Dmrt1 Induces the Male Pathway in a Turtle Species with Temperature

RESEARCH ARTICLE Dmrt1 induces the male pathway in a turtle species with temperature-dependent sex determination Chutian Ge1,*, Jian Ye2,*, Haiyan Zhang1, Yi Zhang1, Wei Sun1, Yapeng Sang1, Blanche Capel3,‡ and Guoying Qian1,‡

ABSTRACT simultaneously exist in the same species (Chen et al., 2014; The molecular mechanism underlying temperature-dependent sex Radder et al., 2008; Yamamoto et al., 2014). Most recently, a determination (TSD) has been a long-standing mystery; in particular, rapid transition from a mixed GSD-TSD system to TSD was the thermosensitive genetic triggers for gonadal sex differentiation experimentally induced in one generation in the dragon lizard are largely unknown. Here, we have characterized a conserved Pogona vitticeps, which exhibits ZZ/ZW heterogamety that is DM domain gene, Dmrt1, in the red-eared slider turtle Trachemys susceptible to sex reversal by temperature (Holleley et al., 2015; scripta (T. scripta), which exhibits TSD. We found that Dmrt1 Quinn et al., 2007). has a temperature-dependent, sexually dimorphic expression Some genes (or gene networks) involved in GSD systems pattern, preceding gonadal sex differentiation, and is capable of have been identified in the gonads of TSD species during the responding rapidly to temperature shifts and aromatase inhibitor thermosensitive period (TSP), and several of those exhibit treatment. Most importantly, loss- and gain-of-function analyses temperature-dependent expression patterns prior to gonadal sex provide solid evidence that Dmrt1 is both necessary and sufficient differentiation (Shoemaker-Daly et al., 2010; Shoemaker et al., to initiate male development in T. scripta. Furthermore, the DNA 2007b). The idea that TSD and GSD systems share common genetic methylation dynamics of the Dmrt1 promoter are tightly correlated components is strongly supported by recent transcriptomes of TSD with temperature and could mediate the impact of temperature on sex taxa from the red-eared slider turtle (Czerwinski et al., 2016), the determination. Collectively, our findings demonstrate that Dmrt1 is a painted turtle (Radhakrishnan et al., 2017) and the alligator (Yatsu candidate master male sex-determining gene in this TSD species, et al., 2016). Although RNA interference has been applied consistent with the idea that DM domain genes are conserved during on in vitro gonads of TSD turtles to knockdown Sox9 expression the evolution of sex determination mechanisms. (Sifuentes-Romero et al., 2013; Shoemaker-Daly et al., 2010), a functional analysis of candidate sex-determining genes has not yet KEY WORDS: Dmrt1, Sex-determining gene, Temperature- been performed in vivo in any reptile with TSD, which is largely due dependent sex determination, Trachemys scripta to lack of efficient genetic manipulation techniques in TSD species. Dmrt1 (doublesex and mab3-related transcription factor 1) INTRODUCTION encodes a transcription factor that contains a DNA-binding motif Temperature-dependent sex determination (TSD) exists in many (DM domain), which is an ancient and conserved component of the reptiles that lack heteromorphic sex chromosomes, in which the vertebrate sex-determining pathway (Matson and Zarkower, 2012). incubation temperature of the developing embryos determines DM-domain genes have been identified as the master gonadal sex (Bull and Vogt, 1979; Charnier, 1966; Ferguson sex-determining gene in several non-mammalian GSD species, and Joanen, 1982; Pieau et al., 1999). However, the molecular including dmy in the medaka, DMRT1 in chicken and dmw in frog mechanism underlying TSD has been a long-standing mystery. In (Matsuda et al., 2002; Smith et al., 2009; Yoshimoto et al., 2008). particular, little is known about (1) which genetic components Although Dmrt1 is not required for male sex determination in the are responsible for triggering the differentiation of bipotential mouse, it is essential for maintaining the Sertoli cell phenotype in primordium into either a testis or ovary; and (2) how the physical postnatal mammalian testes (Matson et al., 2011; Raymond et al., signal of temperature is transduced into a biological signal. 2000). In the red-eared slider turtle Trachemys scripta (T. scripta), a Phylogenetic analyses suggest frequent and repeated evolutionary TSD species, Dmrt1 exhibits a differential expression pattern transitions between TSD and genetic sex determination (GSD) in in the early developing gonad between MPT (male-producing environmentally sensitive lineages, including reptiles (Quinn et al., temperature) and FPT (female-producing temperature) early in the 2011; Sarre et al., 2011). In many cases, both GSD and TSD TSP (from stage 15 to stage 20) (Czerwinski et al., 2016; Kettlewell et al., 2000; Murdock and Wibbels, 2003). The sex-determination period of T. scripta ranges from stage 15 to 19 (Matsumoto and 1Zhejiang Provincial Top Key Discipline of Biological Engineering, Zhejiang Wanli Crews, 2012); therefore, Dmrt1 might be involved in determining University, Ningbo 315100, China. 2HangZhou Aquacultural Technique Extending Centre, Hangzhou 310001, China. 3Department of Cell Biology, Duke University the fate of the bipotential gonad in this TSD species. Although Medical Center, Durham, NC 27710, USA. several investigators have shown that temperature influences Dmrt1 *These authors contributed equally to this work expression, whether Dmrt1 has a critical functional role in the ‡Authors for correspondence ([email protected]; [email protected]) male sex determining pathway has not been investigated in a TSD model system. J.Y., 0000-0001-8308-6492; H.Z., 0000-0002-6292-8280; Y.Z., 0000-0002- We confirmed that Dmrt1 exhibited a temperature-dependent 3593-2321; W.S., 0000-0003-4406-9395; Y.S., 0000-0002-2986-4131; G.Q., 0000- 0003-0917-9839 expression pattern in T. scripta early embryonic gonads prior to sexual differentiation, and showed a rapid response to temperature

Received 15 March 2017; Accepted 28 April 2017 shifts. In addition, the expression of Dmrt1 was induced in FPT DEVELOPMENT

2222 RESEARCH ARTICLE Development (2017) 144, 2222-2233 doi:10.1242/dev.152033 embryonic gonads that were masculinized by aromatase inhibitor MPT gonads. In contrast, Dmrt1 protein signal was not detectable in treatment. We developed an efficient lentiviral vector-mediated FPT gonads throughout embryogenesis (Fig. 1C). These data reveal gene-modulating approach for both in vivo and in vitro functional that the male-specific expression of Dmrt1 precedes the initiation of analysis of the gene. Most importantly, loss- and gain-of-function gonadal sex differentiation in T. scripta, suggesting an upstream role analyses provided strong evidence that Dmrt1 is both necessary for Dmrt1 in this TSD system. and sufficient for testicular differentiation in T. scripta. We show that DNA methylation in the Dmrt1 promoter responds rapidly The temperature sensitivity of Dmrt1 in vivo and in vitro to temperature shifts, and might account for the temperature- To determine the involvement of Dmrt1 in temperature-induced sex dependent dimorphic expression of Dmrt1. Our findings provide the determination, we performed in vivo and in vitro experiments to first functional evidence for a genetic trigger for maleness in a TSD investigate whether Dmrt1 expression was regulated by temperature system, and suggest that further studies to determine whether in T. scripta. In vivo experiments revealed that the mRNA methylation patterns are a cause or consequence of changes in expression of Dmrt1 in turtle gonads at stages 17 and 21 was Dmrt1 expression could lead to insight into the elusive link between thermosensitive. The expression level significantly decreased with temperature and sex determination. the increase in temperature from 27°C to 31°C (Fig. 2A, Fig. S3A). We found a similar temperature-dependent pattern of Dmrt1 RESULTS expression in isolated gonads cultured for 5 and 20 days (Fig. 2B, Characterization of Dmrt1 gene in T. scripta Fig. S3B). In addition, we examined the response of Dmrt1 In a comprehensive transcriptome analysis of MPT and FPT T. expression to temperature shifts from either MPT→FPT or scripta gonads at stages 15-21, Dmrt1 was among a small group of FPT→MPT both in vivo and in vitro. In gonads developing transcripts that showed a consistent MPT-specific pattern from stage in vivo at MPT and transferred to FPT at stage 16 (MPT→FPT), 15 onwards (Czerwinski et al., 2016). The full-length coding Dmrt1 expression responded rapidly to the new FPT temperature. sequence of T. scripta Dmrt1 was obtained by RACE. The complete Expression decreased significantly below MPT-typical levels by cDNA sequence of T. scripta Dmrt1 was 2448 base pairs (bp) early stage 17 and remained at baseline levels from stage 19 (accession number KY945220), with a 241 bp 5′ untranslated onwards. In gonads shifted from FPT to MPT in vivo, significant region (UTR), an 1107 bp 3′ UTR and an open reading frame (ORF) upregulation of Dmrt1 expression occurred rapidly by stage 17, of 1100 bp, which encodes a protein of 369 amino acids (Fig. S1A). reaching the MPT-typical level by stage 18, and stabilizing there for The DM domain that is present in mice and chicken Dmrt1 was also the duration of gonadogenesis (Fig. 2C). In cultured gonads shifted highly conserved in T. scripta Dmrt1. The deduced amino acid from MPT to FPT, Dmrt1 expression was repressed immediately by sequence of T. scripta Dmrt1 shared 80.7%, 75.3% and 53.9% day 2, and dropped to baseline levels typical of constant FPT identity with that of chicken, mice and zebrafish, respectively temperature from day 5 onwards. In the opposite shift (FPT→MPT), (Fig. S1B). The phylogenetic tree showed that T. scripta Dmrt1 was Dmrt1 expression exhibited a twofold increase in response to evolutionarily more closely related to chicken and mice, and temperature shift by day 2, and climbed to peak levels at day 20 distantly related to fish (Fig. S1C). (Fig. 2D, Fig. S3C,D). We also compared the response times of The mRNA and protein expression of Dmrt1 were first examined Dmrt1, Amh and Sox9 expression to temperature shifts in vitro. The in different tissues of the adult turtle. RT-PCR showed that Dmrt1 response times for significant (>twofold) downregulation of Dmrt1, mRNA was abundantly expressed in testis and was not detected in Amh and Sox9 expression in MPT→FPT gonads were day 2, day 3 ovary, heart, liver, spleen, lung, kidney and muscle (Fig. S2A). and day 7, respectively. In FPT→MPT shifts, Dmrt1, Amh and Sox9 Dmrt1 protein was localized in the nucleus of Sertoli cells expression elevated to twofold higher levels by day 2, day 3, and day surrounding the spermatogonia in postnatal testis, and was not 8, respectively. These results indicate a more rapid response of detected in ovary (Fig. S2B). These studies imply that Dmrt1 is Dmrt1 to temperature shifts than Amh or Sox9 (Fig. S3C,D) and that involved in testicular development in T. scripta, as in other species Dmrt1 expression is an upstream responder to temperature shifts in (Raymond et al., 2000; Smith et al., 2009). T. scripta.

