Transcriptional landscape of the embryonic chicken Müllerian duct

Abstract

Background Müllerian ducts are paired embryonic tubes that give rise to the female reproductive tract in vertebrates. Many disorders of female reproduction can be attributed to anomalies of Müllerian duct development. However, the molecular genetics of Müllerian duct formation is poorly understood and most disorders of duct development have unknown etiology. In this study, we describe for the first time the transcriptional landscape of the embryonic Müllerian duct, using the chicken embryo as a model system. RNA sequencing was conducted at 1 day intervals during duct formation to identify developmentally-regulated genes, validated by in situ hybridization. Results This analysis detected hundreds of genes specifically up-regulated during duct morphogenesis. Gene ontology and pathway analysis revealed enrichment for developmental pathways associated with cell adhesion, cell migration and proliferation, ERK and WNT signaling, and, interestingly, axonal guidance. The latter included factors linked to neuronal cell migration or axonal outgrowth, such as Ephrin B2, netrin receptor, SLIT1 and class A semaphorins. A number of transcriptional modules were identified that centred around key hub genes specifying matrix-associated signaling factors; SPOCK1, HTRA3 and ADGRD1. Several novel regulators of the WNT and TFG-β signaling pathway were identified in Müllerian ducts, including APCDD1 and DKK1, BMP3 and TGFBI. A number of novel transcription factors were also identified, including OSR1, FOXE1, PRICKLE1, TSHZ3 and SMARCA2. In addition, over 100 long non-coding RNAs (lncRNAs) were expressed during duct formation. Conclusions This study provides a rich resource of new candidate genes for Müllerian duct development and its disorders. It also sheds light on the molecular pathways engaged during tubulogenesis, a fundamental process in embryonic development.

Mechanistic Drivers of Müllerian Duct Development and Differentiation Into the Oviduct

“…The BMP effector molecule/s is currently unknown, although expression of Bmp2/3/4/7 has been documented during chicken Müllerian development ( Atsuta and Takahashi, 2016 ; Roly et al, 2020b ). Regarding Müllerian progenitors, the term “mesoepithelial” is adopted across species as these progenitors give rise to cells negative for epithelial markers (negative: Ck8 and E -cadherin; weakly positive: pan-cytokeratin) and positive for a mesenchymal marker (vimentin) while showing mesenchymal morphology and arrangement upon the basement membrane (BM), which is an epithelial feature ( Gruenwald, 1941 ; Dohr and Tarmann, 1984 ; Jacob et al, 1999 ; Orvis and Behringer, 2007 ; Prunskaite-Hyyryläinen et al, 2016 ; Ghosh et al, 2020 ; Roly et al, 2020a ). On the other hand, mesenchymal progenitors differentiate into cells of a true mesenchymal phenotype.…”

“…In mouse, Wnt9b controls Müllerian elongation through the canonical WNT pathway and the loss of Gata3 prompts elongation arrest ( Carroll et al, 2005 ; Grote et al, 2008 ). Elongation also depends on the Gpr56 receptor expressed in the mesoepithelial progenitors (chicken), the mesenchymal expression of Wnt4 and Dmrt1 (chicken), the PI3K/Akt pathway (rodents) and coelomic epithelium-derived paracrine signals (chicken) ( Gruenwald, 1941 ; Carroll et al, 2005 ; Kobayashi et al, 2005 ; Pedersen et al, 2005 ; Fujino et al, 2009 ; Atsuta and Takahashi, 2016 ; Prunskaite-Hyyryläinen et al, 2016 ; Roly et al, 2020a ) ( Figure 2B ). This crosstalk between the coelomic epithelium and the elongating tube is evident, given that Wnt4 expression is activated along the chicken coelomic epithelium as elongation advances and that physical disruption of the coelomic epithelium blocks Müllerian development at the mechanical incision level ( Fujino et al, 2009 ; Prunskaite-Hyyryläinen et al, 2016 ).…”

“…Gpr56 is an adhesion G protein-coupled receptor critical for chicken Müllerian elongation ( Roly et al, 2020a ). This protein is coded by the Adgrg1 gene, is first expressed in chicken coelomic epithelium progenitors during invagination, and its expression is maintained in Müllerian luminal cells until later post-developmental stages ( Roly et al, 2020a ).…”

“…Gpr56 is an adhesion G protein-coupled receptor critical for chicken Müllerian elongation ( Roly et al, 2020a ). This protein is coded by the Adgrg1 gene, is first expressed in chicken coelomic epithelium progenitors during invagination, and its expression is maintained in Müllerian luminal cells until later post-developmental stages ( Roly et al, 2020a ). When its expression is knocked down, the number of proliferative cells decreases in vivo , whereas its overexpression in a chicken fibroblastic cell line prompts an increase in cell proliferation and migration ( Roly et al, 2020a ), which suggest Gpr56 control on cell proliferation and migration during Müllerian duct elongation.…”

“…This protein is coded by the Adgrg1 gene, is first expressed in chicken coelomic epithelium progenitors during invagination, and its expression is maintained in Müllerian luminal cells until later post-developmental stages ( Roly et al, 2020a ). When its expression is knocked down, the number of proliferative cells decreases in vivo , whereas its overexpression in a chicken fibroblastic cell line prompts an increase in cell proliferation and migration ( Roly et al, 2020a ), which suggest Gpr56 control on cell proliferation and migration during Müllerian duct elongation. Furthermore, this receptor family could play further or the same roles through different family members given that expression of G protein-coupled receptors is upregulated during Müllerian duct formation, with Adgrd1 showing one of the highest expression levels at the last stages of development ( Roly et al, 2020b ).…”