Chicken Hemogen Homolog Is Involved in the Chicken-Specific Sex

Chicken hemogen homolog is involved in the chicken-speciﬁc sex-determining mechanism

Tomohiro Nakataa, Manabu Ishiguroa, Nana Adumaa, Hiroe Izumib, and Asato Kuroiwaa,b,1

aGraduate School of Life Science, and bLaboratory of Animal Cytogenetics, Department of Biological Sciences, Faculty of Science, Hokkaido University, Hokkaido 060-0810, Japan

Edited by Patricia K. Donahoe, Massachusetts General Hospital, Boston, MA, and approved January 18, 2013 (received for review October 29, 2012) Using a comprehensive transcriptome analysis, a Z chromosome- of male chickens. We present evidence that cHEMGN acts as linked chicken homolog of hemogen (cHEMGN)wasidentified and a transcription factor in the nucleus of (pre)Sertoli cells after the showntobespecifically involved in testis differentiation in early sex-determination period and directly or indirectly triggers the ex- chicken embryos. Hemogen [Hemgn in mice, EDAG (erythroid pression of SOX9, suggesting this gene is specifically involved in differentiation-associated gene protein) in humans] was recently chicken sex determination. characterized as a hematopoietic tissue-specific gene encoding a transcription factor that regulates the proliferation and differen- Results and Discussion tiation of hematopoietic cells in mammals. In chicken, cHEMGN was Identification of Chicken Hemogen cDNA and Chromosome Localization. expressed not only in hematopoietic tissues but also in the early High coverage expression profiling (HiCEP) was used to conduct embryonic gonad of male chickens. The male-specific expression a comprehensive transcriptome analysis (10) comparing male was identified in the nucleus of (pre)Sertoli cells after the sex de- and female gonads at day 5.5–6.5 of incubation. A total of 33,962 termination period and before the expression of SOX9 (SRY-box 9). transcripts were identified. The 18 transcripts that were ex- The expression of cHEMGN was induced in ZW embryonic gonads pressed specifically in males or were more than fivefold higher that were masculinized by aromatase inhibitor treatment. ZW em- in males than females were sequenced, and cHEMGN was among bryos overexpressing cHEMGN, generated by infection with retrovi- these transcripts. The full-length coding sequence of cHEMGN rus carrying cHEMGN, showed masculinized gonads. These findings was obtained by RT-PCR and 5′ and 3′ RACE. The full-length suggest that cHEMGN is a transcription factor specifically involved coding sequence was 543 bp, and the predicted amino acid sequence in chicken sex determination. was 180 aa. A bipartite nuclear localization signal and a coiled-coil domain that are present in mouse HEMGN were also conserved bird | gonadal differentiation in cHEMGN (7) (Fig. S1). The amino acid sequence identities between human and chicken, or mouse and chicken, were both 26%. n birds, as in mammals, sex is genetically determined, but males We performed FISH using cHEMGN cDNA clone as probe. fl Iare the homogametic sex (ZZ), and females are heterogametic The uorescence signals were detected in Zq21 in chicken (ZW). The molecular mechanisms determining sex in birds has chromosomes (Fig. S2). This location corresponded with the been a long-standing mystery. In mammals, the sex-determination information of cHEMGN in a chicken genome database (Ensem- gene SRY (sex determining region Y) acts as a transcription factor ble, www.ensembl.org/index.html, last accessed October 20, 2012). to activate SOX9 (SRY-box 9) expression directly by binding to the cHEMGN SOX9 enhancer in pre-Sertoli cells in the undifferentiated gonads Expression Pattern of in Early Embryonic Gonads. Northern cHEMGN of XY embryos (1). SOX9 functions in Sertoli cell differentiation in blot analysis demonstrated that mRNA was more highly expressed in male gonads than female gonads at day 7.5 mammals and other vertebrates (2). A strong candidate for male (Fig. 1A). Quantitative RT-PCR (qRT-PCR) analysis revealed sex determination in chicken is doublesex and mab-3 (Protein that cHEMGN was expressed in the male gonads from day 5.5 MAB-3) related transcription factor 1 (DMRT1), which is on the Z onward, and expression increased dramatically to a peak at day chromosome (3). DMRT1 has been suggested to activate SOX9 8.5 and was then lost before hatching (Fig. 1B). By contrast, fe- indirectly, because there is a time lag between the expression of male gonads exhibited only very low expression throughout em- DMRT1 and SOX9,whicharefirst expressed on day 4.5 and day 6.5 bryogenesis. The expression in male gonads at day 8.5 was more of incubation, respectively (4–6). Therefore, other factors that are fi than 10-fold higher than in female gonads. The cHEMGN pro- likely to be chicken speci c must be in the molecular cascade be- tein was detected in male gonads from day 6.5 onward (Fig. S3). tween DMRT1 and SOX9. Anti-müllerian hormone (AMH) expression in Sertoli cells is one Herein we show that chicken homolog of hemogen (cHEMGN), of the earliest markers of sex differentiation in chicken embryo also in the Z chromosome linkage group, is a transcription factor fi gonads (11). AMH expression was up-regulated between days 5.5 involved in this chicken-speci c molecular cascade. In mice, hem- and 6.5, similarly to cHEMGN expression (Fig. 1C). SOX9 ex- ogen [Hemgn;alsoknownasEDAG (erythroid differentiation- pression was present from day 6.5 and was up-regulated by day associated gene protein) in humans] is a recently characterized fi 8.5 (Fig. 1C). These results are consistent with previous studies hematopoietic tissue-speci c gene encoding a nuclear protein (7). that reported that AMH was expressed in the male gonad before The expression of Hemgn is restricted to the blood islands of the significant SOX9 expression (5, 12). The RT-PCR analysis here yolk sac and the fetal liver during embryogenesis, as well as the revealed that cHEMGN was also expressed before SOX9. BIOLOGY adult spleen and bone marrow (BM) (7). EDAG shows similar expression patterns. EDAG expression is high in the BM cells in DEVELOPMENTAL

