Gene Expression Patterns 5 (2005) 655–667 www.elsevier.com/locate/modgep Characterization and expression pattern of zebrafish anti-Mu¨llerian hormone (amh) relative to sox9a, sox9b, and cyp19a1a, during gonad development Adriana Rodrı´guez-Marı´, Yi-Lin Yan, Ruth A. BreMiller, Catherine Wilson, Cristian Can˜estro, John H. Postlethwait* Institute of Neuroscience, University of Oregon, 1425 E. 13th Avenue, Eugene, OR 97403, USA Received 28 January 2005; received in revised form 28 February 2005; accepted 28 February 2005 Available online 19 April 2005 Abstract The role of Anti-Mu¨llerian hormone (Amh) during gonad development has been studied extensively in mammals, but is less well understood in other vertebrates. In male mammalian embryos, Sox9 activates expression of Amh, which initiates the regression of the Mu¨llerian ducts and inhibits the expression of aromatase (Cyp19a1), the enzyme that converts androgens to estrogens. To better understand shared features of vertebrate gonadogenesis, we cloned amh cDNA from zebrafish, characterized its genomic structure, mapped it, analyzed conserved syntenies, studied its expression pattern in embryos, larvae, juveniles, and adults, and compared it to the expression patterns of sox9a, sox9b and cyp19a1a. We found that the onset of amh expression occurred while gonads were still undifferentiated and sox9a and cyp19a1a were already expressed. In differentiated gonads of juveniles, amh showed a sexually dimorphic expression pattern. In 31 days post-fertilization juveniles, testes expressed amh and sox9a, but not cyp19a1a, while ovaries expressed cyp19a1a and sox9b, but not amh.In adult testes, amh and sox9a were expressed in presumptive Sertoli cells. In adult ovaries, amh and cyp19a1a were expressed in granulosa cells surrounding the oocytes, and sox9b was expressed in a complementary fashion in the ooplasm of oocytes. The observed expression patterns of amh, sox9a, sox9b, and cyp19a1a in zebrafish correspond to the patterns expected if their regulatory interactions have been conserved with mammals. The finding that zebrafish sox9b and sox8 were not co-expressed with amh in oocytes excludes the possibility that amh expression in zebrafish granulosa cells is directly regulated by either of these two genes. q 2005 Elsevier B.V. All rights reserved. Keywords: Anti-mullerian hormone; Amh; Mullerian inhibiting substance; Mis; TGF-beta; Teleost; Zebrafish; Gonad development; Gonad differentiation; Sox9; Sox9a; Sox9b; Cyp19a1; Cyp19a1a; Aromatase; Cytochrome P450 aromatase; Sox8; Testis; Testes; Sertoli cells; Ovary; Ovaries; Oocyte; Follicle cells; Granulosa cells; Theca cells; Synteny; Sex determination 1. Results and discussion In concert with other factors, including Sf1, Wt1, and Gata4 (Shen et al., 1994; de Santa Barbara et al., 1998; Mammalian embryos initially develop an indifferent Nachtigal et al., 1998; Viger et al., 1998; de Santa gonad that has the potential of becoming an ovary or a testis Barbara et al., 2000), Sox9 activates the Anti- (see for recent reviews (Brennan and Capel, 2004; Park and Mu¨llerian Hormone gene (Amh)(di Clemente et al., 1992; Jameson, 2005)). The Y-chromosome gene Sry stimulates Kent et al., 1996; Morais da Silva et al., 1996; de Santa early testis development (Gubbay et al., 1990; Sinclair et al., Barbara et al., 1998; Rouiller-Fabre et al., 1998; de Santa 1990) and activates the expression of the transcription factor Barbara et al., 2000; Lasala et al., 2004), also known as Sox9 inSertolicellprecursors(Sekido et al., 2004). Mu¨llerian-inhibiting substance (Mis), which encodes a glycoprotein member of the transforming growth factor- beta (TGF-beta) superfamily (Cate et al., 1986; Picard et al., * Corresponding author. Tel.: C1 541 346 4538; fax: C1 541 346 4548. 1986). Amh is produced by Sertoli cells in fetal testes and E-mail address: [email protected] (J.H. Postlethwait). causes the regression of the Mu¨llerian ducts, which in 1567-133X/$ - see front matter q 2005 Elsevier B.V. All rights reserved. females differentiate into Fallopian tubes and uterus doi:10.1016/j.modgep.2005.02.008 (Munsterberg and Lovell-Badge, 1991; Josso et al., 1993). 