Fig. 2: Vax1^AA exhibits defective intercellular transfer but intact transcription factor activity in vivo.

a Intercellular transfer of Vax1 from E14.5 Vax1^+/+ and Vax1^AA/AA littermate mouse OS cells to RGC axons was determined by immunostaining of the embryonic sections with α-Vax1 (green) and α-NF160 (red). b Expression of Vax1 and Vax1^AA mRNA in E14.5 Vax1^+/+ and Vax1^AA/AA littermate mouse eyes and OS was examined by ISH. Boxed areas in the leftmost column are magnified in two right columns. The specificities of the anti-Vax1 antibody (a) and Vax1 ISH probe (b) were determined by the absence of signals in E14.5 Vax1^−/− mice. The ISH signals of the sense probes for Vax1 did not exhibit specific signals (data not shown). The solid lines in the images indicate the boundary of OS, and the dotted lines mark the border between the OS and RGC axon bundles. NR, neural retina; RPE, retinal pigment epithelium; NBL, neuroblast layer; GCL, ganglion cell layer. c The activities of a Vax1 target Pax6 α-enhancer in mouse embryos were measured by detecting H2B-EGFP, the expression of which is induced together with membrane-bound tdTomato (tdTom*) at the R26^tm11 gene locus after α-Cre-dependent excision of the loxP-STOP-loxP cassette. Arrowheads point to the frontlines of the RGC axons. d Binding of Vax1 and Vax1^AA proteins to the Pax6 α-enhancer was determined by PCR detection of Pax6 α-enhancer sequences in DNA fragments isolated from E10.5 mouse embryonic cells by ChIP with the indicated antibodies (see details in Methods). Input, mouse chromosomal DNA; No Ab, no antibody; Rb IgG, preimmune rabbit IgG; α-Vax1, rabbit anti-Vax1 polyclonal antibody. e Relative levels of Pax6 α-enhancer sequences in the ChIPed samples were compared by qPCR. f The numbers on the y-axis are the average 2^-ΔCt values of the samples against the critical threshold (Ct) values of the input. Error bars denote SD (n = 5).