Title: Retinoid signaling controls spermatogonial differentiation by regulating expression of replication-dependent core histone genes

Retinoic acid (RA) signaling is critical for spermatogonial differentiation, which is a key step for spermatogenesis. We explored the mechanisms underlying spermatogonial differentiation by targeting expression of a dominant-negative mutant of RA receptor α (RARα) specifically to the germ cells of transgenic mice to subvert the activity of endogenous receptors. Here we show that (i) inhibition of retinoid signaling in germ cells completely blocked spermatogonial differentiation identical to vitamin A-deficient (VAD) mice; (ii) the blockage of spermatogonial differentiation by impaired retinoid signaling resulted from an arrest of entry of the undifferentiated spermatogonia into S phase; and (iii) retinoid signaling regulated spermatogonial differentiation through controlling expression of its direct target genes including replication-dependent core histone genes. Altogether, our results demonstrate that the action of retinoid signaling on spermatogonial differentiation in vivo is direct through spermatogonia self, and provide the first evidence that this is mediated by regulation of expression of replication-dependent core histone genes. D ev el o pm en t • A dv an ce a rt ic le Introduction Spermatogenesis is a highly organized and complex process that allows for the continuous production of millions of haploid spermatozoa throughout adult male life and for transferring the intact genome and appropriate epigenome from generation to generation (Clermont, 1972, Oatley and Brinster, 2008). The transition of undifferentiated spermatogonia into A1 spermatogonia (termed spermatogonial differentiation) is an initial and irreversible step of spermatogenesis (de Rooij, 2001). The undifferentiated spermatogonia can be subdivided into Asingle (As), Apaired (Ap) and Aaligned (Aal) spermatogonia and contain spermatogonial stem cells (SSCs) and progenitor spermatogonia. This cohort of undifferentiated spermatogonia express self-renewal and proliferation associated genes such as Pou5f1 (Pesce et al., 1998), Lin28a (Tong et al., 2011, Zheng et al., 2009), Mir-21 (Niu et al., 2011), Mir-17~92 (Tong et al., 2012), Foxo1 (Goertz et al., 2011), Nanos2 (Sada et al., 2009, Zhou et al., 2015), Neurog 3 (Ngn3) (Nakagawa et al., 2007), Sox3 (Laronda and Jameson, 2011), Taf4b (Falender et al., 2005), and Zbtb16 (Plzf) (Buaas et al., 2004, Costoya et al., 2004) to maintain the capacity for self-renewal and proliferation. During spermatogonial differentiation, the undifferentiated spermatogonia downregulate those self-renewal associated genes and upregulate genes associated with differentiation such as Sohlh1 (Ballow et al., 2006), Sohlh2 (Hao et al., 2008), Stra8 (Endo et al., 2015, Zhou et al., 2008), Kit (Schrans-Stassen et al., 1999), Ccnd2 (Beumer et al., 2000), and Sall4a (Hobbs et al., 2012, Gely-Pernot et al., 2015). Despite this fact, the molecular mechanisms that govern spermatogonial differentiation remain incomplete. Retinoic acid (RA), an active derivative of vitamin A, is essential for spermatogonial differentiation because (i) the transition of the undifferentiated spermatogonia into A1 spermatogonia is blocked in vitamin A deficient (VAD) rodents, and (ii) RA administration to VAD animals reinitiates spermatogonial differentiation (Clagett-Dame and Knutson, 2011, Griswold et al., 1989, Huang and Hembree, 1979, Morales and Griswold, 1987, Wilson et al., 1953, Wolbach and Howe, 1925, Wolgemuth and Chung, 2007, van Pelt and de Rooij, 1990). There are 12 stages of cycle of seminiferous epithelium (hereafter referred to as epithelial stages I-XII) in the mouse (Clermont, 1972, Hogarth and Griswold, 2010, Oakberg, 1956). Although the undifferentiated spermatogonia in epithelial stages II-VIII are competent for spermatogonial differentiation in the adult mouse testis, D ev el o pm en t • A dv an ce a rt ic le spermatogonial differentiation occurs only in epithelial stages VII/VIII as the RA level reaches its peak (de Rooij, 2001, Endo et al., 2015, Hasegawa and Saga, 2012, Hogarth et al., 2015, Hogarth and Griswold, 2010). Moreover, RA treatment could induce precocious differentiation of the undifferentiated spermatogonia in epithelial stages II-VII into A1 