acute myeloid leukemia, suggesting that EDAG may play a modu- Author contributions: A.K. designed research; T.N., M.I., N.A., and A.K. performed lator role in acute myeloid leukemia (8). Overexpression of Hemgn research; H.I. contributed new reagents/analytic tools; T.N., M.I., N.A., H.I., and A.K. in hematopoietic cells suppresses lymphopoiesis and enhances analyzed data; and T.N. and A.K. wrote the paper. myelopoiesis in transgenic mice, suggesting that Hemgn regulates The authors declare no conﬂict of interest. the proliferation and differentiation of hematopoietic cells (9). This article is a PNAS Direct Submission. However, the gene is not expressed in the gonads during embryo- 1To whom correspondence should be addressed. E-mail: [email protected]. genesis in mammals. In chicken, cHEMGN was expressed not only This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in hematopoietic tissues but also in the early embryonic gonad 1073/pnas.1218714110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1218714110 PNAS | February 26, 2013 | vol. 110 | no. 9 | 3417–3422 Downloaded by guest on September 28, 2021 Fig. 1. cHEMGN is highly expressed in early embryonic male chicken gonads. (A) Northern blot analysis of cHEMGN in embryonic tissues at day 7.5. cHEMGN mRNA was highly expressed in the male gonad. 18S rRNA was used as a loading control. (B) qRT-PCR of male and female gonads. In male embryos, cHEMGN expression was detected by day 5.5 (just after sex determination) and achieved a peak at day 8.5. The expression was gradually reduced and lost at hatching. Filled square, male; ﬁlled circle, female. Data are mean ± SEM; n ≥ 3. (C) RT-PCR of cHEMGN, AMH,andSOX9 in male gonads at day 5.5, 6.5, 7.5, and 8.5. cHEMGN expression was detected before SOX9 expression. GAPDH is the loading control.

cHEMGN expression was detected in the gonads of male the SOX9 signal in the gonadal medulla (Fig. 2 G–J). By contrast, embryos using in situ hybridization of whole embryos (WISH) the cHEMGN signal did not colocalize with that of CVH (Fig. 2 K– (Fig. 2A) and in the gonadal medulla using frozen sections of N). The cHEMGN protein was observed in the nucleus of male embryos (Fig. 2 C and D). No signals were detected in the gonads gonadal cells by immunohistochemistry (IHC) (Fig. 2 O–R). These of female embryos (Fig. 2 B, E,andF). To identify the cells that results suggested that cHEMGN was a nuclear protein expressed in expressed cHEMGN, double-label in situ hybridization was con- Sertoli cells. ducted using SOX9 and chicken vasa homolog [CVH, also known as DDX4 (DEAD box polypeptide 4)] as markers for Sertoli cells and Expression Pattern of cHEMGN in Hematopoietic Tissues. The ex- germ cells (13), respectively. The cHEMGN signal colocalized with pression of cHEMGN in chicken hematopoietic tissues was

