Regulation of Hedgehog Signaling in Chicken Embryonic Stem Cells Differentiation Into Male Germ Cells (Gallus)

The study aims to analyze the key signaling pathways in regulating the process of embryonic stem cells (ESCs) differentiation into spermatogonial stem cells (SSCs). Based on RNA Sequencing result, we further explored the specific regulating mechanisms of Hedgehog (HH) signaling in this process. HH signaling was found to be a crucial signaling pathway participating in the differentiation process of ESCs to SSCs. In Retinoic acid (RA) induced in vitro differentiation assay, the expression of two germ cell marker genes, integrin 6, and integrin 1, was observed to significantly increase, while it decreased dramatically when IHH was knocked down. Fluorescence activated cell sorting analysis showed that the proportion of integrin 6(+) and integrin 1(+) cells in the RA group was significantly higher than that in the RA+siRNA- Indian Hedgehog (IHH) group. In in vivo situations, siRNA-IHH could stably express in fertilized chicken embryos and significantly down-regulate the IHH expression. With real-time quantitative PCR and western blot, we identified that integrin 6 and integrin 1 expression was significantly suppressed along with IHH inhibition in vivo. We concluded that Hedgehog signaling pathway positively regulates the differentiation of ESCs to male germ cells through signal transduction by IHH. J. Cell. Biochem. 118: 1379-1386, 2017. (C) 2016 Wiley Periodicals, Inc.