Chicken hemogen homolog is involved in thechicken-specific sex-determining mechanismTomohiro 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-linked chicken homolog of hemogen (cHEMGN) was identified andshown to be specifically involved in testis differentiation in earlychicken embryos. Hemogen [Hemgn in mice, EDAG (erythroiddifferentiation-associated gene protein) in humans] was recentlycharacterized as a hematopoietic tissue-specific gene encodinga transcription factor that regulates the proliferation and differen-tiation of hematopoietic cells in mammals. In chicken, cHEMGNwasexpressed not only in hematopoietic tissues but also in the earlyembryonic gonad of male chickens. The male-specific expressionwas identified in the nucleus of (pre)Sertoli cells after the sex de-termination period and before the expression of SOX9 (SRY-box 9).The expression of cHEMGN was induced in ZW embryonic gonadsthat were masculinized by aromatase inhibitor treatment. ZW em-bryos overexpressing cHEMGN, generated by infection with retrovi-rus carrying cHEMGN, showed masculinized gonads. These findingssuggest that cHEMGN is a transcription factor specifically involvedin chicken sex determination.

bird | gonadal differentiation

In birds, as in mammals, sex is genetically determined, but malesare the homogametic sex (ZZ), and females are heterogametic

(ZW). The molecular mechanisms determining sex in birds hasbeen a long-standing mystery. In mammals, the sex-determinationgene SRY (sex determining region Y) acts as a transcription factorto activate SOX9 (SRY-box 9) expression directly by binding to theSOX9 enhancer in pre-Sertoli cells in the undifferentiated gonadsof XY embryos (1). SOX9 functions in Sertoli cell differentiation inmammals and other vertebrates (2). A strong candidate for malesex determination in chicken is doublesex and mab-3 (ProteinMAB-3) related transcription factor 1 (DMRT1), which is on the Zchromosome (3). DMRT1 has been suggested to activate SOX9indirectly, because there is a time lag between the expression ofDMRT1 and SOX9, which are first expressed on day 4.5 and day 6.5of incubation, respectively (4–6). Therefore, other factors that arelikely to be chicken specific must be in the molecular cascade be-tween DMRT1 and SOX9.Herein we show that chicken homolog of hemogen (cHEMGN),

also in the Z chromosome linkage group, is a transcription factorinvolved in this chicken-specific molecular cascade. In mice, hem-ogen [Hemgn; also known as EDAG (erythroid differentiation-associated gene protein) in humans] is a recently characterizedhematopoietic tissue-specific gene encoding a nuclear protein (7).The expression of Hemgn is restricted to the blood islands of theyolk sac and the fetal liver during embryogenesis, as well as theadult spleen and bone marrow (BM) (7). EDAG shows similarexpression patterns. EDAG expression is high in the BM cells inacute myeloid leukemia, suggesting that EDAG may play a modu-lator role in acute myeloid leukemia (8). Overexpression ofHemgnin hematopoietic cells suppresses lymphopoiesis and enhancesmyelopoiesis in transgenic mice, suggesting that Hemgn regulatesthe proliferation and differentiation of hematopoietic cells (9).However, the gene is not expressed in the gonads during embryo-genesis in mammals. In chicken, cHEMGN was expressed not onlyin hematopoietic tissues but also in the early embryonic gonad

of male chickens. We present evidence that cHEMGN acts asa transcription factor in the nucleus of (pre)Sertoli cells after thesex-determination period and directly or indirectly triggers the ex-pression of SOX9, suggesting this gene is specifically involved inchicken sex determination.

Results and DiscussionIdentification of Chicken Hemogen cDNA and Chromosome Localization.High coverage expression profiling (HiCEP) was used to conducta comprehensive transcriptome analysis (10) comparing maleand female gonads at day 5.5–6.5 of incubation. A total of 33,962transcripts were identified. The 18 transcripts that were ex-pressed specifically in males or were more than fivefold higherin males than females were sequenced, and cHEMGN was amongthese transcripts. The full-length coding sequence of cHEMGNwas obtained by RT-PCR and 5′ and 3′ RACE. The full-lengthcoding sequence was 543 bp, and the predicted amino acid sequencewas 180 aa. A bipartite nuclear localization signal and a coiled-coildomain that are present in mouse HEMGN were also conservedin cHEMGN (7) (Fig. S1). The amino acid sequence identities be-tween human and chicken, or mouse and chicken, were both 26%.We performed FISH using cHEMGN cDNA clone as probe.