Fig. 2. cHEMGN is expressed in the nucleus of Sertoli cells within the medulla of the male gonads. (A and B) WISH of male and female embryos at day 7.5. cHEMGN was expressed throughout the male gonads. The dashed lines indicate the gonads. (Scale bar, 300 μm.) (C–F) cHEMGN in situ hybridization in frozen sections of male and female gonads at day 7.5. cHEMGN expression was localized to the medulla of the male gonad. The negative control used a sense probe for hybridization. (Scale bar, 100 μm.) (G–N) Dual-labeled in situ hybridization of frozen sections of male and female gonads at day 8.5. cHEMGN expression colocalized with SOX9 expression in Sertoli cells. (Scale bars, 100 μm.) (O–Q) IHC of day-8.5 male gonads using a cHEMGN antibody. cHEMGN was expressed in the nucleus of Sertoli cells in the medulla of the male gonad. (Scale bar, 100 μm.) (R) Higher-magniﬁcation view of the area indicated by the box in Q. Arrows indicate signals in the nucleus. (Scale bar, 100 μm.)

3418 | www.pnas.org/cgi/doi/10.1073/pnas.1218714110 Nakata et al. Downloaded by guest on September 28, 2021 masculinized ZW embryos (Fig. 4 J–O). The cHEMGN expression level was compared among ZW female, ZZ male, and masculinized ZW embryo gonads by qRT-PCR. An increase in cHEMGN expression was detected in masculinized ZW embryos by day 8.5 (Fig. 4P), suggesting that cHEMGN associated with testis differentiation.

Masculinization of the ZW Gonads Overexpressing cHEMGN. Trans- genic embryos overexpressing cHEMGN were produced by in- fecting chicken embryos with an avian retroviral vector, RCAS.A (17), carrying the cHEMGN gene (RCAS.A.cHEMGN). The number of embryos used in experiments is shown in Table S1. Fig. 3. cHEMGN is expressed in hematopoietic tissues, similar to mammals. Embryos infected with RCAS.A. carrying the enhanced green (A) Northern blot analysis of cHEMGN in spleen, BM, and blood of male and fluorescent protein (RCAS.A.eGFP) and uninfected embryos were female embryos at day 8.5. Loading control, 18S rRNA. High cHEMGN expression was detected in blood; weak expression was detected in spleen and used as negative controls. The embryos overexpressing cHEMGN BM at longer exposure times. (B–D) IHC of day-8.5 embryo blood cells. The showed significantly deficient growth compared with the two neg- cHEMGN signal was localized to the nucleus. (Scale bar, 10 μm.) (E) qRT-PCR ative controls (Fig. 5A and Fig. S4 A and B). The average body of spleen, BM, and blood from day-8.5 embryos. White bars, females; black weights of control, RCAS.A.eGFP, and RCAS.A.cHEMGN em- bars, males. Expression was two- to threefold higher in males than females. bryos were 1,211, 1,128, and 308 mg, respectively. Furthermore, ± ≥ Data are mean SEM; n 3. embryos overexpressing cHEMGN exhibited early lethality. Sur- vival to day 8.5 was 36.4% (63 of 173; Table S1), and there were no determined. Northern blot analysis identified cHEMGN expression in the spleen and BM, as well as in the blood, of both sexes, similar to mammals (7) (Fig. 3A). The cHEMGN protein was localized in the nuclei of blood cells by IHC (Fig. 3 B–D). The expression levels were compared between sexes by qRT-PCR, and males expressed two- to threefold higher levels than females in the spleen, BM, and blood (Fig. 3E). The chicken Z chromosome has no gene dosage compensation system such as X chromosome in- activation in mammals, with the net result that a large number of genes on the avian Z chromosome are expressed at a higher level in males (ZZ) than in females (ZW). Previous studies measured the male to female (M:F) ratio of Z-linked genes in chicken and reported that Z genes had M:F expression ratios ranging from 0.4 to 2.7 (14–16). Thus, the differences in expression of cHEMGN between sexes in the hematopoietic tissues may reflect the Z- linked gene dosage of cHEMGN. Furthermore, the 10-fold higher expression in male embryonic gonads relative to female (Fig. 1B) suggests the existence of a specific enhancer for expression in male gonads.

