BMP4/SMAD8 signaling pathway regulated granular cell proliferation to promote follicle development in Wanxi white goose.

Granular cells proliferation in goose regulated by bone morphogenetic proteins (BMPs) signaling pathway is still unknown. In this experiment, BMPs and their receptor, and receptor activated mothers against decapentaplegic homologs (SMADs) were quantitatively expressed in granular cell layer of pre-hierarchycal and hierarchycal follicles in Wanxi White goose. The screened BMP was then used for construction of overexpressed and knockdown vectors and transfected into granular cells of goose to assess the cell proliferation and apoptosis. Granular cells with BMP-overexpressed were then used for ChIP-Seq analysis to elucidate the molecular mechanism of BMP affecting granular cell proliferation. The results showed that the mRNA expression of BMP4 was significantly expressed in pre-hierarchical follicles, and also highly expressed in hierarchical follicles than other BMPs, while the Ⅰ and Ⅱ type of BMP receptors were expressed in basic level. The mRNA expression of SMAD8 was significantly elevated in pre-hierarchical follicles. Overexpression of BMP4 could promote the proliferation of granular cells and inhibited the expression of BMP4 caused a higher cell apoptosis. ChIP-Seq identified multiple regulatory targets of SMAD4, which were mostly related to cell cycle and lipid metabolism according to the GO and KEGG pathway enrichment. From the five most significant binding motif and quantitative expression verification, the activin membrane binding inhibitor (BAMBI) was down regulated in BMP4 overexpressed granular cells. In conclusion, the BMP4 was highly expressed in granular cells and phosphorylates SMAD8, the activated SMAD8 combined with SMAD4 transfers into nucleus to regulate the expression of BAMBI to promote lipid synthesis.