Sexually dimorphic expression of Dmrt1 in early embryonic The induction of Dmrt1 expression in masculinized FPT gonads of T. scripta gonads of T. scripta embryos qRT-PCR analysis showed that the Dmrt1 transcript was expressed Previous studies demonstrated that sex determination in the T. in MPT gonads throughout the period of sex determination, from as scripta embryo is susceptible to application of exogenous estrogen early as stage 15 to stage 17, during which time Dmrt1 transcript and to the aromatase inhibitor (AI) during the TSP (Crews, 1994; was not detected in FPT gonads. From stage 19, Dmrt1 expression Crews and Bergeron, 1994). Masculinized FPT embryos induced by increased dramatically, reaching a peak at stage 23. By contrast, FPT AI exhibited male-like morphology with a dense medulla and a gonads exhibited extremely low expression of Dmrt1 throughout degenerated cortex (Fig. 3A-F). A significant increase in Amh and embryogenesis (Fig. 1A), consistent with other transcriptome Sox9, and downregulation of Cyp19a1 and Foxl2 were detected in results (Czerwinski et al., 2016). A highly MPT-specific Dmrt1 masculinized FPT embryos at stage 25 by qRT-PCR (Fig. S4). mRNA localization in gonadal medulla at stage 21 was observed via Immunochemistry showed that aromatase (Cyp19a1) was highly in situ hybridization (Fig. 1B). We also examined the cellular expressed in the medulla of FPT gonads at stage 25; however, its localization of Dmrt1 protein in turtle embryonic gonads. expression decreased dramatically and disappeared in masculinized Immunofluorescence revealed that Dmrt1 protein was detected in FPT gonads induced by AI (Fig. 3G-H). Sox9 and Dmrt1 proteins MPT gonads as early as stage 14. From stage 15 to 17, Dmrt1 were robustly expressed in the medulla of masculinized FPT protein was strongly expressed throughout the medulla of MPT gonads, similar to levels in MPT gonads (Fig. 3J-O). The response gonads, whereas it was not detected in FPT gonads (Fig. 1C). Dmrt1 timecourse analysis revealed that Dmrt1 expression responded protein was abundantly expressed and mainly localized in the nuclei rapidly to AI treatment and was significantly upregulated from of precursor Sertoli cells surrounding the primordial germ cells in stage 17 onwards (Fig. 3P). These observations indicate that Dmrt1 DEVELOPMENT

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Fig. 1. The sexually dimorphic expression of Dmrt1 in early embryonic gonads of T. scripta. (A) The mRNA expression of Dmrt1 in gonads of different stages (15-26) at MPT (26°C) and FPT (32°C), determined by qRT-PCR analysis; Gapdh was used as a reference gene. Dmrt1 exhibited a highly MPT-specific expression pattern in early embryonic gonads. Data are mean±s.d.; n≥3. (B) Localization of Dmrt1 mRNA in gonadal sections of stage 21 embryos at MPT and FPT, assessed by in situ hybridization with an antisense probe. Dmrt1 was robustly expressed in the medullary region of MPT gonads. (C) Immunofluorescence detection of Dmrt1 protein in the gonad at different stages (14-25) at MPT and FPT. Dmrt1 protein was localized in the nucleus of pre-Sertoli cells in the medulla of the MPT gonad. pre-sc, pre-Sertoli cells; pgc, primordial germ cells. Scale bars: 50 μm.

is an early responder to the induction of male differentiation in embryos showed global GFP reporter expression, including T. scripta. widespread expression in gonad tissue (Fig. S5B-F). Robust GFP immunofluorescence was detected in gonadal sections, indicating Establishment of an efficient lentivirus-mediated gene- effective lentivirus infection (Fig. S5G-I). We next examined Dmrt1 modulating method in T. scripta expression in MPT gonads carrying LV-Dmrt1-shRNAs to determine To solve the problem of the lack of available genetic manipulation whether lentivirus-mediated RNA interference efficiently knocked techniques in T. scripta, we established an efficient gene-modulating down target gene expression. qRT-PCR showed that Dmrt1- method for functional analysis. We used lentiviral vectors carrying shRNA#1 exerted the most powerful repression at stages 19 and Dmrt1-specific shRNAs with a GFP reporter gene to knockdown 21 (Fig. S6A). Dmrt1 mRNA in MPT gonads with LV-Dmrt1- endogenous Dmrt1 transcripts in T. scripta embryos (Fig. S5A). To shRNA#1 was significantly downregulated from early stage 16 test the efficacy of lentivirus delivery, mortality rate and GFP onwards, compared with Dmrt1 mRNA in control MPT gonads expression were examined in turtle embryos injected with virus at (LV-NC-shRNA). The average decrease in Dmrt1 expression stage 14. Approximately 38% (305/800) of treated embryos throughout gonadogenesis induced by lentivirus-mediated RNA survived after stage 21, and 55% (167/305) of these survival interference was 73.7% (Fig. S6B). Immunochemistry further DEVELOPMENT