Expression in Masculinized ZW Gonads by Aromatase Inhibitor Treatment. cHEMGN was expressed in the nucleus of (pre)Ser- toli cells, suggesting that this gene functioned on testis differentiation. However, there was a possibility that this expression was insigniﬁcant, because mammalian hemogen did not function on testis differentiation in humans and mice. Therefore, we analyzed the expression of cHEMGN in masculinized ZW embryo to prove that the expression actually associated with testis differentiation. Day-10.5 masculinized ZW embryos were produced by fadrozole treatment. In females, the right gonad regresses early in embryonic development, and asymmetric gonadal development is observed (Fig. 4A). By contrast, bilateral development is observed in early Fig. 4. Expression of cHEMGN is induced in the gonads of masculinized ZW embryonic development in males (Fig. 4B). The gonads in mascu- embryos. Gonads on top of the mesonephros of female (A), male (B), and masculinized ZW (C) embryos at day 10.5. The gonads of masculinized ZW linized ZW embryos showed the bilateral development character- embryos showed bilateral development similar to male gonads. Dashed lines istic of the male morphology (Fig. 4C). The left gonad of female indicate gonads. (Scale bar, 1 mm.) (D–F) H&E staining of gonad sections from embryos possesses a thickened outer cortex and fragmented me- female, male, and masculinized ZW embryos. The left gonad of masculinized BIOLOGY dulla (Fig. 4D). The gonads of male embryos are characterized by ZW has a testis-like phenotype with a dense medulla and thin cortex, although a dense medulla with seminiferous cords and a reduced cortex (Fig. a slight fragmented medulla was observed. The dashed line indicates the DEVELOPMENTAL 4E). A section from the left gonad of a masculinized ZW embryo border between the cortex and medulla in the female gonad. (Scale bar, 100 exhibited the male-like morphology with dense medulla and a re- μm.) (G–O) CYP19A1, SOX9, and cHEMGN in situ hybridization in male, female, and masculinized ZW gonad frozen sections at day 10.5. Aromatase was duced cortex, although a slight fragmented medulla was observed ﬁ (Fig. 4F). The expression of cytochrome P450, family 19, subfamily identi ed in female gonads, but no expression was observed in male or masculinized ZW gonads. By contrast, the expression of SOX9 and cHEMGN was A, polypeptide 1 (CYP19A1, also known as aromatase) was de- not detected in female gonads but was present in male and masculinized ZW tected in ZW female embryos but not in ZZ male or masculinized gonads. (P) qRT-PCR of cHEMGN in gonads from female (black bars), mascu- ZW embryos (Fig. 4 G–I) by in situ hybridization. SOX9 and linized ZW (gray bars), and male (white bars) embryos at days 6.5, 8.5, and 10.5. cHEMGN were expressed in the gonads of ZZ male and The expression of cHEMGN was induced in masculinized ZW gonads by day 8.5.