The fluorescence signals were detected in Zq21 in chickenchromosomes (Fig. S2). This location corresponded with theinformation of cHEMGN in a chicken genome database (Ensem-ble, www.ensembl.org/index.html, last accessed October 20, 2012).

Expression Pattern of cHEMGN in Early Embryonic Gonads. Northernblot analysis demonstrated that cHEMGN mRNA was morehighly expressed in male gonads than female gonads at day 7.5(Fig. 1A). Quantitative RT-PCR (qRT-PCR) analysis revealedthat cHEMGN was expressed in the male gonads from day 5.5onward, and expression increased dramatically to a peak at day8.5 and was then lost before hatching (Fig. 1B). By contrast, fe-male gonads exhibited only very low expression throughout em-bryogenesis. The expression in male gonads at day 8.5 was morethan 10-fold higher than in female gonads. The cHEMGN pro-tein was detected in male gonads from day 6.5 onward (Fig. S3).Anti-müllerian hormone (AMH) expression in Sertoli cells is oneof the earliest markers of sex differentiation in chicken embryogonads (11). AMH expression was up-regulated between days 5.5and 6.5, similarly to cHEMGN expression (Fig. 1C). SOX9 ex-pression was present from day 6.5 and was up-regulated by day8.5 (Fig. 1C). These results are consistent with previous studiesthat reported that AMH was expressed in the male gonad beforesignificant SOX9 expression (5, 12). The RT-PCR analysis hererevealed that cHEMGN was also expressed before SOX9.

Author contributions: A.K. designed research; T.N., M.I., N.A., and A.K. performedresearch; H.I. contributed new reagents/analytic tools; T.N., M.I., N.A., H.I., and A.K.analyzed data; and T.N. and A.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: asatok@sci.hokudai.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218714110/-/DCSupplemental.

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cHEMGN expression was detected in the gonads of maleembryos using in situ hybridization of whole embryos (WISH)(Fig. 2A) and in the gonadal medulla using frozen sections ofembryos (Fig. 2 C and D). No signals were detected in the gonadsof female embryos (Fig. 2 B, E, and F). To identify the cells thatexpressed cHEMGN, double-label in situ hybridization was con-ducted using SOX9 and chicken vasa homolog [CVH, also known asDDX4 (DEAD box polypeptide 4)] as markers for Sertoli cells andgerm cells (13), respectively. The cHEMGN signal colocalized with

the SOX9 signal in the gonadal medulla (Fig. 2 G–J). By contrast,the cHEMGN signal did not colocalize with that of CVH (Fig. 2 K–N). The cHEMGN protein was observed in the nucleus of malegonadal cells by immunohistochemistry (IHC) (Fig. 2 O–R). Theseresults suggested that cHEMGN was a nuclear protein expressed inSertoli cells.

Expression Pattern of cHEMGN in Hematopoietic Tissues. The ex-pression of cHEMGN in chicken hematopoietic tissues was

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. cHEMGNmRNA 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, cHEMGNexpression 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, and SOX9 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.

Fig. 2. cHEMGN is expressed in the nucleus of Sertoli cells within themedulla of themale gonads. (A and B) WISH of male and female embryos at day 7.5. cHEMGNwas expressed throughout themale gonads. The dashed lines indicate the gonads. (Scale bar, 300 μm.) (C–F) cHEMGN in situ hybridization in frozen sections ofmaleand female gonads at day 7.5. cHEMGN expressionwas localized to themedulla of themale gonad. The negative control used a sense probe for hybridization. (Scalebar, 100 μm.) (G–N) Dual-labeled in situ hybridization of frozen sections ofmale and female gonads at day 8.5. cHEMGN expression colocalizedwith SOX9 expressionin Sertoli cells. (Scale bars, 100 μm.) (O–Q) IHC of day-8.5male gonads using a cHEMGN antibody. cHEMGNwas expressed in the nucleus of Sertoli cells in themedullaof themale gonad. (Scale bar, 100 μm.) (R) Higher-magnification viewof thearea indicatedby the box inQ. Arrows indicate signals in the nucleus. (Scale bar, 100 μm.)

