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Succinate dehydrogenase inhibitor (SDHI) fungicides are increasingly used in agricultureto combat molds and fungi, two major threats to both food supply and public health. However,the essential requirement for the succinate dehydrogenase (SDH) complex—the molecular target ofSDHIs—in energy metabolism for almost all extant eukaryotes and the lack of species specificity ofthese fungicides raise concerns about their toxicity toward off-target organisms and, more generally,toward the environment. Herein we review the current knowledge on the toxicity toward zebrafish(Brachydanio rerio) of nine commonly used SDHI fungicides: bixafen, boscalid, fluxapyroxad, flutolanil,isoflucypram, isopyrazam, penthiopyrad, sedaxane, and thifluzamide. The results indicatethat these SDHIs cause multiple adverse effects in embryos, larvae/juveniles, and/or adults, sometimesat developmentally relevant concentrations. Adverse effects include developmental toxicity,cardiovascular abnormalities, liver and kidney damage, oxidative stress, energy deficits, changes inmetabolism, microcephaly, axon growth defects, apoptosis, and transcriptome changes, suggestingthat glycometabolism deficit, oxidative stress, and apoptosis are critical in the toxicity of most ofthese SDHIs. However, other adverse outcome pathways, possibly involving unsuspected moleculartargets, are also suggested. Lastly, we note that because of their recent arrival on the market, thenumber of studies addressing the toxicity of these compounds is still scant, emphasizing the need tofurther investigate the toxicity of all SDHIs currently used and to identify their adverse effects andassociated modes of action, both alone and in combination with other pesticides., Les fongicides inhibiteurs de la succinate déshydrogénase (SDHI) sont de plus en plus utilisés en agriculturepour lutter contre les moisissures et les champignons, deux menaces majeures pour l'approvisionnement alimentaire et la santé publique. Cependant,l'exigence essentielle pour le complexe succinate déshydrogénase (SDH) - la cible moléculaire deSDHI - dans le métabolisme énergétique pour presque tous les eucaryotes existants et le manque de spécificité d'espèce deces fongicides soulèvent des inquiétudes quant à leur toxicité vis-à-vis des organismes non ciblés et, plus généralement,envers l'environnement. Ici, nous passons en revue les connaissances actuelles sur la toxicité envers le poisson zèbre(Brachydanio rerio) de neuf fongicides SDHI couramment utilisés : bixafen, boscalid, fluxapyroxad, flutolanil,isolucypram, isopyrazam, penthiopyrad, sedaxane et thifluzamide. Les résultats indiquentque ces SDHI provoquent de multiples effets indésirables chez les embryons, les larves/juvéniles et/ou les adultes, parfoisà des concentrations pertinentes pour le développement. Les effets indésirables comprennent la toxicité pour le développement,anomalies cardiovasculaires, lésions hépatiques et rénales, stress oxydatif, déficits énergétiques, modifications demétabolisme, microcéphalie, défauts de croissance axonale, apoptose et modifications du transcriptome, suggérantque le déficit du glycométabolisme, le stress oxydatif et l'apoptose sont critiques dans la toxicité de la plupart desces SDHI. Cependant, d'autres voies de résultats indésirables, impliquant peut-être des molécules moléculaires insoupçonnéescibles, sont également suggérées. Enfin, notons qu'en raison de leur arrivée récente sur le marché, lesnombre d'études portant sur la toxicité de ces composés est encore faible, soulignant la nécessité deétudier plus avant la toxicité de tous les SDHI actuellement utilisés et identifier leurs effets indésirables etmodes d'action associés, à la fois seuls et en combinaison avec d'autres pesticides.

International audience; Succinate dehydrogenase inhibitor (SDHI) fungicides are increasingly used in agricultureto combat molds and fungi, two major threats to both food supply and public health. However,the essential requirement for the succinate dehydrogenase (SDH) complex—the molecular target ofSDHIs—in energy metabolism for almost all extant eukaryotes and the lack of species specificity ofthese fungicides raise concerns about their toxicity toward off-target organisms and, more generally,toward the environment. Herein we review the current knowledge on the toxicity toward zebrafish(Brachydanio rerio) of nine commonly used SDHI fungicides: bixafen, boscalid, fluxapyroxad, flutolanil,isoflucypram, isopyrazam, penthiopyrad, sedaxane, and thifluzamide. The results indicatethat these SDHIs cause multiple adverse effects in embryos, larvae/juveniles, and/or adults, sometimesat developmentally relevant concentrations. Adverse effects include developmental toxicity,cardiovascular abnormalities, liver and kidney damage, oxidative stress, energy deficits, changes inmetabolism, microcephaly, axon growth defects, apoptosis, and transcriptome changes, suggestingthat glycometabolism deficit, oxidative stress, and apoptosis are critical in the toxicity of most ofthese SDHIs. However, other adverse outcome pathways, possibly involving unsuspected moleculartargets, are also suggested. Lastly, we note that because of their recent arrival on the market, thenumber of studies addressing the toxicity of these compounds is still scant, emphasizing the need tofurther investigate the toxicity of all SDHIs currently used and to identify their adverse effects andassociated modes of action, both alone and in combination with other pesticides.; Les fongicides inhibiteurs de la succinate déshydrogénase (SDHI) sont de plus en plus utilisés en agriculturepour lutter contre les moisissures et les champignons, deux menaces majeures pour l'approvisionnement alimentaire et la santé publique. Cependant,l'exigence essentielle pour le complexe succinate déshydrogénase (SDH) - la cible moléculaire deSDHI - dans le métabolisme énergétique pour presque tous les eucaryotes existants et le manque de spécificité d'espèce deces fongicides soulèvent des inquiétudes quant à leur toxicité vis-à-vis des organismes non ciblés et, plus généralement,envers l'environnement. Ici, nous passons en revue les connaissances actuelles sur la toxicité envers le poisson zèbre(Brachydanio rerio) de neuf fongicides SDHI couramment utilisés : bixafen, boscalid, fluxapyroxad, flutolanil,isolucypram, isopyrazam, penthiopyrad, sedaxane et thifluzamide. Les résultats indiquentque ces SDHI provoquent de multiples effets indésirables chez les embryons, les larves/juvéniles et/ou les adultes, parfoisà des concentrations pertinentes pour le développement. Les effets indésirables comprennent la toxicité pour le développement,anomalies cardiovasculaires, lésions hépatiques et rénales, stress oxydatif, déficits énergétiques, modifications demétabolisme, microcéphalie, défauts de croissance axonale, apoptose et modifications du transcriptome, suggérantque le déficit du glycométabolisme, le stress oxydatif et l'apoptose sont critiques dans la toxicité de la plupart desces SDHI. Cependant, d'autres voies de résultats indésirables, impliquant peut-être des molécules moléculaires insoupçonnéescibles, sont également suggérées. Enfin, notons qu'en raison de leur arrivée récente sur le marché, lesnombre d'études portant sur la toxicité de ces composés est encore faible, soulignant la nécessité deétudier plus avant la toxicité de tous les SDHI actuellement utilisés et identifier leurs effets indésirables etmodes d'action associés, à la fois seuls et en combinaison avec d'autres pesticides.

