(83) Müllerian Duct Maintenance at the Cranial Region is Dependent Upon GATA2

Shuai Jia¹, Fei Zhao¹

¹Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA

Abstract Text:

Congenital anomalies of the female reproductive tract (FRT), also known as Müllerian anomalies, are a major cause of female infertility. However, our understanding of normal FRT development and the etiology of Müllerian anomalies remains incomplete. The FRT organs, including the oviduct, uterus, cervix, and the upper vagina, originate from the same embryonic progenitor, the Müllerian duct (MD). The MD is surrounded by multiple layers of undifferentiated connective tissues known as the mesenchyme, which governs the MD maintenance and differentiation. During sexual differentiation, the fetal testis in male embryos produces anti-Müllerian hormone (AMH), which binds to its receptor AMHR2 expressed in the mesenchyme to induce MD regression. Conversely, ovaries in female embryos do not produce AMH; as a result, MDs are maintained. Traditionally, MD maintenance in the female embryo has been viewed as a passive outcome due to the lack of testicular AMH. In contrast to this prevailing view, our study provided genetic evidence that GATA2 actively promoted MD maintenance in the cranial region. Gata2, a member of the GATA family of transcription factors, is known to play crucial roles in morphogenesis and differentiation of other tubular organs and is uniquely expressed in the MD mesenchyme. We hypothesize that mesenchymal Gata2 plays critical roles in MD development. To test this hypothesis, we created conditional Gata2 knockout mice (Osr2-Cre;Gata2-flox/flox, Gata2^cKO), where Gata2 was deleted specifically in MD mesenchyme. Gata2^cKO females exhibited a shorter FRT and a complete loss of the cranial MD, which forms the oviductal infundibulum and proximal ampulla essential for oocyte pickup and fertilization. Deletion of Gata2 in the MD epithelium using Pax8-Cre did not lead to any morphological defects, confirming that the loss of cranial MD is due to Gata2 deficiency within the mesenchyme but not within the epithelium. To uncover the molecular mechanism, we performed bulk RNA-seq on collected cranial MD region from control and mesenchymal Gata2^cKO female embryos at the onset of ductal degeneration (E16.5). The differentially expressed genes (DEGs) did not significantly overlap with the AMH-induced genes, indicating that the cranial MD regression in Gata2^cKO female embryos was not caused by ectopic AMH signaling. It is established that mesenchyme-derived secreted ligands mediate the instructive role of the mesenchyme in epithelial fate and differentiation. Among 296 DEGs, we identified 13 secreted ligands, with a particular focus on the downregulated growth factor Nrg1 (Neuregulin 1). Nrg1 is vital for the growth of various organs, including the heart and testis. Its receptors (Erbb2 and Erbb3) are expressed in MD epithelium. NRG1 supplementation in cultured Gata2^cKO MDs partially restored cranial MD maintenance, highlighting the role of reduced NRG1 signaling in the Gata2^cKO MDs phenotype. In summary, our study challenges the notion that MD maintenance is a passive outcome and demonstrates that cranial MD maintenance is actively promoted by mesenchymal GATA2, possibly through inducing Nrg1. Our research advances our understanding of FRT development and the underlying causes of Müllerian anomalies.