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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).
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