Nakata et al. PNAS | February 26, 2013 | vol. 110 | no. 9 | 3419 Downloaded by guest on September 28, 2021 embryos that survived past day 9.5. These results suggest that forced expression of cHEMGN throughout the embryo results in abnormal transcriptionincellsandcausesgrowthdeficiency and early lethality. Such abnormalities have not been reported in transgenic Hemgn mice in which the overexpression was restricted to hematopoietic tissues as a result of using the human CD11a promoter (9). DMRT1 overexpression experiments in chicken using a retroviral system caused embryo lethality by day 4, and this early lethality was proposed to be due to the global effects of the transcription factor (3). The gonads in ZW embryos overexpressing cHEMGN showed the bilateral development characteristic of the male morphology (Fig. S4C). We performed histological analysis by H&E staining in sections of gonads from male (Fig. 5 B and C), female (Fig. 5 D and E), and ZW overexpressing cHEMGN at day 8.5 (Fig. 5 F and G). After this, male and female embryos uninfected with any viruses were used as negative controls in all experiments. Al- though a little difference was observed in gonads at day 8.5 between control male and female compared with gonads at day 10.5 (Fig. 4 D and E), The ZW gonad overexpressing cHEMGN showed a male-like morphology characterized by a dense medulla with seminiferous cords (Fig. 5 F and G). The expression of male and female markers in the gonads of the embryos overexpressing cHEMGN was examined. Key markers of testicular differentiation are DMRT1 and SOX9, whereas markers of ovarian development are forkhead box L2 (FOXL2)and CYP19A1. DMRT1 has been proposed as a putative testis-determining gene in birds, and high-level expression of the gene is necessary for testis differentiation during embryogenesis after sex determination (18). A conserved role for FOXL2 has been indicated in chicken embryos because this gene is activated female- specifically just before gonad differentiation (19, 20). Furthermore, the temporal and spatial colocalization profiles suggest that FOXL2 activates the estrogen-synthesizing enzyme aromatase (20, 21). At the mRNA level, DMRT1 and SOX9 expression increased, and FOXL2 and CYP19A1 expression decreased, in ZW gonads overexpressing cHEMGN relative to controls (Fig. 5H). In control male embryos at day 8.5, the cHEMGN and SOX9 proteins were expressed normally in the nuclei of Sertoli cells (Fig. 5 I and L), but control female gonads lacked the expression of these proteins (Fig. 5 J and M). The ZW embryos overexpressing cHEMGN possessed high levels of cHEMGN protein throughout the gonad (Fig. 5K), whereas SOX9 protein was found only in the medullary region of the gonad (Fig. 5N). CVH staining showed distribution of germ cells within the interior of control male gonads (Fig. 5O), but control female gonads exhibited cortical distribution of germ cells (Fig. 5P). The ZW embryos overexpressing cHEMGN showed male-like (in- Fig. 5. Masculinization of ZW embryos after overexpression of cHEMGN.(A) terior) distribution of germ cells in the gonads (Fig. 5Q). These Comparison of body weight among controls (black bar), embryos over- results indicate that the ZW gonads were masculinized as a result of expressing eGFP (RCAS.A.eGFP, white bar), and embryos overexpressing high cHEMGN expression. Four of six ZW embryos overexpressing cHEMGN (RCAS.A.cHEMGN, gray bar). The body weights of embryos over- cHEMGN showed clear expression of SOX9 and male-like distri- expressing cHEMGN were significantly reduced. Data presented are mean ± SD; bution of germ cells demonstrated by CVH expression (Table S1). *P < 0.001; n = 40. H&E staining of left and right gonad sections from male (B In the remaining two embryos, significant signals could not be and C), female (D and E), and ZW overexpressing cHEMGN (F and G). The ZW detected by IHC, because the gonads were very fragile and good gonad overexpressing cHEMGN showed a male-like morphology characterized by a dense medulla with seminiferous cords. (Scale bar, 100 μm.) (H)qRT-PCRof sections of gonads could not be obtained in the two embryos. DMRT1, SOX9, CYP19A1,andFOXL2 in gonads of female (black bars), cHEMGN Expression of DMRT1 starts from day 4.5 and is before ZW overexpressing (RCAS.A.cHEMGN ZW, dark gray bars), male (white bars), cHEMGN expression, suggesting that cHEMGN operates down- and cHEMGN ZZ overexpressing (RCAS.A.cHEMGN ZZ, light gray bars) embryos stream of DMRT1. The expression of DMRT1 is kept in Sertoli at day 8.5. The expression of DMRT1 and SOX9 was increased and expression of cells after sex determination and the gene becomes to express in CTP19A1 and FOXL2 was reduced in ZW embryos overexpressing cHEMGN germ cells with progress of developmental stage (18). These facts compared with control female embryos. Data presented are mean ± SEM; *P < indicate that DMRT1 has multiple functions in embryonic gonads 0.001; n ≥ 3. (I–Q) IHC of cHEMGN, SOX9, and CVH in gonad sections of male, and at least two functions in Sertoli cells: triggering of testis dif- female, and cHEMGN ZW overexpressing (RCAS.A.cHEMGN ZW) embryos at day ferentiation and testis differentiation after sex determination. 8.5. cHEMGN was detected in male gonads and at very high levels in the gonad of cHEMGN overexpressing ZW embryos. SOX9 protein was also detected in Therefore, we supposed that each expression was independently gonads of both male and cHEMGN overexpressing ZW embryos. The gonad of regulated, resulting from the masculinization by overexpression of cHEMGN overexpressing embryo showed male-like (interior) distribution of cHEMGN-induced DMRT1 expression in gonads of ZW embryo. germ cells. (Scale bar, 100 μm.) Male and female embryos uninfected with any This report has shown that cHEMGN is a gene involved spe- viruses were used as negative controls (B–E, H–J, L, M, O,andP). cifically in early events in sex determination in chicken and that

