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Oocyte-specific maternal Slbp2 is required for replication-dependent histone storage and early nuclear cleavage in zebrafish oogenesis and embryogenesis

Stem–loop binding protein (SLBP) is required for replication-dependent histone mRNA metabolism in mammals. Zebrafish possesses two slbps, and slbp1 is necessary for retinal neurogenesis. However, the detailed expression and function of slbp2 in zebrafish are still unknown. In this study, we first identified zebrafish slbp2 as an oocyte-specific maternal factor and then generated a maternal-zygotic slbp2 F3 homozygous mutant (MZslbp2Δ4−/−) using CRISPR/Cas9. The depletion of maternal Slbp2 disrupted early nuclear cleavage, which resulted in developmental arrest at the MBT stage. The developmental defects could be rescued in slbp2 transgenic MZslbp2Δ4−/− embryos. However, homozygous mutant MZslbp1Δ1−/− developed normally, indicating slbp1 is dispensable for zebrafish early embryogenesis. Through comparative proteome and transcriptome profiling between WT and MZslbp2Δ4−/− embryos, we identified many differentially expressed proteins and genes. In comparison with those in WT embryos, four replication-dependent histones, including H2a, H2b, H3, and H4, all reduced their expression, while histone variant h2afx significantly increased in MZslbp2Δ4−/− embryos at the 256-cell stage and high stage. Zebrafish Slbp2 can bind histone mRNA stem–loop in vitro, and the defects of MZslbp2Δ4−/− embryos can be partially rescued by overexpression of H2b. The current data indicate that maternal Slbp2 plays a pivotal role in the storage of replication-dependent histone mRNAs and proteins during zebrafish oogenesis.


INTRODUCTION
Replication-dependent histones are tightly regulated in the cell cycle and are necessary for packaging the newly replicated chromosomal DNA (Gunjan et al. 2006;Marzluff et al. 2008;Marzluff and Koreski 2017). In metazoan, replication-dependent histone mRNAs are not polyadenylated but instead terminated with a conserved stemloop (SL), which possesses similar functions of poly(A) tail in translational efficiency and mRNA stability (Hentschel and Birnstiel 1981;Jackson and Standart 1990;Marzluff 1992). Stem-loop binding protein (SLBP) plays a central role at all steps of histone mRNA metabolism (Bernstein and Ross 1989;Gallie et al. 1996) via interactions between the SL and the RNA binding domain (RBD) of SLBP .
SLBP, also known as hairpin-binding protein (HBP), was initially and partially purified from Xenopus oocyte nuclear extracts (Hanson et al. 1996), then identified from human, mouse, and frog by using yeast three-hybrid systems (Wang et al. 1996;Martin et al. 1997). During mouse oocyte growth and maturation, SLBP accumulates and regulates the synthesis of replication-dependent histones (Allard et al. 2002(Allard et al. , 2005, which are the only known targets (Townley-Tilson et al. 2006). Due to insufficient histones H3 and H4, most of the mouse embryos derived from SLBP-depleted oocytes arrested at the two-cell stage (Arnold et al. 2008). The conserved functions of SLBP in histone mRNA metabolism and early embryo development were also revealed in Drosophila (Sullivan et al. 2001) and Caenorhabditis elegans (Pettitt et al. 2002).
Two SLBPs, named as xSLBP1 and xSLBP2, were identified from Xenopus oocytes and showed differential expression during oogenesis and early embryogenesis.
xSLBP1 is the mammalian ortholog of SLBP and participates in pre-mRNA processing, while xSLBP2 is oocytespecific and is supposed to be involved in storage (Wang et al. 1999). xSLBP2 has no effect on translation (Sánchez and Marzluff 2002) and is degraded, paralleling the activation of histone mRNA translation after oocyte maturation (Sánchez and Marzluff 2004). Considering that only the SLBP2 pseudogene is found in mouse, human, and rabbit genomes (Thelie et al. 2012), the sole SLBP appears to regulate replication-dependent histone synthesis in these mammalian animals (Allard et al. 2002(Allard et al. , 2005. However, the SLBP2 transcripts were isolated from some other mammals, such as bovine, rat, dog, horse, and pig. Additionally, bovine SLBP2 and xSLBP2 display differences in their expression and localization during oogenesis (Thelie et al. 2012). Although the functions of SLBP in histone mRNA metabolism have been well revealed (Marzluff and Koreski 2017), the reports about SLBP2 are very scarce (Wang et al. 1999;Thelie et al. 2012;Liu et al. 2015) and the function of SLBP2 is still mostly unknown.
