathways cooperate in the anterior-posterior specification and positioning of cardiac progenitor cells.
Jiandong, J. Liu ; Li, L. Qian ; Robert J, RJ. Wessells ; Yannick, Y. Bidet ; Krzysztof, K. Jagla ; Rolf, R. Bodmer.
The Burnham Institute, Center for Neurosciences and Aging, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
The Drosophila heart is a highly ordered structure with only a limited number of cell types, which are arranged in a stereotyped metameric pattern. Ras signaling has previously been implicated in contributing to heart formation, but how positional information is integrated with this pathway to specify, distinguish and precisely position individual cardiac progenitors within the presumptive heart-forming region are not known. Here, we present evidence that the striped pattern of the secreted factor Hedgehog (Hh), in combination with the RAS pathway, specifies and positions neighboring groups of cardiac progenitors within each segment: the anterior ladybird (lbe)- and the posterior even skipped (eve)-expressing cardiac progenitors. Loss of hh function (while maintaining wg activity) results in the absence of the Eve cells, whereas the Lbe cells are expanded within the cardiac mesoderm. Overexpressing the repressor form of Cubitus interruptus (Ci), a Hh pathway antagonist, also results in expansion of Lbe at the expense of Eve, as does lowering Ras signaling. Conversely, overexpression of Hh or increasing Ras signaling eliminates Lbe expression while expanding Eve within the cardiogenic mesoderm. Increasing Ras signaling in the absence of Hh suggests that the Ras pathway is in part epistatic to Hh. Hh controls dorsal mesodermal Ras signaling by transcriptional regulation of the EGF receptor ligand protease, encoded by rhomboid (rho). Conversely, Hh overexpression can fully inhibit Lbe even when Ras signaling is much reduced, suggesting that Hh also acts in parallel to Ras. We propose that the Eve precursors next to the Hh stripe are distinguished from more distant Lbe precursors by locally augmenting Ras signaling via elevating rho transcripts. Thus, the spatial precision of cell type specification within an organ depends on multiple phases of inductive interaction between the ectoderm and the mesoderm.Mapping Dmef2-binding regulatory modules by using a ChIP-enriched in silico targets approach.
Guillaume, G. Junion ; Teresa, T. Jagla ; Sebastien, S. Duplant ; Romain, R. Tapin ; Jean-Philippe, JP. Da Ponte ; Krzysztof, K. Jagla.
Institut National de la Santé et de la Recherche Médicale Unité 384, Faculté de Médecine, 28 Place Henri Dunant, 63000 Clermont-Ferrand, France.
Mapping the regulatory modules to which transcription factors bind in vivo is a key step toward understanding of global gene expression programs. We have developed a chromatin immunoprecipitation (ChIP)-chip strategy for identifying factor-specific regulatory regions acting in vivo. This method, called the ChIP-enriched in silico targets (ChEST) approach, combines immunoprecipitation of cross-linked protein-DNA complexes (X-ChIP) with in silico prediction of targets and generation of computed DNA microarrays. We report the use of ChEST in Drosophila to identify several previously unknown targets of myocyte enhancer factor 2 (MEF2), a key regulator of myogenic differentiation. Our approach was validated by demonstrating that the identified sequences act as enhancers in vivo and are able to drive reporter gene expression specifically in MEF2-positive muscle cells. Presented here, the ChEST strategy was originally designed to identify regulatory modules in Drosophila, but it can be adapted for any sequenced and annotated genome.Coordinated development of muscles and tendons of the Drosophila leg.
Cédric, C. Soler ; Malgorzata, M. Daczewska ; Jean Philippe, JP. Da Ponte ; Bernard, B. Dastugue ; Krzysztof, K. Jagla.
INSERM U.384, Faculté de Médecine, 28 Place Henri Dunant, 63001 Clermont Ferrand, France.
Since Miller's morphological description, the Drosophila leg musculature and its formation has not been revisited. Here, using a set of GFP markers and confocal microscopy, we analyse Drosophila leg muscle development, and describe all the muscles and tendons present in the adult leg. Importantly, we provide for the first time evidence for tendons located internally within leg segments. By visualising muscle and tendon precursors, we demonstrate that leg muscle development is closely associated with the formation of internal tendons. In the third instars discs, in the vicinity of tendon progenitors, some Twist-positive myoblasts start to express the muscle founder cell marker dumbfounded (duf). Slightly later, in the early pupa, epithelial tendon precursors invaginate inside the developing leg segments, giving rise to the internal string-like tendons. The tendon-associated duf-lacZ-expressing muscle founders are distributed along the invaginating tendon precursors and then fuse with surrounding myoblasts to form syncytial myotubes. At mid-pupation, these myotubes grow towards their epithelial insertion sites, apodemes, and form links between internally located tendons and the leg epithelium. This leads to a stereotyped pattern of multifibre muscles that ensures movement of the adult leg.The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells.
Fabienne, F. De Graeve ; Teresa, T. Jagla ; Jean-Philippe, JP. Daponte ; Christof, C. Rickert ; Bernard, B. Dastugue ; Joachim, J. Urban ; Krzysztof, K. Jagla.
Faculté de Médecine, UMR Inserm 384, 63000 Clermont-Ferrand, France. Fabienne.DEGRAEVE@inserm.u-clermont1.fr
In Drosophila, neurons and glial cells are produced by neural precursor cells called neuroblasts (NBs), which can be individually identified. Each NB generates a characteristic cell lineage specified by a precise spatiotemporal control of gene expression within the NB and its progeny. Here we show that the homeobox genes ladybird early and ladybird late are expressed in subsets of cells deriving from neuroblasts NB 5-3 and NB 5-6 and are essential for their correct development. Our analysis revealed that ladybird in Drosophila, like their vertebrate orthologous Lbx1 genes, play an important role in cell fate specification processes. Among those cells that express ladybird are NB 5-6-derived glial cells. In ladybird loss-of-function mutants, the NB 5-6-derived exit glial cells are absent while overexpression of these genes leads to supernumerary glial cells of this type. Furthermore, aberrant glial cell positioning and aberrant spacing of axonal fascicles in the nerve roots observed in embryos with altered ladybird function suggest that the ladybird genes might also control directed cell movements and cell-cell interactions within the developing Drosophila ventral nerve cord.Patterning of the cardiac outflow region in Drosophila.
Institut National de la Santé et de la Recherche Médicale, Unité 384, Faculté de Médecine, 28 Place Henri Dunant, 63001 Clermont-Ferrand, France.
Specification of bilateral cardiac primordia and formation of the linear heart tube are highly conserved from Drosophila to humans. However, subsequent heart morphogenesis involving nonmesodermal neural crest cells was thought to be specific for vertebrates. Here, we provide evidence that a group of nonmesodermal cells that we have named heart-anchoring cells (HANCs) contribute to heart morphogenesis in Drosophila. We show that the homeobox genes ladybird (lb) known to be involved in diversification of cardiac precursors are expressed in HANCs and required for their specification. Interestingly, the HANCs selectively contact the anterior cardiac cells, which express lb as well. Direct interaction between HANCs and cardiac cells is assisted by a pair of cardiac outflow muscles (COMs), each of which selectively attaches to both the lb-expressing cardiac cells and HANCs. COM muscles seem to ensure ventral bending of the heart tip and together with HANCs determine the spatial positioning of the cardiac outflow region. Experimentally depleted cardiac lb expression leads to the disruption of the contact between the tip of the heart and either the COM muscles or the HANC cells, indicating a pivotal morphogenetic role for the lb expression within the heart.Modifiers of muscle and heart cell fate specification identified by gain-of-function screen in Drosophila.
Yannick, Y. Bidet ; Teresa, T. Jagla ; Jean-Philippe, JP. Da Ponte ; Bernard, B. Dastugue ; Krzysztof, K. Jagla.
UMR INSERM U384-Faculté de Médecine, 28 Place Henri Dunant, 63001 Clermont-Ferrand, France.
The homeobox genes ladybird in Drosophila and their vertebrate counterparts Lbx1 genes display restricted expression patterns in a subset of muscle precursors and are both implicated in diversification of muscle cell fates. In order to gain new insights into mechanisms controlling conserved aspects of cell fate specification, we have performed a gain-of-function (GOF) screen for modifiers of the mesodermal expression of ladybird genes using a collection of EP element carrying Drosophila lines. Amongst the identified genes, several have been previously implicated in cell fate specification processes, thus validating the strategy of our screen. Observed GOF phenotypes have led us to identification of an important number of candidate genes, whose myogenic and/or cardiogenic functions remain to be investigated. Amongst them, the EP insertions close to rhomboid, yan and rac2 suggest new roles for these genes in diversification of muscle and/or heart cell lineages. The analysis of loss and GOF of rhomboid and yan reveals their new roles in specification of ladybird-expressing precursors of adult muscles (LaPs) and ladybird/tinman-positive pericardial cells. Observed phenotypes strongly suggest that rhomboid and yan act at the level of progenitor and founder cells and contribute to the diversification of mesodermal fates. Our analysis of rac2 phenotypes clearly demonstrates that the altered mesodermal level of Rho-GTPase Rac2 can influence specification of a number of cardiac and muscular cell types including those expressing ladybird. Finding that in rac2 mutants ladybird and even skipped-positive muscle founders are overproduced, indicate a new early function for this gene during segregation of muscle progenitors and/or specification of founder cells. Intriguingly, rhomboid, yan and rac2 act as conserved components of Receptor Tyrosine Kinases (RTKs) signalling pathways, suggesting that RTK signalling constitutes a part of a conserved regulatory network governing diversification of muscle and heart cell types.Cross-repressive interactions of identity genes are essential for proper specification of cardiac and muscular fates in Drosophila.
Teresa, T. Jagla ; Yannick, Y. Bidet ; Jean Philippe, JP. Da Ponte ; Bernard, B. Dastugue ; Krzysztof, K. Jagla.
INSERM U.384, Faculté de Médecine, 28, Place Henri Dunant, 63001 Clermont Ferrand, France.
In Drosophila embryos, founder cells that give rise to cardiac precursors and dorsal somatic muscles derive from dorsally located progenitors. Individual fates of founder cells are thought to be specified by combinatorial code of transcription factors encoded by identity genes. To date, a large number of identity genes have been identified; however, the mechanisms by which these genes contribute to cell fate specification remain largely unknown. We have analysed regulatory interactions of ladybird (lb), msh and even skipped (eve), the three identity genes specifying a subset of heart and/or dorsal muscle precursors. We show that deregulation of each of them alters the number of cells that express two other genes, thus changing the ratio between cardiac and muscular cells, and the ratio between different cell subsets within the heart and within the dorsal muscles. Specifically, we demonstrate that mutation of the muscle identity gene msh and misexpression of the heart identity gene lb lead to heart hyperplasia with similar cell fate modifications. In msh mutant embryos, the presumptive msh-muscle cells switch on lb or eve expression and are recruited to form supernumerary heart or dorsal muscle cells, thus indicating that msh functions as a repressor of lb and eve. Similarly, overexpression of lb represses endogenous msh and eve activity, hence leading to the respecification of msh and eve positive progenitors, resulting in the overproduction of a subset of heart cells. As deduced from heart and muscle phenotypes of numb mutant embryos, the cell fate modifications induced by gain-of-function of identity genes are not lineage restricted. Consistent with all these observations, we propose that the major role of identity genes is to maintain their restricted expression by repressing other identity genes competent to respond positively to extrinsic signals. The cross-repressive interactions of identity genes are likely to ensure their localised expression over time, thus providing an essential element in establishing cell identity.A cluster of Drosophila homeobox genes involved in mesoderm differentiation programs.
INSERM U.384, Clermont Ferrand Cedex, France.
Although genes involved in common developmental programs are usually scattered throughout the metazoan genome, there are some important examples of functionally interconnected regulatory genes that display close physical linkage. In particular the homeotic genes, which determine the identities of body parts, are clustered in the Hox complexes and clustering is thought to be crucial for the proper execution of their developmental programs. Here we describe the organization and functional properties of a more recently identified cluster of six homeobox genes at 93DE on the third chromosome of Drosophila. These genes, which include tinman, bagpipe, ladybird early, ladybird late, C15, and slouch, all participate in mesodermal patterning and differentiation programs and show multiple regulatory interactions among each other. We propose that their clustering, through unknown mechanisms, is functionally significant and discuss the similarities and differences between the 93DE homeobox gene cluster and the Hox complexes.Two novel Drosophila TAF(II)s have homology with human TAF(II)30 and are differentially regulated during development.
S, S. Georgieva ; D B, DB. Kirschner ; T, T. Jagla ; E, E. Nabirochkina ; S, S. Hanke ; H, H. Schenkel ; C, C. de Lorenzo ; P, P. Sinha ; K, K. Jagla ; B, B. Mechler ; L, L. Tora.
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, F-67404 Illkirch Cedex, CU de Strasbourg, France.
TFIID is a multiprotein complex composed of the TATA binding protein (TBP) and TBP-associated factors (TAF(II)s). The binding of TFIID to the promoter is the first step of RNA polymerase II preinitiation complex assembly on protein-coding genes. Yeast (y) and human (h) TFIID complexes contain 10 to 13 TAF(II)s. Biochemical studies suggested that the Drosophila (d) TFIID complexes contain only eight TAF(II)s, leaving a number of yeast and human TAF(II)s (e.g., hTAF(II)55, hTAF(II)30, and hTAF(II)18) without known Drosophila homologues. We demonstrate that Drosophila has not one but two hTAF(II)30 homologues, dTAF(II)16 and dTAF(II)24, which are encoded by two adjacent genes. These two genes are localized in a head-to-head orientation, and their 5' extremities overlap. We show that these novel dTAF(II)s are expressed and that they are both associated with TBP and other bona fide dTAF(II)s in dTFIID complexes. dTAF(II)24, but not dTAF(II)16, was also found to be associated with the histone acetyltransferase (HAT) dGCN5. Thus, dTAF(II)16 and dTAF(II)24 are functional homologues of hTAF(II)30, and this is the first demonstration that a TAF(II)-GCN5-HAT complex exists in Drosophila. The two dTAF(II)s are differentially expressed during embryogenesis and can be detected in both nuclei and cytoplasm of the cells. These results together indicate that dTAF(II)16 and dTAF(II)24 may have similar but not identical functions.Plasticity within the lateral somatic mesoderm of Drosophila embryos.
