Fig. 6. Morphological and cellular basis of the forebrain defects in Cnbp^-/-. (A,B) Disorganization of the axial mesendoderm and ANE region of cells (arrow) in E7.5 Cnbp^-/- mutants (A) compared with that (arrow) of wild-type littermates (B). (C,D) Lack of ANE and characteristic headfold structure in E8.5 Cnbp^-/- embryos compared with that of wild-type littermates. (E-H) Evidence for decreased proliferation rate in mutant head plate by BrdU incorporation analysis in adjacent sections of wild-type (E,G) and Cnbp^-/- mutant embryos (F,H). Arrow in F indicates that BrdU-positive nuclei was rarely seen in the anterior region of the mutant compared with that in wild-type embryo (arrow in E). Note that the head regions of wild-type embryos exhibit a high density of BrdU-positive nuclei throughout the axial mesendoderm and ANE regions at E7.5, and in prechordal mesoderm and headfold regions at E8.5. The mutants showed much fewer BrdU-positive nuclei at the same regions. (I-L) TUNEL apoptosis assays in the histologically normal and mutant E7.5 and 8.5 embryos. TUNEL assays showed there was no significant difference of apoptosis in the anterior region of wild-type (I,K) and mutant (J,L) E7.5 and E8.5 embryos. (M-P) E6.0 Cnbp^-/- mutant embryos and wild-type littermates were examined for general morphology (M,N) and cell division using a BrdU incorporation assay (O,P). BrdU-positive nuclei in mutant embryos were absent in the AVE of E6.0 mutant embryos (arrow in P). By contrast, the AVE region in normal E6.0 embryos showed the greatest density of BrdU-positive nuclei (arrow in O). (Q) Quantification of BrdU-positive nuclei in the anterior region of E7.5 embryos. The percent of BrdU-positive nuclei was 84% in wild-type embryos compared with 28% in null-mutant embryos. Error bars represent s.e., counts were made of three wild-type embryos (blue bar) and three null-mutant embryos (red bar). These values were determined to be statistically significant (P<0.001).

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