To further verify the results of RT-qPCR, we performed PH3 staining to assess the proliferation of neuroepithelial cells by the use of immunohistochemical assay, which showed that PH3 staining in NTD embryos (Fig. 5b) was less obvious than that in control embryos (Fig. 5a) (P < 0.05). Here, PHC3 is linked to neural tube defect.