spermatogonia (Hogarth et al., 2015, Endo et al., 2015). However, the mechanisms underlying RA-induced spermatogonial differentiation remain largely unknown. The action of RA on expression of target genes is mediated through two families of nuclear hormone receptors, the RA receptors (RARs) and the retinoid X receptors (RXRs), each with three subtypes: α, β, and γ, which are encoded by distinct genes (Chambon, 1996). RAR/RXR usually function as RAR/RXR heterodimers, which bind to RA-response elements (RAREs) in regulatory regions of the target genes (Bastien and Rochette-Egly, 2004). RAREs are typically composed of two direct repeats of a core hexameric motif, PuG(G/T)TCA separated by a 5 bp spacer sequence (referred to as DR5) (Bastien and Rochette-Egly, 2004). Several subtypes of RARs and RXRs are expressed in both Sertoli cells and germ cells including spermatogonia and exert redundant functions (Vernet et al., 2006b, Gaemers et al., 1998). Ikami et al. showed that ectopic expression of Rarg could induce the differentiation of RARG-negative undifferentiated spermatogonia by RA (Ikami et al., 2015). Global inactivation of individual Rar such as Rara results in male sterility and aberrant spermatogenesis (Lufkin et al., 1993). Several lines of compound mutants lacking multiple RARs or RXRs have been studied in testis, suggesting that retinoid signaling plays a critical role in spermatogonial differentiation (Gely-Pernot et al., 2012, Gely-Pernot et al., 2015). However, the RA target genes implicated in spermatogonial differentiation need to be identified. To address these questions in the current study, we used conditional dominantnegative mouse models to block retinoid signaling specifically in germ cells. We demonstrate that impaired retinoid signaling in germ cells resulted in a complete blockage of spermatogonial differentiation. One of the major biological functions of RA is to inhibit cell proliferation (Bohnsack and Hirschi, 2004, Clagett-Dame and Knutson, 2011); however, RA is capable of stimulating cell proliferation in some type of cells such as neural crest-derived mesenchyme in the forebrain (Schneider et al., 2001) and neonatal germ cells (Busada et al., 2014). We report here that RAinduced entry into S phase of the undifferentiated spermatogonia could be critical for D ev el o pm en t • A dv an ce a rt ic le spermatogonial differentiation. We further show that retinoid signaling could directly control expression of replication-dependent core histone genes that is essential for entry into S phase during spermatogonial differentiation. These findings thus provide novel insights into the molecular mechanisms by which retinoid signaling could regulate expression of replication-dependent core histone genes, thereby control spermatogonial differentiation in vivo. D ev el o pm en t • A dv an ce a rt ic le RESULTS Inactivation of Retinoid Signaling in Spermatogonia Impaired Spermatogenesis. To directly determine whether retinoid signaling in germ cells controls spermatogenesis, we employed a conditional dominant-negative mutant of RARα403 (dnRAR) transgene strategy (Rosselot et al., 2010). RARα403, which is a truncated form of human RARα, retains the ability to dimerize and bind the RARE but lose its transcriptional activation function (Damm et al., 1993). Previous studies have demonstrated that RARα403 can completely block wild-type RARs/RXRs function in a dose-dependent manner (Damm et al., 1993). A dnRAR transgene was inserted into the ROSA26R locus and was preceded by a floxed STOP sequence that is excised in cells expressing the CRE, activating the expression of dnRAR (Fig. S1A) (Rosselot et al., 2010). We conditionally expressed dnRAR in a subset of spermatogonia using a Stra8-Cre transgenic line with Cre expression in germ cells starting at ~3dpp (Sadate-Ngatchou et al., 2008). Throughout this study, we referred to hereafter two different genotypes of mice as: germ cell mutant (dnRAR, Stra8-Cre), heterozygous germ cell mutant (dnRAR, Stra8-Cre). To test whether the expression of dnRAR can impair retinoid signaling, we used a RARElacZ reporter line containing a RARE driven lacZ transgene, which allows the distribution of retinoid signaling to be visualized by 5-bromo-4-chloro-3-indolyl-β-Dgalactoside (X-gal) staining (Rossant et al., 1991). No lacZ activity was detected