RESUMO
Teleost fish represent one of the largest and most diverse clades of vertebrates, which makes them great models in various research areas such as ecology and evolution. Recent sequencing endeavors provided high-quality genomes for species covering the main fish evolutionary lineages, opening up large-scale comparative genomics studies. However, transcriptomic data across fish species and organs are heterogenous and have not been integrated with newly sequenced genomes making gene expression quantification and comparative analyses particularly challenging. Thus, resources integrating genomic and transcriptomic data across fish species and organs are still lacking. Here, we present FEVER, a web-based resource allowing evolutionary transcriptomics across species and tissues. First, based on query genes FEVER reconstructs gene trees providing orthologous and paralogous relationships as well as their evolutionary dynamics across 13 species covering the major fish lineages, and 4 model species as evolutionary outgroups. Second, it provides unbiased gene expression across 11 tissues using up-to-date fish genomes. Finally, genomic and transcriptomic data are combined together allowing the exploration of gene expression evolution following speciation and duplication events. FEVER is freely accessible at https://fever.sk8.inrae.fr/.
Assuntos
Evolução Molecular , Peixes , Internet , Transcriptoma , Animais , Peixes/genética , Transcriptoma/genética , Perfilação da Expressão Gênica/métodos , Bases de Dados Genéticas , Filogenia , Genômica/métodos , Software , Genoma/genéticaRESUMO
In contrast to most fishes, salmonids exhibit the unique ability to hold their eggs for several days after ovulation without significant loss of viability. During this period, eggs are held in the body cavity in a biological fluid, the coelomic fluid (CF) that is responsible for preserving egg viability. To identify CF proteins responsible for preserving egg viability, a proteomic comparison was performed using 3 salmonid species and 3 non-salmonid species to identify salmonid-specific highly abundant proteins. In parallel, rainbow trout CF fractions were purified and used in a biological test to estimate their egg viability preservation potential. The most biologically active CF fractions were then subjected to mass spectrometry analysis. We identified 50 proteins overabundant in salmonids and present in analytical fractions with high egg viability preservation potential. The identity of these proteins illuminates the biological processes participating in egg viability preservation. Among identified proteins of interest, the ovarian-specific expression and abundance in CF at ovulation of N-acetylneuraminic acid synthase a (Nansa) suggest a previously unsuspected role. We show that salmonid CF is a complex biological fluid containing a diversity of proteins related to immunity, calcium binding, lipid metabolism, proteolysis, extracellular matrix and sialic acid metabolic pathway that are collectively responsible for preserving egg viability.
Assuntos
Ovário , Salmonidae , Animais , Feminino , Ovário/metabolismo , Salmonidae/metabolismo , Óvulo/metabolismo , Proteínas de Peixes/metabolismo , Proteômica/métodos , Líquidos Corporais/metabolismo , Oncorhynchus mykiss/metabolismoRESUMO
Understanding microRNA (miRNA) functions has been hampered by major difficulties in identifying their biological target(s). Currently, the main limitation is the lack of a suitable strategy to identify biologically relevant targets among a high number of putative targets. Here we provide a proof of concept of successful de novo (i.e. without prior knowledge of its identity) miRNA phenotypic target (i.e. target whose de-repression contributes to the phenotypic outcomes) identification from RNA-seq data. Using the medaka mir-202 knock-out (KO) model in which inactivation leads to a major organism-level reproductive phenotype, including reduced egg production, we introduced novel criteria including limited fold-change in KO and low interindividual variability in gene expression to reduce the list of 2853 putative targets to a short list of 5. We selected tead3b, a member of the evolutionarily-conserved Hippo pathway, known to regulate ovarian functions, due to its remarkably strong and evolutionarily conserved binding affinity for miR-202-5p. Deleting the miR-202-5p binding site in the 3' UTR of tead3b, but not of other Hippo pathway members sav1 and vgll4b, triggered a reduced egg production phenotype. This is one of the few successful examples of de novo functional assignment of a miRNA phenotypic target in vivo in vertebrates.