INSERM U.384, Faculté de Médecine, Clermont Ferrand, France.
Each of 30 Drosophila larval somatic muscles has its individual shape, insertion sites and innervation. From the very beginning, the formation of individual muscles is controlled by a set of muscle identity genes. The four lateral transverse muscles (LT1-LT4) are thought to be specified by the combinatorial activity of Krüppel (Kr), apterous (ap) and muscle specific homeobox (msh) genes whilst the activity of the ladybird (lb) genes is required for proper formation of the neighbouring segmental border muscle (SBM). We have recently shown that ectopic expression of lb changes the identity of Kr-expressing lateral muscle precursors and recruits them to form enlarged or duplicated SBMs. Here we report that loss of msh function leads to a similar transformation resulting in the overproduction of SBMs. Inversely, in msh gain of function embryos, the prospective SBM myoblasts change their identity resulting in the formation of enlarged lateral transverse muscles. These data indicate a key role for the msh and lb genes in the specification and diversification of myoblast lineages from the lateral domain, and reveal a plasticity of cell fate within the somatic mesoderm of Drosophila.ladybird determines cell fate decisions during diversification of Drosophila somatic muscles.
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, CU de Strasbourg, France. email@example.com
In the mesoderm of Drosophila embryos, a defined number of cells segregate as progenitors of individual body wall muscles. Progenitors and their progeny founder cells display lineage-specific expression of transcription factors but the mechanisms that regulate their unique identities are poorly understood. Here we show that the homeobox genes ladybird early and ladybird late are expressed in only one muscle progenitor and its progeny: the segmental border muscle founder cell and two precursors of adult muscles. The segregation of the ladybird-positive progenitor requires coordinate action of neurogenic genes and an interplay of inductive Hedgehog and Wingless signals from the overlying ectoderm. Unlike so far described progenitors but similar to the neuroblasts, the ladybird-positive progenitor undergoes morphologically asymmetric division. We demonstrate that the ectopic ladybird expression is sufficient to change the identity of a subset of progenitor/founder cells and to generate an altered pattern of muscle precursors. When ectopically expressed, ladybird transforms the identity of neighbouring, Krüppel-positive progenitors leading to the formation of giant segmental border muscles and supernumerary precursors of lateral adult muscles. In embryos lacking ladybird gene function, specification of two ladybird-expressing myoblast lineages is affected. The segmental border muscles do not form or have abnormal shapes and insertion sites while the number of lateral precursors of adult muscles is dramatically reduced. Altogether our results provide new insights into the genetic control of diversification of muscle precursors and indicate a further similarity between the myogenic and neurogenic pathways.ladybird, a new component of the cardiogenic pathway in Drosophila required for diversification of heart precursors.
Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, Collège de France, CU de Strasbourg. firstname.lastname@example.org
The embryonic heart precursors of Drosophila are arranged in a repeated pattern of segmental units. There is growing evidence that the development of individual elements of this pattern depends on both mesoderm intrinsic patterning information and inductive signals from the ectoderm. In this study, we demonstrate that two homeobox genes, ladybird early and ladybird late, are involved in the cardiogenic pathway in Drosophila. Their expression is specific to a subset of cardioblast and pericardial cell precursors and is critically dependent on mesodermal tinman function, epidermal Wingless signaling and the coordinate action of neurogenic genes. Negative regulation by hedgehog is required to restrict ladybird expression to two out of six cardioblasts in each hemisegment. Overexpression of ladybird causes a hyperplasia of heart precursors and alters the identity of even-skipped-positive pericardial cells. Loss of ladybird function leads to the opposite transformation, suggesting that ladybird participates in the determination of heart lineages and is required to specify the identities of subpopulations of heart cells. We find that both early Wingless signaling and ladybird-dependent late Wingless signaling are required for proper heart formation. Thus, we propose that ladybird plays a dual role in cardiogenesis: (i) during the early phase, it is involved in specification of a segmental subset of heart precursors as a component of the cardiogenic tinman-cascade and (ii) during the late phase, it is needed for maintaining wingless activity and thereby sustaining the heart pattern process. These events result in a diversification of heart cell identities within each segment.ladybird, a tandem of homeobox genes that maintain late wingless expression in terminal and dorsal epidermis of the Drosophila embryo.
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, C.U. de Strasbourg, France.
ladybird early and ladybird late genes, tandemly located in the Drosophila 93E homeobox gene cluster, encode highly related homeodomain-containing transcription factors. Here we report the cloning of the complete cDNA sequences of both genes and a study of their expression and regulatory interactions with the segment polarity gene wingless in the epidermis. ladybird genes are co-expressed with wingless in epidermal cells close to the posterior parasegmental boundaries and in terminal regions of the body. In mutant embryos with altered wingless function, transcription of ladybird early and ladybird late is changed; it disappears completely from the epidermis in wingless-embryos, indicating wingless-dependence. After 6 hours of development, wingless expression is maintained by gooseberry in the ventral epidermis. However, in the dorsal epidermis and the terminal regions of the body, expression of wingless is independent of gooseberry but requires a wingless-ladybird regulatory feedback loop. Loss of ladybird function reduces the number of wingless-expressing cells in dorsal epidermis and leads to complete inactivation of wingless in the anal plate. Consequently, mutant ladybird embryos fail to develop anal plates and ubiquitous embryonic expression of either one or both ladybird genes leads to severe defects of the dorsal cuticle. Lack of late wingless expression and anal plate formation can be rescued with the use of a heat-shock-ladybird transgene.Mouse Lbx1 and human LBX1 define a novel mammalian homeobox gene family related to the Drosophila lady bird genes.
K, K. Jagla ; P, P. Dollé ; M G, MG. Mattei ; T, T. Jagla ; B, B. Schuhbaur ; G, G. Dretzen ; F, F. Bellard ; M, M. Bellard.
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS, INSERM, Université Louis Pasteur, Strasbourg, France.
We have cloned two novel homeobox genes which are the mouse (Lbx1) and human (LBX1) homologs of the Drosophila lady bird genes. They are highly related not only within the coding region but also in 5' and 3' untranslated regions. Several amino acid residues inside and around the homeodomain, have been conserved between the mammalian Lbx genes and their Drosophila counterparts. The mouse Lbx1 gene is located on chromosome 19 (region D) and the human LBX1 gene maps to the related q24 region of chromosome 10, known as a breakpoint region in translocations t(7;10) and t(10;14) involved in T-cell leukemias. Thus, LBX1 and the protooncogene HOX11 map to a common chromosomal region, as do their Drosophila counterparts, the lady bird and 93Bal genes. The mouse Lbx1 gene is specifically expressed during embryogenesis. From 10.5 days of gestation, Lbx1 expression is detected in the central nervous system and some developing muscles. In the CNS, Lbx1 transcripts are expressed in the dorsal part of the mantle layer of the spinal cord and hindbrain, up to a sharp boundary within the developing metencephalon. Thus, Lbx1 may be inolved in spinal cord and hindbrain differentiation and/or patterning, and its restricted expression pattern could depend upon evolutionarily conserved inductive signals involving some mammalian Wnt and Pax genes, as is the case for Drosophila lady bird genes and wingless or gooseberry.A distinct class of homeodomain proteins is encoded by two sequentially expressed Drosophila genes from the 93D/E cluster.
Laboratoire de Génétique Moléculaire des Eukaryotes du CNRS, Unité 184 de Biologie Moléculaire et de Génie Génétique de l'INSERM, Faculté de Médecine, Strasbourg, France.
Homeodomains appear to be one of the most frequently employed DNA-binding domains in a superfamily of transacting factors. It is likely that during evolution several sub-types of homeodomain have evolved from a common ancestral domain, resulting in distinct but closely related DNA-binding preferences. Here we describe the conservation of a distinct type of homeodomain encoded by the Drosophila lady-bird-late (lbl) gene, previously named nkch4 (1). Using degenerate PCR primers corresponding to the most divergent regions of the first and third helix of the Lbl homeodomain we have amplified, from genomic DNA of the fly, a lady-bird-like homeobox fragment. The Drosophila PCR products contained both the lbl (1) and a highly related homeobox sequence, which we named lady-bird-early (lbe). This new Drosophila gene resides directly upstream to lbl and together with tinman/NK4 (2, 3, 4, 5), bagpipe/NK3 (2, 4) S59/NK1 (4, 6) and 93Bal (7) compose the 93D/E homeobox gene cluster. Ibe and lbl are transcribed from the same strand and in a temporal order corresponding to their 5'-3' chromosomal location. Transcripts of both genes are found in the epiderm of Drosophila embryos, in cells known to express a segment polarity gene wingless (8), and their spatial and temporal colinearity of expression strongly suggests that they cooperate during segmentation. The amino-acid composition of both Lady-bird homeodomains differ from that of Antp-type at several positions involved in DNA recognition. These substitutions appear to modify DNA-binding preferences since Lbl homeodomain is unable to recognize the most common homeodomain binding TAAT motif in gel retardation experiments.GEBF-I activates the Drosophila Sgs3 gene enhancer by altering a positioned nucleosomal core particle.
P, P. Georgel ; G, G. Dretzen ; K, K. Jagla ; F, F. Bellard ; E, E. Dubrovsky ; V, V. Calco ; M, M. Bellard.
Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Institut de Chimie Biologique, Faculté de Médecine, Strasbourg, France.
The enhancer region of the Drosophila melanogaster ecdysone-regulated glue gene, Sgs3, shows dramatic modifications of chromatin structure in strict correlation with changes in gene expression during development. We show that there is a positioned nucleosomal core particle over the enhancer which is displaced or disrupted during gene activation. This transition is prevented in Drosophila larvae mutated in the ecdysone-dependent 2B5 locus, in which Sgs3 is inactive and GEBF-I, a Glue Enhancer Binding Factor, is missing. We have defined the GEBF-I binding sites in vitro and shown that mutation of these sequences abolishes the enhancer activity in vivo. This combined in vitro and in vivo approach reveals new aspects of the dynamic organization of a regulatory element during development and highlights the potential of this model for studies of the relation between chromatin structure and gene activity.A novel homeobox nkch4 gene from the Drosophila 93E region.
Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Biologie Moléculaire et de Génie Génétique de l'INSERM, Faculté de Médecine, Strasbourg, France.
We have cloned a Drosophilia melanogaster homeobox gene that maps to bands 93D9-E2 on the right arm of the third chromosome, in the proximal region of the NK-homeobox gene cluster. Like NK-1 and nine other known homeobox genes, nkch4 (NK-cluster homeobox 4) contains an intron between the homeodomain codons for Glu44 and Val45. The nkch4 homeodomain sequence is most related to that of the human HOX11 (tcl3) T-cell oncogene (57% homology), but differs from all other homeobox genes at several conserved residues in the third helix of the homeodomain, known to be important for DNA recognition. Low levels of nkch4 transcripts were detected during late stages of embryogenesis as well as in third instar larvae and pupae. In late embryos nkch4 is expressed in the developing CNS.GEBF-I in Drosophila species and hybrids: the co-evolution of an enhancer and its cognate factor.
Laboratoire de Génétique Moléculaire des Eucaryotes, Centre National de la Recherche Scientifique, Faculté de Médecine, Strasbourg, France.
The activation of the Drosophila melanogaster salivary gland secretion protein gene Sgs-3 is marked by important changes in chromatin structure in the distal regulatory region at -600 bp from the Sgs-3 start site. A stage- and tissue-specific glue enhancer binding factor, GEBF-I, binds in vitro to sequences from this region. Previous studies have revealed considerable variation in the DNA sequences of comparable regions in the related Drosophila species, D. simulans, D. erecta and D. yakuba. We detected GEBF-I-like proteins in these species, which appear to evolve as rapidly as the corresponding DNA sequences, and studied in detail the binding characteristics of the GEBF-I proteins of the two most closely related species, D. melanogaster and D. simulans. In crosses between these species, certain strains produce hybrid larvae which, unexpectedly, synthesised a single intermediate form of the protein. This suggests that the factor is subject to species-specific post-transcriptional modifications. In these hybrid larvae, which carry one D. melanogaster and one D. simulans Sgs-3 gene, the hybrid GEBF-I protein appears equally effective in the induction of both target genes.
Characterization of Enhancers Active in the Mouse Embryonic Cerebral Cortex Suggests Sox/Pou cis-Regulatory Logics and Heterogeneity of Cortical Progenitors.
Amandine, A. Bery ; Ben, B. Martynoga ; François, F. Guillemot ; Jean-Stéphane, JS. Joly ; Sylvie, S. Rétaux.
Equipe Development and Evolution of the Forebrain.
We aimed to identify cis-regulatory elements that control gene expression in progenitors of the cerebral cortex. A list of 975 putative enhancers were retrieved from a ChIP-Seq experiment performed in NS5 mouse stem cells with antibodies to Sox2, Brn2/Pou3f2, or Brn1/Pou3f3. Through a selection pipeline including gene ontology and expression pattern, we reduced the number of candidate enhancer sequences to 20. Ex vivo electroporation of green fluorescent pProtein (GFP) reporter constructs in the telencephalon of mouse embryos showed that 35% of the 20 selected candidate sequences displayed enhancer activity in the developing cortex at E13.5. In silico transcription factor binding site (TFBS) searches and mutagenesis experiments showed that enhancer activity is related to the presence of Sox/Pou TFBS pairs in the sequence. Comparative genomic analyses showed that enhancer activity is not related to the evolutionary conservation of the sequence. Finally, the combination of in utero electroporation of GFP reporter constructs with immunostaining for Tbr2 (basal progenitor marker) and phospho-histoneH3 (mitotic activity marker) demonstrated that each enhancer is specifically active in precise subpopulations of progenitors in the cortical germinal zone, highlighting the heterogeneity of these progenitors in terms of cis-regulation.Zebrafish midbrain slow-amplifying progenitors exhibit high levels of transcripts for nucleotide and ribosome biogenesis.
