Fig. 5. Analysis of apoptosis and cell proliferation in Chrd^-/-;Nog^+/- mutants. (A-D) TUNEL/proliferation assay on sections of wild-type (A,C) and Chrd^-/-;Nog^+/- mutant (B,D) embryos. E8.5 Chrd^-/-;Nog^+/- mutant (B) shows a 36% decrease in proliferation (labeled in red, P=0.018), while differences in apoptosis (labeled in green) were not observed in section. E9.5 mutant (D) shows a nearly 10-fold increase in apoptotic cells (947%; P=0.019) in the neural ectoderm rostral to the optic vesicles (ov) Increased apoptosis is also seen in rostral mesenchyme (mc) and trigeminal ganglion (V). Proliferation at E9.5 is not significantly different from wild type. Similar results were seen in three or more independent experiments. (E-H). Nile Blue Sulfate staining of wild-type (E,G) and Chrd^-/-;Nog^+/- mutant (F,H) embryos. (F) Lateral aspect of eight- to 10-somite mutant showing expansion of apoptosis in dorsal domains (arrowheads), and loss from the ventral midline (arrow; n=3). (H) Frontal aspect of 20-25 somite mutant showing expanded apoptosis in the dorsal midline (arrows) and frontonasal mass (n=5). (I-L) Rostral expression of Msx1 in wild-type (I,K) and Chrd^-/-;Nog^+/- mutants (J,L). (J) In severely affected mutants, rostral domains of Msx1 are expanded at eight to 10 somites (n=2). At E9.5 Chrd^-/-;Nog^+/- expression domains have shifted toward the midline (L, arrow) though expression levels are comparable with those in wild type (n=3). Scale bars: 0.2 mm.

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