Nucleoplasmin 2(npm2) is an essential maternal-effect gene that mediates early embryonic events through its function as a histone chaperone that remodels chromatin. Here we report the existence of twonpm2(npm2aandnpm2b) genes in zebrafish. We examined the evolution ofnpm2aandnpm2bin a variety of vertebrates, their potential phylogenetic relationships, and their biological functions using knockout models via the CRISPR/cas9 system. We demonstrated that the twonpm2duplicates exist in a wide range of vertebrates, including sharks, ray-finned fish, amphibians, and sauropsids, whilenpm2awas lost in Coelacanth and mammals, as well as some specific teleost lineages. Using phylogeny and synteny analyses, we traced their origins to the early stages of vertebrate evolution. Our findings suggested thatnpm2aandnpm2bresulted from an ancient local gene duplication, and their functions diverged although key protein domains were conserved. We then investigated their functions by examining their tissue distribution in a wide variety of species and found that they shared ovarian-specific expression, a key feature of maternal-effect genes. We also showed that bothnpm2aandnpm2bare maternally-inherited transcripts in vertebrates. Moreover, we used zebrafish knockouts to demonstrate thatnpm2aandnpm2bplay essential, but distinct, roles in early embryogenesis.npm2afunctions very early during embryogenesis, at or immediately after fertilization, whilenpm2bis involved in processes leading up to or during zygotic genome activation. These novel findings will broaden our knowledge on the evolutionary diversity of maternal-effect genes and underlying mechanisms that contribute to vertebrate reproductive success.Author SummaryThe protein and transcript of thenpm2gene have been previously demonstrated as maternal contributions to embryos of several vertebrates. Recently, twonpm2genes, denoted here asnpm2aandnpm2b, were discovered in zebrafish. This study was conducted to explore the evolutionary origin and changes that occurred that culminated in their current functions. We found that an ancient local duplication of the ancestralnpm2gene created the current two forms, and while most vertebrates retained both genes, notably, mammals and certain species of fish lostnpm2aand, albeit rarely, bothnpm2aandnpm2b. Our functional analyses showed thatnpm2aandnpm2bhave diverse but essential functions during embryogenesis, asnpm2amutants failed to undergo development at the earliest stage whilenpm2bmutants developed, although abnormally, until the zygotic genome activation stage after which their development was arrested followed subsequently by death. Our study is the first to clearly demonstrate the evolution, diversification, and functional analyses of thenpm2genes, which are essential maternal factors that are required for proper embryonic development and survival.

Whole genome duplications (WGD) are important evolutionary events. Our understanding of underlying mechanisms, including the evolution of duplicated genes after WGD, however remains incomplete. Teleost fish experienced a common WGD (teleost-specific genome duplication, or TGD) followed by a dramatic adaptive radiation leading to more than half of all vertebrate species. The analysis of gene expression patterns following TGD at the genome level has been limited by the lack of suitable genomic resources. The recent concomitant release of the genome sequence of spotted gar (a representative of holosteans, the closest lineage of teleosts that lacks the TGD) and the tissue-specific gene expression repertoires of over 20 holostean and teleostean fish species, including spotted gar, zebrafish and medaka (the PhyloFish project), offered a unique opportunity to study the evolution of gene expression following TGD in teleosts. We show that most TGD duplicates gained their current status (loss of one duplicate gene or retention of both duplicates) relatively rapidly after TGD (i.e. prior to the divergence of medaka and zebrafish lineages). The loss of one duplicate is the most common fate after TGD with a probability of approximately 80%. In addition, the fate of duplicate genes after TGD, including subfunctionalization, neofunctionalization, or retention of two ‘similar’ copies occurred not only before, but also after the radiation of species tested, in consistency with a role of the TGD in speciation and/or evolution of gene function. Finally, we report novel cases of TGD ohnolog subfunctionalization and neofunctionalization that further illustrate the importance of these processes.