Assuntos
Via de Sinalização Hippo , MicroRNAs , Oryzias , Animais , Sítios de Ligação , MicroRNAs/genética , MicroRNAs/metabolismo , Fenótipo , RNA-Seq , Oryzias/metabolismoRESUMO
Accurate species phylogenies are a prerequisite for all evolutionary research. Teleosts are the largest and most diversified group of extant vertebrates, but relationships among their three oldest extant lineages remain unresolved. On the basis of seven high-quality new genome assemblies in Elopomorpha (tarpons, eels), we revisited the topology of the deepest branches of the teleost phylogeny using independent gene sequence and chromosomal rearrangement phylogenomic approaches. These analyses converged to a single scenario that unambiguously places the Elopomorpha and Osteoglossomorpha (arapaima, elephantnose fish) in a monophyletic sister group to all other teleosts, i.e., the Clupeocephala lineage (zebrafish, medaka). This finding resolves more than 50 years of controversy on the evolutionary relationships of these lineages and highlights the power of combining different levels of genome-wide information to solve complex phylogenies.
Assuntos
Evolução Biológica , Peixes , Animais , Enguias/classificação , Enguias/genética , Peixes/classificação , Peixes/genética , Genoma , Filogenia , Peixe-Zebra/classificação , Peixe-Zebra/genéticaRESUMO
In the last decade, a plethora of microRNAs (miRNAs) has been reported in a wide variety of physiological processes, including reproduction, in many aquatic organisms. However, miRNAome alterations occurred by environmental cues due to water temperature increment have not yet been elucidated. With the aim to identify epigenetic regulations mediated by miRNAs in the gonads in a climate change scenario, the animal model zebrafish (Danio rerio) were subjected to high temperatures during sex differentiation, a treatment that results in male-skewed sex ratios in the adulthood. Once the fish reached adulthood, gonads were sequenced by high-throughput technologies and a total of 23 and 1 differentially expressed miRNAs in ovaries and testes, respectively, were identified two months after the heat treatment. Most of these heat-recorder miRNAs were involved in human sex-related cancer and about 400 predicted-target genes were obtained, some with reproduction-related functions. Their synteny in the zebrafish genome was, for more than half of the predicted target genes, in the chromosomes 7, 2, 4, 3 and 11 in the ovaries, chromosome 4 being the place where the sex-associated-region (sar) is localized in wild zebrafish. Further, spatial localization in the gonads of two selected heat-recorder miRNAs (miR-122-5p and miR-146-5p) showed exclusive expression in the ovarian germ cells. The present study expands the catalog of sex-specific miRNAs and deciphers, for the first time, thermosensitive miRNAs in the zebrafish gonads that might be used as potential epimarkers to predict environmental past events.
Assuntos
MicroRNAs , Peixe-Zebra , Animais , Feminino , Masculino , Humanos , Adulto , Peixe-Zebra/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Temperatura Alta , Gônadas/metabolismo , Diferenciação Sexual/genética , Perfilação da Expressão GênicaRESUMO
The AUTS2 gene plays major roles during brain development and is associated with various neuropathologies including autism. Data in non-mammalian species are scarce, and the aim of our study was to provide a comprehensive analysis of auts2 evolution in teleost fish, which are widely used for in vivo functional analysis and biomedical purposes. Comparative genomics in 78 species showed that auts2a and auts2b originate from the teleost-specific whole genome duplication (TGD). auts2a, which is highly similar to human AUTS2, was almost systematically retained following TGD. In contrast, auts2b, which encodes for a shorter protein similar to a short human AUTS2 isoform, was lost more frequently and independently during evolution. RNA-seq analysis in 10 species revealed a highly conserved profile with predominant expression of both genes in the embryo, brain, and gonads. Based on protein length, conserved domains, and expression profiles, we speculate that the long human isoform functions were retained by auts2a, while the short isoform functions were retained by auts2a and/or auts2b, depending on the lineage/species. auts2a showed a burst in expression during medaka brain formation, where it was expressed in areas of the brain associated with neurodevelopmental disorders. Together, our data suggest a strong conservation of auts2 functions in vertebrates despite different evolutionary scenarios in teleosts.
