Fig. 7. TUNEL analysis of whole-mount and sectioned preparations specimens reveals similar patterns of abnormal neural crest apoptosis in Fgf8 hypopmorphs, Fgf8/AP2-IRESCre and Fgf8/HoxA3-IRESCre mutants. Both experiments were repeated three times with comparable results. The whole-mount experiment was performed with all embryos in the same tube; the sectioned specimens were all processed simultaneously. PAs are numbered; o, otocyst. (A-H) Right and left views of 25-26 ss stage-matched embryos. (A,E) A wild-type embryo has minimal apoptosis in the region of PAs 3-6 (white ovals, white arrowhead). The white line indicates plane of section for fluorescent immunohistochemical analysis shown in I-L. (B,F) An Fgf8 hypomorph replicates our previous finding of abnormal domains of apoptosis in PAs 3-6 (yellow arrowheads) (Frank et al., 2002). (C,G) Fgf8/AP2-IRESCre and (D,H) Fgf8/hoxa3-IRESCre mutants have increased NC apoptosis in the same regions noted in the hypomorph (yellow arrowheads). (I-L) Fluorescent immunohistochemistry was performed cryosectioned 25 ss, stage-matched control versus hypomorphic and domain-specific mutants using a combination of anti-AP2/FITC (green fluorescence) to detect neural crest cells (NC) and TUNEL/Texas red (red fluorescence). Embryos were sectioned transversely in parallel with the third PA (see white line in A). Each row shows a representative section from a single embryo, proceeding from anterior to posterior through the postotic region (from the third PAA to the developing fourth and sixth arch region). Sections were carefully matched to represent the same region of each embryo in each column, taking into consideration the profoundly perturbed anatomy and severe pharyngeal hypoplasia of Fgf8 hypmorphic and Fgf8/AP2-IRESCre mutants. White asterisks indicate the third PAA in parallel (not present in all sections), yellow arrowheads indicate regions of abnormal apoptosis and dying NC. The dorsal aorta is labeled (ao). (I) A control embryo has minimal apoptosis in NC migrating into the third PA or region of the developing fourth and sixth PAs. (J) An Fgf8 hypomorph has large domains of abnormal apoptosis. Double-labeled NC are migrating from rhombomere 6 into the lateral third arch. Note that the pharynx is poorly segmented and that the PAs and third PAA are hypoplastic. (K) Fgf8/AP2-IRESCre and (L) Fgf8/hoxa3-IRESCre mutants have NC apoptosis in the same domains as the hypomorph. In these examples, the third PAA is pathologically enlarged (see also Fig. 5C,D).