Background Egg quality can be defined as the egg ability to be fertilized and subsequently develop into a normal embryo. Previous research has shed light on factors that can influence egg quality. Large gaps however remain including a comprehensive view of what makes a bad egg. Initial development of the embryo relies on maternally-inherited molecules, such as transcripts, deposited in the egg during its formation. Bad egg quality is therefore susceptible to be associated with alteration or dysregulation of maternally-inherited transcripts. We performed transcriptome analysis on a large number (N = 136) of zebrafish egg clutches, each clutch being split to monitor developmental success and perform transcriptome analysis in parallel. We aimed at drawing a molecular portrait of the egg in order to characterize the relation between egg transcriptome and developmental success and to subsequently identify new candidate genes involved in fertility. Results We identified 66 transcript that were differentially abundant in eggs of contrasted phenotype (low or high developmental success). Statistical modeling using partial least squares regression and genetics algorithm demonstrated that gene signatures from transcriptomic data can be used to predict developmental success. The identity and function of differentially expressed genes indicate a major dysregulation of genes of the translational machinery in poor quality eggs. Two genes, otulina and slc29a1a, predominantly expressed in the ovary and dysregulated in poor quality eggs were further investigated using CRISPR/Cas9 mediated genome editing. Mutants of each gene revealed remarkable subfertility whereby the majority of their eggs were unfertilizable. The Wnt pathway appeared to be dysregulated in the otulina mutant-derived eggs. Conclusions Here we show that egg transcriptome contains molecular signatures, which can be used to predict developmental success. Our results also indicate that poor egg quality in zebrafish is associated with a dysregulation of (i) the translational machinery genes and (ii) novel fertility genes, otulina and slc29a1a, playing an important role for fertilization. Together, our observations highlight the diversity of the possible causes of egg quality defects and reveal mechanisms of maternal origin behind the lack of fertilization and early embryonic failures that can occur under normal reproduction conditions. Electronic supplementary material The online version of this article (10.1186/s12864-019-5930-8) contains supplementary material, which is available to authorized users.

A large amount of chemicals are released to the environment each year. Among them, bisphenol A (BPA) is of utmost concern since it interferes with the reproductive system of wild organisms due to its capacity to bind to hormone receptors. Additionally, BPA epigenotoxic activity is known to affect basic processes during embryonic life. However, its effects on primordial germ cells (PGCs) proliferation and migration, both mechanisms being crucial for gametogenesis, remain unknown. To investigate the effects of BPA on PGCs migration and eventual testicle development, zebrafish embryos were exposed to 100, 2000 and 4000 &micro, g/L BPA during the first 24 h of development. Vasa immunostaining of PGCs revealed that exposure to 2000 and 4000 &micro, g/L BPA impaired their migration to the genital ridge. Two pivotal genes of PGCs migration (cxcr4b and sdf1a) were highly dysregulated in embryos exposed to these doses, whereas DNA methylation and epigenetic marks in PGCs and their surrounding somatic cells were not altered. Once embryos reached adulthood, the morphometric study of their gonads revealed that, despite the reduced number of PGCs which colonized the genital ridges, normal testicles were developed. Although H3K9ac decreased in the sperm from treated fishes, it did not affect the progeny development.

Background Previously, we have demonstrated that genes involved in ovarian function are highly conserved throughout evolution. In this study, we aimed to document the conservation of genes involved in spermatogenesis from flies to vertebrates and their expression profiles in vertebrates. Results We retrieved 379 Drosophila melanogaster genes that are functionally involved in male reproduction according to their mutant phenotypes and listed their vertebrate orthologs. 83% of the fly genes have at least one vertebrate ortholog for a total of 625 mouse orthologs. This conservation percentage is almost twice as high as the 42% rate for the whole fly genome and is similar to that previously found for genes preferentially expressed in ovaries. Of the 625 mouse orthologs, we selected 68 mouse genes of interest, 42 of which exhibited a predominant relative expression in testes and 26 were their paralogs. These 68 mouse genes exhibited 144 and 60 orthologs in chicken and zebrafish, respectively, gathered in 28 groups of paralogs. Almost two thirds of the chicken orthologs and half of the zebrafish orthologs exhibited a relative expression ≥50% in testis. Finally, our focus on functional in silico data demonstrated that most of these genes were involved in the germ cell process, primarily in structure elaboration/maintenance and in acid nucleic metabolism. Conclusion Our work confirms that the genes involved in germ cell development are highly conserved across evolution in vertebrates and invertebrates and display a high rate of conservation of preferential testicular expression among vertebrates. Among the genes highlighted in this study, three mouse genes (Lrrc46, Pabpc6 and Pkd2l1) have not previously been described in the testes, neither their zebrafish nor chicken orthologs. The phylogenetic approach developed in this study finally allows considering new testicular genes for further fundamental studies in vertebrates, including model species (mouse and zebrafish). Electronic supplementary material The online version of this article (10.1186/s12862-019-1462-8) contains supplementary material, which is available to authorized users.

Oviparous vertebrates produce multiple forms of vitellogenin (Vtg), the major source of yolk nutrients, but little is known about their individual contributions to reproduction and development. This study employed a CRISPR/Cas9 genome editing to assess essentiality and functionality of zebrafish (Danio rerio) type-I and -III Vtgs. The multiple CRISPR approach employed to knock out (KO) all genes encoding type-Ivtgs(vtg1, 4, 5, 6,and7) simultaneously (vtg1-KO), and the type-IIIvtg(vtg3) individually (vtg3-KO). Results of PCR genotyping and sequencing, qPCR, LC-MS/MS and Western blotting showed that onlyvtg6andvtg7escaped Cas9 editing. In fish whose remaining type-Ivtgswere incapacitated (vtg1-KO), and invtg3-KO fish, significant increases in Vtg7 transcript and protein levels occurred in liver and eggs, a heretofore-unknown mechanism of genetic compensation to regulate Vtg homeostasis. Fecundity was more than doubled invtg1-KO females, and fertility was ~halved invtg3-KO females. Substantial mortality was evident invtg3-KO eggs/embryos after only 8 h of incubation and invtg1-KO embryos after 5 d. Hatching rate and timing were markedly impaired invtgmutant embryos and pericardial and yolk sac/abdominal edema and spinal lordosis were evident in the larvae, with feeding and motor activities also being absent invtg1-KO larvae. By late larval stages,vtgmutations were either completely lethal (vtg1-KO) or nearly so (vtg3-KO). These novel findings offer the first experimental evidence that different types of vertebrate Vtg are essential and have disparate requisite functions at different times during both reproduction and development.