Assuntos
Duplicação Gênica , Oryzias , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Transtorno do Espectro Autista/genética , Genoma/genética , Genômica , Oryzias/genética , Proteínas , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Teleost fishes are ancient tetraploids descended from an ancestral whole-genome duplication that may have contributed to the impressive diversification of this clade. Whole-genome duplications can occur via self-doubling (autopolyploidy) or via hybridization between different species (allopolyploidy). The mode of tetraploidization conditions evolutionary processes by which duplicated genomes return to diploid meiotic pairing, and subsequent genetic divergence of duplicated genes (cytological and genetic rediploidization). How teleosts became tetraploid remains unresolved, leaving a fundamental gap in the interpretation of their functional evolution. As a result of the whole-genome duplication, identifying orthologous and paralogous genomic regions across teleosts is challenging, hindering genome-wide investigations into their polyploid history. Here, we combine tailored gene phylogeny methodology together with a state-of-the-art ancestral karyotype reconstruction to establish the first high-resolution comparative atlas of paleopolyploid regions across 74 teleost genomes. We then leverage this atlas to investigate how rediploidization occurred in teleosts at the genome-wide level. We uncover that some duplicated regions maintained tetraploidy for more than 60 million years, with three chromosome pairs diverging genetically only after the separation of major teleost families. This evidence suggests that the teleost ancestor was an autopolyploid. Further, we find evidence for biased gene retention along several duplicated chromosomes, contradicting current paradigms that asymmetrical evolution is specific to allopolyploids. Altogether, our results offer novel insights into genome evolutionary dynamics following ancient polyploidizations in vertebrates.
RESUMO
The evolution of sex determination (SD) in teleosts is amazingly dynamic, as reflected by the variety of different master sex-determining genes identified. Pangasiids are economically important catfishes in South Asian countries, but little is known about their SD system. Here, we generated novel genomic resources for 12 Pangasiids and characterized their SD system. Based on a Pangasianodon hypophthalmus chromosome-scale genome assembly, we identified an anti-Müllerian hormone receptor type â ¡ gene (amhr2) duplication, which was further characterized as being sex-linked in males and expressed only in testes. These results point to a Y chromosome male-specific duplication (amhr2by) of the autosomal amhr2a. Sequence annotation revealed that the P. hypophthalmus Amhr2by is truncated in its N-terminal domain, lacking the cysteine-rich extracellular part of the receptor that is crucial for ligand binding, suggesting a potential route for its neofunctionalization. Reference-guided assembly of 11 additional Pangasiids, along with sex-linkage studies, revealed that this truncated amhr2by duplication is a male-specific conserved gene in Pangasiids. Reconstructions of the amhr2 phylogeny suggested that amhr2by arose from an ancient duplication/insertion event at the root of the Siluroidei radiation that is dated to ~100 million years ago. Together these results bring multiple lines of evidence supporting that amhr2by is an ancient and conserved master sex-determining gene in Pangasiids, a finding that highlights the recurrent use of the transforming growth factor ß pathway, which is often used for the recruitment of teleost master SD genes, and provides another empirical case towards firther understanding of dynamics of SD systems.
Assuntos
Peixes-Gato , Animais , Peixes-Gato/genética , Masculino , Filogenia , Receptores de Peptídeos/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Cromossomo Y/genéticaRESUMO
MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression involved in countless biological processes and are widely studied across metazoans. Although miRNA research continues to grow, the large community of fish miRNA researchers lacks exhaustive resources consistent among species. To fill this gap, we developed FishmiRNA, an evolutionarily supported miRNA annotation and expression database for ray-finned fishes: www.fishmirna.org. The self-explanatory database contains detailed, manually curated miRNA annotations with orthology relationships rigorously established by sequence similarity and conserved syntenies, and expression data provided for each detected mature miRNA. In just few clicks, users can download the annotation and expression database in several convenient formats either in its entirety or a subset. Simple filters and Blast search options also permit the simultaneous exploration and visual comparison of expression data for up to any ten mature miRNAs across species and organs. FishmiRNA was specifically designed for ease of use to reach a wide audience.