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expression by gonadal histology, immunofluorescence and qRT- PCR. Control MPT embryos treated with the non-silencing scrambled virus exhibited the typical male phenotype, consisting of round, cylindrical testes with degenerated oviducts, whereas scrambled control FPT embryos displayed typical long and flat ovaries along with oviducts located in the mesonephric tissue (Fig. 4A,D). In MPT embryos infected with LV-Dmrt1-shRNA and showing high GFP expression, gonads became elongated and nonvascularized, which was accompanied by the differentiation of oviducts in the mesonephros, exhibiting varying degrees of female- like morphology (Fig. 4B,C, Fig. S8A). Hematoxylin and Eosin stains of gonadal sections showed that control MPT gonads had a dense medulla with seminiferous cords and a reduced cortex (Fig. 4E,E′), whereas the gonads of control FPT embryos showed a well-developed outer cortex, populated with primordial germ cells, and a degenerated medulla (Fig. 4H,H′). In contrast, most of the MPT gonads with Dmrt1 knockdown were strongly feminized, characterized by a thickened outer cortex with a number of primordial germ cells and a vacuolated medulla (Fig. 4G,G′, Fig. 2. The temperature sensitivity of Dmrt1 in vivo and in vitro. (A,B) qRT- Fig. S8B). Interestingly, some Dmrt1-knockdown MPT gonads PCR analysis of Dmrt1 in gonads at stage 17 or day 5, which were developed in exhibited partial redirection of sexual trajectory (ovotestis), showing vivo (A) or cultured in vitro (B) at different temperatures. The mRNA expression an ovarian-like developed cortex and a dense medulla with testis of Dmrt1 in embryonic gonads exhibited temperature dependence, prior to cords simultaneously present (Fig. 4F,F′, Fig. S8B). Overall, 54 (44 gonadal sexual differentiation. (C) Timecourse response of Dmrt1 expression to temperature shifts from either MPT→FPT or FPT→MPT in vivo. Embryos ovaries and 10 ovotestes) out of 63 knockdown MPT embryos were shifted at stage 16, and gonads were dissected for qRT-PCR analysis at (85.71%) exhibited varying degrees of male to female shift in sexual stages 16-26. Dmrt1 expression in gonads with temperature shifts responded trajectory (Table 1). We also performed the Dmrt1 knockdown rapidly to the new temperatures, with rapid expression changes occurring by experiments in turtle eggs incubated at the threshold or pivotal stage 17. (D) The expression of Dmrt1 in response to temperature shifts temperature (PvT) that produces an even ratio of males and females. in vitro, examined by qRT-PCR at different culture times (days 5-30). Dmrt1 At PvT, 95% of embryos treated with LV-Dmrt1-shRNA showed → expression in FPT MPT gonads displayed a strong and rapid increase above phenotypic evidence of female development (38/40, 31 ovaries and FPT levels by day 5. In the opposite shift (MPT→FPT), Dmrt1 expression also responded quickly and was significantly downregulated below MPT levels by seven ovotestes) (Table S1). day 5. Data are mean±s.d.; n≥3. P<0.05. To confirm the activation of the female pathway in MPT embryos with Dmrt1 knockdown, we analyzed the expression of sex-specific demonstrated that in MPT gonads with LV-Dmrt1-shRNA#1, no marker genes and germ cell distribution patterns at stages 19, 21, 23, Dmrt1 protein expression was detected in the obviously feminized 25 and 26. At the mRNA level, strong downregulation of testicular cortical region, and Dmrt1 protein signal was weak or almost lost in differentiation markers Amh and Sox9, and significant upregulation of the medulla (Fig. S6C). ovarian development regulators Cyp19a1 and Foxl2, were observed Overexpression of Dmrt1 in turtle FPT embryos was also in Dmrt1-knockdown MPT gonads relative to controls at different performed using a lentivirus-mediated expression system; however, developmental stages (Fig. 4I, Fig. S9A). At the protein level, Sox9 no embryos survived to stage 19, presumably owing to global protein was expressed specifically in the nuclei of precursor Sertoli effects. As an alternative approach to investigate whether cells in control MPT gonads, whereas control FPT gonads lacked overexpression of Dmrt1 was sufficient to drive male Sox9 expression. In most MPT gonads following knockdown of development, we used in vitro electroporation of cultured FPT Dmrt1, Sox9 protein expression was sharply reduced (Fig. 4J,J′,M,M′, gonads with lentiviral vector carrying the Dmrt1 ORF (LV-Dmrt1- Fig. S9B). However, some Sox9 protein was still present in the OE). Fifty-four percent (188/350) of cultured gonads removed from medulla of partially sex-reversed MPT gonads (Fig. 4K,K′). stage 16 embryos were viable to day 20 and 65% (123/188) of these Following Dmrt1 knockdown, MPT embryos simultaneously survival gonads were GFP positive. qRT-PCR showed that ectopic exhibited female-like cortical expression of γH2ax, a protein that is expression of Dmrt1 in FPT cultured gonads carrying LV-Dmrt1- expressed in female germ cells entering meiotic prophase and is not OE was dramatically increased by day 5 of culture, and remained present in male germ cells throughout embryogenesis (Fig. 4J,J′,M,M′). more than fivefold higher than FPT-typical levels during 30 days of Aromatase, the key enzyme involved in estrogen synthesis, was gonad culture in vitro (Fig. S7A). Ectopic activation of Dmrt1 strongly expressed in the medullary region of control FPT gonads, protein was found in the medulla of FPT-cultured gonads infected and was never detected in control MPT gonads. However, Dmrt1- with LV-Dmrt1-OE (Fig. S7B). These results indicate that we knockdown MPT embryos showed ectopic activation of aromatase successfully developed an effective gene up- and downmodulating in the gonadal medulla (Fig. 4N-Q′). In some MPT embryos with method in T. scripta that works both in vitro and in vivo. partial feminization, a small amount of aromatase was ectopically expressed in medullary cells outside the remaining testis cord Feminization of T. scripta MPT embryos following Dmrt1 structures (Fig. 4O,O′). Vasa staining showed medullary cord knockdown in ovo distribution of germ cells in control MPT gonads, whereas control To verify that Dmrt1 plays a role in sex determination or gonadal FPT gonads displayed cortical localization of germ cells. In MPT differentiation in T. scripta, we infected embryos at stage 14 with embryos with Dmrt1 knockdown, Vasa-positive germ cells a scrambled virus shRNA (LV-NC-shRNA) or a silencing virus exhibited a female-like distribution pattern, mainly enriched in the

(LV-Dmrt1-shRNA) and compared phenotype and marker gene developed outer cortex with few germ cells localized in the medulla DEVELOPMENT

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the lentiviral vector carrying the Dmrt1 gene survived past the period of gonadal differentiation. Therefore, we used a whole-organ in vitro culture system to analyze the effects of overexpression of Dmrt1 on FPT gonadal development. Gonads from stage 16 embryos incubating in ovo at FPT or PvT were dissected, electroporated with the lentiviral vector carrying the Dmrt1 ORF (LV-Dmrt1) and cultured either at FPT or PvT (in accordance with previous in ovo culture temperature). At day 30 of in vitro culture, the control gonads cultured at FPT with empty lentiviral vector exhibited an oval shape, a thickened cortical region populated with germ cells and a reduced medulla with no evidence of sex cords. FPT-cultured gonads overexpressing Dmrt1 showed an obvious female-to-male redirection of gonadal morphology, characterized by well-organized testis cord structure in the medulla and a degenerated cortex, similar to normal males (Fig. 5A-D′). Of gonads cultured at FPT, 42 out of 51 (82%) infected with LV-Dmrt1-OE displayed complete or partial female-to-male redirection of sexual trajectory (Table 2). Under the condition of PvT, 51 out of 54 (94%) gonads overexpressing Dmrt1 developed into testes or ovotestes (Table S2). qRT-PCR for male and female marker genes showed that Amh and Sox9 expression increased, and Cyp19a1 and Foxl2 expression decreased relative to controls in cultured FPT gonads overexpressing Dmrt1 at days 10, 15, 20, 25 and 30 (Fig. 5E). Maintenance of Sox9 protein in sex cords of Dmrt1-overexpressing FPT-cultured gonads was confirmed by immunofluorescence (Fig. 5F-I′). γH2ax protein expression (a marker of female meiosis) was totally lost in completely sex-reversed FPT gonads. Although overexpression of Dmrt1 in FPT gonads resulted in the relocalization of many germ cells from the cortical region to sex cords within the medulla (Fig. 5J-M), some γH2ax-positive germ cells were still present in the remaining cortex in partially sex reversed FPT gonads (Fig. 5F-I′). These findings suggest that Dmrt1 is sufficient to initiate male development in the TSD system.