Supplementary data to this article can be found online at : https://doi.org/10.1016/j.cbpb.2018.11.005; The understanding of muscle tissue formation and regeneration is essential for the development of therapeutic approaches to treat muscle diseases or loss of muscle mass and strength during ageing or cancer. One of the critical steps in muscle formation is the fusion of muscle cells to form or regenerate muscle fibres.To identify new genes controlling myoblast fusion, we performed a siRNA screen in c2c12 myoblasts. The genes identified during this screen were then studiedi n vivo by knock down in zebrafish using morpholino. We found that N-alpha-acetyl transferase 15 (Naa15) knockdown enhanced c2c12 myoblast fusion, suggesting that Naa15 negatively regulates myogenic cell fusion. We identified two Naa15 orthologous genes in the zebrafish genome: Naa15a and Naa15b. These two orthologs were expressed in the myogenic domain of the somite. Knockdown of zebrafish Naa15a and Naa15b genes induced a“U”-shaped segmentation of the myotome and alteration of myotome boundaries, resulting in the formation of abnormally long myofibres spanning adjacent somites. Taken together, these results show that Naa15 regulates myotome formation and myogenesis in fish.

Introduction: Fsh is a pituitary gonadotropin that regulates both gonadal functions, gametogenesis and steroidogenesis, in fish. The biological actions of Fsh are mediated through its binding to a membrane receptor (Fshr) that belongs to the superfamily of the G protein-coupled receptor (GPCR). Previous reports on in vivo disruption of the Fshr gene showed that Fsh signaling pathway was dispensable for male fertility but was required for oogenesis. In the present study we generated new mutant zebrafish lines to further investigate the role of fshr in reproductive and growth performances. Methods: We disrupted the fshr gene in zebrafish using the CRISPR/Cas9 method. We selected three CRISPR sites in the 10th exon of the gene to cause a large deletion easily detectable by a PCR-based genotyping method. Two mutant lines were generated from independent founders mated with transgenic vasa:eGFP females. The mutations were characterized by DNA sequencing in both lines. The deletions led to predicted truncated proteins enable to anchor to the membrane and to transduce Fsh signal. Results and Discussion: Heterozygous mutant males and females developed an apparently normal gametogenesis and were fertile. However heterozygous zebrafish population (fshr+/-) showed a female biased sexratio compared to the non mutated siblings. At 27 days post fertilization heterozygous mutants showed higher relative abundance of the gonadal aromatase gene (cyp19a1a) and increased levels of transcripts involved in granulosa cell differentiation (foxl2a2, gsdf, fshr, sox9a). In addition, transcripts accumulated during late oocyte growth (vasa, sox9b and nanos2) were also detected at higher levels. The relative abundance of sycp1 was unchanged indicating that meiosis initiation was normal. In contrast, in homozygous mutant meiosis was altered as revealed by decreased expression levels of sycp1. In addition, oocytes did not progress beyond the pre-follicle phase of primary growth. All adult homozygous mutants became males suggesting that impaired oogenesis during early ontogenesis caused sex reversal. Additional new phenotypes were observed in the homozygous males including lower sperm count and viability. This could result from 11-KT deficiency as revealed by decreased expression levels of cyp11b2. Perturbation of steroidogenesis could also explain the increased body weight and length observed in homozygous mutants. Conclusion: The marked phenotypic perturbations induced by the Fshr mutation confirm that Fsh and Lh have distinct functions. We showed that the loss of one fshr gene copy favours the differentiation of granulosa cells and oocyte growth. Fsh signalling pathway is dispensable for Sertoli cell differentiation but remains important for sperm quality and excretion.

Accepted manuscript; In zebrafish, there exists a clear need for new tools to study sex differentiation dynamic and its perturbation by endocrine disrupting chemicals. In this context, we developed and characterized a novel transgenic zebrafish line expressing green fluorescent protein (GFP) under the control of the zebrafish cyp19a1a (gonadal aromatase) promoter. In most gonochoristic fish species including zebrafish, cyp19a1a, the enzyme responsible for the synthesis of estrogens, has been shown to play a critical role in the processes of reproduction and sexual differentiation. This novel cyp19a1a-eGFP transgenic line allowed a deeper characterization of expression and localization of cyp19a1a gene in zebrafish gonads both at the adult stage and during development. At the adult stage, GFP expression was higher in ovaries than in testis. We showed a perfect co-expression of GFP and endogenous Cyp19a1a protein in gonads that was mainly localized in the cytoplasm of peri-follicular cells in the ovary and of Leydig and germ cells in the testis. During development, GFP was expressed in all immature gonads of 20 dpf-old zebrafish. Then, GFP expression increased in early differentiated female at 30 and 35 dpf to reach a high GFP intensity in well-differentiated ovaries at 40 dpf. On the contrary, males consistently displayed low GFP expression as compared to female whatever their stage of development, resulting in a clear dimorphic expression between both sexes. Interestingly, fish that undergoes ovary-to-testis transition (35 and 40 dpf) presented GFP levels similar to males or intermediate between females and males. In this transgenic line our results confirm that cyp19a1a is expressed early during development, before the histological differentiation of the gonads, and that the down-regulation of cyp19a1a expression is likely responsible for the testicular differentiation. Moreover, we show that although cyp19a1a expression exhibits a clear dimorphic expression pattern in gonads during sexual differentiation, its expression persists whatever the sex suggesting that estradiol synthesis is important for gonadal development of both sexes. Monitoring the expression of GFP in control and exposed-fish will help determine the sensitivity of this transgenic line to EDCs and to refine mechanistic based-assays for the study of EDCs. In fine, this transgenic zebrafish line will be a useful tool to study physiological processes such as reproduction and sexual differentiation, and their perturbations by EDCs.