Assuntos
MicroRNAs , Animais , Peixes/genética , Peixes/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
BACKGROUND: Sexual maturation causes loss of fish muscle mass and deterioration of fillet quality attributes that prevent market success. We recently showed that fillet yield and flesh quality recover in female trout after spawning. To gain insight into the molecular mechanisms regulating flesh quality recovery, we used an Agilent-based microarray platform to conduct a large-scale time course analysis of gene expression in female trout white muscle from spawning to 33 weeks post-spawning. RESULTS: In sharp contrast to the situation at spawning, muscle transcriptome of female trout at 33 weeks after spawning was highly similar to that of female trout of the same cohort that did not spawn, which is consistent with the post-spawning flesh quality recovery. Large-scale time course analysis of gene expression in trout muscle during flesh quality recovery following spawning led to the identification of approximately 3340 unique differentially expressed genes that segregated into four major clusters with distinct temporal expression profiles and functional categories. The first cluster contained approximately 1350 genes with high expression at spawning and downregulation after spawning and was enriched with genes linked to mitochondrial ATP synthesis, fatty acid catabolism and proteolysis. A second cluster of approximately 540 genes with transient upregulation 2 to 8 weeks after spawning was enriched with genes involved in transcription, RNA processing, translation, ribosome biogenesis and protein folding. A third cluster containing approximately 300 genes upregulated 4 to 13 weeks after spawning was enriched with genes encoding ribosomal subunits or regulating protein folding. Finally, a fourth cluster that contained approximately 940 genes with upregulation 8 to 24 weeks after spawning, was dominated by genes encoding myofibrillar proteins and extracellular matrix components and genes involved in glycolysis. CONCLUSION: Overall, our study indicates that white muscle tissue restoration and flesh quality recovery after spawning are associated with transcriptional changes promoting anaerobic ATP production, muscle fibre hypertrophic growth and extracellular matrix remodelling. The generation of the first database of genes associated with post-spawning muscle recovery may provide insights into the molecular and cellular mechanisms controlling muscle yield and fillet quality in fish and provide a useful list of potential genetic markers for these traits.
Assuntos
Oncorhynchus mykiss , Animais , Feminino , Perfilação da Expressão Gênica , Humanos , Análise em Microsséries , Músculos , Oncorhynchus mykiss/genética , TranscriptomaRESUMO
BACKGROUND: Circulating miRNAs (c-miRNAs) are found in most, if not all, biological fluids and are becoming well-established non-invasive biomarkers of many human pathologies. However, their features in non-pathological contexts and whether their expression profiles reflect normal life history events have received little attention, especially in non-mammalian species. The aim of the present study was to investigate the potential of c-miRNAs to serve as biomarkers of reproductive and metabolic states in fish. RESULTS: The blood plasma was sampled throughout the reproductive cycle of female rainbow trout subjected to two different feeding regimes that triggered contrasting metabolic states. In addition, ovarian fluid was sampled at ovulation, and all samples were subjected to small RNA-seq analysis, leading to the establishment of a comprehensive miRNA repertoire (i.e., miRNAome) and enabling subsequent comparative analyses to a panel of RNA-seq libraries from a wide variety of tissues and organs. We showed that biological fluid miRNAomes are complex and encompass a high proportion of the overall rainbow trout miRNAome. While sharing a high proportion of common miRNAs, the blood plasma and ovarian fluid miRNAomes exhibited strong fluid-specific signatures. We further revealed that the blood plasma miRNAome significantly changed depending on metabolic and reproductive states. We subsequently identified three evolutionarily conserved muscle-specific miRNAs or myomiRs (miR-1-1/2-3p, miR-133a-1/2-3p, and miR-206-3p) that accumulated in the blood plasma in response to high feeding rates, making these myomiRs strong candidate biomarkers of active myogenesis. We also identified miR-202-5p as a candidate biomarker for reproductive success that could be used to predict ovulation and/or egg quality. CONCLUSIONS: Together, these promising results reveal the high potential of c-miRNAs, including evolutionarily conserved myomiRs, as physiologically relevant biomarker candidates and pave the way for the use of c-miRNAs for non-invasive phenotyping in various fish species.
Assuntos
MicroRNAs , Oncorhynchus mykiss , Animais , Biomarcadores , Feminino , Humanos , MicroRNAs/genética , Oncorhynchus mykiss/genética , Reprodução/genéticaRESUMO
The aim of this study was to investigate the respective contribution of maternally-inherited mRNAs and proteins to egg molecular cargo and to its developmental competence in fish using pikeperch as a model. Our study provides novel insights into the understanding of type-specific roles of maternally-inherited molecules in fish. Here we show, for the first time, that transcripts and proteins have distinct, yet complementary, functions in the egg of teleost fish. Maternally-inherited mRNAs would shape embryo neurodevelopment, while maternally-inherited proteins would rather be responsible for protecting the embryo against pathogens. Additionally, we observed that processes directly preceding ovulation may considerably affect the reproductive success by modifying expression level of genes crucial for proper embryonic development, being novel fish egg quality markers (e.g., smarca4 or h3f3a). These results are of major importance for understanding the influence of external factors on reproductive fitness in both captive and wild-type fish species.