in testes from the mice expressing both alleles of the dnRAR transgene (dnRAR, Stra8-Cre, RARElacZ), whereas strong lacZ staining was seen in the control testes (Fig. S1B). However, in those animals expressing a single allele of dnRAR (dnRAR, Stra8-Cre, RARElacZ) testes, lacZ activity was reduced but still detectable (Fig. S1B), suggesting that the dnRAR in this line can block endogenous retinoid signaling in a dose-dependent manner as previously shown (Rosselot et al., 2010). Histologically, the seminiferous epithelium in control testes contained lacZpositive spermatogonia and spermatocytes (Fig. S1C). Significantly, no lacZ-positive cells can be found in testes from the mice expressing both alleles of the dnRAR transgene (Fig. S1C), but more than 40% of seminiferous tubes contained lacZpositive spermatogonia in the testes from the mice expressing a single allele of the dnRAR (Fig. S1C). Germ cell mutant males were sterile but they exhibited normal copulating behavior. Germ cell mutant testes were much smaller than control littermate testes D ev el o pm en t • A dv an ce a rt ic le [Fig. 1A, B, at age of 2 wk, control = 0.231  0.016 (mean testis weight/body weight × 100  s.d.), germ cell mutants = 0.132  0.006; P<0.001, n = 12; At age of 12 wk, control = 0.368  0.014, germ cell mutants = 0.079  0.012; P<0.001, n = 8]. Histological examinations of adult germ cell mutants showed severe defects in spermatogenesis (Fig. 1E, F). In contrast to control seminiferous tubules that contained the full complement of germ cells (Fig. 1C, D), adult germ cell mutant seminiferous tubules showed a reduced diameter and contained only morphologically normal Sertoli cells and undifferentiated spermatogonia cells (Fig. 1E, F). Moreover, compared with control testes, seminiferous epithelium were also devoid of differentiated germ cells, containing only Sertoli cells and undifferentiated spermatogonia at the basal membrane in 2or 3-week-old germ cell mutants (Fig. S2A-D). Germ Cell Mutants Exhibits Complete Blockage of Spermatogonial Differentiation. To further characterize the remaining spermatogonia in germ cell mutants, we employed immunostaining with antibodies to spermatogonial markers. By immunostaining for STRA8, a marker for differentiated spermatogonia, STRA8 positive germ cells were not observed in adult germ cell mutant testes (Fig. 2B), whereas control testes showed many seminiferous tubules with STRA8 positive germ cells (Fig. 2A), demonstrating that the undifferentiated spermatogonia in adult germ cell mutants fail to differentiate. Consistent with this, KIT positive germ cells were rarely seen in germ cell mutant testes in contrast to control testes that contained many seminiferous tubules with KIT positive germ cells (Fig. S3A, B). We next examine the undifferentiated spermatogonial markers, LIN28 and PLZF, in both control and germ cell mutant testes. The LIN28-expressing (Fig. 2C, D) or PLZFexpressing (Fig. S3C, D) undifferentiated spermatogonia in germ cell mutants were similar to that in controls. Coimmunostaining for SOX9, a marker for Sertoli cell, showed normal Sertoli cell development in adult germ cell mutant testes (Fig. 2C, D and Fig. S3C, D). These results revealed complete blockage of spermatogonial differentiation in adult germ cell mutants. Furthermore, immunostaining with spermatogonial markers showed that seminiferous tubules were depletion of STRA8expressing differentiated spermatogonia (Fig. S2E-H) and had normal LIN28expressing undifferentiated spermatogonia (Fig. S2I-L) at 2or 3-week-old, indicating that inactivation of retinoid signaling in spermatogonia also causes impaired D ev el o pm en t • A dv an ce a rt ic le spermatogonial differentiation during the first wave of spermatogenesis. Taken together, the observed defects in germ cell mutant testes are identical to the abnormalities present in VAD animals. We further found that the defects in spermatogonial differentiation first occurred by the 4.5-day-old (Fig. 2E-G). We did not observe significant difference in apoptosis of PLZF-expressing spermatogonia between control and germ cell mutant testes using a TUNEL assay (Fig. S4A-C). Thus, we conclude that inactivation of retinoid signaling in germ cells causes complete blockage of spermatogonial differentiation in both the first wave of spermatogenesis and adult