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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.

The family of forkhead box (Fox) transcription factors regulate gonadogenesis and embryogenesis, but the role of foxr1/foxn5 in reproduction is unknown. Evolution of foxr1 in vertebrates was examined and the gene found to exist in most vertebrates, including mammals, ray-finned fish, amphibians, and sauropsids. By quantitative PCR and RNA-seq, we found that foxr1 had an ovarian-specific expression in zebrafish, a common feature of maternal-effect genes. In addition, it was demonstrated using in situ hybridization that foxr1 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 foxr1 in female reproduction using a zebrafish CRISPR/Cas9 knockout model. It was observed that embryos from the foxr1-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 a dramatically increased level of p21, a cell cycle inhibitor, and reduced rictor, 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 foxr1 is an essential maternal-effect gene and is 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 fitness.

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.

Maternal-effect genes and egg quality: usual suspects and new players. 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

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.

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.