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determined. Northern blot analysis identified cHEMGN expres-sion in the spleen and BM, as well as in the blood, of both sexes,similar to mammals (7) (Fig. 3A). The cHEMGN protein waslocalized in the nuclei of blood cells by IHC (Fig. 3 B–D). Theexpression levels were compared between sexes by qRT-PCR, andmales expressed two- to threefold higher levels than females in thespleen, BM, and blood (Fig. 3E). The chicken Z chromosome hasno gene dosage compensation system such as X chromosome in-activation in mammals, with the net result that a large number ofgenes on the avian Z chromosome are expressed at a higher levelin males (ZZ) than in females (ZW). Previous studies measuredthe male to female (M:F) ratio of Z-linked genes in chicken andreported that Z genes had M:F expression ratios ranging from 0.4to 2.7 (14–16). Thus, the differences in expression of cHEMGNbetween sexes in the hematopoietic tissues may reflect the Z-linked gene dosage of cHEMGN. Furthermore, the 10-fold higherexpression in male embryonic gonads relative to female (Fig. 1B)suggests the existence of a specific enhancer for expression inmale gonads.

Expression in Masculinized ZW Gonads by Aromatase InhibitorTreatment. cHEMGN was expressed in the nucleus of (pre)Ser-toli cells, suggesting that this gene functioned on testis differen-tiation. However, there was a possibility that this expression wasinsignificant, because mammalian hemogen did not function ontestis differentiation in humans and mice. Therefore, we analyzedthe expression of cHEMGN in masculinized ZW embryo to provethat the expression actually associated with testis differentiation.Day-10.5 masculinized ZWembryos were produced by fadrozole

treatment. In females, the right gonad regresses early in embryonicdevelopment, and asymmetric gonadal development is observed(Fig. 4A). By contrast, bilateral development is observed in earlyembryonic development in males (Fig. 4B). The gonads in mascu-linized ZW embryos showed the bilateral development character-istic of the male morphology (Fig. 4C). The left gonad of femaleembryos possesses a thickened outer cortex and fragmented me-dulla (Fig. 4D). The gonads of male embryos are characterized bya dense medulla with seminiferous cords and a reduced cortex (Fig.4E). A section from the left gonad of a masculinized ZW embryoexhibited the male-like morphology with dense medulla and a re-duced cortex, although a slight fragmented medulla was observed(Fig. 4F). The expression of cytochrome P450, family 19, subfamilyA, polypeptide 1 (CYP19A1, also known as aromatase) was de-tected in ZW female embryos but not in ZZ male or masculinizedZW embryos (Fig. 4 G–I) by in situ hybridization. SOX9 andcHEMGN were expressed in the gonads of ZZ male and

masculinized ZW embryos (Fig. 4 J–O). The cHEMGN ex-pression level was compared among ZW female, ZZ male, andmasculinized ZW embryo gonads by qRT-PCR. An increase incHEMGN expression was detected in masculinized ZW em-bryos by day 8.5 (Fig. 4P), suggesting that cHEMGN associatedwith 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). Thenumber of embryos used in experiments is shown in Table S1.Embryos infected with RCAS.A. carrying the enhanced greenfluorescent protein (RCAS.A.eGFP) and uninfected embryos wereused as negative controls. The embryos overexpressing cHEMGNshowed significantly deficient growth compared with the two neg-ative controls (Fig. 5A and Fig. S4 A and B). The average bodyweights of control, RCAS.A.eGFP, and RCAS.A.cHEMGN em-bryos were 1,211, 1,128, and 308 mg, respectively. Furthermore,embryos overexpressing cHEMGN exhibited early lethality. Sur-vival to day 8.5 was 36.4% (63 of 173; Table S1), and there were no