Zebrafish (Danio rerio) is an important vertebrate model animal for research on development and disease (Grunwald and Eisen 2002). In zebrafish slbp1 mutant, retinal neurogenesis is severely delayed (Imai et al. 2014). Recently, we identified oocyte-specific slbp2 from gibel carp (Carassius gibelio) (Gui and Zhou 2010;Zhou and Gui 2017) and analyzed its dynamic expression pattern during oogenesis and embryogenesis (Liu et al. 2015). Imai et al. (2014) also detected maternal slbp2 in early zebrafish embryos. However, the detailed expression pattern and function of slbp2 in early embryogenesis have not been revealed. In this study, we first characterized zebrafish Slbp2 as an oocyte-specific maternal protein. Then its role in zebrafish oogenesis and early embryogenesis was revealed by knockout using CRISPR/Cas9. Finally, we confirmed the expression changes of histone proteins influenced by Slbp2 deficiency and explored the interaction between replication-dependent histones and Slbp2.

Oocyte-specific and maternal expression pattern of zebrafish slbp2
Both zebrafish slbp2 transcripts and proteins were specifically detected in the ovary, not in other analyzed tissues (Fig. 1A). During oogenesis, Slbp2 was expressed in primary growth oocytes (stage I), reached a peak in cortical alveolar oocytes (stage II), and then decreased from vitellogenic stage oocytes (stage III). After oocyte maturation, the Slbp2 protein was slightly reduced in mature eggs (stage V) (Fig. 1B). Consistent with Cgslbp2 in gibel carp (Liu et al. 2015), zebrafish slbp2 transcripts and proteins were detected only in unfertilized eggs and early embryos before the 50% epiboly stage (Fig. 1C), indicating that zebrafish Slbp2 is a maternal factor and no zygotic product is synthesized during embryogenesis. The distribution of zebrafish slbp2 in the ovary is also the same as that of Cgslbp2 in gibel carp (Liu et al. 2015). Zebrafish slbp2 was specifically expressed in the oocytes, not in somatic cells surrounding oocytes (Fig. 1D,E). The slbp2 transcript signals were intensively observed in the cytoplasm of primary growth oocytes (I), cortical alveolus oocytes (II), and vitellogenic oocytes (III), and subsequently reduced and concentrated in peripheral cytoplasm of maturing oocytes (IV) (Fig. 1D). The protein signals were weak in primary growth oocytes (I), and distributed strongly and uniformly in the cytoplasm of cortical alveolar oocytes (II). Along with the oocyte growth, Slbp2 began to reduce its abundance in vitellogenic oocytes (III). In maturing oocytes (IV), the signal was faint owing to the reduction and diffusion of Slbp2 protein along with oocyte growth (Fig. 1E).
The data indicate that zebrafish Slbp2 is a maternal factor expressed specifically in oocytes.
Different from the oocyte-specific expression pattern of zebrafish slbp2, zebrafish slbp1 was ubiquitously expressed in adult tissues, and abundant transcripts were detected in ovary, testis, kidney, and brain (Fig. 1A). In addition, a few of maternal slbp1 transcripts were detected in unfertilized eggs, and the zygotic mRNA slbp1 level was strikingly activated at the mid-blastula transition and maintained a relatively stable expression level during the following embryogenesis embryos (Fig. 1C).

Establishment of zebrafish slbp2 knockout mutant lines
To explore the function of slbp2, we generated slbp2 gene-disrupted zebrafish using CRISPR/Cas9. The targeting site was chosen in the sixth exon to disrupt the RBD domain (Fig. 2B). The sequencing results showed several types of mutations (data not shown), of which two mutated types modified at the target site; a 4-bp deletion (slbp2Δ4) and a 5-bp insertion (slbp2+5) (Fig. 2C) were chosen to construct mutant lines. The mutations in slbp2Δ4 and slbp2+5 caused a reading frame shift and created a premature stop codon. The two slbp2 knockout mutant lines both altered the RBD domain ( Fig. 2A,D).