Gaëlle, G. Recher ; Julia, J. Jouralet ; Alessandro, A. Brombin ; Aurélie, A. Heuzé ; Emilie, E. Mugniery ; Jean-Michel, JM. Hermel ; Sophie, S. Desnoulez ; Thierry, T. Savy ; Philippe, P. Herbomel ; Franck, F. Bourrat ; Nadine, N. Peyriéras ; Françoise, F. Jamen ; Jean-Stéphane, JS. Joly.
CNRS, UPR3294 Unité Neurobiologie et Développement, F-91198 Gif-sur-Yvette, France.
Investigating neural stem cell (NSC) behaviour in vivo, which is a major area of research, requires NSC models to be developed. We carried out a multilevel characterisation of the zebrafish embryo peripheral midbrain layer (PML) and identified a unique vertebrate progenitor population. Located dorsally in the transparent embryo midbrain, these large slow-amplifying progenitors (SAPs) are accessible for long-term in vivo imaging. They form a neuroepithelial layer adjacent to the optic tectum, which has transitory fast-amplifying progenitors (FAPs) at its margin. The presence of these SAPs and FAPs in separate domains provided the opportunity to data mine the ZFIN expression pattern database for SAP markers, which are co-expressed in the retina. Most of them are involved in nucleotide synthesis, or encode nucleolar and ribosomal proteins. A mutant for the cad gene, which is strongly expressed in the PML, reveals severe midbrain defects with massive apoptosis and sustained proliferation. We discuss how fish midbrain and retina progenitors might derive from ancient sister cell types and have specific features that are not shared with other SAPs.Molecular evolution of peptidergic signaling systems in bilaterians.
Unité propre de Recherche 3294, Centre National de la Recherche Scientifique and Institut National de la Recherche Agronomique, 91198 Gif-sur-Yvette, France. email@example.com
Peptide hormones and their receptors are widespread in metazoans, but the knowledge we have of their evolutionary relationships remains unclear. Recently, accumulating genome sequences from many different species have offered the opportunity to reassess the relationships between protostomian and deuterostomian peptidergic systems (PSs). Here we used sequences of all human rhodopsin and secretin-type G protein-coupled receptors as bait to retrieve potential homologs in the genomes of 15 bilaterian species, including nonchordate deuterostomian and lophotrochozoan species. Our phylogenetic analysis of these receptors revealed 29 well-supported subtrees containing mixed sets of protostomian and deuterostomian sequences. This indicated that many vertebrate and arthropod PSs that were previously thought to be phyla specific are in fact of bilaterian origin. By screening sequence databases for potential peptides, we then reconstructed entire bilaterian peptide families and showed that protostomian and deuterostomian peptides that are ligands of orthologous receptors displayed some similarity at the level of their primary sequence, suggesting an ancient coevolution between peptide and receptor genes. In addition to shedding light on the function of human G protein-coupled receptor PSs, this work presents orthology markers to study ancestral neuron types that were probably present in the last common bilaterian ancestor.Monoaminergic modulation of photoreception in ascidian: evidence for a proto-hypothalamo-retinal territory.
Florian, F. Razy-Krajka ; Euan R, ER. Brown ; Takeo, T. Horie ; Jacques, J. Callebert ; Yasunori, Y. Sasakura ; Jean-Stéphane, JS. Joly ; Takehiro G, TG. Kusakabe ; Philippe, P. Vernier.
Neurobiology and Development, UPR, Institut de Neurobiologie Alfred Fessard, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
The proximal promoter region of the zebrafish gsdf gene is sufficient to mimic the spatio-temporal expression pattern of the endogenous gene in Sertoli and granulosa cells.
BACKGROUND:The retina of craniates/vertebrates has been proposed to derive from a photoreceptor prosencephalic territory in ancestral chordates, but the evolutionary origin of the different cell types making the retina is disputed. Except for photoreceptors, the existence of homologs of retinal cells remains uncertain outside vertebrates.
RESULTS:We show that many molecular characteristics of dopamine-synthesizing cells located in the vicinity of photoreceptors in the sensory vesicle of the ascidian Ciona intestinalis are similar to those of amacrine dopamine cells of the vertebrate retina. The ascidian dopamine cells share with vertebrate amacrine cells the expression of the key-transcription factor Ptf1a, as well as that of dopamine-synthesizing enzymes. Surprisingly, the ascidian dopamine cells accumulate serotonin via a functional serotonin transporter, as some amacrine cells also do. Moreover, dopamine cells located in the vicinity of the photoreceptors modulate the light-off induced swimming behavior of ascidian larvae by acting on alpha2-like receptors, instead of dopamine receptors, supporting a role in the modulation of the photic response. These cells are located in a territory of the ascidian sensory vesicle expressing genes found both in the retina and the hypothalamus of vertebrates (six3/6, Rx, meis, pax6, visual cycle proteins).
CONCLUSION:We propose that the dopamine cells of the ascidian larva derive from an ancestral multifunctional cell population located in the periventricular, photoreceptive field of the anterior neural tube of chordates, which also gives rise to both anterior hypothalamus and the retina in craniates/vertebrates. It also shows that the existence of multiple cell types associated with photic responses predates the formation of the vertebrate retina.
Aude, A. Gautier ; Frédéric, F. Sohm ; Jean-Stéphane, JS. Joly ; Florence, F. Le Gac ; Jean-Jacques, JJ. Lareyre.
INRA, UR1037 SCRIBE, Testicular Physiology and Puberty Research Team, IFR140, Ouest-BioGenOuest, Rennes, France.
The gonadal soma-derived factor (GSDF) is a new member of the transforming growth factor beta (TGF-beta) superfamily that regulates the proliferation of the primordial germ cells (PGC) in developing embryos and spermatogonia in juvenile male trout. The gsdf transcripts are expressed in the somatic cells supporting germ cell development. In zebrafish, we show that GSDF is encoded by a single copy gene that generates polymorphic transcripts and proteins. We determined that gsdf gene expression occurs before gonadal differentiation and is restricted to the gonads. Gene expression is maintained in adult granulosa cells and Sertoli cells but decreases in the cells that are in contact with meiotic and postmeiotic germ cells. Using zebrafish transgenic lines, we demonstrate that the 2-kb proximal promoter region of the gsdf gene targets high levels of transgene expression in the Sertoli and granulosa cells, and is sufficient to mimic the temporal expression pattern of the endogenous gsdf gene from 16 days postfertilization onward. We identified within the first 500 bp evolutionarily conserved DNA motifs that may be involved in Sertoli and granulosa cell-specific expression. However, the 2-kb proximal promoter region failed to drive efficient expression of the transgene in the gonads in four transgenic medaka lines. We propose that the proximal promoter region can be used to target candidate gene deregulation in zebrafish granulosa and Sertoli cells. Furthermore, the green fluorescent protein-expressing zebrafish lines produced in the present study are new valuable models for cell lineage tracing during sex differentiation and gametogenesis.Genome-wide analysis of the POU genes in medaka, focusing on expression in the optic tectum.
Alessandro, A. Brombin ; Jean-Philippe, JP. Grossier ; Aurélie, A. Heuzé ; Zlatko, Z. Radev ; Franck, F. Bourrat ; Jean-Stéphane, JS. Joly ; Françoise, F. Jamen.
MSNC INRA group, UPR 3294 NeD, Institut Fessard, CNRS, Gif-sur-Yvette, France.
The highly conserved POU genes encode homeodomain transcription factors involved in various developmental events, with some, the Brn genes, playing key roles in neurogenesis. We investigated the evolutionary relationships between these genes, by studying the POU gene complement of a model teleost, the medaka (Oryzias latipes). We identified 17 POU genes and carried out a comprehensive in situ hybridization analysis focusing on the optic tectum, a cortical structure of the mesencephalon, in which cell positions and their differentiation states are spatially and temporally correlated. Six POU genes displayed patterned expression in the optic tectum: two genes were expressed in the center of the organ (a zone with differentiated neurons), two in an intermediate zone in which cells exit the cell cycle and two in the peripheral proliferation zone. These results suggest that POU genes may play key roles in both late neurogenesis and in multipotent neural progenitors.When needles look like hay: how to find tissue-specific enhancers in model organism genomes.
U1126 MSNC INRA Group, UPR3294 NED, Institut Fessard, CNRS, Gif-sur-Yvette, France. firstname.lastname@example.org
A major prerequisite for the investigation of tissue-specific processes is the identification of cis-regulatory elements. No generally applicable technique is available to distinguish them from any other type of genomic non-coding sequence. Therefore, researchers often have to identify these elements by elaborate in vivo screens, testing individual regions until the right one is found. Here, based on many examples from the literature, we summarize how functional enhancers have been isolated from other elements in the genome and how they have been characterized in transgenic animals. Covering computational and experimental studies, we provide an overview of the global properties of cis-regulatory elements, like their specific interactions with promoters and target gene distances. We describe conserved non-coding elements (CNEs) and their internal structure, nucleotide composition, binding site clustering and overlap, with a special focus on developmental enhancers. Conflicting data and unresolved questions on the nature of these elements are highlighted. Our comprehensive overview of the experimental shortcuts that have been found in the different model organism communities and the new field of high-throughput assays should help during the preparation phase of a screen for enhancers. The review is accompanied by a list of general guidelines for such a project.The ANISEED database: digital representation, formalization, and elucidation of a chordate developmental program.
Olivier, O. Tassy ; Delphine, D. Dauga ; Fabrice, F. Daian ; Daniel, D. Sobral ; François, F. Robin ; Pierre, P. Khoueiry ; David, D. Salgado ; Vanessa, V. Fox ; Danièle, D. Caillol ; Renaud, R. Schiappa ; Baptiste, B. Laporte ; Anne, A. Rios ; Guillaume, G. Luxardi ; Takehiro, T. Kusakabe ; Jean-Stéphane, JS. Joly ; Sébastien, S. Darras ; Lionel, L. Christiaen ; Magali, M. Contensin ; Hélène, H. Auger ; Clément, C. Lamy ; Clare, C. Hudson ; Ute, U. Rothbächer ; Michael J, MJ. Gilchrist ; Kazuhiro W, KW. Makabe ; Kohji, K. Hotta ; Shigeki, S. Fujiwara ; Nori, N. Satoh ; Yutaka, Y. Satou ; Patrick, P. Lemaire.
Institut de Biologie du Développement de Marseille-Luminy, UMR6216 CNRS, F-13288 Marseille, Cedex 9, France.
Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions.Formation of oral and pharyngeal dentition in teleosts depends on differential recruitment of retinoic acid signaling.
Yann, Y. Gibert ; Laure, L. Bernard ; Melanie, M. Debiais-Thibaud ; Franck, F. Bourrat ; Jean-Stephane, JS. Joly ; Karen, K. Pottin ; Axel, A. Meyer ; Sylvie, S. Retaux ; David W, DW. Stock ; William R, WR. Jackman ; Pawat, P. Seritrakul ; Gerrit, G. Begemann ; Vincent, V. Laudet.
Molecular Zoology Group, Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, CNRS, INRA, UCB Lyon 1, Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France.
One of the goals of evolutionary developmental biology is to link specific adaptations to changes in developmental pathways. The dentition of cypriniform fishes, which in contrast to many other teleost fish species possess pharyngeal teeth but lack oral teeth, provides a suitable model to study the development of feeding adaptations. Here, we have examined the involvement of retinoic acid (RA) in tooth development and show that RA is specifically required to induce the pharyngeal tooth developmental program in zebrafish. Perturbation of RA signaling at this stage abolished tooth induction without affecting the development of tooth-associated ceratobranchial bones. We show that this inductive event is dependent on RA synthesis from aldh1a2 in the ventral posterior pharynx. Fibroblast growth factor (FGF) signaling has been shown to be critical for tooth induction in zebrafish, and its loss has been associated with oral tooth loss in cypriniform fishes. Pharmacological treatments targeting the RA and FGF pathways revealed that both pathways act independently during tooth induction. In contrast, we find that in Mexican tetra and medaka, species that also possess oral teeth, both oral and pharyngeal teeth are induced independently of RA. Our analyses suggest an evolutionary scenario in which the gene network controlling tooth development obtained RA dependency in the lineage leading to the cypriniforms. The loss of pharyngeal teeth in this group was cancelled out through a shift in aldh1a2 expression, while oral teeth might have been lost ultimately due to deficient RA signaling in the oral cavity.Evidence for neural stem cells in the medaka optic tectum proliferation zones.
Alessandro, A. Alunni ; Jean-Michel, JM. Hermel ; Aurélie, A. Heuzé ; Franck, F. Bourrat ; Françoise, F. Jamen ; Jean-Stéphane, JS. Joly.
MSNC INRA Group, UPR3294 NED, Institut Fessard, CNRS, 91 198 Gif-sur-Yvette, France.
Few adult neural stem cells have been characterized in vertebrates. Although teleosts continually generate new neurons in many regions of the brain after embryogenesis, only two types of neural stem cells (NSCs) have been reported in zebrafish: glial cells in the forebrain resembling mammalian NSCs, and neuroepithelial cells in the cerebellum. Here, following our previous studies on dividing progenitors (Nguyen et al. : J Comp Neurol 413:385-404.), we further evidenced NSCs in the optic tectum (OT) of juvenile and adult in the medaka, Oryzias latipes. To detect very slowly cycling progenitors, we did not use the commonly used BrdU/PCNA protocol, in which PCNA may not be present during a transiently quiescent state. Instead, we report the optimizations of several protocols involving long subsequent incubations with two thymidine analogs (IdU and CldU) interspaced with long chase times between incubations. These protocols allowed us to discriminate and localize fast and slow cycling cells in OT of juvenile and adult in the medaka. Furthermore, we showed that adult OT progenitors are not glia, as they express neither brain lipid-binding protein (BLBP) nor glial fibrillary acidic protein (GFAP). We also showed that expression of pluripotency-associated markers (Sox2, Musashi1 and Bmi1) colocalized with OT progenitors. Finally, we described the spatio-temporally ordered population of NSCs and progenitors in the medaka OT. Hence, the medaka appears as an invaluable model for studying neural progenitors that will open the way to further exciting comparative studies of neural stem cells in vertebrates.A cis-regulatory signature for chordate anterior neuroectodermal genes.