Temperature-dependent differential methylation of the Dmrt1 promoter in gonads of T. scripta DNA methylation in gene promoters is a conserved epigenetic modification that influences transcript expression. Recently, differences in methylation patterns at MPT and FPT have been Fig. 3. The induction of Dmrt1 expression in the gonads of masculinized shown to correlate with the sex-specific expression of genes in FPT embryos. (A-C) Gonads (outlined by yellow dotted lines) on top of organisms with TSD (Piferrer, 2013). To investigate whether mesonephros of FPT, masculinized FPT and MPT embryos at stage 25. Gd, gonad; Ovi, oviduct. Scale bars: 1 mm. (D-F) Hematoxylin and Eosin staining of temperature is correlated with the methylation pattern of the Dmrt1 gonadal sections from FPT, masculinized FPT and MPT embryos at stage 25 promoter in T. scripta, we analyzed the DNA methylation signature showing that the masculinized FPT embryonic gonads have a well-developed in the promoter region of Dmrt1 in T. scripta gonads incubated at medullary region with seminiferous cord-like structure and thin cortex. The MPT versus FPT. Bisulfite sequencing revealed that the overall dashed line indicates the border between medulla and cortex. Scale bars: percentage of CpG methylation in the Dmrt1 promoter was 50 μm. (G-O) Protein localization of aromatase (Arom), Sox9 and Dmrt1 in FPT, significantly lower in MPT gonads relative to FPT gonads from masculinized FPT and MPT gonadal sections at stage 25. Dmrt1 expression was stage 15 onwards throughout the TSP (Fig. 6A). To test whether the highly induced in masculinized FPT gonads. Scale bars: 50 μm. (P) qRT-PCR analysis of Dmrt1 in gonads from FPT, masculinized FPT and MPT embryos at DNA methylation signature shifts in accordance with temperature stages 17, 19, 21, 23 and 25, and hatchling. Dmrt1 exhibited a rapid response to shifts during the TSP, eggs were shifted either MPT→FPT or aromatase inhibitor (letrozole) treatment and was upregulated by stage 17. Data FPT→MPT at stage 16 and the methylation status of Dmrt1 are mean±s.d.; *P<0.05; **P<0.01; ***P<0.001; n.s., not significant; n≥3. promoter was examined at stages 17, 18, 19, 20 and 21. Results showed that the methylation level of the Dmrt1 promoter in (Fig. 4R-U′, Fig. S9C). These results provide solid evidence that MPT→FPT gonads was dramatically increased by stage 17 relative Dmrt1 is required for testis determination in T. scripta. to that of MPT gonads. By contrast, FPT→MPT gonads exhibited a rapid decrease in DNA methylation of Dmrt1 promoter region by Masculinization of cultured FPT gonads overexpressing stage 17 in response to temperature shifts (Fig. 6B). These data Dmrt1 in vitro revealed that the Dmrt1 transcript expression fluctuation was To determine whether Dmrt1 was sufficient to initiate the male negatively correlated with the methylation dynamics of Dmrt1 pathway in this TSD system, we investigated whether ectopic promoter region during the temperature shifts (Fig. 2B), raising the activation of Dmrt1 in FPT individuals would result in a redirection possibility that DNA methylation directly responds to temperature of sexual trajectory. Unfortunately, no embryos infected in ovo with and is responsible for the temperature regulation of Dmrt1. DEVELOPMENT

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Fig. 4. Feminization of MPT embryos following Dmrt1 knockdown in vivo. (A-D) Representative images of the gonad-mesonephros complexes from MPT, MPT+ Dmrt1-RNAi and FPT embryos at stage 25. Gd, gonad; Ovi, oviduct. Scale bar: 1 mm. (E-H′) Hematoxylin and Eosin staining of gonadal sections from MPT, MPT+Dmrt1-RNAi and FPT embryos at stage 25. The MPT gonads with Dmrt1 knockdown displayed an ovary-like phenotype, characterized by a thickened outer cortex and degenerated or absent testis cords. The dashed black line indicates the border between medulla and cortex. pgc, primordial germ cells; sc, seminiferous cord; Cor, cortex; Med, medulla. Scale bars: 50 μm. (I) qRT-PCR of Sox9, Amh, Foxl2 and Cyp19a1 in gonads form MPT, MPT with Dmrt1 knockdown and FPT embryos at stage 25. Strong downregulation of Amh and Sox9 expression and significant upregulation of Cyp19a1 and Foxl2 expression were observed in stage 25 MPT gonads with Dmrt1 knockdown. Data are mean±s.d.; *P<0.05; **P<0.01; ***P<0.001; n≥3. (J-Q′) Immunohistochemistry of Sox9, γH2ax, aromatase (Arom) and β-catenin in gonadal sections of MPT, MPT following Dmrt1 knockdown and FPT embryos at stage 25. Sox9 protein expression was strikingly decreased and aromatase was robustly expressed in MPT gonads following Dmrt1 knockdown. (R-U′) A female-typical distribution of germ cells was observed in MPT gonads with Dmrt1 knockdown at stage 25, determined by Vasa and β-catenin immunostaining. Scale bars: 50 μm.

DISCUSSION that Dmrt1 has a temperature-dependent dimorphic expression Even though TSD has been studied for several decades, the pattern preceding the initiation of gonadal sex differentiation. Using molecular mechanism underlying this mode of sex determination anovelin vivo viral transduction system, we provide solid functional has remained elusive. Here, we demonstrate that Dmrt1 can act as a evidence that Dmrt1 is both necessary and sufficient to trigger male sex-determining gene in the TSD species T. scripta. We show testicular differentiation. This is the first such functional study to

Table 1. Phenotypes of stage 25 embryos with Dmrt1 knockdown in ovo Incubation Number of embryos with Number of developing Number of developing Number of temperature Viral treatment high GFP expression testes ovaries ovotestes MPT (26°C) LV-NC-shRNA 54 54 0 0 LV-Dmrt1-shRNA 63 9 44 10 FPT (32°C) LV-NC-shRNA 50 0 50 0

Phenotype of embryos was assessed by gonadal histology, immunofluorescent marker expression or qRT-PCR. DEVELOPMENT

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Fig. 5. Masculinization of cultured FPT gonads overexpressing Dmrt1 in vitro. (A-D′) Histological images of FPT, PPT+Dmrt1-overexpression (OE) and MPT gonads cultured in vitro for 30 days. Cultured FPT gonads overexpressing Dmrt1 showed varying degrees of male-like morphology, with apparent testis cords being located in an organized medulla. The dashed black line indicates the border between medulla and cortex. pgc, primordial germ cells; sc, seminiferous cord. Scale bars: 50 μm. (E) qRT-PCR of Sox9, Amh, Foxl2 and Cyp19a1 in FPT, FPT+Dmrt1-OE and MPT gonads after 10-30 days culture. The expression of Sox9 and Amh was increased and expression of Foxl2 and Cyp19a1 was reduced in FPT gonads overexpressing Dmrt1, relative to control FPT gonads. Data are mean±s.d.; *P<0.05; **P<0.01; ***P<0.001; n.s., not significant; n≥3. (F-I′) Immunohistochemistry of Sox9 and γH2ax in transverse sections of FPT, FPT +Dmrt1-OE and MPT gonads at day 30. Sox9 protein was induced in the medulla of FPT gonads overexpressing Dmrt1, whereas γH2ax variably labeled meiotic germ cells in the cortex. (J-M) Immunohistochemistry of Vasa in FPT, FPT+Dmrt1-OE and MPT gonads at day 30. Ectopic expression of Dmrt1 in FPT gonads led to male-like distribution of germ cells within seminiferous cords in many samples. Scale bars: 50 μm. identify and test a genetic factor that can determine the fate of the primordial germ cells in the MPT gonad as early as stage 14. Pre- bipotential gonad in a TSD species. Sertoli cells are the first cell type to differentiate in the developing A number of genes involved in the GSD system have been testis and send organizing signals to other nascent cell types, previously identified in the gonads of some TSD species (Rhen et al., thereby directing testis formation. These features of Dmrt1 2007; Shoemaker-Daly et al., 2010; Shoemaker et al., 2007a; expression suggest that it is important for both primary sex Czerwinski et al., 2016). In the red-eared slider turtle, Dmrt1 is a gene determination and subsequent gonadal differentiation in T. scripta. that exhibits an early sexually dimorphic expression pattern in the To determine whether Dmrt1 is a master TSD gene, we examined developing gonad between MPT and FPT (Kettlewell et al., 2000; the effect of temperature on Dmrt1 expression both in vivo and in Shoemaker et al., 2007a). In this study, MPT-specific expression of vitro. Dmrt1 transcript expression in individual embryos was highly Dmrt1 transcript was detected as early as stage 15, just at the onset of temperature dependent between 25 and 33°C, during the sex- sex determination and the TSP. Sexually dimorphic expression of determination period and TSP. The temperature-shift assays showed Dmrt1 preceded Amh and Sox9, two well-known factors for male a rapid response of Dmrt1 to temperature shifts from MPT to FPT or sexual development, and was maintained throughout the gonadal FPT to MPT both in vivo and in vitro, preceding changes in Amh and differentiation period, which is very similar to the expression pattern Sox9, which is consistent with previous reports (Shoemaker-Daly of Dmrt1 in chicken (Lambeth et al., 2014; Smith et al., 2009). et al., 2010; Shoemaker et al., 2007b). These findings confirm the Interestingly, we found that Dmrt1 also exhibited early male-specific thermosensitivity of Dmrt1 in T. scripta gonadal cells. embryonic expression before the onset of gonadal sex differentiation In T. scripta, exogenous estrogen and its synthetase aromatase in the Chinese soft-shelled turtle, a GSD species with ZZ/ZW sex can override the temperature effect if applied during the TSP chromosomes (W. Sun, H. Cai, G. Zhang, H. Zhang, H. Bao, L. (Matsumoto and Crews, 2012; Ramsey and Crews, 2009). In Wang, J. Ye, G. Qian and C. Ge, unpublished). We show that the chicken, ZW eggs treated with aromatase inhibitor (AI) exhibit Dmrt1 protein is present in nuclei of pre-Sertoli cells surrounding upregulation of Dmrt1, leading to masculinization of ZW embryos