Maternal Slbp2 depletion disrupts early nuclear cleavage
In comparison with WT zebrafish (Fig. 4A), MZslbp2Δ4 −/− embryos appeared to develop normally during cleavage stage and blastula stage, but they did not initiate epiboly and arrested at the MBT stage (Fig. 4B), and all embryos died after 8 hpf. In order to visualize the dynamic cell behavior, in vitro transcribed H2B-GFP mRNAs were injected into the one-cell WT and MZslbp2Δ4 −/− embryos, respectively. The first and second cycles of MZslbp2Δ4 −/− embryos appeared somewhat normal, but they showed obvious disruptions of nuclear cleavage from the eightcell stage. The mitotic nuclei in WT and MZslbp1Δ1 −/− embryos condensed at anaphase (Fig. 4F,H), while the nuclei in MZslbp2Δ4 −/− embryos had aberrant morphologies, forming a bridge connecting the two mitotic daughter nuclei (Fig. 4G).
To confirm the defects in MZslbp2Δ4 −/− embryos caused by the deficiency of maternal Slbp2, plasmid slbp2-GFP containing slbp2 ORF driven by β-actin promoter (Wang et al. 2001) was injected into the one-cell embryos produced by crossing heterozygous F1 female and male (slbp2Δ4 +/− ), and the female Zslbp2Δ4 −/− mutants were screened by PCR. As described above, Zslbp2Δ4 −/− developed and normally spawned (Fig. 3A). Subsequently, 10 females of slbp2 transgenic Zslbp2Δ4 −/− fish were randomly selected to cross with males of Zslbp2Δ4 −/− . Owing to the germline mosaic pattern in F0 transgenic fish, only ∼2%-15% of one-cell embryos exhibited the GFP fluorescent, indicating that these embryos expressed Slbp2. Compared to the MZslbp2Δ4 −/− embryos, all of slbp2 transgenic MZslbp2Δ4 −/− embryos completed epiboly (Fig. 4D), which suggested that the defects of early developmental arrest might be rescued by the expression of Slbp2. However, only 30%-55% of these embryos developed normally and grew to fertile adults. The others grew abnormally and showed a series of morphological deformations, including yolk sac deformity, head or tail hypoplasia, or growth retardation (data not shown). Additionally, the nuclear cleavage of slbp2 transgenic MZslbp2Δ4 −/− exhibited normally (Fig. 4I). The injection of in vitro transcribed slbp2-GFP mRNAs or plasmid slbp2-GFP into MZslbp2Δ4 −/− could not rescue the defects (data not shown). Therefore, the very early developmental arrest and the disruption of early nuclear cleavage in MZslbp2Δ4 −/− embryos may have resulted from the specific depletion of maternal Slbp2. Comparative proteomics and transcriptome profiling between WT and MZslbp2Δ4 −/− embryos To reveal the proteins influenced by slbp2 deficiency, iTRAQ was used to assess proteome changes between WT and MZslbp2Δ4 −/− embryos at the 256-cell stage (MZslbp2Δ4 −/− -256 versus WT-256) and high stage (MZslbp2Δ4 −/− -high versus WT-high), respectively. A total of 2944 proteins were identified and annotated in six public databases (Supplemental Table S1). We applied a threshold of >1.2-fold change and a P-value of <0.05 to identify differentially expressed proteins (DEPs). Additionally, only DEPs that were detected in at least two replicates were defined as significant DEPs. Based on these three criteria, 38 and 41 DEPs were identified from MZslbp2Δ4 −/− -256 versus WT-256 and MZslbp2Δ4 −/− -high versus WT-high, respectively (Supplemental Table S2). A fish-egg, lectin-like and histone H2a were the most highly down-regulated proteins in MZslbp2Δ4 −/− -256 versus WT-256 and MZ slbp2Δ4 −/− -high versus WT-high, respectively. Thirteen commonly down-regulated DEPs were screened, including H2a, H4, vitellogenin 3, tubulin, alpha 4-like, and others. Compared to their contents in WT embryos, H2a reduced by two-thirds and H4 decreased by one-third, while H3 slightly reduced expression. Additionally, H2b was also detected only in one of three biological replicates and displayed the reduction by 60%. Eosinophil chemotactic cytokine, the glycyl-tRNA synthetase, and acyl-CoA dehydrogenase medium chain were commonly up-regulated in MZslbp2Δ4 −/− embryos at the 256-cell and high stages compared to WT embryos.
