In mammals, the developing reproductive tract primordium of male and female fetuses consists of the Wolffian duct and the Müllerian duct (MD), two epithelial tube pairs surrounded by mesenchyme. During male development, mesenchyme-epithelia interactions mediate MD regression to prevent its development into a uterus, oviduct, and upper vagina. It is well established that transforming growth factor-β family member anti-Müllerian hormone (AMH) secreted from the fetal testis and its type 1 and 2

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... eptors expressed in MD mesenchyme regulate MD regression. However, little is known about the molecular network regulating downstream actions of AMH signaling. To identify potential AMH-induced genes and regulatory networks controlling MD regression in a global nonbiased manner, we examined transcriptome differences in MD mesenchyme between males (AMH signaling on) and females (AMH signaling off) by RNA-seq analysis of purified fetal MD mesenchymal cells. This analysis found 82 genes up-regulated in males during MD regression and identified Osterix (Osx)/Sp7, a key transcriptional regulator of osteoblast differentiation and bone formation, as a downstream effector of AMH signaling during MD regression. Osx/OSX was expressed in a malespecific pattern in MD mesenchyme during MD regression. OSX expression was lost in mutant males without AMH signaling. In addition, transgenic mice ectopically expressing human AMH in females induced a male pattern of Osx expression. Together, these results indicate that AMH signaling is necessary and sufficient for Osx expression in the MD mesenchyme. In addition, MD regression was delayed in Osx-null males, identifying Osx as a factor that regulates MD regression. Osterix | Müllerian duct regression | reproductive tract development | anti-Müllerian hormone | sex differentiation I n mammals, the Wolffian ducts (WDs) differentiate into the male epididymides, vas deferens, and seminal vesicles, whereas the Müllerian ducts (MDs) develop into the female oviducts, uterus, and upper vagina. Reproductive tract differentiation in amniotes is unique because initially both WD and MD are generated in the embryo independent of genetic sex. Sex-specific signaling results in loss of the WD in females and regression of the MD in males (1, 2). MD regression requires transforming growth factor-β family member anti-Müllerian hormone (AMH) secreted from the Sertoli cells of the fetal testis and its type 1 and 2 receptors present in MD mesenchyme (3-7). Following AMH binding, AMH type 2 receptor (AMHR2) recruits a type 1 receptor into a heteromeric complex. Within the complex, AMHR2 transphosphorylates and activates the type 1 receptor kinase. This activation results in the phosphorylation of an R-Smad and formation of an R-SMAD/SMAD-4 complex that translocates into the nucleus to transcriptionally activate AMH signaling pathway target genes. AMH type 1 receptors ACVR1 and BMPR1A, and AMH R-Smad effectors (SMAD1, SMAD5, and SMAD8) function redundantly for MD regression and are shared with the bone morphogenetic protein (BMP) pathway (8, 9). While the upstream components (AMH, type 1 and 2 receptors, and R-Smads) of AMH signaling are well known, the downstream transcriptional effectors of AMH signaling are still largely unidentified. To date, only the WNT pathway effector β-catenin (Ctnnb1) has been shown to be required for AMH-induced MD regression in vivo (10). The requirement of Ctnnb1 for MD regression suggests WNT signaling is important for the downstream actions of AMH during MD regression in males. However, candidate gene approaches have failed to identify an individual WNT, WNT effector, or WNT regulator whose in vivo loss blocks MD regression (10-12). Because of the limited success of candidate gene approaches in uncovering AMH signaling effectors, in the current study we undertook a nonbiased global approach using next-generation transcriptome sequencing. To elucidate potential gene networks and novel AMH signaling targets, we used RNA-seq analysis of yellow fluorescent protein (YFP)-positive MD mesenchymal cells flow sorted from embryonic day 14.5 (E14.5) Amhr2 Cre/+ ; R26R yfp/+ reproductive tracts to identify transcriptome differences between males and females during regression. This analysis identified Osterix (Osx)/Sp7 as a downstream effector of AMH signaling during MD Significance In mammals, each embryo forms both male and female reproductive tract progenitor tissues. Anti-Müllerian hormone (AMH) secreted by fetal testes acts on mesenchyme cells adjacent to Müllerian duct (MD) epithelium, the progenitor tissue of female reproductive tract, to induce MD epithelial regression. While AMH and early AMH signaling components are elucidated, downstream gene networks directing this process are largely unknown. A global nonbiased approach using whole-transcriptome sequencing of fetal MD mesenchymal cells identified 82 factors as potential target genes of AMH including Osterix (Osx). Our findings provide in vivo evidence that Osx is an AMH-induced gene that regulates MD regression. Identification of Osx may provide key insights into gene-regulatory networks underlying MD regression, male sex differentiation, and mesenchyme-epithelial interactions.