Table 2. Phenotypes of day 30 cultured gonads overexpressing Dmrt1 in vitro Incubation Viral Number of gonads Number of developing Number of developing Number of temperature treatment with high GFP expression testes ovaries ovotestes FPT (32°C) LV-empty 39 0 39 0 LV-Dmrt1 51 32 10 9 MPT (26°C) LV-empty 33 33 0 0

Phenotype of gonads was assessed by histology, immunofluorescent marker expression or qRT-PCR. DEVELOPMENT

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Fig. 6. Temperature-dependent differential methylation of the Dmrt1 promoter in gonads of T. scripta. (A) Total percent DNA methylation of the Dmrt1 promoter region in gonads at constant MPT and FPT from stages 15 to 21. DNA methylation levels were significantly lower at MPT relative to FPT from stage 15 onwards (onset time of sex determination). P<0.01. (B) Response of DNA methylation levels of the Dmrt1 promoter to temperature shifts from either MPT→FPT or FPT→MPT in vivo. Eggs were shifted at stage 16, and gonadal genomic DNA was extracted for PCR and bisulphate sequencing at stages 16, 17, 18, 19, 20 and 21. DNA methylation levels of the Dmrt1 promoter responded rapidly to both MPT→FPT and FPT→MPT, and the earliest significant changes occurred by stage 17 in both cases. Data are mean±s.d.; *P<0.05; **, ##P<0.01; n≥3.

(Smith et al., 2003). It has been proposed that exogenous steroid et al., 2009). In medaka, XY Dmy knockout fish exhibit a fertile hormones may redirect the gonadal trajectory by interacting with the male-to-female sex reversal (Matsuda et al., 2002). Most candidate sex-determining genes (Matsumoto and Crews, 2012). In importantly, our gain-of-function experiment revealed that ectopic this study, turtle embryos incubating at FPT exhibited a rapid expression of Dmrt1 in cultured FPT turtle gonads resulted in a increase in Dmrt1 transcript and protein expression in response to female-to-male redirection of sexual trajectory, evidenced by AI treatment. This response of Dmrt1 expression occurred at early formation of sex cord-like structures. Likewise, overexpression of stages of TSP, clearly preceding the first signs of morphological Dmy in XX medaka results in testicular differentiation (Matsuda differentiation of testes or ovaries in T. scripta. The change of et al., 2007), and ZW chicken gonads overexpressing Dmrt1 display Dmrt1 transcript in response to AI treatment was earlier than that of an evident masculinized morphology, as well as activation of Amh Sox9, which occurred at stage 19 (Matsumoto et al., 2013b). By and Sox9 (Lambeth et al., 2014). These findings demonstrate that, contrast, treatment of MPT eggs with estrogen caused a rapid similar to its role in chicken and medaka, Dmrt1 is both necessary downregulation of Dmrt1 transcript expression in T. scripta and sufficient for testicular differentiation in T. scripta. (Murdock and Wibbels, 2006), which preceded downregulation of Both Amh and Sox9 were upregulated following overexpression Sox9 (Barske and Capel, 2010). It is possible that the reversed of Dmrt1 in FPT-cultured gonads, raising the possibility that these expression pattern of Dmrt1 induced by exogenous ligands is twogenesrespondtoelevatedDmrt1. The order of their responsible for the ultimate sex-reversal in T. scripta. At the least, expression at MPT also implies that Dmrt1 lies upstream of Amh these results suggest that Dmrt1 acts upstream in the testis pathway. and Sox9 in T. scripta, similar to chicken. Recently, the Dmrt1 Genetic research in TSD species has been hindered by lack of ortholog Doublesex (Dsx) has been identified as a key regulator of available genetic manipulation techniques. A number of studies the male phenotype in the branchiopod crustacean Daphina have focused on establishing effective loss- and gain-of-function magna, which exhibits environmental sex determination (ESD) methods in turtle species. Sifuentes-Romero et al. reported that (Kato et al., 2011). In beetles, Dsx is involved in nutrition- transfection of in vitro cultured MPT gonads of the olive ridley sea dependent male sexual trait development (Gotoh et al., 2014; turtle with siRNAs specific to Sox9 caused a significant reduction of Kijimoto et al., 2012). All these observations suggest that, in spite Sox9 mRNA and protein expression (Sifuentes-Romero et al., of the diversity and plasticity of sex-determination mechanisms, 2013). On the other hand, in vitro cultured gonads of T. scripta DM domain genes are highly conserved in male sexual electroporated with a fusion GFP:Sox9 plasmid exhibited a certain differentiation pathways between GSD, TSD and the broader amount of ectopic expression of Sox9 at FPT (Shoemaker-Daly category of ESD systems. et al., 2010). However, no in vivo genetic modification has been We have demonstrated that Dmrt1 is a strong candidate master achieved in turtle embryos to date. In this study, we have developed TSD gene in T. scripta, as evidenced by temperature sensitivity of both in vivo and in vitro methods to efficiently knockdown and Dmrt1 during the sex-determination period, and that it is necessary overexpress Dmrt1 transcript in the gonads of T. scripta, achieved and sufficient for testis differentiation. However, the mechanism by by injection of embryos in ovo with lentiviral vectors carrying which temperature regulates Dmrt1 remains unclear. Dmrt1 might Dmrt1-specific shRNAs and in vitro electroporation of cultured not directly respond to temperature because, in some TSD species, gonads with lentiviral vector carrying Dmrt1 ORF. To our lower temperatures produce males, whereas in others, higher or knowledge, this is the first time that an in ovo gene-modulating intermediate temperatures promote males. It is possible that there is approach has been established in turtle embryos, which opens the a temperature sensor or chromatin regulator present during the TSD door to elucidate the function of the molecular cascade underlying initiation period that is responsible for regulating temperature- TSD. Using this approach, we have clearly demonstrated that Dmrt1 induced activation or repression of sex-determining genes. A recent knockdown caused complete feminization of MPT embryos. study in the American alligator revealed that the male gonad-typical Similarly, knockdown of Dmrt1 in chicken embryos leads to TRPV4 channel may influence the male gonadal sex determination feminization of genetic male individuals, with a decline in Sox9 pathway by modifying Amh and Sox9 expression during TSP (Yatsu expression and an increase in Cyp19a1 and Foxl2 expression (Smith et al., 2015). However, TRPV4 alone was not sufficient to initiate DEVELOPMENT