Fig. 3. cHEMGN is expressed in hematopoietic tissues, similar to mammals.(A) Northern blot analysis of cHEMGN in spleen, BM, and blood of male andfemale embryos at day 8.5. Loading control, 18S rRNA. High cHEMGN ex-pression was detected in blood; weak expression was detected in spleen andBM at longer exposure times. (B–D) IHC of day-8.5 embryo blood cells. ThecHEMGN signal was localized to the nucleus. (Scale bar, 10 μm.) (E) qRT-PCRof spleen, BM, and blood from day-8.5 embryos. White bars, females; blackbars, males. Expression was two- to threefold higher in males than females.Data are mean ± SEM; n ≥ 3.

Fig. 4. Expression of cHEMGN is induced in the gonads of masculinized ZWembryos. Gonads on top of the mesonephros of female (A), male (B), andmasculinized ZW (C) embryos at day 10.5. The gonads of masculinized ZWembryos showed bilateral development similar to male gonads. Dashed linesindicate gonads. (Scale bar, 1 mm.) (D–F) H&E staining of gonad sections fromfemale, male, and masculinized ZW embryos. The left gonad of masculinizedZWhas a testis-like phenotypewith a densemedulla and thin cortex, althougha slight fragmented medulla was observed. The dashed line indicates theborder between the cortex and medulla in the female gonad. (Scale bar, 100μm.) (G–O) CYP19A1, SOX9, and cHEMGN in situ hybridization inmale, female,and masculinized ZW gonad frozen sections at day 10.5. Aromatase wasidentified in female gonads, but no expression was observed in male or mas-culinized ZW gonads. By contrast, the expression of SOX9 and cHEMGN wasnot detected in female gonads but was present in male and masculinized ZWgonads. (P) qRT-PCR of cHEMGN in gonads from female (black bars), mascu-linized ZW (gray bars), andmale (white bars) embryos at days 6.5, 8.5, and 10.5.The expression of cHEMGNwas induced inmasculinized ZWgonads by day 8.5.

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embryos that survived past day 9.5. These results suggest that forcedexpression of cHEMGN throughout the embryo results in abnormaltranscription in cells and causes growth deficiency and early lethality.Such abnormalities have not been reported in transgenic Hemgnmice in which the overexpression was restricted to hematopoietictissues as a result of using the human CD11a promoter (9).DMRT1 overexpression experiments in chicken using a retroviralsystem caused embryo lethality by day 4, and this early lethalitywas proposed to be due to the global effects of the transcriptionfactor (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 stainingin sections of gonads from male (Fig. 5 B and C), female (Fig. 5D and E), and ZW overexpressing cHEMGN at day 8.5 (Fig. 5 Fand G). After this, male and female embryos uninfected with anyviruses were used as negative controls in all experiments. Al-though a little difference was observed in gonads at day 8.5 be-tween control male and female compared with gonads at day10.5 (Fig. 4 D and E), The ZW gonad overexpressing cHEMGNshowed a male-like morphology characterized by a dense me-dulla 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 oftesticular differentiation are DMRT1 and SOX9, whereas markersof ovarian development are forkhead box L2 (FOXL2) andCYP19A1. DMRT1 has been proposed as a putative testis-de-termining gene in birds, and high-level expression of the gene isnecessary for testis differentiation during embryogenesis after sexdetermination (18). A conserved role for FOXL2 has been in-dicated 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 FOXL2activates the estrogen-synthesizing enzyme aromatase (20, 21). Atthe mRNA level, DMRT1 and SOX9 expression increased, andFOXL2 and CYP19A1 expression decreased, in ZW gonads over-expressing cHEMGN relative to controls (Fig. 5H). In control maleembryos at day 8.5, the cHEMGN and SOX9 proteins wereexpressed normally in the nuclei of Sertoli cells (Fig. 5 I and L), butcontrol female gonads lacked the expression of these proteins (Fig.5 J and M). The ZW embryos overexpressing cHEMGN possessedhigh levels of cHEMGN protein throughout the gonad (Fig. 5K),whereas SOX9 protein was found only in the medullary region ofthe gonad (Fig. 5N). CVH staining showed distribution of germ cellswithin the interior of control male gonads (Fig. 5O), but controlfemale gonads exhibited cortical distribution of germ cells (Fig. 5P).The ZW embryos overexpressing cHEMGN showed male-like (in-terior) distribution of germ cells in the gonads (Fig. 5Q). Theseresults indicate that the ZWgonads weremasculinized as a result ofhigh cHEMGN expression. Four of six ZW embryos overexpressingcHEMGN showed clear expression of SOX9 and male-like distri-bution of germ cells demonstrated by CVH expression (Table S1).In the remaining two embryos, significant signals could not bedetected by IHC, because the gonads were very fragile and goodsections of gonads could not be obtained in the two embryos.Expression of DMRT1 starts from day 4.5 and is before

