Double maternal effect: duplicated nucleoplasmin 2 genes, npm2a and npm2b, are shared by fish and tetrapods, and have distinct and essential roles in early embryogenesis

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. Author Summary The protein and transcript of the npm2 gene have been previously demonstrated as maternal contributions to embryos of several vertebrates. Recently, two npm2 genes, denoted here as npm2a and npm2b, 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 ancestral npm2 gene created the current two forms, and while most vertebrates retained both genes, notably, mammals and certain species of fish lost npm2a and, albeit rarely, both npm2a and npm2b. Our functional analyses showed that npm2a and npm2b have diverse but essential functions during embryogenesis, as npm2a mutants failed to undergo development at the earliest stage while npm2b mutants 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 the npm2 genes, which are essential maternal factors that are required for proper embryonic development and survival.

genes are at two distinct loci located on the same chromosomic region (Fig. 2). These results 2 0 3 strongly suggested that npm2a and npm2b genes arose from local gene duplication rather than 2 0 4 a whole genome (or chromosome) duplication event (Fig. 3).

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The teleost ancestor experienced an extra WGD event (TGD) , but in all 2 0 6 investigated teleosts, we observed a maximum of one npm2a ortholog and one npm2b 2 0 7 ortholog (except in salmonids) (Fig. 1). In addition, npm2a and npm2b are located on two 2 0 8 TGD ohnologous regions on chromosomes 8 and 10, respectively (Fig. 2), in zebrafish, 2 0 9 1 0 which is consistent with the loss of one of the npm2a duplicates from one region and the loss 2 1 0 of one npm2b duplicate from the corresponding ohnologous region. Thus, npm2 diversity is 2 1 1 most likely due to the early loss of one of the two npm2a and npm2b TGD ohnologs (Fig. 3).

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This observation in zebrafish strengthened the hypothesis that TGD did not impact the npm2a 2 1 3 and npm2b diversity in teleosts. In addition, the lack of npm2a and npm2b in neoteleostei 2 1 4 species, such as tetraodon, suggested that additional gene losses occurred early in the 2 1 5 evolutionary history of this group (Fig. 2).

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In contrast, we identified only one npm2a gene in all investigated salmonids suggesting that 2 2 0 SaGD did not have any impact on the current salmonid npm2a diversity mostly due to an 2 2 1 early loss of the SaGD ohnolog of this gene (Fig. 3).

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In addition to the early gene losses after WGD, various other independent and 2 2 3 phylum-specific gene losses may have contributed to shape the current diversity of npm2a 2 2 4 and npm2b in vertebrates. In fact, although both npm2a and npm2b have been globally 2 2 5 conserved in sarcopterygians and actinopterygians, some phyla in each group lack at least one 2 2 6 of the genes. In sarcopterygians, npm2a is conserved in amphibians such as Xenopus 2 2 7 tropicalis and laevis, and in sauropsids such as Chinese alligator and chicken (Figs. 1-3). In 2 2 8 contrast, we did not find any npm2a genes in the Coelacanth genome (Figs. 1-3), which could 2 2 9 be due to the lower assembly quality of the concerned genomic region (see Supplemental 2 3 0 . Their expression profiles 2 8 9 are typical features of maternally-inherited mRNAs, which highly suggested that the novel 2 9 0 npm2a is also a maternal-effect gene. . As previously 2 9 6 demonstrated, npm2b is a maternal-effect gene whose transcripts are accumulated in the 2 9 7 growing oocyte and maternally-inherited by the zygote, where it functions as a histone 2 9 8 chaperone to decondense sperm DNA as well as reorganize chromatin, thus, it has also been 2 9 9 suggested to contribute to ZGA as well [8,14,27,28]. Npm2b is thought to be activated by Lastly, we performed functional analysis of these two Npm2 proteins by genetic 3 2 5 knockout using the CRISPR/cas9 system. One-cell staged embryos were injected with the 3 2 6 CRISPR/cas9 guides that targeted either npm2a or npm2b and allowed to grow to adulthood.

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Mosaic founder mutant females (F0) were identified by fin clip genotyping and subsequently 3 2 8 mated with wild-type (WT) males, and embryonic development of the F1 fertilized eggs was 3 2 9 recorded. Since the mutagenesis efficiency of the CRISPR/cas9 system was very high, as versus 0% in controls) (Fig.6B), which included smaller size, enlarged yolk to membrane 3 4 0 ratio, and small yolk to membrane ratio.  and mortality at the earliest stages of embryogenesis.

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In consequence, the dominancy of the mutant allele combined with the strong 3 7 7 maternal effect of these genes, which led to massive early embryonic mortality such that none Since two annotated genes corresponding to npm2, which is known as an essential 3 8 7 maternal-effect gene in mammals and amphibians, were recently found in zebrafish, we set 3 8 8 out to investigate their evolution and function. In this study, we demonstrated that the two 3 8 9 duplicates of npm2, npm2a and npm2b, exist in a wide range of vertebrate species, including 3 9 0 ray-finned fish, amphibians, and bird. We also found that the mammalian npm2 gene is in 3 9 1 fact an ortholog of npm2b. Using phylogeny and synteny analyses, we traced the origins of 3 9 2 those two duplicates to the early stages of vertebrate evolution. Our findings indicate that 3 9 3 npm2a and npm2b genes resulted from a local gene duplication that may have occurred 3 9 4 between VGD2 and the divergence of ray-finned fish and tetrapods (~450-500 Mya). This  The following genomic data were extracted and investigated from the ENSEMBL genomic

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Predictions of npm2 genes 4 3 5 The peptidic sequences of zebrafish Npm2a and Npm2b were used as query in TBLASTN   "clustal", and the Yang and Nielsen dN/dS substitution models were used.

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CrispR-cas9 genetic knockout 4 9 4 CRISPR/cas9 guide RNA (gRNA) were designed using the ZiFiT online software and were 4 9 5 made against 3 targets within each gene to generate large genomic deletions, ranging from 4 9 6 250-1600 base pairs, that span exons which allow the formation of non-functional proteins.

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Nucleotide sequences containing the gRNA were ordered, annealed together, and cloned into 4 9 8 the DR274 plasmid. In vitro transcription of the gRNA from the T7 initiation site was 4 9 9 performed using the Maxiscript T7 kit (Applied Biosystems), and their purity and integrity 5 0 0 were assessed using the Agilent RNA 6000 Nano Assay kit and 2100 Bioanalyzer (Agilent in Paris, France). The embryos were allowed to grow to adulthood, and genotyped using fin 5 0 5 clip and PCR that detected the deleted regions. The PCR bands of the mutants were then sent 5 0 6 for sequencing to verify the deletion. Once confirmed, the mutant females were mated with 5 0 7 2 2 wildtype males to produce F1 embryos, whose phenotypes were subsequently recorded.