BMP4 knockdown of NCSCs leads to aganglionosis in the middle embryonic stage

Abstract

Transplacental bone morphogenetic protein (BMP)4 RNA interference (RNAi) is a technique used to knockdown genes in embryos. BMP4 are essential for the development of nervous system in the differentiation of neural crest stem cells (NCSCs). The failure of differentiation and migration of NCSCs may lead to aganglionosis. In the present study, pregnant mice were divided into three groups: Ringer's group, pSES group and RNAi-BMP4 group. In order to silence the BMP4 gene in the first generation (F1), 11.5 day pregnant mice were injected with the small interfering RNA BMP4 plasmid, pSES or Ringer's solution via the tail vein. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)and western blotting were employed to ensure the downregulation of BMP4. Finally, X-rays were performed following a barium enema. Aganglionosis was diagnosed by general anatomy and immunohistochemistry. Compared with the control group, transplacental RNAi was able to downregulate the BMP4-Smad4 of 11.5 day embryos, as determined by semi-quantitative RT-PCR and western blotting. The megacolons of the mice were demonstrated by X-ray and confirmed by general anatomy. Aganglionosis of colonic mucosa and submucosa were diagnosed by pathology, and immunohistochemistry. Knockdown of BMP4 in pregnant mice at the middle embryonic stage led to aganglionosis. It was therefore demonstrated that BMP-Smad was essential to the NCSCs of middle stage embryos. BMP-Smad served important roles in the generation of aganglionosis. This technique of knockdown BMP4 gene may be used to establish an aganglionosis mouse model.