Figure 5: Jmjd3 is required for Shh-dependent CGNP proliferation.

(a) Global changes of modified histone levels during cerebellum development evaluated by western blot. (b) Immunostaining of sections of E16.5 and P5 cerebella with antibodies against modified histones and EZH2. The EGL is the dense cell layer outlining the cerebellum. (c) Local changes of H3H27me3 and H3K4me3 at Gli1 regulatory region in E16.5 and P5 cerebella (means±s.d., n=3). (d) RT–qPCR analysis of Jmjd3 in NIH3T3 and MEF cells and in cerebellum (CB) and medulloblastoma (MB) samples (means±s.d., n=3). (e) In situ hybridization of P5 CB with Jmjd3 antisense or sense probes. Note the enriched Jmjd3 expression in EGL. (f) H&E staining of cerebella from E18.5 wild-type and Jmjd3^−/− mice. The average length of EGL per cerebellum section is shown on the right (means±s.d., n=4). (g) Inhibition of Jmjd3 expression in cultured CGNPs results in defective Shh-induced gene expression as shown by RT–qPCR. (h) Jmjd3 knockdown in cultured CGNPs decreases proliferation (as shown by BrdU incorporation) (means±s.d., n=3). (i) Western blots indicating global histone modification levels in CGNP cultures in the absence and presence of Shh treatment with or without Jmjd3 shRNA treatment. (j) Immunostaining of sections of E18.5 wild-type and Jmjd3^−/− cerebella with antibodies against modified histones. (k) Decrease in Jmjd3 levels in Shh-treated CGNP cultures by lentiviral-expressed shRNA resulted in significant changes of histone modifications at the Gli1 regulatory region as shown by ChIP-qPCR. Histone H3 ChIP was used for normalization (means±s.d., n=3). Significance was determined by Student’s t-test; ** indicates P<0.01 and * indicates P<0.05.