INRA group, UPR3294, Institute of Neurosciences Alfred Fessard, CNRS, Gif-sur-Yvette, France.
One of the striking findings of comparative developmental genetics was that expression patterns of core transcription factors are extraordinarily conserved in bilaterians. However, it remains unclear whether cis-regulatory elements of their target genes also exhibit common signatures associated with conserved embryonic fields. To address this question, we focused on genes that are active in the anterior neuroectoderm and non-neural ectoderm of the ascidian Ciona intestinalis. Following the dissection of a prototypic anterior placodal enhancer, we searched all genomic conserved non-coding elements for duplicated motifs around genes showing anterior neuroectodermal expression. Strikingly, we identified an over-represented pentamer motif corresponding to the binding site of the homeodomain protein OTX, which plays a pivotal role in the anterior development of all bilaterian species. Using an in vivo reporter gene assay, we observed that 10 of 23 candidate cis-regulatory elements containing duplicated OTX motifs are active in the anterior neuroectoderm, thus showing that this cis-regulatory signature is predictive of neuroectodermal enhancers. These results show that a common cis-regulatory signature corresponding to K50-Paired homeodomain transcription factors is found in non-coding sequences flanking anterior neuroectodermal genes in chordate embryos. Thus, field-specific selector genes impose architectural constraints in the form of combinations of short tags on their target enhancers. This could account for the strong evolutionary conservation of the regulatory elements controlling field-specific selector genes responsible for body plan formation.Regeneration of oral siphon pigment organs in the ascidian Ciona intestinalis.
INRA MSNC Group, DEPSN, Institut A. Fessard, CNRS, 1 Avenue de la Terrasse, 91198 Gif-Sur-Yvette, France.
Ascidians have powerful capacities for regeneration but the underlying mechanisms are poorly understood. Here we examine oral siphon regeneration in the solitary ascidian Ciona intestinalis. Following amputation, the oral siphon rapidly reforms oral pigment organs (OPO) at its distal margin prior to slower regeneration of proximal siphon parts. The early stages of oral siphon reformation include cell proliferation and re-growth of the siphon nerves, although the neural complex (adult brain and associated organs) is not required for regeneration. Young animals reform OPO more rapidly after amputation than old animals indicating that regeneration is age dependent. UV irradiation, microcautery, and cultured siphon explant experiments indicate that OPOs are replaced as independent units based on local differentiation of progenitor cells within the siphon, rather than by cell migration from a distant source in the body. The typical pattern of eight OPOs and siphon lobes is restored with fidelity after distal amputation of the oral siphon, but as many as 16 OPOs and lobes can be reformed following proximal amputation near the siphon base. Thus, the pattern of OPO regeneration is determined by cues positioned along the proximal distal axis of the oral siphon. A model is presented in which columns of siphon tissue along the proximal-distal axis below pre-existing OPO are responsible for reproducing the normal OPO pattern during regeneration. This study reveals previously unknown principles of oral siphon and OPO regeneration that will be important for developing Ciona as a regeneration model in urochordates, which may be the closest living relatives of vertebrates.Similar regulatory logic in Ciona intestinalis for two Wnt pathway modulators, ROR and SFRP-1/5.
Hélène, H. Auger ; Clément, C. Lamy ; Maximilian, M. Haeussler ; Pierre, P. Khoueiry ; Patrick, P. Lemaire ; Jean-Stéphane, JS. Joly.
INRA "Morphogenèse du Système Nerveux des Chordés" Group, DEPSN, UPR2197, Institut Fessard, CNRS, 1 Avenue de la Terrasse, 91198 GIF SUR YVETTE, France.
Anteroposterior patterning of the ectoderm in the invertebrate chordate Ciona intestinalis first relies on key zygotic activators, such as FoxA, and later on the coordinated responses to inducing signals from the underlying mesendoderm. Here, we focus on a mechanism of coordination of these responses by looking at the cis-regulatory logics of Ci-Rora and Ci-Rorb, which are coding for putative non-canonical transmembrane Wnt receptors, and are present in tandem along the C. intestinalis chromosome 08q. We showed that during cleavage stages, both Ci-Rora and Ci-Rorb genes are initially expressed in all blastomeres of the anterior ectoderm (a-line), as sFRP1/5 (Lamy, C., Rothbächer, U., Caillol, D., Lemaire, P., 2006. Ci-FoxA-a is the earliest zygotic determinant of the ascidian anterior ectoderm and directly activates Ci-sFRP1/5. Development 133, 2835-2844.). We then carried out a functional analysis of cis-regulatory regions and showed that both genes have elements enriched in Ci-FoxA-a binding sites. We dissected one of these early enhancers, and showed that it is directly activated by Ci-FoxA-a, as one sFRP1/5 cis-regulatory element. We also showed that although FoxA binding sites are abundant in genomes, dense clusters of these sites are found upstream from very few genes, and have a high predictive value of a-line expression. These data indicate an important role for FoxA in anterior specification, via the transcriptional regulation of target genes belonging to various signalling pathways.Refining the Ciona intestinalis model of central nervous system regeneration.
Carl, C. Dahlberg ; Hélène, H. Auger ; Sam, S. Dupont ; Yasunori, Y. Sasakura ; Mike, M. Thorndyke ; Jean-Stéphane, JS. Joly.
Department of Marine Ecology, Göteborg University, Fiskebäckskil, Sweden. email@example.com
Trunk lateral cells are neural crest-like cells in the ascidian Ciona intestinalis: insights into the ancestry and evolution of the neural crest.
BACKGROUND:New, practical models of central nervous system regeneration are required and should provide molecular tools and resources. We focus here on the tunicate Ciona intestinalis, which has the capacity to regenerate nerves and a complete adult central nervous system, a capacity unusual in the chordate phylum. We investigated the timing and sequence of events during nervous system regeneration in this organism.
William R, WR. Jeffery ; Takuto, T. Chiba ; Florian Razy, FR. Krajka ; Carole, C. Deyts ; Nori, N. Satoh ; Jean-Stéphane, JS. Joly.
Department of Biology, University of Maryland, College Park, MD 20742, USA. Jeffery@umd.edu
Neural crest-like cells (NCLC) that express the HNK-1 antigen and form body pigment cells were previously identified in diverse ascidian species. Here we investigate the embryonic origin, migratory activity, and neural crest related gene expression patterns of NCLC in the ascidian Ciona intestinalis. HNK-1 expression first appeared at about the time of larval hatching in dorsal cells of the posterior trunk. In swimming tadpoles, HNK-1 positive cells began to migrate, and after metamorphosis they were localized in the oral and atrial siphons, branchial gill slits, endostyle, and gut. Cleavage arrest experiments showed that NCLC are derived from the A7.6 cells, the precursors of trunk lateral cells (TLC), one of the three types of migratory mesenchymal cells in ascidian embryos. In cleavage arrested embryos, HNK-1 positive TLC were present on the lateral margins of the neural plate and later became localized adjacent to the posterior sensory vesicle, a staging zone for their migration after larval hatching. The Ciona orthologues of seven of sixteen genes that function in the vertebrate neural crest gene regulatory network are expressed in the A7.6/TLC lineage. The vertebrate counterparts of these genes function downstream of neural plate border specification in the regulatory network leading to neural crest development. The results suggest that NCLC and neural crest cells may be homologous cell types originating in the common ancestor of tunicates and vertebrates and support the possibility that a putative regulatory network governing NCLC development was co-opted to produce neural crest cells during vertebrate evolution.Comparison of the expression of medaka (Oryzias latipes) pitx genes with other vertebrates shows high conservation and a case of functional shuffling in the pituitary.
Yan, Y. Jaszczyszyn ; Maximilian, M. Haeussler ; Aurélie, A. Heuzé ; Mélanie, M. Debiais-Thibaud ; Didier, D. Casane ; Franck, F. Bourrat ; Jean-Stéphane, JS. Joly.
MSNC INRA Group, UPR2197 DEPSN Institut Fessard, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France.
With the availability of an increasing number of whole genome sequences in chordates, exhaustive comparisons of multigene families become feasible. Relationships of orthology/paralogy can not only be inferred from sequence similarity but also by comparing synteny conservation on chromosomes. More accurate scenarios for gene and expression domain gain or loss can now be proposed. Here, we take benefit from the recent release of the medaka (Oryzias latipes) genome to analyse the orthology relationships and expression patterns of the three different sub-families of the pitx homeobox genes belonging to the paired class. They are involved in a wide variety of developmental processes and have pleiotropic expression patterns, especially in the case of the pitx2 sub-family. The emerging picture is a strong conservation of expression domains, suggesting that most functions have been present in the common ancestor of actinopterygians and sarcopterygians. Almost all pitx genes are expressed in anterior placodes in all species studied so far, including medaka. It has previously been shown that in mammals, pitx1 and 2 are expressed in the pituitary. Interestingly we demonstrate here that only pitx3 is expressed in medaka pituitary. It will be interesting to analyze what are the corresponding changes in the regulatory elements of pitx genes.Evolutionary modification of mouth position in deuterostomes.
Lionel, L. Christiaen ; Yan, Y. Jaszczyszyn ; Marina, M. Kerfant ; Shungo, S. Kano ; Violette, V. Thermes ; Jean-Stéphane, JS. Joly.
Center for Integrative Genomics, Molecular & Cell Biology Department, University of California, Berkeley, CA 94720, USA. firstname.lastname@example.org
In chordates, the oral ectoderm is positioned at the anterior neural boundary and is characterized by pituitary homeobox (Pitx) and overlapping Dlx and Six3 expressions. Recent studies have shown that the ectoderm molecular map is also conserved in hemichordates and echinoderms. However, the mouth develops in a more posterior position in these animals, in a domain characterized by Nkx2.1 and Goosecoid expression, in a manner similar to that observed in protostomes. Furthermore, BMP signaling antagonizes mouth development in echinoderms and hemichordates, but seems to promote oral ectoderm specification in chordates. Conversely, Nodal signaling appears to be required for oral ectoderm specification in sea urchins but not in chordates. The Nodal/BMP antagonism at work during ectoderm patterning thus seems to constitute a conserved feature in deuterostomes, and mouth relocation may have been accompanied by a change in the influence of BMP/Nodal signals on oral ectoderm specification. We suggest that the mouth primordium was located at the anterior neural boundary, in early chordate evolution. In extant chordate embryos, subsequent mouth positioning differ between urochordates and vertebrates, presumably as a consequence of surrounding tissues remodelling. We illustrate these morphogenetic movements by means of morphological data obtained by the confocal imaging of ascidian tailbud embryos, and provide a table for determining the tailbud stages of this model organism.Windows of the brain: towards a developmental biology of circumventricular and other neurohemal organs.
Jean-Stéphane, JS. Joly ; Joana, J. Osório ; Alessandro, A. Alunni ; Hélène, H. Auger ; Shungo, S. Kano ; Sylvie, S. Rétaux.
U1126/INRA Morphogenèse du système nerveux des chordés group, DEPSN, UPR2197, Institut Fessard, CNRS, 1 Avenue de la Terrasse, 91198 GIF SUR YVETTE, France. email@example.com
We review the anatomical and functional features of circumventricular organs in vertebrates and their homologous neurohemal organs in invertebrates. Focusing on cyclostomes (lamprey) and urochordates (ascidians), we discuss the evolutionary origin of these organs as a function of their cell type specification and morphogenesis.Ol-insm1b, a SNAG family transcription factor involved in cell cycle arrest during medaka development.
Eva, E. Candal ; Alessandro, A. Alunni ; Violette, V. Thermes ; Françoise, F. Jamen ; Jean-Stéphane, JS. Joly ; Franck, F. Bourrat.
INRA MSNC Group, DEPSN, Institut Fessard, CNRS, 1 Avenue de la Terrasse, 91198 GIF-SUR-YVETTE, France. firstname.lastname@example.org
Through whole-mount in situ hybridisation screen on medaka (Oryzias latipes) brain, Ol-insm1b, a member of the Insm1/Mlt1 subfamily of SNAG-domain containing genes, has been isolated. It is strongly expressed during neurogenesis and pancreas organogenesis, with a pattern that suggests a role in cell cycle exit. Here, we describe Ol-insm1b expression pattern throughout development and in adult brain, and we report on its functional characterisation. Our data point to a previously unravelled role for Ol-insm1b as a down-regulator of cell proliferation during development, as it slows down the cycle without triggering apoptosis. Clonal analysis demonstrates that this effect is cell-autonomous, and, through molecular dissection studies, we demonstrate that it is likely to be non-transcriptional, albeit mediated by zinc-finger domains. Additionally, we report that Ol-insm1b mRNA, when injected in one cell of two-cell stage embryos, exhibits a surprising behaviour: it does not spread uniformly amongst daughter cells but remains cytoplasmically localised in the progeny of the injected blastomere. Our experiments suggest that Insm1 is a negative regulator of cell proliferation, possibly through mechanisms that do not involve modulation of transcription.Culture of Ciona intestinalis in closed systems.
Jean-Stéphane, JS. Joly ; Shungo, S. Kano ; Terumi, T. Matsuoka ; Helene, H. Auger ; Kazuko, K. Hirayama ; Nori, N. Satoh ; Satoko, S. Awazu ; Laurent, L. Legendre ; Yasunori, Y. Sasakura.
INRA MSNC group, DEPSN, UPR2197, Institut Fessard, CNRS, Gif-sur-Yvette, France. email@example.com
Improvements in closed-system culturing methods for marine invertebrates are important prerequisites for the generalized use of transgenic lines. We discuss here the effects of several closed-system conditions on the growth and survival of the solitary ascidian, Ciona intestinalis. In Shimoda, close to the sea, a small-tank system was used to ensure that tanks and systems were reasonably equipped, water exchange was rapid, and animals separated to minimize the risk of infection. In Gif-sur-Yvette, an inland site, we tried to determine the optimal conditions to limit handling operations, and to save artificial seawater by avoiding water pollution. A mixture of at least two types of live algae was better than any single-organism diet. With these maintenance protocols, we were able to obtain several generations of Ciona intestinalis, including several transgenic lines. Because these systems make it easier to rear Ciona intestinalis in laboratories, they increase the potentialities of this model organism for research.Expanded expression of Sonic Hedgehog in Astyanax cavefish: multiple consequences on forebrain development and evolution.