Subsequently, we performed comparative transcriptome analysis of MZslbp2Δ4 −/− -256 versus WT-256 and MZslbp2Δ4 −/− -high versus WT-high, respectively. Three biological replicates were carried out. In comparison with WT embryos at the 256-cell and high stages, a total of 165 and   Table S3). Zebrafish slbp1 kept similar expression levels between WT and MZslbp2Δ4 −/− embryos at the 256-cell stage (Supplemental Table S4). Owing to the absence of poly(A) tails, replication-dependent histones were not credibly and repeatedly detected from transcriptomic profiles obtained by using the Illumina HiSeq 4000 platform. Four transcripts of h2a variants and five transcripts of h3 variants were screened from transcriptomic profiles. Compared to WT embryos, only h2afx expression increased up to 7.36-and 9.13-fold in MZslbp2Δ4 −/− embryos at the 256cell stage and high stage, respectively (Supplemental Table S4).
Dynamic expression changes of histones between WT and MZslbp2Δ4 −/− embryos To confirm dynamic expression changes of histones between WT and Zslbp2Δ4 −/− ovary, and WT and MZslbp2Δ4 −/− embryos, the replication-dependent histones and histone variants identified from proteomic and transcriptomic profiles were selected to perform qPCR analyses. Compared to their expression levels in wild-type ovary, zebrafish h2a, h2b, h3, and h4 mRNAs all largely decreased, while many histone proteins were detected in Zslbp2Δ4 −/− ovary (Fig. 5A). In comparison with their expression levels in wild-type embryos, traces of zebrafish h2a, h2b, h3, and h4 mRNA were detected in MZ slbp2Δ4 −/− embryos at the two-cell, 256-cell, and high stages by qPCR (Fig. 5A). The results of western blot confirmed the large reduction of H2b and H3 proteins in MZslbp2Δ4 −/− embryos at two-cell and high stages (Fig.  5B). In contrast, the expression levels of h2afx mRNA in MZslbp2Δ4 −/− embryos increased up to 3.2-and 3.6fold at the 256-cell stage and high stage, respectively. Additionally, other histone variants, such as h2afva and h3f3a, kept similar expression levels between WT and MZslbp2Δ4 −/− embryos (Fig. 5C).

Maternal Slbp2 is required for replicationdependent histone storage in zebrafish oogenesis
The significant reduction of replication-dependent histones (Fig. 5A,B) suggests that maternal Slbp2 might be required for replication-dependent histone mRNA and protein metabolism during zebrafish oogenesis and early embryogenesis. To confirm the assumption, the binding ability of zebrafish Slbp2 with the SL of replication-dependent histone mRNAs was assessed. Replication-dependent histone mRNAs share a conserved SL (Marzluff et al. 2008). Therefore, the SL of zebrafish h2a mRNA was selected to perform an electrophoretic mobility shift assay (EMSA) to determine its binding ability with a recombinant Slbp2 protein with MBP tag in vitro. As shown in Figure 5D, the Slbp2-MBP fusion protein could bind to the biotin-labeled h2a mRNA SL, and the excessive amount of unlabeled competitors effectively competed for this binding. Under the same condition, MBP protein did not bind to h2a mRNA SL. These results indicate that zebrafish Slbp2 can interact with the SL of replication-dependent histone mRNAs.
To verify whether or not the defects of MZslbp2Δ4 −/− embryos can be rescued by overexpression of replicationdependent histones, the plasmid H2B-GFP containing human H2B ORF driven by β-actin promoter was injected into the one-cell embryos produced by crossing heterozygous F1 female and male (slbp2Δ4 +/− ). The H2B transgenic Zslbp2Δ4 −/− and MZslbp2Δ4 −/− fish were screened by the similar procedure used in the establishment of slbp2 transgenic Zslbp2Δ4 −/− and MZslbp2Δ4 −/− fish. Compared to the WT embryos (Fig. 4A), all of the H2B transgenic MZslbp2Δ4 −/− embryos exhibited similar defects to MZslbp2Δ4 −/− embryos (Fig. 4B) and also did not initiate epiboly (Fig. 4E). However, the defects of nuclear cleavage in H2B transgenic MZslbp2Δ4 −/− embryos started at the 64cell stage (Fig. 4J), which is later than that observed in MZslbp2Δ4 −/− embryos at the eight-cell stage (Fig. 4G). The nuclei bridges were formed in a few mitotic daughter cells of H2B transgenic MZslbp2Δ4 −/− embryos at the 64cell stage (Fig. 4J). The injection of in vitro transcribed replication-dependent histone mRNAs into MZslbp2Δ4 −/− could not rescue the defects (data not shown), indicating that maternal H2b might partially rescue the defects of MZslbp2Δ4 −/− embryos. The results confirm the role of Slbp2 in the storage of replication-dependent histone mRNAs and proteins during zebrafish oogenesis.