Genome editing reveals developmental dependencies on specific types of vitellogenesis in Zebrafish (Danio rerio). 11. International Symposium on Reproductive Physiology of Fish

International audience; Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis essential for the control of intermediary metabolism. So far, the absence of any identifiable LAMP2A—a necessary and limiting protein for CMA—outside of the tetrapod clade, led to the paradigm that this cellular function was (presumably) restricted to mammals and birds. However, after we identified expressed sequences displaying high sequence homology with the mammalian LAMP2A in several fish species, our findings challenge that view and suggest that CMA likely appeared much earlier during evolution than initially thought. Hence, our results do not only shed an entirely new light on the evolution of CMA, but also bring new perspectives on the possible use of complementary genetic models, such as zebrafish or medaka for studying CMA function from a comparative angle/view.

Oviparous vertebrates produce multiple forms of vitellogenin (Vtg), the major source of yolk nutrients, but little is known about their individual contributions to reproduction and development. This study employed a CRISPR/Cas9 genome editing to assess essentiality and functionality of zebrafish (Danio rerio) type-I and -III Vtgs. The multiple CRISPR approach employed to knock out (KO) all genes encoding type-I vtgs (vtg1, 4, 5, 6, and 7) simultaneously (vtg1-KO), and the type-III vtg (vtg3) individually (vtg3-KO). Results of PCR genotyping and sequencing, qPCR, LC-MS/MS and Western blotting showed that only vtg6 and vtg7 escaped Cas9 editing. In fish whose remaining type-I vtgs were incapacitated (vtg1-KO), and in vtg3-KO fish, significant increases in Vtg7 transcript and protein levels occurred in liver and eggs, a heretofore-unknown mechanism of genetic compensation to regulate Vtg homeostasis. Fecundity was more than doubled in vtg1-KO females, and fertility was ~halved in vtg3-KO females. Substantial mortality was evident in vtg3-KO eggs/embryos after only 8 h of incubation and in vtg1-KO embryos after 5 d. Hatching rate and timing were markedly impaired in vtg mutant embryos and pericardial and yolk sac/abdominal edema and spinal lordosis were evident in the larvae, with feeding and motor activities also being absent in vtg1-KO larvae. By late larval stages, vtg mutations were either completely lethal (vtg1-KO) or nearly so (vtg3-KO). These novel findings offer the first experimental evidence that different types of vertebrate Vtg are essential and have disparate requisite functions at different times during both reproduction and development.

Background Nucleoplasmin 2 (npm2) is an essential maternal-effect gene that mediates early embryonic events through its function as a histone chaperone that remodels chromatin. Recently, two npm2 (npm2a and npm2b) genes have been annotated in zebrafish. Thus, we examined the evolution of npm2a and npm2b in a variety of vertebrates, their potential phylogenetic relationships, and their biological functions using knockout models via the CRISPR/cas9 system. Results We demonstrated that the two npm2 duplicates exist in a wide range of vertebrates, including sharks, ray-finned fish, amphibians, and sauropsids, while npm2a was lost in coelacanth and mammals, as well as some specific teleost lineages. Using phylogeny and synteny analyses, we traced their origins to the early stages of vertebrate evolution. Our findings suggested that npm2a and npm2b resulted from an ancient local gene duplication, and their functions diverged although key protein domains were conserved. We then investigated their functions by examining their tissue distribution in a wide variety of species and found that they shared ovarian-specific expression, a key feature of maternal-effect genes. We also demonstrated that both npm2a and npm2b are maternally-inherited transcripts in vertebrates, and that they play essential, but distinct, roles in early embryogenesis using zebrafish knockout models. Both npm2a and npm2b function early during oogenesis and may play a role in cortical granule function that impact egg activation and fertilization, while npm2b is also involved in early embryogenesis. Conclusion These novel findings will broaden our knowledge on the evolutionary history of maternal-effect genes and underlying mechanisms that contribute to vertebrate reproductive success. In addition, our results demonstrate the existence of a newly described maternal-effect gene, npm2a, that contributes to egg competence, an area that still requires further comprehension. Electronic supplementary material The online version of this article (10.1186/s12862-018-1281-3) contains supplementary material, which is available to authorized users.

Supplemental information for this article can be found online at:http://dx.doi.org/10.7717/peerj.5534#supplemental-information; The family of forkhead box (Fox) transcription factors regulates gonadogenesis and embryogenesis, but the role of in reproduction is unknown. Evolutionary history of in vertebrates was examined and the gene was found to exist in most vertebrates, including mammals, ray-finned fish, amphibians, and sauropsids. By quantitative PCR and RNA-seq, we found that had an ovarian-specific expression in zebrafish, a common feature of maternal-effect genes. In addition, it was demonstrated using in situ hybridization that was a maternally-inherited transcript that was highly expressed even in early-stage oocytes and accumulated in the developing eggs during oogenesis. We also analyzed the function of in female reproduction using a zebrafish CRISPR/cas9 knockout model. It was observed that embryos from the -deficient females had a significantly lower survival rate whereby they either failed to undergo cell division or underwent abnormal division that culminated in growth arrest at around the mid-blastula transition and early death. These mutant-derived eggs contained dramatically increased levels of , a cell cycle inhibitor, and reduced , a component of mTOR and regulator of cell survival, which were in line with the observed growth arrest phenotype. Our study shows for the first time that is an essential maternal-effect gene and may be required for proper cell division and survival via the p21 and mTOR pathways. These novel findings will broaden our knowledge on the functions of specific maternal factors stored in the developing egg and the underlying mechanisms that contribute to reproductive success.