3420 | www.pnas.org/cgi/doi/10.1073/pnas.1218714110 Nakata et al. Downloaded by guest on September 28, 2021 cHEMGN functions in the molecular cascade between DMRT1 Preparation of Recombinant Protein and Rabbit Antibodies. Details of the and SOX9 as a transcription factor in (pre)Sertoli cells. Our data procedure are provided in SI Materials and Methods. indicate that cHEMGN acts as a transcription factor in the pre- Sertoli cells to induce directly or indirectly SOX9 expression Western Blot Analysis. Details of the procedure are provided in SI Materials after sex determination. In the 5′-flanking region of mouse and and Methods. human Hemgn/EDAG, binding sites for GATA1 (GATA binding protein 1) and HOXB4 (homeobox B4) (only reported in Immunohistochemistry Analysis. The primary antibodies, rabbit anti-SOX9 Hemgn) are present, and Hemgn/EDAG has been shown to be and rat anti-CVH, were a kind gift from R. Lovell-Badge (London, UK) and M.-A. Hattori (Fukuoka, Japan), respectively. Details of the procedure are a direct transcriptional target of these genes in hematopoetic provided in SI Materials and Methods. cells (22–25). Future studies should determine the presence of fi agonad-speci c enhancer and transcription factors that me- Aromatase Inhibitor Treatments. To obtain sex reversal chickens (female to diate the gonadal expression of cHEMGN,aswellasidentify male), we performed aromatase inhibitor treatments according to a previous direct targets of cHEMGN. Further investigation of the role report (18), with slight modifications. Details of the procedure are provided fi of cHEMGN will reveal the chicken-speci c mechanisms of in SI Materials and Methods. sex determination. Preparation of RCAS.A.cHEMGN and RCAS.A.eGFP Virus. The cHEMGN trans- Materials and Methods genic chicken embryos were produced by infection with Replication- Chicken Strains. Fertilized chicken eggs (Gallus gallus domesticus)were Competent Avian Leucosis Sarcoma virus LTR with Splice acceptor, and purchased from Takeuchi Hatchery(Nara, Japan). This study used the Hy- subgroup An env (envelope protein) gene (RCAS.A). RCAS.A proviral DNA Line Maria chicken strain. Fertilized eggs were incubated at 37.8 °C. All was the kind gift of Stephen H. Hughes (National Institutes of Health, the animal experiments in this study were performed in accordance with Bethesda, MD) (17). Both primers included an artificial ClaI site for cloning the Guidelines for the Care and Use of Laboratory Animals, Hokkaido into the RCAS.A. The products were subcloned using the pGEM T-easy University. Vector System (Promega). The identity of the insert was confirmed by se- quencing, and the pGEM T-easy-cHEMGN plasmid DNA was subsequently HiCEP Screening. We performed HiCEP screening according to a previous digested with ClaI and cloned into ClaI-digested RCAS.A. report (10). Details of the procedure are provided in SI Materials and Methods. For negative controls, RCAS.A.eGFP was used, as previously reported (30). This construct was provided by ARK-Genomics, The Roslin Institute. Cloning of Chicken cHEMGN cDNA. A detailed procedure is provided in SI Materials and Methods, and the sequences of primers used are shown in Table S2. Injection of Embryos with RCAS.A.cHEMGN. Embryo injections were performed as described in a previous study (31), with a slight modification. Details of the Chromosome Preparation and FISH Mapping. The preparation of R-banded chromosomes and FISH were performed as previously described (26, 27). procedure are provided in SI Materials and Methods. Details of the procedures are provided in SI Materials and Methods. Measurement of Body Weight. Details of the procedure are provided in SI RT-PCR and qRT-PCR. Details of the procedure are provided in SI Materials and Materials and Methods. Methods, and the sequences of primers used are shown in Table S2. Accession Codes. Homo sapiens EDAG, NM_018437.3; Mus musculus Hemgn, Northern Blot Analysis. Details of the procedure are provided in SI Materials NM_053149.2; Gallus gallus HEMGN, XM_430508.3. and Methods. ACKNOWLEDGMENTS. We thank H. Yunokawa and Y. Mikami for help with WISH and in Situ Hybridization. Chicken embryos were fixed in 4% (wt/vol) HiCEP screening; S. H. Hughes and A. Ferris for providing RCAS vectors; R. Lovell-Badge, S. Guioli and M.-A. Hattori for providing the SOX9 and CVH pareformaldehyde and processed for WISH according to a previous report antibody, respectively; and H. Yoshioka, Y. Ishimaru, Y. Atsumi, A. P. Kimura, (28). The urogenital tissue of chicken embryos was slowly frozen in tissue-tek Y. Matsuda, and C. Nishida-Umehara for helpful suggestions regarding experi- − (Sakura Finetek USA) and kept at 80 °C until use and processed for in situ mental techniques and this research. This work was supported by Grant-in-Aid hybridization, as previously reported (29). Details of the procedures are for Scientific Research 23132501 and the F3 Project Support office for female shown in SI Materials and Methods. researchers at Hokkaido University.

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