2229 RESEARCH ARTICLE Development (2017) 144, 2222-2233 doi:10.1242/dev.152033 gonadal sex determination, because activation of the TRPV4 To summarize, we have characterized a conserved DM domain channel in FPT gonads did not cause redirection of gonadal sexual gene, Dmrt1, in the red-eared slider turtle, and have found that trajectory. Schroeder et al. reported that a SNP in the cold-inducible Dmrt1 exhibits a temperature-dependent sexually dimorphic RNA-binding protein (CIRBP) loci was highly associated with expression pattern from the earliest stages of the sex- gonadal sex phenotype in the snapping turtle with TSD, indicating determination period, and is capable of responding rapidly to CIRBP is involved in gonadal differentiation (Schroeder et al., temperature shifts and aromatase inhibitor treatment. Most 2016). Nevertheless, additional factors are likely involved in importantly, in ovo and in vitro loss- and gain-of-function sensing or transducing the thermal influence because there was analyses provide solid evidence that Dmrt1 is both necessary and not perfect concordance between CIRBP genotype and sex. sufficient to initiate the male development in T. scripta. Considering the diversity of TSD patterns, it is probable that the Furthermore, DNA methylation of the Dmrt1 promoter might act transduction of temperature into a molecular signal (gene) for as a crucial mediator in the regulation of Dmrt1 by temperature. gonadal differentiation is polygenic and complex, as opposed to These findings demonstrate that Dmrt1 is a candidate master TSD being dependent on a single common factor. gene in T. scripta, consistent with the strong conservation of this Recently, epigenetic mechanisms have been demonstrated to DM domain gene during the evolution of sex-determination regulate genes involved in sex determination and gonadal mechanisms (Herpin and Schartl, 2015; Matson and Zarkower, differentiation (Kuroki et al., 2013; Piferrer, 2013; Shao et al., 2012). In short, we have functionally identified the first genetic 2014; Tachibana, 2015; Zhang et al., 2013). Epigenetic trigger for maleness in a TSD system, thereby shedding new light on mechanisms, including DNA methylation, histone modification the elusive TSD molecular mechanism. and non-coding RNAs, can integrate environmental information with the regulation of gene expression, and have been emerging as MATERIALS AND METHODS a promising regulatory mechanism for TSD (Matsumoto et al., Turtle eggs 2013a; Navarro-Martín et al., 2011; Parrott et al., 2014; Piferrer, Freshly laid red-eared slider turtle (T. scripta) eggs were obtained from the 2013; Venegas et al., 2016). The first example of an epigenetic Hanshou Institute of Turtles (Hunan, China). Fertilized eggs were mechanism mediating temperature effects on sexual development randomized in trays of moist vermiculite and placed in incubators at 26°C in a vertebrate came from a study on the European sea bass, a fish (MPT) or 32°C (FPT), with humidity maintained at 70-80%. In this species, incubation of eggs at 26°C (MPT) produces all males, whereas incubation at with a polygenic system of sex determination where temperature 32°C (FPT) generates all females. For in vivo temperature-shift experiments, and genetics contribute equally to sexual fate. In this example, 200 eggs were shifted at developmental stage 16 from an incubator kept at DNA methylation of the gonadal aromatase (Cyp19a1) promoter 26°C to an incubator kept at 32°C, and vice versa. For in vivo experiments was associated with temperature-induced sex ratio shifts (Navarro- regarding temperature-dependent expression of Dmrt1, groups of 200 eggs Martín et al., 2011). In T. scripta, the DNA methylation level of were placed in humidified incubators held at 25°C, 26°C, 27°C, 28°C, 29°C, the gonadal Cyp19a1 promoter was higher at MPT than FPT by 30°C, 31°C, 32°C and 33°C. The temperature-sensitive period (TSP) in stage 19 onwards, and was reduced in response to temperature T. scripta extends from approximately stage 15 to stage 20, when the embryo shifts from MPT to FPT (Matsumoto et al., 2013a). Similarly, is environmentally sensitive and when sex determination occurs (Wibbels differential incubation temperatures resulted in dimorphic DNA et al., 1991). Embryos were staged according to criteria established by methylation patterning of the Cyp19a1 and Sox9 promoters in Greenbaum (2002). At stages 14, 15, 16, 17, 19, 21, 23 and 25, embryos incubated at different temperatures were removed from eggshells, gonads of American alligator embryos (Parrott et al., 2014). We decapitated and placed in PBS for dissection. Gonads of stage 16 show that the DNA methylation status of the Dmrt1 promoter in embryos incubated at different temperatures were dissected for whole- gonads of T. scripta displayed significant temperature-dependent gonad in vitro organ culture at the constant or shifted temperatures. dimorphism from stage 15 onwards, and responded rapidly to Experiments were carried out according to a protocol approved by the temperature shifts both from MPT to FPT and FPT to MPT. This Zhejiang Wanli University. temperature-induced dimorphic DNA methylation patterning of the Dmrt1 promoter occurred at the beginning of the TSP, prior to Cloning of Dmrt1 cDNA the initial time of differential DNA methylation in the Cyp19a1 Total RNA isolated from MPT embryonic gonads at stage 25 was treated promoter in the middle of TSP. These findings raise the possibility with DNase I and then reverse transcribed using SuperScript III reverse that DNA methylation functions as a key mediator that integrates transcriptase and oligo(dT). Based on the published partial sequence of temperature into a molecular trigger that determines sex in the Dmrt1 (Accession Number AY316537.1), a pair of PCR primers were designed to amplify a fragment of turtle Dmrt1 cDNA. 5′ RACE and 3′ TSD system. However, the DNA methylation status of the Dmrt1 RACE were performed according to the manufacturers’ protocols for the and Cyp19a1 promoters at both constant and shifted temperatures SMART RACE cDNA Amplification kit (Clontech) and a CapFishing were perfectly correlated with transcript expression patterns; Full-length cDNA Premix kit (Seegene). The sequences of primers for thus, it is unclear whether changes in methylation are a cause amplification are listed in Table S3. or consequence of gene expression changes. If temperature can modulate the DNA methylation status of a putative sex- RNA extraction and qRT-PCR determining gene, elucidation of the mechanism will be a key Gonads from embryos in each group were microdissected from the next step. mesonephros, and individual pairs of gonads were harvested for RNA Recent transcriptomes of TSD taxa show a series of genes extraction using TRIzol (Invitrogen) or RNeasy Plus Micro kit (Qiagen). The cDNA was generated from 0.5-2 μg RNA using the SuperScript First- exhibiting earlier differential expression between MPT and FPT, ’ before the onset of TSP or even the formation of a gonad, such as Sf1 Strand Synthesis System (Fermantas) based upon the manufacturer s protocol, followed by DNase treatment. Real-time PCR was carried out (Valenzuela et al., 2006; Valenzuela, 2008), Igf1r, Insr in triplicate with a SYBR Green Supermix (Bio-Rad) in a Bio-Rad (Radhakrishnan et al., 2017), Fdxr, Pcsk6, Kdm6b, Twist1, Hsp6b iCycler system. After normalization with Gapdh, relative RNA levels in and TRP channel genes (Czerwinski et al., 2016). These genes samples were calculated by the comparative threshold cycle (Ct) method might be involved in regulating temperature-induced activation or (Schmittgen and Livak, 2008). The sequences of primers for PCR are listed repression of putative sex-determining genes. in Table S3. DEVELOPMENT