Arnaud, A. Menuet ; Alessandro, A. Alunni ; Jean-Stéphane, JS. Joly ; William R, WR. Jeffery ; Sylvie, S. Rétaux.
CNRS-UPR2197 DEPSN, Institut Fessard, Avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France.
Ventral midline Sonic Hedgehog (Shh) signalling is crucial for growth and patterning of the embryonic forebrain. Here, we report how enhanced Shh midline signalling affects the evolution of telencephalic and diencephalic neuronal patterning in the blind cavefish Astyanax mexicanus, a teleost fish closely related to zebrafish. A comparison between cave- and surface-dwelling forms of Astyanax shows that cavefish display larger Shh expression in all anterior midline domains throughout development. This does not affect global forebrain regional patterning, but has several important consequences on specific regions and neuronal populations. First, we show expanded Nkx2.1a expression and higher levels of cell proliferation in the cavefish basal diencephalon and hypothalamus. Second, we uncover an Nkx2.1b-Lhx6-GABA-positive migratory pathway from the subpallium to the olfactory bulb, which is increased in size in cavefish. Finally, we observe heterochrony and enlarged Lhx7 expression in the cavefish basal forebrain. These specific increases in olfactory and hypothalamic forebrain components are Shh-dependent and therefore place the telencephalic midline organisers in a crucial position to modulate forebrain evolution through developmental events, and to generate diversity in forebrain neuronal patterning.Morphological and gene expression similarities suggest that the ascidian neural gland may be osmoregulatory and homologous to vertebrate peri-ventricular organs.
Carole, C. Deyts ; Didier, D. Casane ; Philippe, P. Vernier ; Franck, F. Bourrat ; Jean-Stéphane, JS. Joly.
INRA MSNC group, DEPSN, UPR2197, Institut Fessard, CNRS, 1 Avenue de la Terrasse, 91 198 Gif-sur-Yvette, France.
Summary The central nervous system (cerebral ganglion) of adult ascidians is linked to the neural gland complex (NGC), which consists of a dorsal tubercle, a ciliated duct and a neural gland. The function of the NGC has been the subject of much debate. The recent publication of the complete genomic sequence of Ciona intestinalis provides new opportunities to examine the presence and distribution of protein families in this basal chordate. We focus here on the ascidian neuropeptide G-protein-coupled receptors (GPCRs), the vertebrate homologues of which are involved in homeostasis. In situ hybridization revealed that five Ciona GPCRs [vasopressin receptor, somatostatin receptor, CRH (corticotropin-releasing hormone) receptor, angiotensin receptor and tachykinin receptor] are expressed in the NGC of adult ascidians. These findings, together with histological and ultrastructural data, provide evidence to support a role for the ascidian NGC in maintaining ionic homeostasis. We further speculate about the potential similarities between the ascidian NGC and the vertebrate choroid plexus, a neural peri-ventricular organ.Precraniate origin of cranial motoneurons.
Héloïse D, HD. Dufour ; Zoubida, Z. Chettouh ; Carole, C. Deyts ; Renaud, R. de Rosa ; Christo, C. Goridis ; Jean-Stéphane, JS. Joly ; Jean-François, JF. Brunet.
Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8542, Ecole Normale Supérieure, 46 Rue d'Ulm, 75005 Paris, France.
The craniate head is innervated by cranial sensory and motor neurons. Cranial sensory neurons stem from the neurogenic placodes and neural crest and are seen as evolutionary innovations crucial in fulfilling the feeding and respiratory needs of the craniate "new head." In contrast, cranial motoneurons that are located in the hindbrain and motorize the head have an unclear phylogenetic status. Here we show that these motoneurons are in fact homologous to the motoneurons of the sessile postmetamorphic form of ascidians. The motoneurons of adult Ciona intestinalis, located in the cerebral ganglion and innervating muscles associated with the huge "branchial basket," express the transcription factors CiPhox2 and CiTbx20, whose vertebrate orthologues collectively define cranial motoneurons of the branchiovisceral class. Moreover, Ciona's postmetamorphic motoneurons arise from a hindbrain set aside during larval life and defined as such by its position (caudal to the prosensephalic sensory vesicle) and coexpression of CiPhox2 and CiHox1, whose orthologues collectively mark the vertebrate hindbrain. These data unveil that the postmetamorphic ascidian brain, assumed to be a derived feature, in fact corresponds to the vertebrate hindbrain and push back the evolutionary origin of cranial nerves to before the origin of craniates.Medaka simplet (FAM53B) belongs to a family of novel vertebrate genes controlling cell proliferation.
Violette, V. Thermes ; Eva, E. Candal ; Alessandro, A. Alunni ; Guillaume, G. Serin ; Franck, F. Bourrat ; Jean-Stéphane, JS. Joly.
INRA MSNC Group, DEPSN, Institut A. Fessard, CNRS, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
The identification of genes that regulate proliferation is of great importance to developmental biology, regenerative medicine and cancer research. Using an in situ screen on a cortical structure of the medaka fish brain, we identified the simplet gene (smp), which is homologous to the human FAM53B gene. smp was expressed in actively proliferating cells of the CNS throughout embryogenesis. It belongs to a family of vertebrate-specific genes with no characterized biochemical domains. We showed that FAM53B bound 14-3-3 chaperones, as well as SKIIP proteins, adaptor proteins connecting DNA-binding proteins to modulators of transcription. smp inactivation with morpholinos led to delayed epiboly and reduced embryonic size. Absence of Smp activity did not induce apoptosis, but resulted in a reduced cell proliferation rate and enlarged blastomeres. Moreover, smp was shown to control the expression of the pluripotency-associated oct4/pou5f1 gene. We propose that smp is a novel vertebrate-specific gene needed for cell proliferation and that it is probably associated with the maintenance of a pluripotent state.An automated in situ hybridization screen in the Medaka to identify unknown neural genes.
INRA/CNRS Group, DEPSN, Institut Fessard, CNRS, Gif-sur-Yvette, France.
Despite the fact that a large body of factors that play important roles in development are known, there are still large gaps in understanding the genetic pathways that govern these processes. To find previously unknown genes that are expressed during embryonic development, we optimized and performed an automated whole-mount in situ hybridization screen on medaka embryos at the end of somitogenesis. Partial cDNA sequences were compared against public databases and identified according to similarities found to other genes and gene products. Among 321 isolated genes showing specific expression in the central nervous system in at least one of five stages of development, 55.14% represented genes whose functions are already documented (in fish or other model organisms). Additionally, 16.51% were identified as conserved unknown genes or genes with unknown function. We provide new data on eight of these genes that presented a restricted expression pattern that allowed for formulating testable hypotheses on their developmental roles, and that were homologous to mammalian molecules of unknown function. Thus, gene expression screening in medaka is an efficient tool for isolating new regulators of embryonic development, and can complement genome-sequencing projects that are producing a high number of genes without ascribed functions.Expression domains suggest cell-cycle independent roles of growth-arrest molecules in the adult brain of the medaka, Oryzias latipes.
JE INRA U1126, INRA/CNRS Group, UPR 2197 DEPSN, CNRS, Institut de Neurosciences A. Fessard, Gif-sur-Yvette, France. firstname.lastname@example.org
Teleost fish are unique for their enormous potential to produce new neurons in the adult brain. Nevertheless, the regulation of this adult neurogenesis remains to be characterized. Does it resort to the same molecular mechanisms as those at play in embryonic development? Here, we analyse the expression of the neurogenic gene Ol-DeltaA in the brain of medaka (Oryzias latipes) embryos and adults. To determine the relationships between neurogenic and growth-arrest genes in the adult brain, we compare the expression domains of Ol-DeltaA with those of Ol-KIP and Ol-Gadd45gamma, two well-characterized genes involved in cell-cycle arrest and growth inhibition. While it is widely assumed that genes controlling cell-cycle exit show restricted expression domains next to proliferating cells (in the sites of prospective cell differentiation), we observe highly particular expression domains of Ol-KIP and Ol-Gadd45gamma not associated to proliferating areas of the adult brain, suggesting locally different and cell-cycle independent roles of these molecules in the adult brain.The dopamine-synthesizing cells in the swimming larva of the tunicate Ciona intestinalis are located only in the hypothalamus-related domain of the sensory vesicle.
Frédéric, F. Moret ; Lionel, L. Christiaen ; Carole, C. Deyts ; Maryline, M. Blin ; Jean-Stéphane, JS. Joly ; Philippe, P. Vernier.
Development, Evolution, Plasticity of the Nervous System, UPR 2197, Institut de Neurobiologie Alfred Fessard, C.N.R.S., 1, ave de la Terrasse, F-91198 Gif-sur-Yvette, France. email@example.com
Dopamine is a major neuromodulator synthesized by numerous cell populations in the vertebrate forebrain and midbrain. Owing to the simple organization of its larval nervous system, ascidian tunicates provide a useful model to investigate the anatomy, neurogenesis and differentiation of the dopaminergic neural network underlying the stereotypical swimming behaviour of its chordate-type larva. This study provides a high-resolution cellular analysis of tyrosine hydroxylase (TH)-positive and dopamine-positive cells in Ciona intestinalis embryos and larvae. Dopamine cells are present only in the sensory vesicle of the Ciona larval brain, which may be an ancestral chordate feature. The dopamine-positive cells of the ascidian sensory vesicle are located in the expression domain of homologues of vertebrate hypothalamic markers. We show here that the larval coronet cells also arise from this domain. As a similar association between coronet cells and the hypothalamus was reported in bony and cartilaginous fishes, we propose that part of the ascidian ventral sensory vesicle is the remnant of a proto-hypothalamus that may have been present in the chordate ancestor. As dopaminergic cells are specified in the hypothalamus in all vertebrates, we suggest that the mechanisms of dopamine cell specification are conserved in the hypothalamus of Ciona and vertebrates. To test this hypothesis, we have identified new candidate regulators of dopaminergic specification in Ciona based on their expression patterns, which can now be compared with those in vertebrates.Embryonic versus blastogenetic development in the compound ascidian Botryllus schlosseri: insights from Pitx expression patterns.
Stefano, S. Tiozzo ; Lionel, L. Christiaen ; Carole, C. Deyts ; Lucia, L. Manni ; Jean-Stéphane, JS. Joly ; Paolo, P. Burighel.
Dipartimento di Biologia, Università di Padova, Via U. Bassi 58/B 35121 Padova, Italy. firstname.lastname@example.org
The colonial ascidians reproduce either sexually or asexually, having evolved a rich variety of modes of propagative development. During embryogenesis, the fertilized egg develops into a swimming tadpole larva that subsequently metamorphoses into a sessile oozooid. Clonal individuals (blastozooids), resembling oozooids, are formed from few bud-forming multipotent somatic cells, following a wide range of ways that seem to characterize each family of this class. Here, we compare these two developmental processes in the compound ascidian species Botryllus schlosseri to determine whether similar gene activities are used during embryogenesis/metamorphosis and recruited in the asexual development. We analyzed expression of Pitx, a Paired-related homeobox gene. Pitx genes are key developmental genes in vertebrates, and their expression is reported to be conserved in chordate stomodea and in the establishment of left/right asymmetries. Here, we report full-length cDNA cloning of a B. schlosseri Pitx ortholog (Bs-Pitx) and expression analysis during both embryo/metamorphosis and blastogenesis. During organogenesis of both developmental sequences, Bs-Pitx was detected in identical domains: the stomodeum/neural complex and asymmetrically in the left digestive system. In striking contrast, expression patterns at early stages differ deeply. These observations provide the first evidence for a key developmental gene being deployed in essentially similar ways in two different developmental sequences that eventually give rise to similar zooids.Regulatory gene expressions in the ascidian ventral sensory vesicle: evolutionary relationships with the vertebrate hypothalamus.
Frédéric, F. Moret ; Lionel, L. Christiaen ; Carole, C. Deyts ; Maryline, M. Blin ; Philippe, P. Vernier ; Jean-Stéphane, JS. Joly.
Development, Evolution and Plasticity of the Nervous System, Institut de Neurobiologie Alfred Fessard, Centre National de la Recherche Scientifique, UPR2197, 1 ave de la terrasse, F-91198 Gif-sur-Yvette, France.
In extant chordates, the overall patterning along the anteroposterior and dorsoventral axes of the neural tube is remarkably conserved. It has thus been proposed that four domains corresponding to the vertebrate presumptive forebrain, midbrain-hindbrain transition, hindbrain, and spinal cord were already present in the common chordate ancestor. To obtain insights on the evolution of the patterning of the anterior neural tube, we performed a study aimed at characterizing the expression of regulatory genes in the sensory vesicle of Ciona intestinalis, the anteriormost part of the central nervous system (CNS) related to the vertebrate forebrain, at tailbud stages. Selected genes encoded primarily for homologues of transcription factors involved in vertebrate forebrain patterning. Seven of these genes were expressed in the ventral sensory vesicle. A prominent feature of these ascidian genes is their restricted and complementary domains of expression at tailbud stages. These patterning markers thus refine the map of the developing sensory vesicle. Furthermore, they allow us to propose that a large part of the ventral and lateral sensory vesicle consists in a patterning domain corresponding to the vertebrate presumptive hypothalamus.A modular cis-regulatory system controls isoform-specific pitx expression in ascidian stomodaeum.
INRA junior group, UPR2197 DEPSN, INAF, CNRS, 1 Avenue De La Terrasse, F-91198 Gif-sur-Yvette, France.