un poster présenté; International audience; Good quality or developmentally competent fish eggs are successfully fertilized and develop normally as viable, non-malformed embryos. However, the detailed mechanisms involved in egg quality remain poorly understood and, at present, no predictive markers of egg quality exist. Early developmental success relies on maternally-inherited molecules deposited in the oocyte throughout oogenesis, and over the last few years, our work has aimed at drawing the molecular picture of poorly competent (i.e. bad quality) fish eggs in order to (1) further uncover the molecular mechanisms associated with low developmental success, (2) reveal new maternal-effect genes that play important roles during early development, and (3) possibly obtain molecular signatures based on maternal mRNA profiles in fertilized eggs that could be used as predictive markers of good quality eggs. A large transcriptomic analysis was performed in zebrafish (Danio rerio) using a high number of egg clutches obtained under normal husbandry conditions that reflect naturally occurring variability in egg quality. The differentially expressed gene profile revealed dysregulation of genes involved in translation and protein synthesis. Interestingly, translation was also one of the main processes dysregulated in a similar study conducted in Sea Bass (Dicentrarchus labrax). Analysis of a smaller number of zebrafish females that consistently produced bad eggs demonstrated a much lower variability in expression profiles that resulted in a higher number of differentially expressed genes identified between good and bad quality eggs. These indicate that while common/general traits of bad quality egg exist, a multiplicity of other factors can dysregulate specific processes that ultimately lead to reduced egg quality. To gain insight into their functions, genome editing using the CRISPR/Cas9 technology was performed to create mutants of two new potential maternal-effect genes, otulina (OTU deubiquitinase with linear linkage specificity a) and slc29a1a (solute carrier family 29, member 1a), that are especially abundant in the ovary. Due to the high efficiency of this technique, mutations could not be transmitted to the next generation and the phenotype was analyzed in F0 females. We observed that both otulina and slc29a1a mutant-derived eggs had very low developmental success. Eggs from mutant females crossed with vasa:eGFP males do not contain GFP, suggesting that they cannot be fertilized. These novel findings showed for the first time that otulina and slc29a1a are essential for the developmental competence of eggs, therefore, are crucial maternal-effect genes. Finally, we applied statistical modeling using Partial Least Square (PLS) regression and genetic algorithm to our data to search for possible molecular signatures. We observed the presence of strong gene signatures, which were statistically robust both in terms of reproducibility and validation by pseudo-data, to link gene expression to the survival rate of eggs in our transcriptomic data. In summary, the molecular mechanisms that control egg quality are complex and probably variable even though some core processes including translation and proteins synthesis appear to play a key role. Yet, some maternaleffect genes have been identified that are important for fertilization. Finally, it appears possible to statistically predict developmental success based on the molecular portrait of the egg.

International audience; The use of aquatic model organisms has been greatly diversified in laboratories. Zebrafish is the most advanced aquatic species for the use of Crispr-Cas9 in laboratories. Because of the simplicity and broad applicability of this later system, knock-out is now efficiently performed at medium scale. Forward genetics in zebrafish can now be performed by CRISPR-based F0 screening using high speed and high content phenotyping for example by confocal imaging. As zebrafish, marine model organisms have the prominent advantage to be transparent, all the more at young stages (embryos and larvae) or when fixed samples are cleared by novel methods. The Cripsr-Cas9 system is routinely used in the ascidian Ciona intestinalis. It also starts to be used in many other marine models, such as the medusa Clythia hemispherica. We provide at the end of this review a list of aquatic model species and some examples of questions on the origin of our nervous system that can be coped with these models, where the possibility to perform genome editing would constitute a major advance.

Session IV. Gamete "Omics". Un poster présenté; International audience

The invalidation of the FSHR gene in zebrafish leads to a gain of body weight, a biased sex-steroid and a decrease in sperm quality. 6. International Workshop on the Biology of Fish Gametes

Egg transcriptome reveals molecular signature that can predict developmental competence and novel maternal-effect genes. 10. European Zebrafish Meeting

Récolte de sperme sur des poissons zèbres mutés pour analyse en cytométrie de flux. 2. Journées des Installations Expérimentales LPGP

Supplementary materials can be found at www.mdpi.com/1422-0067/18/3/560/s1; Zebrafish were exposed through diet to two environmentally relevant polycyclic aromatic hydrocarbons (PAHs) mixtures of contrasted compositions, one of pyrolytic (PY) origin and one from light crude oil (LO). Monoamine concentrations were quantified in the brains of the fish after six month of exposure. A significant decrease in noradrenaline (NA) was observed in fish exposed to both mixtures, while a decrease in serotonin (5HT) and dopamine (DA) was observed only in LO-exposed fish. A decrease in metabolites of 5HT and DA was observed in fish exposed to both mixtures. Several behavioural disruptions were observed that depended on mixtures, and parallels were made with changes in monoamine concentrations. Indeed, we observed an increase in anxiety in fish exposed to both mixtures, which could be related to the decrease in 5HT and/ or NA, while disruptions of daily activity rhythms were observed in LO fish, which could be related to the decrease in DA. Taken together, these results showed that (i) chronic exposures to PAHs mixtures disrupted brain monoamine contents, which could underlie behavioural disruptions, and that (ii) the biological responses depended on mixture compositions.