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In situ hybridization Aromatase inhibitor treatments Gonad-mesonephros complexes were dissected from turtle embryos at stages A non-steroidal aromatase inhibitor letrozole (PHR1540, Sigma) was 23 and 25, immediately frozen in OCT embedding medium, stored at −80°C, administered to eggs incubating at FPT (32°C). Letrozole was dissolved in and subsequently sectioned at 20 μm and thaw-mounted onto SuperFrost Plus 95% ethanol at a concentration of 10 μg/μl, and 10 μl of the drug was slides (Erie Scientific). Sections were fixed in ice-cold 4% paraformaldehyde applied topically to the eggshell in the region adjacent to the embryo at stage (PFA)/PBS, and incubated in 0.25% acetic anhydride /triethanolamine. After 15. Controls were treated with 10 μl of 95% ethanol. Gonad-mesonephros washes in 2× standard saline citrate (SSC), slides were dehydrated through a complexes were dissected from treated and control embryos at stage 25 for series of ethanol solutions, air dried, and stored at −80°C. In situ histology and immunohistochemistry. Gonads were separated from the hybridization for Dmrt1 mRNA expression was performed as described adjacent mesonephros at stages 17, 19, 21, 23 and 25 and hatching time, and previously (Shoemaker et al., 2007a). Briefly, sections were rehydrated, pre- preserved for qRT-PCR analysis. hybridized for 2 h at 65°C and then hybridized overnight under the same conditions in the presence of a digoxygenin-labeled Dmrt1-specific antisense Preparation of lentivector-Dmrt1-shRNA constructs riboprobe, generated from MPT gonadal cDNA. After RNase A treatment at Three shRNAs targeting turtle Dmrt1 mRNA were designed to give rise 37°C, sections were washed in a decreasing series of SSC and equilibrated in to siRNA, using the shRNA designer website (http://rnaidesigner. Tris buffer at room temperature before incubation in 1:5000 anti-DIG- thermofisher.com/rnaiexpress/design.do). The lentivirus vector was used alkaline phosphatase Fab fragments (Roche) for 2 h. Sections were washed in to deliver shRNAs directed specifically against turtle Dmrt1 mRNA. The 100 mM Tris and incubated in 5 mM levamisole. Chromogenic product was designed shRNA construct contained a unique 21 nt double-stranded Dmrt1 formed using BCIP/NBT in 100 mM Tris (Sangon, Shanghai) at 30°C until sequence that presented as an inverted complementary repeat, a loop the desired darkness was achieved, then terminated simultaneously for all sequence (5′-CTCGAG-3′) and the RNA Pol-II terminator (5′-TTTTTT-3′). slides. Sense controls did not show any color reaction. Sections were Annealed oligonucleotides were ligated into pGP-U6 (GenePharma) dehydrated, delipidated, mounted in AquaMount and photographed. between the BbsI and XhoI sites by T4 DNA ligase (TaKaRa) to produce pGP-U6-Dmrt1-shRNA. The pGP-U6-Dmrt1-shRNA construct was Immunofluorescence staining digested with AgeI-EcoRI and inserted into the EcoRI site of pGLV-U6- Gonad-mesonephros complexes or cultured gonads were fixed in 4% PFA GFP (GenePharma). The recombinant vector pGLV-GFP-Dmrt1-shRNA overnight at 4°C, then embedded in paraffin wax and sectioned. Paraffin was termed LV-Dmrt1-shRNA. The negative control vector (pGLV-GFP- sections (5-8 μm) were deparaffinized prior to immersion in 10 mM sodium NC-shRNA, termed LV-NC-shRNA) contained a nonsense shRNA insert in citrate buffer for 15 min for antigen retrieval at a sub-boiling temperature order to control any effects caused by non-RNAi mechanisms. The (99°C). Sections were covered with primary antibodies and incubated sequences of the shRNA are as follows: Dmrt1-shRNA#1,5′-GGTGGCA- overnight at 4°C. The primary antibodies used in this analysis included GCTCCTGTTTATTG-3′; Dmrt1-shRNA#2, 5′-GGATGCTCATTCAGG- rabbit anti-Dmrt1 (produced privately by Sangon Biotech, 1:250), rabbit ACATTC-3′; Dmrt1-shRNA#3, 5′-GCAGTCAAGATTCTGGCTTAA-3′; anti-Sox9 (Chemicon, AB5535, 1:1000), mouse anti-β-catenin (Sigma, negative control, 5′-TTCTCCGAACGTGTCACGTAT-3′. C7207, 1:250), rabbit anti-aromatase (Abcam, ab18995, 1:150), rabbit anti- For the generation of lentivirus, 293T producer cells were transfected with Vasa (Abcam, ab13840, 1:50) and mouse anti-γH2AX (Abcam, ab26350, optimized packaging plasmids (pGag/Pol, pRev and pVSV-G) along with 1:250). Primary antibodies were detected using secondary antibodies Alexa pGLV-Dmrt1-shRNA or pGLV-NC-shRNA expression clone construct by Fluor 488 donkey anti-rabbit IgG (Invitrogen, A21206), Alexa Fluor 488 lipofectamine. Twenty-four hours post-transfection, the transfection mix donkey anti-mouse IgG (Invitrogen, A21202), Alexa Fluor 594 donkey anti- was replaced with a fresh culture medium (without antibiotics). The virus- rabbit IgG (Invitrogen, A21207) and Alexa Fluor 594 donkey anti-mouse containing supernatant was harvested 72 h post-transfection, cleared by IgG (Invitrogen, A21203), all diluted at 1:250. Nuclei were stained with centrifugation (1000 g for 15 min at 4°C) and then filtered through a DAPI. Gonad sections were imaged using a fluorescence microscope (Ti-E, 0.45 μm filter (Millipore). Viruses were titrated by adding serial dilutions to Nikon) or confocal microscope (A1 Plus, Nikon). fresh 293T and assessing GFP expression after 48 h. Viral titers of ∼4×108 infectious units/ml were obtained. Lentivirus aliquots were stored at −80°C Gonad culture before infection of turtle embryos. Turtle gonad culture in vitro was carried out mainly according to the methods described previously (Mork and Capel, 2013; Shoemaker-Daly et al., 2010). Preparation of lentivector-Dmrt1 overexpression construct Briefly, gonad-mesonephros complexes were immediately removed from Total RNA was isolated from MPT embryonic gonads at stage 25, stage 16 embryos incubating in vivo at different temperatures. Gonads were whereupon reverse transcription was carried out to prepare cDNA. A full- carefully dissected from the adjacent mesonephros and placed on 0.4 μm length turtle Dmrt1 open reading frame (1107 bp) was PCR amplified from transparent, low-protein-binding Biopore membrane (Millipore) floating on cDNA using forward primer 5′-CCCCAAATTGTAGAGGCGAACC-3′ 2 ml of Leibovitz’s L-15 medium (Gibco) supplemented with 10% charcoal- and reverse primer 5′-TGAGGGCAGGGCAGAGGAGG-3′. The PCR stripped fetal bovine serum (FBS) and 0.2% penicillin-streptomycin solution. product was digested with EcoRI and cloned to pGLV-EF1a-GFP (LV-4, Isolated gonads were cultured in sterile culture plate wells (Corning) placed in GenePharma). The recombinant vector pGLV-GFP-Dmrt1 was named LV- cell incubators, in which the same temperatures were maintained as before Dmrt1. The empty vector pGLV-GFP-empty was used as a negative control dissection, monitored daily with HOBO data loggers and verified with (LV-empty). High-quality proviral DNA was used to transfect 293T cells. calibrated thermometers. Culture medium was refreshed by replacing 750 μl Virus propagation was carried out as described above. A viral titer of 4×108 every day for the duration of culture. Gonads were grown at either constant infectious units/ml was obtained for in vitro electroporation of cultured temperatures or shifted temperatures (26°C→32°C and 32°C→26°C). Using gonads. this approach, we cultured gonads explanted at stage 16 for up to 30 days. On day5ofin vitro culture, the gonad was found to have reached late stage 18. Infection of turtle embryos Gonads cultured for 10, 15, 20, 25 and 30 days at FPT were found to have A high-titer virus of LV-Dmrt1-shRNA (at least 1×108 infectious units/ml) reached stages early 20, late 21, late 22, 23 and late 23, respectively. Gonads was injected into stage 13.5-14 turtle embryos at MPT (26°C) or PvT (29.2°C, cultured at MPT for 5, 10, 15, 20, 25 and 30 days corresponded to stages late a threshold temperature that produces an even ratio of males and females). 17, late 18, late 19, late 20, 21 and late 21, respectively. Following 1, 2, 3, 4, 5, Eggs were swabbed with alcohol swabs prior to injection using a fine 10, 15, 20, 25 and 30 days of culture, gonads at MPT (26°C) and FPT (32°C) (0-25 µl) metal Hamilton needle. Approximately 5 μl was injected per were collected for histology, immunohistochemistry and gene expression embryo and a total of 500 eggs were injected in each treated group. Three- analysis. For in vitro experiments regarding the relationship between Dmrt1 hundred control embryos at MPT, PvT and FPT were injected with expression and temperature, isolated gonads were cultured in cell incubators at scrambled control virus of LV-NC-shRNA. Eggs were sealed with parafilm different temperatures (23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, and incubated for the indicated time points (stage 23 and 25). Ratio of

31°C, 32°C, 33°C), and harvested on day 5 or 20 for RNA extraction. survival to stage 25 was 40-60%. Embryos showing GFP fluorescence in the DEVELOPMENT

2231 RESEARCH ARTICLE Development (2017) 144, 2222-2233 doi:10.1242/dev.152033 urogenital system were chosen for further analysis. More than 30 pairs of Varieties (2016C02055-4) and National Natural Science Foundation of China GFP+ gonads were sampled per treatment. (31101884).