Pituitary homeobox (pitx) genes have been identified in vertebrates as critical molecular determinants of various craniofacial ontogenetic processes including pituitary organogenesis. Accordingly, a prominent conserved feature of pitx genes in chordates is their early expression in the anterior neural boundary (ANB) and oral ectoderm, also known as the stomodaeum. Here we used the ascidian model species Ciona intestinalis to investigate pitx gene organization and cis-regulatory logic during early stages of oral development. Two distinct Ci-pitx mRNA variants were found to be expressed in mutually exclusive embryonic domains. Ci-pitx and vertebrate pitx2 genes display remarkably similar exon usage and organization, suggesting ancestry of the pitx transcriptional unit and regulation in chordates. We next combined phylogenetic footprinting, transient transgenesis, and confocal imaging methods to study the Ci-pitx cis-regulatory system, with special emphasis on the regulation of isoform-specific ANB/stomodaeal expression. Among 10 conserved noncoding sequences (CNSs) interspersed in C. intestinalis and Ciona savignyi pitx loci, we identified two separate cis-regulatory modules (CRMs) that drive ANB/stomodaeal expression in complementary spatiotemporal patterns. We discuss the developmental relevance of these data that provide an entry point to investigate the gene regulatory networks (GRNs) that position and shape oral structures in chordates.Highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI.
Developmental Biology Program, European Molecular Biology Laboratory (EMBL), 69117-Heidelberg, Germany.
The colonial ascidians reproduce either sexually or asexually, having evolved a rich variety of modes of propagative development. During embryogenesis, the fertilized egg develops into a swimming tadpole larva that subsequently metamorphoses into a sessile oozooid. Clonal individuals (blastozooids), resembling oozooids, are formed from few bud-forming multipotent somatic cells, following a wide range of ways that seem to characterize each family of this class. Here, we compare these two developmental processes in the compound ascidian species Botryllus schlosseri to determine whether similar gene activities are used during embryogenesis/metamorphosis and recruited in the asexual development. We analyzed expression of Pitx, a Paired-related homeobox gene. Pitx genes are key developmental genes in vertebrates, and their expression is reported to be conserved in chordate stomodea and in the establishment of left/right asymmetries. Here, we report full-length cDNA cloning of a B. schlosseri Pitx ortholog (Bs-Pitx) and expression analysis during both embryo/metamorphosis and blastogenesis. During organogenesis of both developmental sequences, Bs-Pitx was detected in identical domains: the stomodeum/neural complex and asymmetrically in the left digestive system. In striking contrast, expression patterns at early stages differ deeply. These observations provide the first evidence for a key developmental gene being deployed in essentially similar ways in two different developmental sequences that eventually give rise to similar zooids.Neurogenic and non-neurogenic placodes in ascidians.
Lucia, L. Manni ; Nancy J, NJ. Lane ; Jean-Stéphane, JS. Joly ; Fabio, F. Gasparini ; Stefano, S. Tiozzo ; Federico, F. Caicci ; Giovanna, G. Zaniolo ; Paolo, P. Burighel.
Dipartimento di Biologia, Università di Padova, I-35121 Padova, Italy. email@example.com
The late differentiation of the ectodermal layer is analysed in the ascidians Ciona intestinalis and Botryllus schlosseri, by means of light and electron microscopy, in order to verify the possible presence of placodal structures. Cranial placodes, ectodermal regions giving rise to nonepidermal cell types, are classically found exclusively in vertebrates; however, data are accumulating to demonstrate that the nonvertebrate chordates possess both the genetic machinery involved in placode differentiation, and ectodermal structures that are possible homologues of vertebrate placodes. Here, the term "placode" is used in a broad sense and defines thickenings of the ectodermal layer that can exhibit an interruption of the basal lamina where cells delaminate, and so are able to acquire a nonepidermal fate. A number of neurogenic placodes, ones capable of producing neurons, have been recognised; their derivatives have been analysed and their possible homologies with vertebrate placodes are discussed. In particular, the stomodeal placode may be considered a multiple placode, being composed of different sorts of placodes: part of it, which differentiates hair cells, is discussed as homologous to the octavo-lateralis placodes, while the remaining portion, giving rise to the ciliated duct of the neural gland, is considered homologous to the adenohypophyseal placode. The neurohypophyseal placode may include the homologues of the hypothalamus and vertebrate olfactory placode; the rostral placode, producing the sensorial papillae, may possibly be homologous to the placodes of the adhesive gland of vertebrates.Culture of adult ascidians and ascidian genetics.
Carolyn, C. Hendrickson ; Lionel, L. Christiaen ; Karine, K. Deschet ; Di, D. Jiang ; Jean-Stéphane, JS. Joly ; Laurent, L. Legendre ; Yuki, Y. Nakatani ; Jason, J. Tresser ; William C, WC. Smith.
Neuroscience Research Institute, Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106, USA.
The colonial ascidians reproduce either sexually or asexually, having evolved a rich variety of modes of propagative development. During embryogenesis, the fertilized egg develops into a swimming tadpole larva that subsequently metamorphoses into a sessile oozooid. Clonal individuals (blastozooids), resembling oozooids, are formed from few bud-forming multipotent somatic cells, following a wide range of ways that seem to characterize each family of this class. Here, we compare these two developmental processes in the compound ascidian species Botryllus schlosseri to determine whether similar gene activities are used during embryogenesis/metamorphosis and recruited in the asexual development. We analyzed expression of Pitx, a Paired-related homeobox gene. Pitx genes are key developmental genes in vertebrates, and their expression is reported to be conserved in chordate stomodea and in the establishment of left/right asymmetries. Here, we report full-length cDNA cloning of a B. schlosseri Pitx ortholog (Bs-Pitx) and expression analysis during both embryo/metamorphosis and blastogenesis. During organogenesis of both developmental sequences, Bs-Pitx was detected in identical domains: the stomodeum/neural complex and asymmetrically in the left digestive system. In striking contrast, expression patterns at early stages differ deeply. These observations provide the first evidence for a key developmental gene being deployed in essentially similar ways in two different developmental sequences that eventually give rise to similar zooids.Medaka as a model system for the characterisation of cell cycle regulators: a functional analysis of Ol-Gadd45gamma during early embryogenesis.
INRA/CNRS Group, DEPSN, Institut Fessard, CNRS, 1 Avenue de la Terrasse, 91 198 Gif-sur-Yvette, France. firstname.lastname@example.org
Numerous studies, mostly performed on mammalian cell cultures, have implicated the Gadd45 family of small acidic proteins in cell cycle control (arrest and/or engagement in the apoptotic pathway). We report here the cloning, detailled expression pattern and functional characterisation in embryonic development of Ol-Gadd45gamma, the Oryzias latipes ortholog of mammalian Gadd45gamma. Its expression pattern, notably in the developing brain (optic tectum) strongly suggests that it is involved in cell cycle exit. Gain-of-function experiments (through mRNA injection) slowed down early development, and produced embryos clearly reduced in size, while morpholino knockdowns resulted in small embryos over-sensitive to DNA damage (UV irradiation). We further demonstrated that, following Ol-Gadd45gamma overexpression, cells are proliferation-arrested before both G1/S and G2/M cell cycle checkpoints, while in the MO-Ol-Gadd45 loss-of-function experiments cells are engaged in apoptosis rather than prevented from proliferating. These results show that Ol-Gadd45gamma is likely to play an important role in coordinating cell fate decisions during neurogenesis; they also demonstrate that the medakafish is a promising model to analyse in vivo the developmental control of the cell cycle.Effects of microcystin-LR on development of medaka fish embryos (Oryzias latipes).
Claire, C. Jacquet ; Violette, V. Thermes ; Amaury, A. de Luze ; Simone, S. Puiseux-Dao ; Cécile, C. Bernard ; Jean-Stéphane, JS. Joly ; Franck, F. Bourrat ; Marc, M. Edery.
USM 0505 Ecosystèmes et Interactions Toxiques, Muséum National d'Histoire Naturelle, 12 rue Buffon, F-75231, Paris cedex 05, France.
Chronic and subchronic toxicity from exposure to microcystins, cyclic hepatotoxic heptapeptides from cyanobacteria, receives increasing attention as a public human health biohazard. So far, the effects of microcystin on fish have been studied mainly in adults, rather than during early life stages. Limitations of direct ambient exposure experiments to fish egg have resulted from the difficult access of microcystin through the egg chorion. Using a microinjection technology, we have introduced microcystin-LR (MC-LR) directly into one-cell stage embryos or into the vitellus of late neurula embryos (stage 19) or into the vitellus of stage 25 embryos of medaka (Oryzias latipes) at the onset of the liver anlage. Microinjection (100 pl; stage 1 or 2 nl; stage 19 or 25) of MC-LR resulted in a dose dependent mortality of embryos. Survival rates were reduced up to 90% with microcystin concentrations of 10 or 1 microg/ml (corresponding to 1-20 pg or 0.1-2 pg of toxin injected), injected either at stages 1, 19 or 25. Also, a dose dependent advanced embryonic hatching processing was observed; hatching being brought forward from 2 or 3 days compared to controls in most of the microcystin injected groups. In agreement with the known hepatotoxic effects of microcystin, injected embryos consistently displayed hepatobiliary abnormalities such as liver hypertrophy and hepatic hemorrhage, also evidenced in post-hatching juveniles. Thus, the methodology presented in this paper should be valuable tool to analyze the effects of toxins on the development of aquatic vertebrate embryos.I-SceI meganuclease mediates highly efficient transgenesis in fish.
Violette, V. Thermes ; Clemens, C. Grabher ; Filomena, F. Ristoratore ; Franck, F. Bourrat ; André, A. Choulika ; Jochen, J. Wittbrodt ; Jean-Stéphane, JS. Joly.
INRA Junior Group, UPR2197, Institut de Neurobiologie A. Fessard, CNRS, Avenue de la Terrasse, 91 198 Gif-Sur-Yvette, France.
The widespread use of fish as model systems is still limited by the mosaic distribution of cells transiently expressing transgenes leading to a low frequency of transgenic fish. Here we present a strategy that overcomes this problem. Transgenes of interest were flanked by two I-SceI meganuclease recognition sites, and co-injected together with the I-SceI meganuclease enzyme into medaka embryos (Oryzias latipes) at the one-cell stage. First, the promoter dependent expression was strongly enhanced. Already in F0, 76% of the embryos exhibited uniform promoter dependent expression compared to 26% when injections were performed without meganuclease. Second, the transgenesis frequency was raised to 30.5%. Even more striking was the increase in the germline transmission rate. Whereas in standard protocols it does not exceed a few percent, the number of transgenic F1 offspring of an identified founder fish reached the optimum of 50% in most lines resulting from meganuclease co-injection. Southern blot analysis showed that the individual integration loci contain only one or few copies of the transgene in tandem. At a lower rate this method also leads to enhancer trapping effects, novel patterns that are likely due to the integration of the transgene in the vicinity of enhancer elements. Meganuclease co-injection thus provides a simple and highly efficient tool to improve transgenesis by microinjection.Pitx genes in Tunicates provide new molecular insight into the evolutionary origin of pituitary.
Lionel, L. Christiaen ; Paolo, P. Burighel ; William C, WC. Smith ; Philippe, P. Vernier ; Franck, F. Bourrat ; Jean-Stéphane, JS. Joly.
INRA Junior Group Morphogenèse du Système Nerveux des Chordés, UPR2197 DEPSN, Institut de Neurobiologie Alfred Fessard, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France. email@example.com
We have initiated a project aimed at documenting molecular and cellular changes underlying the emergence of the hypothalamo-hypophyseal axis in Chordates. Considering the phylogenetic position of Tunicates and the 'pan-hypophyseal' expression pattern of Pitx genes in Vertebrate pituitary, we searched for a Pitx-related homeobox gene in the ascidian Ciona intestinalis, and identified Ci-Pitx (ona intestinalis uitary homeobo gene). We also isolated Cs-Pitx and Bs-Pitx, the Ci-Pitx respective counterparts of Ciona savignyi and Botryllus schlosseri, two other Tunicate species. Ci-Pitx mRNA encodes a putative protein exhibiting the diagnostic K50-Paired-class homeodomain and a conserved C-terminal Aristaless domain. Embryonic expression pattern of Ci-Pitx revealed a conserved expression domain in the anterior neural ridge and subsequently in the pharyngeal primordium, defined in Vertebrates as the stomodeal ectomere, which encompasses the presumptive pituitary territory. This shows that expression at early steps of pituitary development is a feature of Pitx-related genes that was already present in the last common ancestor of Chordates.
Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis.
M, M. Crispo ; M, M. Vilariño ; P C, PC. Dos Santos-Neto ; R, R. Núñez-Olivera ; F, F. Cuadro ; N, N. Barrera ; A P, AP. Mulet ; T H, TH. Nguyen ; I, I. Anegón ; A, A. Menchaca.
Unidad de Animales Transgénicos y de Experimentación (UATE), Institut Pasteur de Montevideo, Mataojo 2020, 11400, Montevideo, Uruguay.
Lentiviral technology has been recently proposed to generate transgenic farm animals more efficiently and easier than traditional techniques. The objective was to evaluate several parameters of lambs obtained by lentiviral transgenesis in comparison with non-transgenic counterparts. In vitro produced embryos were microinjected (TG group) at two-cell stage with a lentiviral construct containing enhanced green fluorescent protein (eGFP) gene, while embryos produced by in vitro fertilization (IVF group) or intrauterine insemination (IUI group) were not microinjected. Microinjection technique efficiently generated eight-cell transgenic embryos (97.4 %; 114/117). Development rate on day 5 after fertilization was similar for TG (39.3 %, 46/117) and IVF embryos (39.6 %, 44/111). Pregnancy rate was detected in 50.0 % (6/12) of recipient ewes with TG embryos, in 46.7 % (7/15) with IVF embryos, and in 65.0 % (13/20) of IUI ewes (P = NS). Nine lambs were born in TG group, six lambs in IVF group, and 16 lambs in IUI group. All TG lambs (9/9) were GFP positive to real-time PCR and eight (88.9 %) showed a strong and evident GFP expression in mucosae, eyes and keratin tissues. Fetal growth monitored every 15 day by ultrasonography did not show significant differences. Transgenic lambs neither differ in morphometric variables in comparison with non transgenic IVF lambs within 3 months after birth. Transmission of the transgene to the progeny was observed in green fluorescent embryos produced by IVF using semen from the TG founder lambs. In conclusion, this study demonstrates the high efficiency of lentiviral technology to produce transgenic sheep, with no clinic differences in comparison with non transgenic lambs."Transgenesis, recent technical developments and applications" Nantes, 8th June 2009.
