Adhesion G-protein-coupled receptor, GPR56, is required for Müllerian duct development in the chick

Abstract

The embryonic Müllerian ducts give rise to the female reproductive tract (fallopian tubes, uterus and upper vagina in humans, the oviducts in birds). Embryonic Müllerian ducts initially develop in both sexes, but later regress in males under the influence of anti-Müllerian hormone. While the molecular and endocrine control of duct regression in males have been well studied, early development of the ducts in both sexes is less well understood. Here, we describe a novel role for the adhesion G protein-coupled receptor, GPR56, in development of the Müllerian ducts in the chicken embryo. GPR56 is expressed in the ducts of both sexes from early stages. The mRNA is present during the elongation phase of duct formation, and it is restricted to the inner Müllerian duct epithelium. The putative ligand, Collagen III, is abundantly expressed in the Müllerian duct at the same developmental stages. Knockdown of GPR56 expression using in ovo electroporation results in variably truncated ducts, with a loss of expression of both epithelial and mesenchymal markers of duct development. Over-expression of GPR56 in vitro results in enhanced cell proliferation and cell migration. These results show that GPR56 plays an essential role in avian Müllerian duct development through the regulation of duct elongation.

Transcriptional landscape of the embryonic chicken Müllerian duct

“…In rodent and chicken embryos, the Müllerian duct elongates through cell proliferation and active migration at the tip of the caudally progressing duct epithelium [ 23 , 29 , 39 , 51 ]. Epithelial-matrix interactions appear to be important for this process, as we recently described a requirement for the cell adhesion-linked factor, GPR56 , in chicken Müllerian duct development [ 52 ].…”

“…These large membrane-bound proteins transduce extra-cellular matrix signals through G proteins [ 76 ]. We recently demonstrated a requirement for one such family member, GPR56, in the chicken Müllerian duct [ 52 ]. SPOCK1 (testican) also has a matrix association, encoding proteoglycan containing chondroitin- and heparan-sulfate chains, with unclear function [ 77 ].…”

“…We and others have used in ovo electroporation to manipulate endogenous gene expression in the embryonic chicken Müllerian duct [ 21 , 23 , 40 ]. TOL2 transposons that integrate into the genome can be electroporated into the nascent duct, delivering transgene over-expression or shRNA-mediated knockdown [ 52 ]. These approaches can be used for rapid functional analysis of the novel genes and pathways identified here.…”

TGIF1is required for chicken ovarian cortical development and generation of the juxtacortical medulla

“…Their ability to knock down TGIF1 expression was assessed in vitro in chicken fibroblastic DF-1 cells. Firstly, DF-1 cells were transfected with the plasmids containing BFP-T2A and a non-specific shRNA (firefly sh774) (Roly et al, 2020) or with 4 different putative shRNA designed for TGIF1 knockdown (sh318, sh364, sh416 and sh998), following the Lipofectamine 2000 protocol (Life Technologies). After all cells were BFP positive, they were transfected with the TOL2-GFP-T2A-TGIF1 overexpression plasmid, a transposase expressing plasmid and a TOL2 plasmid expressing mCherry (as a transfection control) following the Lipofectamine 2000 protocol (Life Technologies).…”

“…TGIF1 shRNA design and electroporation: TGIF1 shRNA design, validation and cloning was performed as previously described (Roly et al, 2020, Major et al, 2019. Four different shRNAs were designed against TGIF1 ORF, ranked for effectiveness (Clarke et al, 2017) and cloned into RCAS(D)-nBFP plasmid.…”