In situ staining of mRNA for down-regulated genes in E11.5 rat whole embryos. (A) Whole mount in situ hybridization (WISH) using an antisense RNA probe for Pax6 . Localization of Pax6 mRNA (blue-violet) was detected in the forebrain, hindbrain and spinal cord of all embryos of the three genotypes. (B) Immunostaining of E11.5 rat embryos with the monoclonal antibody against the PD of Pax6 . The localization of the Pax6 protein (brown) is nearly identical to that of Pax6 mRNA in WT and rSey2/+ embryos. The expression of the Pax6 protein is reduced in the rSey2/+ embryo and is not detected in the rSey2/rSey2 embryo. (C-F) WISH using probes for the down-regulated genes. Expression of Fabp7 (C) and Unc5h1 (E) is reduced in rSey2/+ embryos and not present in rSey2/rSey2 embryos. While there were no significant differences found between WT and rSey2/+ embryos with respect to the expression of Dbx1 (D) and Cyp26b1 (F), the expression of these mRNAs was markedly decreased in the hindbrain (D, F) and spinal cord (D) of the rSey2/rSey2 embryo. (G) WISH using a probe for RARβ. The expression of RARβ is up-regulated in the rSey2/+ embryo and is up-regulated to a greater extent in the rSey2/rSey2 embryo. Scale bar: 1 mm.