Séverine, S. Ménoret ; Laurent, L. Tesson ; Séverine, S. Remy ; Claire, C. Usal ; Anne-Laure, AL. Iscache ; Ignacio, I. Anegon.
Plate-Forme Transgenese Rat IBiSA-CNRS, 30 Bd Jean Monnet, 44093, Nantes, France. Severine.Menoret@univ-nantes.fr
Lentiviral vectors are now well recognized as good vehicles for gene delivery. This is because they can efficiently transduce both dividing and post-mitotic cells, and stably integrate into the host genome allowing for long-term expression of the transgene. Their potential utility for the generation of transgenic animals has been recognized as an attractive and promising alternative to the conventional DNA-microinjection method which lacks efficiency. The initial success of lentiviral transgenesis in mice considerably broadened its use in other species, in which classical transgenic techniques are difficult, such as in the rat.In this chapter, we describe detailed procedures for both the production of human immunodeficiency virus-1 (HIV-1)-derived lentiviral vectors and for the generation of transgenic rats by injection of these vectors into the perivitelline space of fertilized one-cell eggs.The use of lentiviral vectors to obtain transgenic rats.
Séverine, S. Remy ; Tuan Huy, TH. Nguyen ; Séverine, S. Ménoret ; Laurent, L. Tesson ; Claire, C. Usal ; Ignacio, I. Anegon.
INSERM, UMR643, Nantes, France.
Lentiviral vectors are now well recognized as good vehicles for gene delivery. This is because they can efficiently transduce both dividing and post-mitotic cells, and stably integrate into the host genome allowing for long-term expression of the transgene. Their potential utility for the generation of transgenic animals has been recognized as an attractive and promising alternative to the conventional DNA-microinjection method which lacks efficiency. The initial success of lentiviral transgenesis in mice considerably broadened its use in other species, in which classical transgenic techniques are difficult, such as in the rat.In this chapter, we describe detailed procedures for both the production of human immunodeficiency virus-1 (HIV-1)-derived lentiviral vectors and for the generation of transgenic rats by injection of these vectors into the perivitelline space of fertilized one-cell eggs.Transgenic modifications of the rat genome.
Laurent, L. Tesson ; Jean, J. Cozzi ; Séverine, S. Ménoret ; Séverine, S. Rémy ; Claire, C. Usal ; Alexandre, A. Fraichard ; Ignacio, I. Anegon.
Institut de Transplantation et de Recherche en Transplantation (ITERT), F-44093, Nantes, France.
The laboratory rat (R. norvegicus) is a very important experimental animal in several fields of biomedical research. This review describes the various techniques that have been used to generate transgenic rats: classical DNA microinjection and more recently described techniques such as lentiviral vector-mediated DNA transfer into early embryos, sperm-mediated transgenesis, embryo cloning by nuclear transfer and germline mutagenesis. It will also cover techniques associated to transgenesis such as sperm cryopreservation, embryo freezing and determination of zygosity. The availability of several technologies allowing genetic manipulation in the rat coupled to genomic data will allow biomedical research to fully benefit from the rat as an experimental animal.No functional benefit for hDAF-transgenic rat livers despite protection from tissue damage following perfusion with human serum.
Hein, H. Stockmann ; Caroline, C. Verbakel ; Petra, P. Okkema ; Fred, F. Bonthuis ; Severin, S. Menoret ; Ignacio, I. Anegon ; Richard, R. Marquet ; Jan, J. IJzermans.
Department of General Surgery, University Hospital Dijkzigt, 3000 CA Rotterdam, The Netherlands.
Currently, xenogeneic extracorporeal liver perfusion is used in the treatment of acute liver failure. In order to determine whether transgeneity for human regulatory proteins could improve the functional outcome of the ex-vivo liver in relation to the histopathological changes, we studied the effect of the humoral mechanism in xenogeneic isolated rat liver perfusion in normal and transgenic rat livers. Isolated rat liver perfusion was performed for 2 h in normal rat livers with Krebs Henseleit (KH) and human serum (HS), and in livers transgenic for human decay accelerating factor (hDAF; Tg HS). Function of the liver was established by measurement of liver enzymes and bile production, and clearance of bromosulphophthalein (BSP). Tissue specimens taken after perfusion were analysed by routine histology and immunofluorescence staining for C3c deposition. No change in release of liver enzymes could be established throughout the perfusion period. In the 2nd hour, a higher level of bile production was seen for the transgenic group than for the HS group. The transgenic rat livers outperformed the normal livers perfused with HS, when BSP concentration in the bile was measured; however, clearance of BSP from the perfusate was not significantly different. Haematoxylin-eosin (HE) staining of the liver tissue showed evidence of hyperacute rejection in the HS group. There was only mild tissue injury in the transgenic liver. High-intensity fluorescent staining for C3c deposition was seen in the normal livers perfused with HS, and significantly less in the transgenic livers. Although histologically less tissue damage and less C3c deposition was shown, no significantly improved function of the livers transgenic for hDAF was established. These results suggest that for short-term extracorporeal liver perfusion transgenesis offers no functional benefit.Transgenesis in rats: technical aspects and models.
INSERM U437, Institut de Transplantation et Recherche en Transplantation, Nantes, France.
The production of transgenic rats by DNA-microinjection into fertilized ova has now become an established procedure, although fewer than 20 lines have been described during the last 5 years. Overall, transgenic rats remain more difficult to produce than transgenic mice, but satisfactory yields have been obtained by several laboratories. A review of the methods used to generate transgenic rats shows considerable variation between different laboratories, particularly in choice of strain, superovulation protocols and the use of embryo culture before reimplantation. In some instances, the production of transgenic rats has provided data that are new and relevant, compared to data obtained in mice bearing the same transgene. Models have been developed for human diseases such as hypertension and autoimmunity, and applications have been found in the study of carcinogenesis and in pharmacological research. Transgenic rat technology also opens up interesting perspectives for transplantation research, in which microsurgery is an essential procedure. Intensive research is in progress in several laboratories to produce rat embryonic stem (ES) cell lines, but existing lines have not participated in germ line formation a prerequisite for their use in gene knock out experiments.
CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish.
1] Institut Curie, Centre de Recherche, Paris, France.  Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 3215, Paris, France.  Institut National de la Santé de la Recherche Médicale (INSERM) U934, Paris, France. 
Here we present a protocol for the conversion of eGFP-transgenic zebrafish lines into lines expressing Gal4 from the same locus. This conversion allows the in-depth analysis of the former eGFP-expressing cell population; with the Gal4-upstream activating sequence (UAS) system, diverse UAS transgenes can be transactivated. Site-specific targeting of the gene encoding eGFP is achieved using the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. A single-guide RNA (sgRNA) that targets eGFP is injected into embryos together with a donor vector containing an optimized version of Gal4 (KalTA4) to trigger integration of the donor into the targeted eGFP genomic location. To enable screening for successful integration events, injection is performed in a UAS:RFP transgenic background; fish showing mosaic eGFP-to-RFP conversion are raised to adulthood. The progeny of these adult fish are then screened for stable germline transmission, and converted progeny are used to generate stable lines. We have been able to generate two stably converted transgenic lines within 4 months.Editing and investigating genomes with TALE and CRISPR/Cas systems: applications of artificial TALE and CRISPR-Cas systems.
Here we present a protocol for the conversion of eGFP-transgenic zebrafish lines into lines expressing Gal4 from the same locus. This conversion allows the in-depth analysis of the former eGFP-expressing cell population; with the Gal4-upstream activating sequence (UAS) system, diverse UAS transgenes can be transactivated. Site-specific targeting of the gene encoding eGFP is achieved using the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. A single-guide RNA (sgRNA) that targets eGFP is injected into embryos together with a donor vector containing an optimized version of Gal4 (KalTA4) to trigger integration of the donor into the targeted eGFP genomic location. To enable screening for successful integration events, injection is performed in a UAS:RFP transgenic background; fish showing mosaic eGFP-to-RFP conversion are raised to adulthood. The progeny of these adult fish are then screened for stable germline transmission, and converted progeny are used to generate stable lines. We have been able to generate two stably converted transgenic lines within 4 months.Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining.
Hind, H. Ghezraoui ; Marion, M. Piganeau ; Benjamin, B. Renouf ; Jean-Baptiste, JB. Renaud ; Annahita, A. Sallmyr ; Brian, B. Ruis ; Sehyun, S. Oh ; Alan E, AE. Tomkinson ; Eric A, EA. Hendrickson ; Carine, C. Giovannangeli ; Maria, M. Jasin ; Erika, E. Brunet.
Museum National d'Histoire Naturelle, 43 rue Cuvier, F-75005 Paris, France; CNRS, UMR7196, 43 rue Cuvier, F-75005 Paris, France; Inserm, U1154, 43 rue Cuvier, F-75005 Paris, France.
Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4. Translocation junctions had significantly longer deletions and more microhomology, indicative of alt-NHEJ. Thus, unlike mouse cells, translocations in human cells are generated by c-NHEJ. Human cancer translocations induced by paired Cas9 nicks also showed a dependence on c-NHEJ, despite having distinct joining characteristics. These results demonstrate an unexpected and striking species-specific difference for common genomic rearrangements associated with tumorigenesis.Gene targeting in rats using transcription activator-like effector nucleases.
Séverine, S. Ménoret ; Laurent, L. Tesson ; Séverine, S. Rémy ; Claire, C. Usal ; Virginie, V. Thépenier ; Reynald, R. Thinard ; Laure-Hélène, LH. Ouisse ; Anne, A. De Cian ; Carine, C. Giovannangeli ; Jean-Paul, JP. Concordet ; Ignacio, I. Anegon.
Transgenic Rats Nantes IBiSA-Centre National de Recherche Scientifique, F44093 Nantes, France; ITUN, CHU Nantes, F44000 Nantes, France; INSERM UMR 1064-Center for Research in Transplantation and Immunology, France. Electronic address: severine.menoret@uni
The rat is a model of choice to understanding gene function and modeling human diseases. Since recent years, successful engineering technologies using gene-specific nucleases have been developed to gene edit the genome of different species, including the rat. This development has become important for the creation of new rat animals models of human diseases, analyze the role of genes and express recombinant proteins. Transcription activator-like (TALE) nucleases are designed nucleases consist of a DNA binding domain fused to a nuclease domain capable of cleaving the targeted DNA. We describe a detailed protocol for generating knockout rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.
Séverine, S. Remy ; Laurent, L. Tesson ; Séverine, S. Menoret ; Claire, C. Usal ; Anne, A. De Cian ; Virginie, V. Thepenier ; Reynald, R. Thinard ; Daniel, D. Baron ; Marine, M. Charpentier ; Jean-Baptiste, JB. Renaud ; Roland, R. Buelow ; Gregory J, GJ. Cost ; Carine, C. Giovannangeli ; Alexandre, A. Fraichard ; Jean-Paul, JP. Concordet ; Ignacio, I. Anegon.
INSERM UMR 1064-ITUN, CHU de Nantes, Nantes F44093, France; Platform Rat Transgenesis, Nantes F44093, France;
The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.
Thomas O, TO. Auer ; Karine, K. Duroure ; Anne, A. De Cian ; Jean-Paul, JP. Concordet ; Filippo, F. Del Bene.
Institut Curie, Centre de Recherche, Paris F-75248, France;
Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.Cancer translocations in human cells induced by zinc finger and TALE nucleases.
Marion, M. Piganeau ; Hind, H. Ghezraoui ; Anne, A. De Cian ; Lionel, L. Guittat ; Mark, M. Tomishima ; Loic, L. Perrouault ; Oliver, O. René ; George E, GE. Katibah ; Lei, L. Zhang ; Michael C, MC. Holmes ; Yannick, Y. Doyon ; Jean-Paul, JP. Concordet ; Carine, C. Giovannangeli ; Maria, M. Jasin ; Erika, E. Brunet.
Museum National d'Histoire Naturelle, CNRS UMR7196, Inserm U565, 75005 Paris, France.
Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1-FLI1 and NPM1-ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell-derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases.Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.
Charlotte, C. Andrieu-Soler ; Mariana, M. Casas ; Anne-Marie, AM. Faussat ; Christelle, C. Gandolphe ; Marc, M. Doat ; Denis, D. Tempé ; Carine, C. Giovannangeli ; Francine, F. Behar-Cohen ; Jean-Paul, JP. Concordet.
INSERM U598, Institut Biomédical des Cordeliers 15 rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France.
Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor betaPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.
Dopamine inhibits reproduction in female zebrafish (Danio rerio) via three pituitary D2 receptor subtypes.
Romain, R. Fontaine ; Pierre, P. Affaticati ; Kei, K. Yamamoto ; Cécile, C. Jolly ; Charlotte, C. Bureau ; Sylvie, S. Baloche ; Françoise, F. Gonnet ; Philippe, P. Vernier ; Sylvie, S. Dufour ; Catherine, C. Pasqualini.
Centre National de la Recherche Scientifique, Unité Propre de Recherche 3294, Neurobiologie et Développement, Avenue de la Terrasse, bat 5E, Gif-sur-Yvette, 91198 Cedex, France.
In many teleosts, the stimulatory control of gonadotrope axis by GnRH is opposed by an inhibitory control by dopamine (DA). The functional importance of this inhibitory pathway differs widely from one teleostean species to another. The zebrafish (Danio rerio) is a teleost fish that has become increasingly popular as an experimental vertebrate model. However, the role of DA in the neuroendocrine control of its reproduction has never been studied. Here the authors evaluated in sexually regressed female zebrafish the effects of in vivo treatments with a DA D2 receptor (D2-R) antagonist domperidone, or a GnRH agonist, alone and in combination, on the pituitary level of FSHβ and LHβ transcripts, the gonadosomatic index, and the ovarian histology. Only the double treatment with GnRH agonist and domperidone could induce an increase in the expression of LHβ, in the gonadosomatic index, and a stimulation of ovarian vitellogenesis, indicating that removal of dopaminergic inhibition is required for the stimulatory action of GnRH and reactivation of ovarian function to occur. Using double immunofluorescent staining on pituitary, the authors showed in this species the innervation of LH cells by tyrosine-hydroxylase immunoreactive fibers. Finally, using in situ hybridization and immunofluorescence, the authors showed that the three subtypes of zebrafish DA D2-R (D2a, D2b, and D2c) were expressed in LH-producing cells, suggesting that they all may be involved in mediating this inhibition. These results show for the first time that, in zebrafish, DA has a direct and potent inhibitory action capable of opposing the stimulatory effect of GnRH in the neuroendocrine control of reproduction.Development of hypothalamic serotoninergic neurons requires Fgf signalling via the ETS-domain transcription factor Etv5b.