Nucleoplasmin 2 (npm2) is an essential maternal-effect gene that mediates early embryonic events through its function as a histone chaperone that remodels chromatin. Here we report the existence of two npm2 (npm2a and npm2b) genes in zebrafish. We examined the evolution of npm2a and npm2b in a variety of vertebrates, their potential phylogenetic relationships, and their biological functions using knockout models via the CRISPR/cas9 system. We demonstrated that the two npm2 duplicates exist in a wide range of vertebrates, including sharks, ray-finned fish, amphibians, and sauropsids, while npm2a was lost in Coelacanth and mammals, as well as some specific teleost lineages. Using phylogeny and synteny analyses, we traced their origins to the early stages of vertebrate evolution. Our findings suggested that npm2a and npm2b resulted from an ancient local gene duplication, and their functions diverged although key protein domains were conserved. We then investigated their functions by examining their tissue distribution in a wide variety of species and found that they shared ovarian-specific expression, a key feature of maternal-effect genes. We also showed that both npm2a and npm2b are maternally-inherited transcripts in vertebrates. Moreover, we used zebrafish knockouts to demonstrate that npm2a and npm2b play essential, but distinct, roles in early embryogenesis. npm2a functions very early during embryogenesis, at or immediately after fertilization, while npm2b is involved in processes leading up to or during zygotic genome activation. These novel findings will broaden our knowledge on the evolutionary diversity of maternal-effect genes and underlying mechanisms that contribute to vertebrate reproductive success.

Les fibres musculaires sont des cellules multinuclées et sont l'élément cellulaire de base du muscle. Chez l’adulte, la croissance et la régénération du muscle se font grâce à des cellules souches musculaires, les cellules satellites. Une fois activées (alors appelées myoblastes) ces cellules se différencient et fusionnent avec la fibre existante (hypertrophie) ou avec un autre myoblaste pour former une nouvelle fibre (hyperplasie). Contrairement aux animaux d'élevage terrestres, les poissons à croissance rapide comme la truite, ont une croissance musculaire post¬larvaire qui résulte à la fois d'une hypertrophie et d'une hyperplasie. Ainsi chez les poissons, les cellules satellites peuvent s'engager dans la fusion avec une fibre existante ou dans la fusion avec une autre cellule satellite. Notre projet visait à identifier de nouveaux facteurs moléculaires impliqués dans l'engagement d'une cellule satellite dans la fusion avec un myoblaste ou avec un myotube. Les facteurs qui contrôlent la fusion des myoblastes avec une fibre ou avec un autre myoblaste ont un intérêt majeur du point de vue agronomique et thérapeutique pour améliorer la croissance du muscle.

Most farmed fishes exhibit problems producing high quality eggs, which has adverse socioeconomic impacts and endangers global food security. Almost no attention has been paid to imbalance of egg proteins crucial for early development as a potential cause of poor egg quality. The major components of fish eggs are yolk proteins (YPs) derived from circulating precursors, vitellogenins (Vtgs). All fish produce multiple types of Vtg and the different types may play disparate roles in oocyte maturation and embryonic versus larval nutrition. Underlying cytological events involve complex systems of Vtg receptors, endocytotic elements, enzymes and other proteome components. Preliminary evidence that dysfunction of these systems contributes to poor egg quality has been obtained. The proposed research will exploit zebrafish, having a sequenced genome, as a laboratory model for proteomics of fish egg quality. Objectives are; 1) Molecular characterization of multiple Vtg gene structure, expression, product proteins and phylogeny, 2) Quantification of the Vtgs or their derived YPs in maternal females, oocytes, eggs and offspring of good and poor quality spawns, and 3) Identification of the most significant Vtgs and their functions via application of novel vtg gene knock-out technology. This research will reveal fundamental molecular details of vitellogenesis and its relation to egg quality common to zebrafish and important farmed species. The host institution has ideal infrastructure and expertise available to the applicant to carry out the research, and the scientist-in-charge and his group are international leaders in this specific research area. The overall mobility program will endow the applicant with the scientific knowledge, the technical capabilities and other transferable professional skills, and the international network and visibility required for her to develop a superior research career of significant benefit to the European Research Area and associated countries.

International audience; Egg quality is a complex biological trait and a major determinant of reproductive fitness in all animals. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading biomedical model for early development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were sampled immediately after spawning and used to create pooled or replicated sample sets whose protein extracts were subjected to different levels of fractionation before liquid chromatography and tandem mass spectrometry. Obtained spectra were searched against a zebrafish proteome database and detected proteins were annotated, categorized and quantified based on normalized spectral counts. Manually curated and automated enrichment analyses revealed poor quality eggs to be deficient of proteins involved in protein synthesis and energy and lipid metabolism, and of some vitellogenin products and lectins, and to have a surfeit of proteins involved in endo-lysosomal activities, autophagy, and apoptosis, and of some oncogene products, lectins and egg envelope proteins. Results of pathway and network analyses suggest that this aberrant proteomic profile results from failure of oocytes giving rise to poor quality eggs to properly transit through final maturation, and implicated Wnt signaling in the etiology of this defect. Quantitative comparisons of abundant proteins in good versus poor quality eggs revealed 17 candidate egg quality markers. Thus, the zebrafish egg proteome is clearly linked to embryo developmental potential, a phenomenon that begs further investigation to elucidate the root causes of poor egg quality, presently a serious and intractable problem in livestock and human reproductive medicine.

