Fig. 6. Cited2 is a positive regulator for Pax6 expression. (A,B) Expression of Pax6 in Cited2^-/- lens epithelial cells. Immunostaining revealed an appreciable level of Pax6 expression in Cited2^-/- lens epithelial cells at 13.5 dpc (B) compared with that of wild-type littermate controls (A). (C) Pax6 mRNA expression in developing lens at 14.5 dpc was analyzed by real-time PCR, which revealed a 2.5-fold reduction of Pax6 expression in Cited2^-/- lens (n=4) as compared with wild-type littermate controls (n=4). (D,E) Effect of Cited2 on Pax6 autoregulation. Transcriptional activation of reporters containing Pax6 enhancer and promoter fragments, LE9-P0, LE0-P0 and P0, in Cited2- or Pax6-overexpressing cells was measured by reporter assays. -TN4-1 mouse lens epithelial cells were transfected with a reporter plasmid containing the indicated fragment (270 ng) with different combinations of Cited2 (225 ng) and Pax6 (75 ng) expression plasmids. Cited2 overexpression in Pax6-expressing -TN4-1 cells significantly increased the activity of LE9-P0, LE0-P0 and P0 reporters (D). This effect was Pax6-dependent because Cited2 overexpression had no effect on LE9-P0 reporter activity in NMuMG and HEK293 cells, which do not express Pax6, but co-expression of Pax6 and Cited2 significantly increased the reporter activity (E). (F) Cited2 is present on the Pax6 promoter. ChIP assays were performed using 2x10⁶ -TN4-1 cells and antibodies against Cited2 and Pax6. The precipitated DNA was analyzed by PCR with primers covering the LE9 ectoderm enhancer and the P0 promoter region. Normal mouse IgG and PCR amplifying the sequence between LE9 and P0 were also included as negative controls. Input was 10% of the chromatin for immunoprecipitation. Representative pictures show occupancy of Cited2 on LE9 (a) and P0 region (b) as compared with the negative control (NC) (c). (G,H) Histological examination was performed after H&E staining of paraffin-embedded eye serial sections collected from Cited2^-/-;PAX77^- and Cited2^-/-;PAX77⁺ littermates at 14.5 dpc. Abnormal lens stalk formation was consistently detected in Cited2^-/-;PAX77^- embryonic eyes (n=2) (arrowhead in G). However, no lens stalk was detected in any of the serial sections collected from Cited2^-/-;PAX77⁺ mouse embryos (n=3) (H).