New insights into Müllerian duct differentiation provided by single cell transcriptomics in the chicken embryo

In vertebrate embryos, Müllerian ducts give rise to the female reproductive tract. Here, we present a comprehensive analysis of Müllerian duct differentiation using single cell transcriptomics in the chicken embryo, a unique model for duct development. The data reveal greater cellular complexity of Müllerian duct differentiation than previously understood. Transcriptomic profiling of early formed Müllerian ducts identifies molecular signatures for the Müllerian duct Epithelium (MDE) and the Müllerian Duct Mesenchyme (MDM), the two principle compartments of the Müllerian duct. The MDE expresses PAX2, LHX1, WNT7, LGR5, PDGFA/D, and CDH1/2. The MDM is marked by WNT4, EMX2, POSTN, OSR1, VIM,DMRT and the EMT markers ZEB1/2. In the male, the regressing mesenchyme (MDM) has a unique molecular signature, expressing the transcription factors MSX1, MSX2, RUNX2, GATA5, DLX3 and DLX5, and the signaling regulators DKK4 and NRG1 (Neuregulin). Single cell transcript profiling of the regressing male mesenchyme identifies new genes implicated in duct regression, including the WNT antagonist, DKK4. DKK4, responds to Anti-Mullerian Hormone induction and can induce duct regression when ectopically expressed in females. The duct mesenchyme in the female follows a different fate, with a molecular signature comprising SMARCA2/ HMGA2/ZEB1/2/TWIST1/2/DMRT2 and persistent expression of FOXE1, OSR2 and HGF (Hepatocyte Growth Factor). Cell clusters in the developing female ducts reflects spatial pre-patterning of the duct prior to later regionalisation. In contrast to AMH-mediated bilateral Müllerian duct regression in males, female chicken embryos show an unusual pattern of left duct differentiation and contralateral right duct regression. We find that right female duct does not regress in the same manner as in males, being transcriptionally distinct and lacking a population of regressing mesenchyme, despite presumably being mediated by AMH, as in males.