In vitro electroporation of gonads Supplementary information Supplementary information available online at To assess the effects of Dmrt1 overexpression in FPT embryonic gonads, we http://dev.biologists.org/lookup/doi/10.1242/dev.152033.supplemental used in vitro electroporation of FPT-cultured gonads with the lentiviral vector carrying the Dmrt1 ORF (LV-Dmrt1). High-quality LV-Dmrt1 DNA References was prepared and used at final concentration of 1 μg/μl diluted in Barske, L. A. and Capel, B. (2010). Estrogen represses SOX9 during sex electroporation mix (0.16% carboxymethyl cellulose, 1 mM MgCl2 in determination in the red-eared slider turtle Trachemys scripta. Dev. Biol. 341, PBS). In vitro electroporation was performed according to methods 305-314. described previously (Lambeth et al., 2014; Shoemaker-Daly et al., Bull, J. J. and Vogt, R. C. (1979). Temperature-dependent sex determination in 2010). Briefly, gonads from stage 16 embryos that were incubated in vivo turtles. Science 206, 1186-1188. Charnier, M. (1966). Action of temperature on the sex ratio in the Agama agama at FPT or PvT were placed on solidified Sylgard in a sterile petri dish in 10 μl μ μ (Agamidae, Lacertilia) embryo. C. R. Seances Soc. Biol. Fil. 160, 620-622. of 1 g/ l LV-Dmrt1 DNA and electroporated under the following Chen, S., Zhang, G., Shao, C., Huang, Q., Liu, G., Zhang, P., Song, W., An, N., condition: 40 V, three pulses of 50 ms each, with a 100 ms pulse interval. Chalopin, D., Volff, J.-N. et al. (2014). Whole-genome sequence of a flatfish Control gonads at stage 16 at FPT, MPT or PvT were electroporated with provides insights into ZW sex chromosome evolution and adaptation to a benthic LV-empty DNA. After electroporation, each gonad was immediately lifestyle. Nat. Genet. 46, 253-260. transferred to fresh culture medium for 5-10 min. Electroporated gonads Crews, D. (1994). Temperature, steroids and sex determination. J. Endocrinol. were then transferred to a 0.4 μm Millicell membrane (Millipore), and 142, 1-8. Crews, D. and Bergeron, J. M. (1994). Role of reductase and aromatase in sex cultured as above for up to 30 days. More than 30 cultured gonads showing determination in the red-eared slider (Trachemys scripta), a turtle with GFP fluorescence were selected for further analysis. temperature-dependent sex determination. J. Endocrinol. 143, 279-289. Czerwinski, M., Natarajan, A., Barske, L., Looger, L. L. and Capel, B. (2016). A Genomic DNA isolation and bisulphite sequencing timecourse analysis of systemic and gonadal effects of temperature on sexual Genomic DNA from MPT, FPT, MPT→FPT or FPT→MPT individual pairs development of the red-eared slider turtle Trachemys scripta elegans. Dev. Biol. 420, 166-177. of gonads at stages 15, 16, 17, 18, 19, 20 and 21 was isolated using the Ferguson, M. W. J. and Joanen, T. (1982). Temperature of egg incubation QIAamp Fast DNA Tissue kit (Qiagen). Six pairs of gonads were sampled at determines sex in Alligator mississippiensis. Nature 296, 850-853. each stage/temperature for bisulfite sequencing. The isolated DNA was treated Gotoh, H., Miyakawa, H., Ishikawa, A., Ishikawa, Y., Sugime, Y., Emlen, D. J., with Proteinase K and bisulfite and purified from a Methylamp DNA Lavine, L. C. and Miura, T. (2014). Developmental link between sex and nutrition; modification kit (Epigentek) according to the manufacturer’s protocol. PCR doublesex regulates sex-specific mandible growth via juvenile hormone signaling for Dmrt1 promoter CpG sites was carried out on bisulphite-treated DNA in stag beetles. PLoS Genet. 10, e1004098. using primers specific to the converted DNA around the TSS (designed based Greenbaum, E. (2002). A standardized series of embryonic stages for the emydid turtle Trachemys scripta. Can. J. Zool. Rev. Can. Zool. 80, 1350-1370. on the published promoter sequence of T. scripta Dmrt1, Accession Number Herpin, A. and Schartl, M. (2015). Plasticity of gene-regulatory networks controlling KJ583239, the forward primer 5′-TTTTTAGTTTTGGAGTTAAGGTAGTA- sex determination: of masters, slaves, usual suspects, newcomers, and 3′ and reverse primer 5′-AAAAAATAACACTAACCACACCAAC-3′). The usurpators. EMBO Rep. 16, 1260-1274. purified gDNA was amplified by nested PCR under the following PCR Holleley, C. E., O’Meally, D., Sarre, S. D., Marshall Graves, J. A., Ezaz, T., conditions: 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 57°C for Matsubara, K., Azad, B., Zhang, X. and Georges, A. (2015). Sex reversal 30 s, 68°C for 30 s and final extension at 72°C for an additional 10 min period. triggers the rapid transition from genetic to temperature-dependent sex. Nature 523, 79-82. The PCR products were gel-purified and cloned into the pGEM-T vector Kato, Y., Kobayashi, K., Watanabe, H. and Iguchi, T. (2011). Environmental sex (Promega). The resulting sequences were examined for conversion efficiency determination in the branchiopod crustacean Daphnia magna: deep conservation and accuracy, using BiQ analyzer software. Sequences with less than a 97% of a Doublesex gene in the sex-determining pathway. PLoS Genet. 7, e1001345. conversion rate were not analyzed. Ten clones from each pair of gonads were Kettlewell, J. R., Raymond, C. S. and Zarkower, D. (2000). Temperature- sequenced. dependent expression of turtle Dmrt1 prior to sexual differentiation. Genesis 26, 174-178. Kijimoto, T., Moczek, A. P. and Andrews, J. (2012). Diversification of doublesex Statistical analyses function underlies morph-, sex-, and species-specific development of beetle Each experiment was independently repeated at least three times. All data horns. Proc. Natl. Acad. Sci. USA 109, 20526-20531. are expressed as the mean±s.d. Student’s unpaired t-test was used to test Kuroki, S., Matoba, S., Akiyoshi, M., Matsumura, Y., Miyachi, H., Mise, N., Abe, significance (*, #P<0.05; **, ##P<0.01; ***, ###P<0.001; n.s., no K., Ogura, A., Wilhelm, D., Koopman, P. et al. (2013). Epigenetic regulation of significance). mouse sex determination by the histone demethylase Jmjd1a. Science 341, 1106-1109. Lambeth, L. S., Raymond, C. S., Roeszler, K. N., Kuroiwa, A., Nakata, T., Acknowledgements Zarkower, D. and Smith, C. A. (2014). Over-expression of DMRT1 induces the We thank Ceri Weber (Duke University) for her comments on the manuscript, male pathway in embryonic chicken gonads. Dev. Biol. 389, 160-172. and Mr Dongdong Pan, Han Cai and Haisheng Bao (Zhejiang Wanli University) Matson, C. K. and Zarkower, D. (2012). Sex and the singular DM domain: insights for assistance in egg incubation. into sexual regulation, evolution and plasticity. Nat. Rev. Genet. 13, 163-174. Matson, C. K., Murphy, M. W., Sarver, A. L., Griswold, M. D., Bardwell, V. J. and Competing interests Zarkower, D. (2011). DMRT1 prevents female reprogramming in the postnatal The authors declare no competing or financial interests. mammalian testis. Nature 476, 101-104. Matsuda, M., Nagahama, Y., Shinomiya, A., Sato, T., Matsuda, C., Kobayashi, T., Author contributions Morrey, C. E., Shibata, N., Asakawa, S., Shimizu, N. et al. (2002). DMY is Conceptualization: C.G., B.C., G.Q.; Methodology: C.G., J.Y., H.Z., Y.Z., W.S., Y.S., a Y-specific DM-domain gene required for male development in the medaka B.C., G.Q.; Software: C.G.; Validation: C.G., J.Y., H.Z., Y.Z., G.Q.; Formal analysis: fish. Nature 417, 559-563. C.G., J.Y., H.Z., Y.Z., W.S., B.C., G.Q.; Investigation: C.G., J.Y., H.Z., Y.Z., W.S., Y.S., Matsuda, M., Shinomiya, A., Kinoshita, M., Suzuki, A., Kobayashi, T., Paul- B.C., G.Q.; Resources: Y.S.; Data curation: C.G., J.Y., H.Z., W.S., B.C., G.Q.; Writing Prasanth, B., Lau, E.-L., Hamaguchi, S., Sakaizumi, M. and Nagahama, Y. - original draft: C.G., J.Y.; Writing - review & editing: C.G., B.C., G.Q.; Supervision: (2007). DMY gene induces male development in genetically female (XX) medaka B.C., G.Q.; Project administration: C.G., G.Q.; Funding acquisition: G.Q. fish. Proc. Natl. Acad. Sci. USA 104, 3865-3870. Matsumoto, Y. and Crews, D. (2012). Molecular mechanisms of temperature- dependent sex determination in the context of ecological developmental biology. Funding Mol. Cell. Endocrinol. 354, 103-110. This study was supported by the Natural Science Foundation of Zhejiang Province Matsumoto, Y., Buemio, A., Chu, R., Vafaee, M. and Crews, D. (2013a). (LY14C190008), Zhejiang Provincial Top Key Discipline of Biological Engineering Epigenetic control of gonadal aromatase (cyp19a1) in temperature-dependent

(ZS2016007), the Zhejiang Provincial Project of Selective Breeding of Aquatic New sex determination of red-eared slider turtles. PLoS ONE 8, e63599. DEVELOPMENT

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