Fig. 4. Decrease in Pax6, Six3 and Chx10 expression following MeisEnR transfection. (A-H) Expression of Pax6 (A-D), Chx10 (E,F) or Six3 (G,H) in HH15 chick eyes transfected with control-EnR (A,C,E,G) or MeisEnR (B,D,F,H). (A,B,E,F) Wholemounts. (C,D,G,H) Vibratome sections. (I) Quantification of the gene expression changes observed. (J) RT-PCR of two independent experiments (Exp_a, Exp_b) for Pax6 and β-actin of chick HH15 eyes transfected with GFP (control) or M1si_I and M2si_I together (Msi). PCR conditions were: 30 seconds 94°C, 30 seconds 60°C, 1 minute 72°C for 27 cycles. (K) Relative pixel intensity of the ethidium bromide-stained bands from J (Pax6/β-actin). Simultaneous knock-down of Meis1 and Meis2 expression decreases Pax6 transcript levels in the eye. (L,M) Pax6 fails to rescue cyclin D1 expression in MeisEnR-transfected chick retinae. Arrows indicate domains of MeisEnR/Pax6 misexpression and reduced cyclin D1 expression. (M) cyclin D1 expression following transfection of MeisEnR together with Pax6. (N-P) Expression of cyclin D1 (N) or cyclin D2 (P) following Pax6EnR misexpression. (O,L) Transfection control. L,M and N-P are neighboring sections. (Q) Quantification of the observed gene expression changes from Fig. 3F-J and data not shown.