Assuntos
Desenvolvimento Embrionário , Reprodução , Animais , Desenvolvimento Embrionário/genética , Feminino , Sistema Imunitário/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The bowfin (Amia calva) is a ray-finned fish that possesses a unique suite of ancestral and derived phenotypes, which are key to understanding vertebrate evolution. The phylogenetic position of bowfin as a representative of neopterygian fishes, its archetypical body plan and its unduplicated and slowly evolving genome make bowfin a central species for the genomic exploration of ray-finned fishes. Here we present a chromosome-level genome assembly for bowfin that enables gene-order analyses, settling long-debated neopterygian phylogenetic relationships. We examine chromatin accessibility and gene expression through bowfin development to investigate the evolution of immune, scale, respiratory and fin skeletal systems and identify hundreds of gene-regulatory loci conserved across vertebrates. These resources connect developmental evolution among bony fishes, further highlighting the bowfin's importance for illuminating vertebrate biology and diversity in the genomic era.
Assuntos
Evolução Biológica , Evolução Molecular , Genoma/genética , Rajidae/genética , Rajidae/fisiologia , Animais , Cromatina/genética , Peixes , Rajidae/imunologia , Sequenciamento Completo do GenomaRESUMO
MicroRNAs (miRNAs) are important gene expression regulators implicated in many biological processes, but we lack a global understanding of how miRNA genes evolve and contribute to developmental canalization and phenotypic diversification. Whole-genome duplication events likely provide a substrate for species divergence and phenotypic change by increasing gene numbers and relaxing evolutionary pressures. To understand the consequences of genome duplication on miRNA evolution, we studied miRNA genes following the teleost genome duplication (TGD). Analysis of miRNA genes in four teleosts and in spotted gar, whose lineage diverged before the TGD, revealed that miRNA genes were retained in ohnologous pairs more frequently than protein-coding genes, and that gene losses occurred rapidly after the TGD. Genomic context influenced retention rates, with clustered miRNA genes retained more often than nonclustered miRNA genes and intergenic miRNA genes retained more frequently than intragenic miRNA genes, which often shared the evolutionary fate of their protein-coding host. Expression analyses revealed both conserved and divergent expression patterns across species in line with miRNA functions in phenotypic canalization and diversification, respectively. Finally, major strands of miRNA genes experienced stronger purifying selection, especially in their seeds and 3'-complementary regions, compared with minor strands, which nonetheless also displayed evolutionary features compatible with constrained function. This study provides the first genome-wide, multispecies analysis of the mechanisms influencing metazoan miRNA evolution after whole-genome duplication.
Assuntos
Evolução Biológica , Peixes/genética , Genoma , MicroRNAs/genética , Animais , Sequência de Bases , Sequência Conservada , Peixes/metabolismo , Duplicação Gênica , Gônadas/metabolismo , Família Multigênica , Seleção Genética , Especificidade da EspécieRESUMO
Pikeperch, Sander lucioperca, is a species of high interest to the aquaculture. The expansion of its production can only be achieved by furthering domestication level. However, the mechanisms driving the domestication process in finfishes are poorly understood. Transcriptome profiling of eggs was found to be a useful tool allowing understanding of the domestication process in teleosts. In this study, using next-generation sequencing, the first pikeperch transcriptome has been generated as well as pikeperch-specific microarray comprising 35,343 unique probes. Next, we performed transcriptome profiling of eggs obtained from wild and domesticated populations. We found 710 differentially expressed genes that were linked mostly to nervous system development. These results provide new insights into processes that are directly involved in the domestication of finfishes. It can be suggested that all the identified processes were predetermined by the maternally derived set of genes contained in the unfertilized eggs. This allows us to suggest that fish behavior, along with many other processes, can be predetermined at the cellular level and may have significant implications on the adaptation of cultured fish to the natural environment. This also allows to suggest that fish behavior should be considered as a very important pikeperch aquaculture selection trait.