spermatogenesis. Inactivation of Retinoid Signaling Blocks Entry into S Phase in the Undifferentiated Spermatogonia . The above data indicated that impaired retinoid signaling in germ cells causes the blockage at the differentiation of undifferentiated spermatogonia into A1 spermatogonia. To pinpoint the spermatogonial differentiation defects incurred by the impaired retinoid signaling in germ cells, we administered a short-term (4h) 5-ethynyl-2’-deoxyuridine (EdU) pulse to control and germ cell mutant mice. We found that the ratio (EdUPLZF/ PLZF) of both EdU positive and PLZF positive cells (EdUPLZF) to PLZF positive cells in germ cell mutant testes was significantly lower than that of controls (Fig. 3A-C), indicating that germ cell mutants had more undifferentiated spermatogonia in the G0/G1 phase of the cell cycle. Only a subset of undifferentiated spermatogonia, which are arrested in the G0/G1 phase of the cycle, are competent for spermatogonial differentiation (Kluin and de Rooij, 1981, Endo et al., 2015). We thus speculated that inaction of retinoid signaling could result in G1/S phase transition arrest of the undifferentiated spermatogonia, accounting for impaired spermatogonial differentiation observed in germ cell mutant testes. To test this hypothesis, we examined cell cycle progression of the undifferentiated spermatogonia (THY1 spermatogonia) in control and germ cell mutants by fluorescence-activated cell sorting (FACS) analysis. We found that, compared with control testes, impaired retinoid signaling in spermatogonia inhibited cell cycle progression, by significantly increasing the G1 population of the undifferentiated spermatogonia (Fig. 3D,E), suggesting that the undifferentiated spermatogonia in the germ cell mutants underwent an arrest of entry into S phase. To confirm that impaired spermatogonial differentiation results from an arrest of entry of the undifferentiated spermatogonia into S phase, we then injected WIN18,466 to D ev el o pm en t • A dv an ce a rt ic le chemically inhibit RA synthesis and block spermatogonial differentiation in the control mice. As predicted, WIN18,466 lead to a substantial accumulation of the undifferentiated spermatogonia in G1 phase while spermatogonial differentiation is blocked (Fig 3F). Because injected RA induced differentiation of the undifferentiated spermatogonia into A1 spermatogonia, we predicted that RA could rescue the G1/S transition arrest by WIN18,466 treatment. We first found that, after RA injection, WIN18,466 resulted in the accumulation of cells in the G1 phase was significantly reduced (Fig. S5A,B). We then showed that STRA8-positive spermatogonia incorporated EdU in RA-treated mice as previously reported, whereas both mice without RA treatment and germ cell mutant mice with RA administration did not contain STRA8-positive spermatogonia in the testes (Fig. 3G-I; Fig. S5C-E), indicating newly differentiating spermatogonia entry into mitotic S phase through RA induction. Collectively, these findings provide strong evidence that impaired spermatogonial differentiation in the germ cell mutant testes results from an arrest of entry into S phase in the undifferentiated spermatogonia. Retinoid Signaling Controls Spermatogonial Differentiation Through Expression of the Target Genes Including Replication-dependent Core Histone Genes. To investigate alterations in gene expression that result from impaired retinoid signaling, we conducted RNA-seq to profile the transcriptome of germ cell mutant and control THY1 spermatogonia. Gene ontology (GO) analysis of the genes at the top of the ranked genes indicated enrichment in genes associated with roles in reproduction, transcription, and spermatogenesis (Fig. 4A). In total, we identified 1633 and 742 transcripts (Reads Per Kilobase of transcript per Million mapped reads (RPKM) > 1) that were significantly (p-value < 0.05, > 1.5-fold difference) downand up-regulated, respectively, in the germ cell mutants compared with the controls (Table S1). It is of note that a dramatic upregulation of a specific subset of transcripts encoding proteins previously reported to be expressed by the undifferentiated spermatogonia in germ cell mutants relative to controls (Fig. 4B). In contrast, expression of genes known to be involved in spermatogonial differentiation was significantly downregulated