DISCUSSION
The replication-dependent histone synthesis is cell cycleregulated and is tightly controlled by the regulation of the SLBP level during the cell cycle in mammals (Marzluff and Koreski 2017). In this study, we first characterized zebrafish slbp2 as an oocyte-specific maternal factor by analyzing its dynamic expression pattern during oogenesis and embryogenesis (Fig. 1). Then, three slbp2 mutants, Zslbp2Δ4 −/− , Mslbp2Δ4 +/− , and MZslbp2Δ4 −/− , were constructed by CRISPR/Cas9 (Figs. 2B-D, 3). To reveal the function of maternal Slbp2, the detailed comparison between WT and MZslbp2Δ4 −/− embryos, such as early nuclear cleavage, proteome and transcriptome profiling, and dynamic expression of replication-dependent his-tones and histone variants were carried out. Similar to the defects in C. elegans, Drosophila, and mice SLBP-depleted embryos (Sullivan et al. 2001;Pettitt et al. 2002;Arnold et al. 2008), MZslbp2Δ4 −/− embryos arrested development at MBT, which was caused by the abnormalities of nuclear morphology and behavior in early cleavage cells (Fig. 4). Subsequently, we observed a dramatic reduction of H2a and H2b, a moderate decrease of H3 and H4, and a significant increase of h2afx expression in Zslbp2Δ4 −/− ovary and MZslbp2Δ4 −/− embryos (Fig. 5A-C). The partial rescue by maternal H2b in MZslbp2Δ4 −/− embryos might be due to the compensation of some histone variants, such as H2afx, and the residual H3 and H4 protein in MZslbp2Δ4 −/− embryos. Combined with the confirmation of the binding ability between Slbp2 and h2a SL (Fig.  5D), we suggest that maternal Slbp2 should be required for early nuclear cleavage and replication-dependent histone expression in zebrafish embryogenesis.
The knockout of maternal Slbp1 had no effect on zebrafish early embryogenesis and nuclear cleavage (Fig. 4C,G). In the zebrafish rw440 homozygous mutant, a nonsense mutation occurred at the third amino acid of Slbp1. At 3 dpf, the rw440 mutant showed slow retinal cell proliferation and delay of retinal neurogenesis (Imai et al. 2014). The normal early embryogenesis of rw440 mutant and MZslbp1Δ1 −/− indicate that they might be hypomorphs. Interestingly, zebrafish slbp1 and slbp2 showed obvious differences in their expression (Fig. 1), and the zygotic mRNA slbp1 level was strikingly activated at the MBT (Fig. 1C). In zebrafish, zygotic genome activation (ZGA) coincides with the MBT (Vastenhouw et al. 2010;Lindeman et al. 2011). However, the maternal factors that regulate ZGA remain largely unknown in vertebrates (Tadros and Lipshitz 2009;Andersen et al. 2013). The function of slbp1 in ZGA awaits further investigation. Nonetheless, our results indicate that slbp2, not slbp1, plays a conserved and pivotal role in maintaining normal early nuclear cleavage (proper chromatin assembly).