Assuntos
Domesticação , Neurogênese/genética , Óvulo/metabolismo , Percas , Animais , Aquicultura , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Controladores do Desenvolvimento/genética , Masculino , Óvulo/crescimento & desenvolvimento , Percas/embriologia , Percas/genética , Percas/crescimento & desenvolvimento , Transcriptoma/genéticaRESUMO
Dmrt1 is a highly conserved transcription factor, which is critically involved in regulation of gonad development of vertebrates. In medaka, a duplicate of dmrt1-acting as master sex-determining gene-has a tightly timely and spatially controlled gonadal expression pattern. In addition to transcriptional regulation, a sequence motif in the 3' UTR (D3U-box) mediates transcript stability of dmrt1 mRNAs from medaka and other vertebrates. We show here that in medaka, two RNA-binding proteins with antagonizing properties target this D3U-box, promoting either RNA stabilization in germ cells or degradation in the soma. The D3U-box is also conserved in other germ-cell transcripts, making them responsive to the same RNA binding proteins. The evolutionary conservation of the D3U-box motif within dmrt1 genes of metazoans-together with preserved expression patterns of the targeting RNA binding proteins in subsets of germ cells-suggest that this new mechanism for controlling RNA stability is not restricted to fishes but might also apply to other vertebrates.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Oryzias/genética , Processos de Determinação Sexual/genética , Regiões 3' não Traduzidas/genética , Animais , Evolução Biológica , Feminino , Proteínas de Peixes/genética , Células Germinativas/metabolismo , Masculino , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vertebrados/metabolismoRESUMO
Domestication is an evolutionary process during which we expect populations to progressively adapt to an environment controlled by humans. It is accompanied by genetic and presumably epigenetic changes potentially leading to modifications in the transcriptomic profile in various tissues. Reproduction is a key function often affected by this process in numerous species, regardless of the mechanism. The maternal mRNA in fish eggs is crucial for the proper embryogenesis. Our working hypothesis is that modifications of maternal mRNAs may reflect potential genetic and/or epigenetic modifications occurring during domestication and could have consequences during embryogenesis. Consequently, we investigated the trancriptomic profile of unfertilized eggs from two populations of Eurasian perch. These two populations differed by their domestication histories (F1 vs. F7+-at least seven generations of reproduction in captivity) and were genetically differentiated (FST = 0.1055, p<0.05). A broad follow up of the oogenesis progression failed to show significant differences during oogenesis between populations. However, the F1 population spawned earlier with embryos presenting an overall higher survivorship than those from the F7+ population. The transcriptomic profile of unfertilized eggs showed 358 differentially expressed genes between populations. In conclusion, our data suggests that the domestication process may influence the regulation of the maternal transcripts in fish eggs, which could in turn explain differences of developmental success.
Assuntos
Domesticação , Óvulo/metabolismo , Percas/embriologia , Percas/genética , RNA Mensageiro Estocado/genética , Transcriptoma/genética , Animais , Desenvolvimento Embrionário/genéticaRESUMO
Female gamete production relies on coordinated molecular and cellular processes that occur in the ovary throughout oogenesis. In fish, as in other vertebrates, these processes have been extensively studied both in terms of endocrine/paracrine regulation and protein expression and activity. The role of small non-coding RNAs in the regulation of animal reproduction remains however largely unknown and poorly investigated, despite a growing interest for the importance of miRNAs in a wide variety of biological processes. Here, we analyzed the role of miR-202, a miRNA predominantly expressed in male and female gonads in several vertebrate species. We studied its expression in the medaka ovary and generated a mutant line (using CRISPR/Cas9 genome editing) to determine its importance for reproductive success with special interest for egg production. Our results show that miR-202-5p is the most abundant mature form of the miRNA and that it is expressed in granulosa cells and in the unfertilized egg. The knock out (KO) of mir-202 gene resulted in a strong phenotype both in terms of number and quality of eggs produced. Mutant females exhibited either no egg production or produced a dramatically reduced number of eggs that could not be fertilized, ultimately leading to no reproductive success. We quantified the size distribution of the oocytes in the ovary of KO females and performed a large-scale transcriptomic analysis approach to identified dysregulated molecular pathways. Together, cellular and molecular analyses indicate that the lack of miR-202 impairs the early steps of oogenesis/folliculogenesis and decreases the number of large (i.e. vitellogenic) follicles, ultimately leading to dramatically reduced female fecundity. This study sheds new light on the regulatory mechanisms that control the early steps of follicular development, including possible targets of miR-202-5p, and provides the first in vivo functional evidence that a gonad-predominant microRNA may have a major role in female reproduction.
Assuntos
Fertilidade/genética , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/fisiologia , Oogênese/genética , Oryzias/fisiologia , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Feminino , Edição de Genes , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Células da Granulosa , Masculino , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Ovário/citologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismoRESUMO
Whole-genome duplications (WGDs) 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-related 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), offers 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 divergence 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.