Collagen VI (COLVI), a protein ubiquitously expressed in connective tissues, is crucial for structural integrity, cellular adhesion, migration and survival. Six different genes are recognized in mammalians, encoding six COLVI-chains that assemble as two 'short' (α1, α2) and one 'long' chain (theoretically any one of α3-6). In humans, defects in the most widely expressed heterotrimer (α123), due to mutations in the COL6A1-3 genes, cause a heterogeneous group of neuromuscular disorders, collectively termed COLVI-related muscle disorders. Little is known about the function(s) of the recently described α4-6 chains and no mutations have been detected yet. In this study, we characterized two novel COLVI long chains in zebrafish that are most homologous to the mammalian α4 chain; therefore, we named the corresponding genes col6a4a and col6a4b. These orthologues represent ancestors of the mammalian Col6a4-6 genes. By in situ hybridization and RT-qPCR, we unveiled a distinctive expression kinetics for col6a4b, compared with the other col6a genes. Using morpholino antisense oligonucleotides targeting col6a4a, col6a4b and col6a2, we modelled partial and complete COLVI deficiency, respectively. All morphant embryos presented altered muscle structure and impaired motility. While apoptosis was not drastically increased, autophagy induction was defective in all morphants. Furthermore, motoneuron axon growth was abnormal in these morphants. Importantly, some phenotypical differences emerged between col6a4a and col6a4b morphants, suggesting only partial functional redundancy. Overall, our results further confirm the importance of COLVI in zebrafish muscle development and may provide important clues for potential human phenotypes associated with deficiency of the recently described COLVI-chains.

International audience

International audience

The preservation of the genetic resources (mitochondrial and nuclear genomes) and the regeneration of individuals are important economic and ecological issues for the sustainability of the aquaculture sector and conservation of the biodiversity, respectively. In fish, the cryopreservation of the ovocytes and embryos is impossible but the transplantation of cryopreserved diploid spermatogonial stem cells (SSC) in pre-hatching recipient embryos is a promising approach fulfilling the requirements. However, the rareness, and our incapacity to efficiently purify and amplify in vitro the SSCs limit the use and the dissemination of this procedure. In order to unravel mechanisms involved in the control of SSC self-renewal, we have undertaken the identification of paracrine factors that are released from the Sertoli cells within the germinal niche and could act on their cognate membrane bound receptors expressed on SSC. In the present study, we have produced transgenic zebrafish lines that express GFP specifically in the Sertoli cells (Dr_Gsdf:eGFP) or in spermatogonia (Dr_Vasa:eGFP) using promoter fragments of cell-specific genes (gsdf and vasa respectively). The expression pattern of the transgene was validated using whole mount RNA in situ hybridization and immunocytochemistry techniques. Note that transgene expression was not observed in the primordial germ cells (PGCs) of the developing Dr_vasa:eGFP embryos but was mainly detected in isolated or doublets of undifferentiated spermatogonial cells in the testis of pubertal males. The fluorescent cell populations were isolated from adult testes using a differential plating approach followed by a fluorescent assisted cell sorting. The enrichment of the candidate cell types was determined using real time quantitative PCR and a panel of 15 genes representative of different testicular cell types and germ cell stemness. We demonstrate that the transgenic zebrafish lines are valuable models to purify highly enriched populations of Sertoli cells or germ cells expressing high levels of certain but not all germ stem cell markers previously proposed in mammals and in fish.

Session: Fish model species in environmental toxicology (P); International audience

International audience; Multinucleated muscle fibres arise by fusion of precursor cells called myoblasts. We previously showed that CKIP-1 ectopic expression in C2C12 myoblasts increased cell fusion. In this work, we report that CKIP-1 depletion drastically impairs C2C12 myoblast fusion in vitro and in vivo during zebrafish muscle development. Within developing fast-twich myotome, Ckip-1 localises at the periphery of fast precursor cells, closed to the plasma membrane. Unlike wild-type myoblasts that form spatially arrayed multinucleated fast myofibres, Ckip-1-deficient myoblasts show a drastic reduction in fusion capacity. A search for CKIP-1 binding partners identified the ARPC1 subunit of Arp2/3 actin nucleation complex essential for myoblast fusion. We demonstrate that CKIP-1, through binding to plasma membrane phosphoinositides via its PH domain, regulates cell morphology and lamellipodia formation by recruiting the Arp2/3 complex at the plasma membrane. These results establish CKIP-1 as a regulator of cortical actin that recruits the Arp2/3 complex at the plasma membrane essential for muscle precursor elongation and fusion.

After the recent report of the expression of several nme genes in the zebrafish gonads, the present study aimed at further analyzing the expression of nme genes in the ovary with special attention for the nme transcripts that are maternally inherited and could thus participate in the determination of oocyte developmental competence. The expression levels of all groups I and II nme genes were characterized by QPCR in a panel of zebrafish tissues. The nme genes exhibiting an ovarian expression were subsequently monitored throughout oogenesis and early development, and their expression sites characterized using in situ hybridization. Here, we show that nme2b1, nme3, nme4, and nme6 are highly expressed in the ovary and present in the zebrafish oocyte throughout oogenesis. While the four transcripts are maternally inherited, nme3 and nme6 display a typical maternal profile and are detected in the zebrafish early embryo. In contrast to nme3, nme6, abundance exhibits a sharp decrease during early embryogenesis. After zygotic genome activation, we observed an increased expression of nme2b1, nme2b2, nme3, and nme6. The present study provides a comprehensive overview of the expression of nme family members during zebrafish oogenesis and early development. In addition, the maternal origin of two nme transcripts in the early embryo is reported here for the first time in any vertebrate species. Together, our observations suggest an important role of the nme family in oocyte and embryo development in vertebrates.

Programme national de recherche sur les perturbateurs endocriniens

Diplôme : Dr. d'Universite