Replication-dependent histones play an essential role in mitotic chromosome condensation (de la Barre et al. 2000). The defect of early nuclear cleavage and chromosome condensation in zebrafish MZslbp2Δ4 −/− embryos might be due to deficiency of replication-dependent histones. In Zslbp2Δ4 −/− ovary, mRNAs of replication-dependent histones all largely decreased, while a lot of histone proteins were detected (Fig. 5A,B). Since they are not dividing, the other cell types in the ovary will not produce histone mRNAs but have normal levels of histone protein. This accounts for the large drop in histone mRNAs in the Zslbp2Δ4 −/− ovary, while there is still a lot of histone proteins present. qPCR and western blot confirmed the large reduction of replication-dependent histones in MZslbp2Δ4 −/− embryos (Fig. 5A,B). The results indicate that the reduction of histone proteins in the Zslbp2Δ4 −/− ovary is likely due to failure of the oocyte to produce and store histone proteins. A small quantity of H3 proteins detected by western blot might be the H3.3 variant histones. Because of the replication-dependent histone deficiency, the DNA in cleavage cells could not be appropriately packaged into normal chromatin. Consequently, this probably led to the failure of chromosome condensation and impaired nuclear division (Fig. 4G). Compared to WT embryos, the C. elegans and mouse SLBP-depleted embryos had much less H3 and H4 (Pettitt et al. 2002;Arnold et al. 2008), and the protein level of H3 also decreased in the zebrafish slbp1 mutant rw440 embryos at 2 and 3 dpf (Imai et al. 2014). The Drosophila dSLBP mutant embryos showed similar defects observed in MZslbp2Δ4 −/− and accumulated abnormal polyadenylated H3 and H4 mRNAs (Sullivan et al. 2001). Surprisingly, the total amount of H2a and H2b in mouse SLBP-depleted embryos was similar to WT embryos (Arnold et al. 2008), which indicates that SLBP is not necessary for storage of H2a and H2b in mouse oocytes (Arnold et al. 2008). In Xenopus, the storage of replication-dependent histone mRNAs and proteins during oogenesis and its subsequent uses during embryogenesis have been extensively studied (Woodland 1980). Xenopus SLBP2 is oocyte-specific and is supposed to be involved in storage and translational repression of histone mRNA (Wang et al. 1999;Marzluff 2002, 2004). Our results suggest that Slbp2 should be required for the accumulation of four replication-dependent histones in zebrafish oocytes (Fig. 5A,B), which may reflect the Slbp regulatory mechanism differences in histone metabolism among mammal, amphibian, and teleost.
Interestingly, a total of nine histone variants were screened from transcriptomic profiles, and the only h2afx was up-regulated expression in MZslbp2Δ4 −/− embryos at the 256-cell stage and high stage, respectively (Fig. 5C). H2afx, also known as H2ax, is a variant of histone H2a and represents 2%-25% of total H2a (Rogakou et al. 1998). The phosphorylated form of H2AFX (γ-H2AFX) plays a critical role in DNA repair and maintenance of genomic integrity (Rogakou et al. 1998;Bassing et al. 2002;Celeste et al. 2002Celeste et al. , 2003aYuan et al. 2010;Testa et al. 2018). In addition, H2afx is also required for the chromatin remodeling of sex chromosomes and meiotic silencing in mouse (Fernandez-Capetillo et al. 2003;Cloutier et al. 2015). The accumulation of γ-H2afx was observed in XO female mice (Baarends et al. 2005), and the ablation of H2afx could restore oocyte numbers in XO females to XX WT levels (Cloutier et al. 2015). In our previous study, an oocyte-specific histone H2a variant H2af1o was identified in gibel carp and showed significantly higher mobility in nucleosomes than ubiquitous H2afx (Wu et al. 2009). Moreover, H2af1o deficiency disturbed cell synchrony in early cleavages before MBT (Yue et al. 2013). Dramatic global chromatin remodeling events occur in oocyte maturation and early embryogenesis, including changes in histone biosynthesis, modification, and exchange (Becker et al. 2005). The reason why h2afx increases expression levels in zebrafish MZslbp2Δ4 −/− embryos is unclear. The increased expression of h2afx in MZslbp2Δ4 −/− embryos indicated that a more polyadenylated form of this mRNA was produced and might function in the absence of Slbp2.
In summary, this work confirms slbp2 as the key player in teleost histone storage in oogenesis, and establishes a fundamental groundwork for future investigations toward elucidating the regulative mechanisms behind Slbp2 and histone storage.
RNA isolation, reverse transcription PCR (RT-PCR), and quantitative real time PCR (qPCR) Total RNAs were extracted by RNeasy Plus Mini Kit (Qiagen). One microgram of DNase-treated total RNA was transcribed reversely using Goldscript cDNA Synthesis Kit (Invitrogen), as described by the manufacturer. The cDNA templates of replication dependent histones were transcribed by random primer, and others were transcribed by oligo(dT) primer. RT-PCR and qPCR were performed as described previously (Huang et al. 2009;Zhong et al. 2014). β-actin or ef1α was selected as housekeeping gene (Livak and Schmittgen 2001). The primers are listed in Supplemental  Table S5.

