Wnt signaling pathway regulates differentiation of chicken embryonic stem cells into spermatogonial stem cells via Wnt5a

In this study, we investigated the mechanism of signaling pathway-mediated differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs) in chicken. The Wnt signaling pathway was identified based on previous RNA Sequencing results and was proven a crucial signaling pathway that participates in the differentiation of ESCs into SSCs. In retinoic acid (RA) induction experiments in vitro, we found that Wnt signaling expression was inhibited by Wnt5a-shRNA, resulting in decreased expression of corresponding marker genes in SSCs, C-kit, Cvh, integrin 6 and integrin 1, but it was significantly promoted by RA treatment. Immunofluorescence assay showed that percentage of C-kit, Cvh, and integrin 6 and integrin 1-positive cells in RA treatment group and Wnt5a overexpression group was significantly higher than that in Wnt5a signaling interference group. Results of fluorescence-activated cell sorting analysis (FACS) also showed that proportion of germ-like cells was reduced by 14.3% (from 18.3% to 4.0%) at day 4 and 15.4% (from 18.6% to 3.2%) at day 12 after transfection, respectively. In experiments in vivo, shRNA-Wnt5a was stably expressed in fertilized chicken embryos and significantly reduced germ cell formation by 11.3% (from 21.7% to 10.4%) and 3.7% (6.4% from 10.1%). Results of quantitative PCR (qRT-PCR) and western blot assays showed that the expression of some specific germ cell marker genes, integrin 6 and integrin 1, was significantly suppressed following Wnt5a signaling interference in vivo. Taken together, our study suggests that Wnt signaling pathway could regulate positively the differentiation of chicken ESCs into SSCs through Wnt5a.