Adriana, A. Bosco ; Charlotte, C. Bureau ; Pierre, P. Affaticati ; Patricia, P. Gaspar ; Laure, L. Bally-Cuif ; Christina, C. Lillesaar.
Zebrafish Neurogenetics Group, Laboratory of Neurobiology and Development, CNRS UPR3294, Institute of Neurobiology Albert Fessard, 1 Avenue de Terrasse, 91198 Gif-sur-Yvette, France.
Serotonin is a monoamine neurotransmitter that is involved in numerous physiological functions and its dysregulation is implicated in various psychiatric diseases. In all non-placental vertebrates, serotoninergic (5-HT) neurons are present in several regions of the brain, including the hypothalamus. In placental mammals, however, 5-HT neurons are located in the raphe nuclei only. In all species, though, 5-HT neurons constitute a functionally and molecularly heterogeneous population. How the non-raphe 5-HT populations are developmentally encoded is unknown. Using the zebrafish model we show that, in contrast to the raphe populations, hypothalamic 5-HT neurons are generated independently of the ETS-domain transcription factor Pet1 (Fev). By applying a combination of pharmacological tools and gene knockdown and/or overexpression experiments, we demonstrate that Fgf signalling acts via another ETS-domain transcription factor, Etv5b (Erm), to induce hypothalamic 5-HT neurons. We provide evidence that Etv5b exerts its effects by regulating cell cycle parameters in 5-HT progenitors. Our results highlight a novel role for Etv5b in neuronal development and provide support for the existence of a developmental heterogeneity among 5-HT neurons in their requirement for ETS-domain transcription factors.A GAL4-driver line resource for Drosophila neurobiology.
Arnim, A. Jenett ; Gerald M, GM. Rubin ; Teri-T B, TT. Ngo ; David, D. Shepherd ; Christine, C. Murphy ; Heather, H. Dionne ; Barret D, BD. Pfeiffer ; Amanda, A. Cavallaro ; Donald, D. Hall ; Jennifer, J. Jeter ; Nirmala, N. Iyer ; Dona, D. Fetter ; Joanna H, JH. Hausenfluck ; Hanchuan, H. Peng ; Eric T, ET. Trautman ; Robert R, RR. Svirskas ; Eugene W, EW. Myers ; Zbigniew R, ZR. Iwinski ; Yoshinori, Y. Aso ; Gina M, GM. DePasquale ; Adrianne, A. Enos ; Phuson, P. Hulamm ; Shing Chun Benny, SC. Lam ; Hsing-Hsi, HH. Li ; Todd R, TR. Laverty ; Fuhui, F. Long ; Lei, L. Qu ; Sean D, SD. Murphy ; Konrad, K. Rokicki ; Todd, T. Safford ; Kshiti, K. Shaw ; Julie H, JH. Simpson ; Allison, A. Sowell ; Susana, S. Tae ; Yang, Y. Yu ; Christopher T, CT. Zugates.
Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
We established a collection of 7,000 transgenic lines of Drosophila melanogaster. Expression of GAL4 in each line is controlled by a different, defined fragment of genomic DNA that serves as a transcriptional enhancer. We used confocal microscopy of dissected nervous systems to determine the expression patterns driven by each fragment in the adult brain and ventral nerve cord. We present image data on 6,650 lines. Using both manual and machine-assisted annotation, we describe the expression patterns in the most useful lines. We illustrate the utility of these data for identifying novel neuronal cell types, revealing brain asymmetry, and describing the nature and extent of neuronal shape stereotypy. The GAL4 lines allow expression of exogenous genes in distinct, small subsets of the adult nervous system. The set of DNA fragments, each driving a documented expression pattern, will facilitate the generation of additional constructs for manipulating neuronal function.BrainAligner: 3D registration atlases of Drosophila brains.
Hanchuan, H. Peng ; Phuong, P. Chung ; Fuhui, F. Long ; Lei, L. Qu ; Arnim, A. Jenett ; Andrew M, AM. Seeds ; Eugene W, EW. Myers ; Julie H, JH. Simpson.
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA. firstname.lastname@example.org
Analyzing Drosophila melanogaster neural expression patterns in thousands of three-dimensional image stacks of individual brains requires registering them into a canonical framework based on a fiducial reference of neuropil morphology. Given a target brain labeled with predefined landmarks, the BrainAligner program automatically finds the corresponding landmarks in a subject brain and maps it to the coordinate system of the target brain via a deformable warp. Using a neuropil marker (the antibody nc82) as a reference of the brain morphology and a target brain that is itself a statistical average of data for 295 brains, we achieved a registration accuracy of 2 μm on average, permitting assessment of stereotypy, potential connectivity and functional mapping of the adult fruit fly brain. We used BrainAligner to generate an image pattern atlas of 2954 registered brains containing 470 different expression patterns that cover all the major compartments of the fly brain.Refinement of tools for targeted gene expression in Drosophila.
Barret D, BD. Pfeiffer ; Teri-T B, TT. Ngo ; Karen L, KL. Hibbard ; Christine, C. Murphy ; Arnim, A. Jenett ; James W, JW. Truman ; Gerald M, GM. Rubin.
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA. email@example.com
A wide variety of biological experiments rely on the ability to express an exogenous gene in a transgenic animal at a defined level and in a spatially and temporally controlled pattern. We describe major improvements of the methods available for achieving this objective in Drosophila melanogaster. We have systematically varied core promoters, UTRs, operator sequences, and transcriptional activating domains used to direct gene expression with the GAL4, LexA, and Split GAL4 transcription factors and the GAL80 transcriptional repressor. The use of site-specific integration allowed us to make quantitative comparisons between different constructs inserted at the same genomic location. We also characterized a set of PhiC31 integration sites for their ability to support transgene expression of both drivers and responders in the nervous system. The increased strength and reliability of these optimized reagents overcome many of the previous limitations of these methods and will facilitate genetic manipulations of greater complexity and sophistication.Tools for neuroanatomy and neurogenetics in Drosophila.
Barret D, BD. Pfeiffer ; Arnim, A. Jenett ; Ann S, AS. Hammonds ; Teri-T B, TT. Ngo ; Sima, S. Misra ; Christine, C. Murphy ; Audra, A. Scully ; Joseph W, JW. Carlson ; Kenneth H, KH. Wan ; Todd R, TR. Laverty ; Chris, C. Mungall ; Rob, R. Svirskas ; James T, JT. Kadonaga ; Chris Q, CQ. Doe ; Michael B, MB. Eisen ; Susan E, SE. Celniker ; Gerald M, GM. Rubin.
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn VA 20147, USA.
We demonstrate the feasibility of generating thousands of transgenic Drosophila melanogaster lines in which the expression of an exogenous gene is reproducibly directed to distinct small subsets of cells in the adult brain. We expect the expression patterns produced by the collection of 5,000 lines that we are currently generating to encompass all neurons in the brain in a variety of intersecting patterns. Overlapping 3-kb DNA fragments from the flanking noncoding and intronic regions of genes thought to have patterned expression in the adult brain were inserted into a defined genomic location by site-specific recombination. These fragments were then assayed for their ability to function as transcriptional enhancers in conjunction with a synthetic core promoter designed to work with a wide variety of enhancer types. An analysis of 44 fragments from four genes found that >80% drive expression patterns in the brain; the observed patterns were, on average, comprised of <100 cells. Our results suggest that the D. melanogaster genome contains >50,000 enhancers and that multiple enhancers drive distinct subsets of expression of a gene in each tissue and developmental stage. We expect that these lines will be valuable tools for neuroanatomy as well as for the elucidation of neuronal circuits and information flow in the fly brain.Standardized atlas of the brain of the desert locust, Schistocerca gregaria.
Angela E, AE. Kurylas ; Torsten, T. Rohlfing ; Sabine, S. Krofczik ; Arnim, A. Jenett ; Uwe, U. Homberg.
Fachbereich Biologie, Tierphysiologie, Philipps Universität Marburg, 35032, Marburg, Germany.
In order to understand the connectivity of neuronal networks, their constituent neurons should ideally be studied in a common framework. Since morphological data from physiologically characterized and stained neurons usually arise from different individual brains, this can only be performed in a virtual standardized brain that compensates for interindividual variability. The desert locust, Schistocerca gregaria, is an insect species used widely for the analysis of olfactory and visual signal processing, endocrine functions, and neural networks controlling motor output. To provide a common multi-user platform for neural circuit analysis in the brain of this species, we have generated a standardized three-dimensional brain of this locust. Serial confocal images from whole-mount locust brains were used to reconstruct 34 neuropil areas in ten brains. For standardization, we compared two different methods: an iterative shape-averaging (ISA) procedure by using affine transformations followed by iterative nonrigid registrations, and the Virtual Insect Brain (VIB) protocol by using global and local rigid transformations followed by local nonrigid transformations. Both methods generated a standard brain, but for different applications. Whereas the VIB technique was designed to visualize anatomical variability between the input brains, the purpose of the ISA method was the opposite, i.e., to remove this variability. A novel individually labeled neuron, connecting the lobula to the midbrain and deutocerebrum, has been registered into the ISA atlas and demonstrates its usefulness and accuracy for future analysis of neural networks. The locust standard brain is accessible at http://www.3d-insectbrain.com .Multiple memory traces for olfactory reward learning in Drosophila.
Lehrstuhl für Genetik und Neurobiologie, Biozentrum, Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany.
Physical traces underlying simple memories can be confined to a single group of cells in the brain. In the fly Drosophila melanogaster, the Kenyon cells of the mushroom bodies house traces for both appetitive and aversive odor memories. The adenylate cyclase protein, Rutabaga, has been shown to mediate both traces. Here, we show that, for appetitive learning, another group of cells can additionally accommodate a Rutabaga-dependent memory trace. Localized expression of rutabaga in either projection neurons, the first-order olfactory interneurons, or in Kenyon cells, the second-order interneurons, is sufficient for rescuing the mutant defect in appetitive short-term memory. Thus, appetitive learning may induce multiple memory traces in the first- and second-order olfactory interneurons using the same plasticity mechanism. In contrast, aversive odor memory of rutabaga is rescued selectively in the Kenyon cells, but not in the projection neurons. This difference in the organization of memory traces is consistent with the internal representation of reward and punishment.Highly restricted diversity of TCR delta chains of the amphibian Mexican axolotl (Ambystoma mexicanum) in peripheral tissues.
Sébastien, S. André ; Fabienne, F. Kerfourn ; Pierre, P. Affaticati ; Aline, A. Guerci ; Philippe, P. Ravassard ; Julien S, JS. Fellah.
UMR 7622, National Centre for Scientific Research, and Hôpital Pitié Salpêtrière, Pierre and Marie Curie University, Paris, France.
Gammadelta T cells localize at mammalian epithelial surfaces to exert both protective and regulatory roles in response to infections. We have previously characterized the Mexican axolotl (Ambystoma mexicanum) T cell receptor delta (TRD) chain. In this study, TRD repertoires in spleen, liver, intestine and skin from larvae, pre-adult and adult axolotls were examined and compared to the thymic TRD repertoire. A TRDV transcript without N/D diversity, TRDV1S1-TRDJ1, dominates the TRD repertoires until sexual maturation. In adult tissues, this canonical transcript is replaced by another dominant TRDV1S1-TRDJ1 transcript. In the thymus, these two transcripts are detected early in development. Our results suggest that gammadelta T cells that express the canonical TRDV1S1-TRDJ1 transcript emerge from the thymus and colonize the peripheral tissues, where they are selectively expanded by recurrent ligands. This particular situation is probably related to the neotenic state and the slow development of the axolotl. In thymectomized axolotls, TRD repertoires appear different from those of normal axolotls, suggesting that extrathymic gammadelta T cell differentiation could occur. Gene expression analysis showed the importance of the gut in T cell development.Early expression of two TdT isoforms in the hematopoietic system of the Mexican axolotl. Implications for the evolutionary origin of the N-nucleotide addition.
Rachel, R. Golub ; Sébastien, S. André ; Alexandre, A. Hassanin ; Pierre, P. Affaticati ; Mani, M. Larijani ; Julien S, JS. Fellah.
Unité du Développement des Lymphocytes, CNRS URA 1961 Institut Pasteur, 25-28 rue du Docteur Roux, 75724, Paris Cedex 15, France.
Nontemplate (N)-nucleotide addition by the terminal dideoxynucleotidyl transferase (TdT) at the junctions of rearranging V( D) J gene segments greatly contribute to antigen-receptor diversity. TdT has been identified in several vertebrate species, where it is highly conserved. We report here the isolation of two forms of TdT mRNA in an amphibian, the Mexican axolotl. The isoform TdT1 shares all of the conserved structural motifs required for TdT activity and displays an average of 50-58% similarity at the amino acid level with TdT of other species. The second axolotl TdT variant ( TdT2) differs from TdT1 by a 57-amino acid deletion located between amino acids 165-222 of TdT1, including the first helix-hairpin-helix DNA-binding motif. During ontogeny, TdT products are first detected in the head of 6-week-old larvae and further in the head and trunk of 8-month-old larvae. These developmental stages correspond to the first detection of RAG1 and antigen-receptor (TCRbeta and IgHmicro) products in axolotl larvae. Our results suggest that in contrast to mammalian development, N diversity occurs early in axolotl development to diversify the primary repertoire. Phylogenetic analyses reveal that TdT and DNA polymerase mu(Pol mu) genes are closely related, and that both enzymes were already present in the common ancestor of jawed vertebrates.