656 A. Rodrı´guez-Marı´ et al. / Gene Expression Patterns 5 (2005) 655–667 Amh also blocks the ability of cAMP and FSH to cause to that of the zebrafish orthologs of mammalian genes known rat fetal testis to express Cyp19a1, the P450 aromatase to be upstream (Sox9) and downstream (Cyp19a1) of Amh. enzyme that converts androgens to estrogens (Rouiller-Fabre et al., 1998). In accord with these regulatory interactions 1.1. Isolation, characterization, and mapping (Sox9/Amh-jjCyp19a1), the onset of Sox9 expression of zebrafish amh precedes Amh expression in mammalian testes. In contrast to mammals, Amh expression precedes Sox9 expression in To identify a zebrafish ortholog of the mammalian Amh the indifferent gonad of chicken and alligator embryos, gene, we performed a tBLASTn (Altschul et al., 1997)search suggesting that different classes of vertebrates differ in the of the zebrafish genome database (Sanger Institute) using the regulatory interactions of these genes (Smith et al., 1999; mouse AMH protein (NP_031471) as query. The best Western et al., 1999; Oreal et al., 2002). zebrafish hit (ENSDART00000013803) returned Amh as the In addition to the early role in the mammalian bipotential most similar protein among mammals in a BLASTx search of gonad, Amh plays a later role in negatively regulating the NCBI nr database. We used the ENSDART00000013803 gonadal development in both sexes. In females, Amh plays sequence to design RACE primers for amplifying the cDNA an important role in folliculogenesis (Durlinger et al., 2002). sequence of the zebrafish amh gene and submitted the After Mu¨llerian ducts have lost sensitivity to Amh action in sequence to GenBank (AY881649). This sequence provides late fetal life, ovarian granulosa cells produce Amh, thereby a 2275 bp mRNA which includes the complete coding inhibiting the transcription of aromatase (Cyp19a1) and LH sequence for the zebrafish Amh protein. Comparing the receptor (Vigier et al., 1989; Josso et al., 1998). Females mRNA to its genomic sequence (Sanger Institute) revealed maintain high levels of Amh throughout reproductive life that the zebrafish amh gene consists of seven exons and spans a until menopause, so serum levels of Amh provide a good genomic region of 13.6 kb (Fig. 1A). Human AMH marker of ovarian follicle reserve and fertility potential contains just five exons (Cate et al., 1986), and the difference (Fanchin et al., 2005). In males, Amh also influences the in exon number is due to the presence of two additional introns development of adult testes by blocking the differentiation (intron 1 and intron 6) in the zebrafish gene (Fig. 1A). of mesenchymal into Leydig cells and by decreasing the Comparative analysis of the Amh promoter among expression of steroidogenic enzymes (Josso et al., 1998). mammals revealed the presence of conserved binding sites In contrast to mammals, the molecular genetic regulation for Sox9, SF-1 and GATA proteins (Arango et al., 1999; of gonad differentiation in other vertebrates remains largely Watanabe et al., 2000). Our analysis of the zebrafish amh unknown. In fishes, only basally diverging ray-fin fish, such promoter showed the presence of a putative TATA box at as sturgeon, have Mu¨llerian ducts, and these do not undergo -67 nt relative to the ATG start codon, and in accordance regression in males (Wrobel, 2003). Teleost fish, like eel, with consensus sequences described in mammals (Arango zebrafish, salmon, and flounder, do not have Mu¨llerian ducts et al., 1999; Watanabe et al., 2000), we found several (Lasala et al., 2004). Despite the absence of Mu¨llerian ducts, putative transcription factor binding sites: Sox9 binding a gene coding for a protein with high similarity to Amh, sites (TTTGAG) at K865, K3588, and K3832 nt; GATA originally named spermatogenesis preventing substance, has binding sites (TGATAG) at K1752 and K3515 nt; and been recently sequenced in three teleosts: Japanese eel SF-1 binding site (CCAAGGACAC) at K123 nt, and SF-1 (BAB93107), Japanese flounder (BAD37138) and Atlantic binding sites (AGGTCA) at K2074 and K3399 nt. Future salmon (AAU85130). In Japanese eel, expression of this gene experiments are required to test whether these putative was reported in Sertoli cells of immature testes, where it transcription factor binding sites shown to be functional in prevents the initiation of spermatogenesis, but its expression mammals are active in zebrafish. was not investigated in ovaries (Miura et al., 2002). In To determine the genomic location of amh, we mapped Japanese flounder, Amh expression was detected in Sertoli the sequence on both the HS meiotic mapping panel cells in testes but not in ovaries (Yoshinaga et al., 2004). (Woods et al., 2000) and the radiation hybrid mapping In this work, we investigate the poorly understood process panel LN54 (Hukriede et al., 1999). These experiments of gonadal differentiation in zebrafish. Zebrafish, like many revealed that zebrafish amh maps to LG22. There were no other fish species, do not have sex chromosomes (Schreeb et recombinants between amh and markers AI396840, oaz1, al., 1993; Pijnacker and Ferwerda, 1995; Amores and and 34 other markers previously located at 38.9 cM on Postlethwait, 1999). Juvenile zebrafish first develop an LG22 on the HS mapping panel (Fig. 1B). In the current undifferentiated ovary-like gonad containing immature Sanger database, however, zebrafish amh