cHEMGN expression, suggesting that cHEMGN operates down-stream of DMRT1. The expression of DMRT1 is kept in Sertolicells after sex determination and the gene becomes to express ingerm cells with progress of developmental stage (18). These factsindicate that DMRT1 has multiple functions in embryonic gonadsand at least two functions in Sertoli cells: triggering of testis dif-ferentiation and testis differentiation after sex determination.Therefore, we supposed that each expression was independentlyregulated, resulting from the masculinization by overexpression ofcHEMGN-induced DMRT1 expression in gonads of ZW embryo.This report has shown that cHEMGN is a gene involved spe-

cifically in early events in sex determination in chicken and that

Fig. 5. Masculinization of ZW embryos after overexpression of cHEMGN. (A)Comparison of body weight among controls (black bar), embryos over-expressing eGFP (RCAS.A.eGFP, white bar), and embryos overexpressingcHEMGN (RCAS.A.cHEMGN, gray bar). The body weights of embryos over-expressing cHEMGNwere significantly reduced. Data presented aremean± SD;*P < 0.001; n = 40. H&E staining of left and right gonad sections from male (Band C), female (D and E), and ZW overexpressing cHEMGN (F and G). The ZWgonad overexpressing cHEMGN showed a male-like morphology characterizedby a dense medulla with seminiferous cords. (Scale bar, 100 μm.) (H) qRT-PCR ofDMRT1, SOX9, CYP19A1, and FOXL2 in gonads of female (black bars), cHEMGNZW overexpressing (RCAS.A.cHEMGN ZW, dark gray bars), male (white bars),and cHEMGN ZZ overexpressing (RCAS.A.cHEMGN ZZ, light gray bars) embryosat day 8.5. The expression ofDMRT1 and SOX9was increased and expression ofCTP19A1 and FOXL2 was reduced in ZW embryos overexpressing cHEMGNcompared with control female embryos. Data presented are mean ± SEM; *P <0.001; n ≥ 3. (I–Q) IHC of cHEMGN, SOX9, and CVH in gonad sections of male,female, and cHEMGNZWoverexpressing (RCAS.A.cHEMGNZW) embryos at day8.5. cHEMGNwas detected inmale gonads and at very high levels in the gonadof cHEMGN overexpressing ZW embryos. SOX9 protein was also detected ingonads of both male and cHEMGN overexpressing ZW embryos. The gonad ofcHEMGN overexpressing embryo showed male-like (interior) distribution ofgerm cells. (Scale bar, 100 μm.) Male and female embryos uninfected with anyviruses were used as negative controls (B–E, H–J, L, M, O, and P).

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cHEMGN functions in the molecular cascade between DMRT1and SOX9 as a transcription factor in (pre)Sertoli cells. Our dataindicate that cHEMGN acts as a transcription factor in the pre-Sertoli cells to induce directly or indirectly SOX9 expressionafter sex determination. In the 5′-flanking region of mouse andhuman Hemgn/EDAG, binding sites for GATA1 (GATA bindingprotein 1) and HOXB4 (homeobox B4) (only reported inHemgn) are present, and Hemgn/EDAG has been shown to bea direct transcriptional target of these genes in hematopoeticcells (22–25). Future studies should determine the presence ofa gonad-specific enhancer and transcription factors that me-diate the gonadal expression of cHEMGN, as well as identifydirect targets of cHEMGN. Further investigation of the roleof cHEMGN will reveal the chicken-specific mechanisms ofsex determination.