in germ cell mutants compared to controls (Fig. 4B). This finding suggested that the expression program of spermatogonia in germ cell mutants was switched to the undifferentiated spermatogonia program. D ev el o pm en t • A dv an ce a rt ic le The above data showed an arrest of entry of the undifferentiated spermatogonia into S phase in germ cell mutants. Interestingly, we found that the majority of transcripts of replication-dependent core histone genes, histone cluster 1 (Hist1) (Osley, 1991, Kurat et al., 2014, Marzluff et al., 2002) were downregulated in germ cell mutants (Fig. 4C; Table S1). In mammals, the genes for the five histones H1, H2A, H2B, H3 and H4 are clustered in two loci, Hist1 and Hist2 (Osley, 1991, Kurat et al., 2014, Marzluff et al., 2002). Transcription of the Hist1 and Hist2 cluster genes is initiated at the G1/S transition and downregulated soon after completion of genome duplication in the S phase (Kurat et al., 2014, Osley, 1991). The downregulation of individual Hist1 genes was further validated by qRT-PCR on RNA from isolated germ cell mutant and control THY1 spermatogonia. Seven out of eight Hist1 genes examined showed significant decreases in expression in germ cell mutants compared to controls (Fig. 4D). Downregulation of the Hist1 cluster genes is consistent with G1/S transition arrest of the undifferentiated spermatogonia in germ cell mutants. To further investigate whether RA controls the transcription of Hist1 genes, we examined expression of the individual Hist1 genes in THY1 spermatogonia from mice with WIN18,466 treatment alone and WIN18,466 treated mice with RA exposure. As shown in Fig. 4E, RA treatment resulted in a significant increase in Hist1 mRNA levels. Taken together, we conclude that retinoid signaling could play an in vivo role in the regulation of the expression of replication-dependent core histone genes located in the Hist1 cluster. In addition, we found that expression of E2F and Ccnd2, which are crucial for regulation of cell cycle, was significantly suppressed in the undifferentiated spermatogonia of germ cell mutants, whereas, in VAD mice, RA administration stimulated Ccnd2 expression in spermatogonia (Fig. S6), as previously reported. Retinoid Signaling Regulates Expression of Replication-dependent Core Histone Genes. The mouse Hist1 cluster is located on chromosome 13 and the histone genes in the Hist1 cluster are arranged in 3 subclusters (Marzluff et al., 2002) (Fig. 5A). Given that most of Hist1 gene transcripts are coordinately regulated in THY1 spermatogonia by retinoid signaling, we assume that expression of Hist1 genes is directly controlled by retinoid signaling. To test this hypothesis, we found a putative RARE upstream of the histoneencoding genes of the Hist1 cluster. The RARE is located between -671 and -655 D ev el o pm en t • A dv an ce a rt ic le relative to the transcription start site of h2bl, the first Hist1 gene (Fig. 5A). Notably, chromatin immunoprecipitation assays (ChIPs) using a RARG antibody performed on mouse testis at day 5 revealed that RARG is present at the RARE region upstream of the Hist1 cluster similar to the Stra8 promoter (Fig. 5B). Compared with controls, there was a significant decrease in acetylated H4 (H4Ac) levels at the regulatory regions of both Hist1 and Stra8 in the germ cell mutants (Fig. 5C). In addition, RA administration to WIN18,466 treated mice induces a striking upregulation in H4Ac at the regulatory regions of both Hist1 and Stra8 (Fig. 5D). To further examine whether the -671/-655 sequences is a functional RARE, we inserted ~1.5kb upstream regulatory region of Hist1 cluster containing this putative RARE into the luciferase reporter vector and transfected the reporter into RARs/RXRsexpressing cells, P19 cells (Kruyt et al., 1991, Schoorlemmer et al., 1995). As shown in Fig. 5e, luciferase activity was significantly induced by RA treatment compared to vehicle-treated controls; however, RA-induced luciferase activity was significantly inhibited by cotransfected with dnRAR vector. Furthermore, a mutation of the RARE in the reporter significantly disrupted the RA-induced luciferase activity (Fig. 5E). Collectively, these results reveal that a functional RARE is present upstream of the Hist1 cluster and that the Hist1 cluster genes could be direct targets for retinoid