Probe synthesis and in situ hybridization
A 618 bp cDNA fragment of zebrafish slbp2 was amplified by specific primers (Supplemental Table S5). Antisense or sense DIGlabeled RNA probes were synthesized using T7 polymerase by in vitro transcription (Roche). The ovarian tissues were sampled for in situ hybridization, which was performed according to a previous report (Liu et al. 2015). Images were acquired by Zeiss Axio Observer A1 inverted microscope.

Polyclonal antibody preparation, western blot detection, and immunofluorescence localization
The 15 amino acid peptide (CLEKMNTELSDGSEK) that locates at the 222-235 amino acid site in the C terminus of zebrafish Slbp2 was chosen to produce the polyclonal antibody, which is different from zebrafish Slbp1 or other proteins. The peptide was conjugated to the KLH (Keyhole Limpet Hemocyanin) peptide and then used to immunize rabbits to get the polyclonal antibody. Anti-H2b (ab1790) and anti-H3 (ab1791) antibodies were purchased from Abcam Company. Western blot detection and immunofluorescence localization were also performed as described previously (Liu et al. 2015). The results of western blot and immunofluorescence were acquired by ImageQuant LAS 4000mini (GE) and Leica confocal laser scanning microscope, respectively.
In vitro transcribed H2B-GFP mRNAs and microinjection The H2B-GFP was amplified from pCAG-H2B-GFP and inserted into the PCS2+ vector. The plasmid PCS2+-H2B-GFP was linearized by NotI, which was used as a template to in vitro transcribe H2B-GFP mRNAs by Message Machine Kit (Ambion). To visualize the dynamic nuclear cleavage, about 120 pg H2B-GFP mRNA was injected into the zebrafish WT, MZslbp2Δ4 −/− , and slbp2 transgenic MZslbp2Δ4 −/− embryos at the one-cell stage.

Proteome and transcriptome analysis
Zebrafish WT and MZslbp2Δ4 −/− at the 256-cell stage and high stage were sampled to perform iTRAQ and RNA-seq analysis, respectively. Three biological replicates were analyzed per group. The detection of peptides, library construction, sequencing, and bioinformatics analysis was accomplished by the Beijing Genomics Institute (BGI) according to the standard procedures. The raw data of transcriptomes have been submitted to the NCBI database (accession no. SRP126712), and the mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE (Vizcaino et al. 2016) partner repository, with the data set identifier PXD008614.

Recombinant protein production and electrophoretic mobility shift assay (EMSA)
The optimized 981 bp ORF of slbp2 was synthesized and cloned into pMAL-c5x (NEB). Plasmid pMAL-c5x-slbp2 was transformed into BL21(DE3) Condon Plus (Stratagene). Single colonies were screened and cultured in LB medium until OD600 = 0.6, and then 0.2 mM isopropyl thio-β-D-galactoside (IPTG) was added for induction at 20°C for 16 h. E. coli cells were collected and disrupted by ultrasonic. The fused Slbp2-MBP protein was purified from the supernatant by dextran gel separation and ion exchange and detected by SDS-PAGE.
EMSA analysis was performed as described previously (Qiu et al. 2015). In brief, the conserved H2a SL (AAAAAGGUUC UUUUAAGAACCACCCAUUUUU) was synthesized for the RNA probe labeled with 5 ′ -biotin (Thermo Scientific) (Williams et al. 1994;Wang et al. 1999). Five microgams of purified Slbp2-MBP protein was incubated with 1 nmol 5 ′ -biotin-labeled probe RNA and 10 µmol mimic negative control (5 ′ -biotin-unlabeled competitor) in binding buffer for 30 min. Binding reactions were resolved by 6% PAGE and electrophoretically transferred to nylon membrane. The transferred RNA was cross-linked to the nylon membrane by using CL-1000 Ultraviolet Crosslinker (UVP). Biotinlabeled RNA was detected by chemiluminescence and exposed with ChemiDoc XRS+ chemiluminescent imaging analysis system (Bio-Rad).

SUPPLEMENTAL MATERIAL
Supplemental material is available for this article.