Materials and MethodsChicken Strains. Fertilized chicken eggs (Gallus gallus domesticus) werepurchased from Takeuchi Hatchery(Nara, Japan). This study used the Hy-Line Maria chicken strain. Fertilized eggs were incubated at 37.8 °C. Allthe animal experiments in this study were performed in accordance withthe Guidelines for the Care and Use of Laboratory Animals, HokkaidoUniversity.

HiCEP Screening. We performed HiCEP screening according to a previousreport (10). Details of the procedure are provided in SI Materials and Methods.

Cloning of Chicken cHEMGN cDNA. A detailed procedure is provided in SIMaterials andMethods, and the sequences of primers usedare shown inTable S2.

Chromosome Preparation and FISH Mapping. The preparation of R-bandedchromosomes and FISH were performed as previously described (26, 27).Details of the procedures are provided in SI Materials and Methods.

RT-PCR and qRT-PCR. Details of the procedure are provided in SI Materials andMethods, and the sequences of primers used are shown in Table S2.

Northern Blot Analysis. Details of the procedure are provided in SI Materialsand Methods.

WISH and in Situ Hybridization. Chicken embryos were fixed in 4% (wt/vol)pareformaldehyde and processed for WISH according to a previous report(28). The urogenital tissue of chicken embryos was slowly frozen in tissue-tek(Sakura Finetek USA) and kept at −80 °C until use and processed for in situhybridization, as previously reported (29). Details of the procedures areshown in SI Materials and Methods.

Preparation of Recombinant Protein and Rabbit Antibodies. Details of theprocedure are provided in SI Materials and Methods.

Western Blot Analysis. Details of the procedure are provided in SI Materialsand Methods.

Immunohistochemistry Analysis. The primary antibodies, rabbit anti-SOX9and rat anti-CVH, were a kind gift from R. Lovell-Badge (London, UK) andM.-A. Hattori (Fukuoka, Japan), respectively. Details of the procedure areprovided in SI Materials and Methods.

Aromatase Inhibitor Treatments. To obtain sex reversal chickens (female tomale), we performed aromatase inhibitor treatments according to a previousreport (18), with slight modifications. Details of the procedure are providedin SI Materials and Methods.

Preparation of RCAS.A.cHEMGN and RCAS.A.eGFP Virus. The cHEMGN trans-genic chicken embryos were produced by infection with Replication-Competent Avian Leucosis Sarcoma virus LTR with Splice acceptor, andsubgroup An env (envelope protein) gene (RCAS.A). RCAS.A proviral DNAwas the kind gift of Stephen H. Hughes (National Institutes of Health,Bethesda, MD) (17). Both primers included an artificial ClaI site for cloninginto the RCAS.A. The products were subcloned using the pGEM T-easyVector System (Promega). The identity of the insert was confirmed by se-quencing, and the pGEM T-easy-cHEMGN plasmid DNA was subsequentlydigested with ClaI and cloned into ClaI-digested RCAS.A.

For negative controls, RCAS.A.eGFP was used, as previously reported (30).This construct was provided by ARK-Genomics, The Roslin Institute.

Injection of Embryos with RCAS.A.cHEMGN. Embryo injections were performedas described in a previous study (31), with a slight modification. Details of theprocedure are provided in SI Materials and Methods.

Measurement of Body Weight. Details of the procedure are provided in SIMaterials and Methods.

Accession Codes. Homo sapiens EDAG, NM_018437.3; Mus musculus Hemgn,NM_053149.2; Gallus gallus HEMGN, XM_430508.3.

ACKNOWLEDGMENTS. We thank H. Yunokawa and Y. Mikami for help withHiCEP 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 CVHantibody, respectively; and H. Yoshioka, Y. Ishimaru, Y. Atsumi, A. P. Kimura,Y. Matsuda, and C. Nishida-Umehara for helpful suggestions regarding experi-mental techniques and this research. This work was supported by Grant-in-Aidfor Scientific Research 23132501 and the F3 Project Support office for femaleresearchers at Hokkaido University.

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