Alk5/Wnt1-Cre mutants display increased apoptosis of post-migratory neural crest cells
As described above, Alk5/Wnt1-Cre mutants displayed an inadequate amount of AP-septal tissue in the base of the aortic sac between the origins of 4th and 6th PAAs. To analyze whether this phenotype resulted either from defective CNCC proliferation or inappropriate apoptosis, we used BrdU and TUNEL staining, respectively. While CNCC proliferation was not affected in Alk5 mutants (data not shown), we could detect a dramatic increase in the number of TUNEL positive cells in tissues surrounding the aortic sac including the site where the AP-septum forms (Fig. 7A–C). Dual staining for lacZ and TUNEL positive cells demonstrated that these cells were postmigratory CNCCs; this phenotype was already clearly detectable at E10.5. These results were confirmed by using immunostaining for cleaved caspase-3, another marker for apoptosis (Fig. 7I,J). In the chick, apoptotic neural crest-derived cells have also been found at the sites, where the prongs of the AP septum penetrate into the OFT cushion mesenchyme [25,26]. Thus, we compared apoptosis patterns also on the more proximal level, but found no detectable differences at E11.0 between Alk5/Wnt1-Cre mutants and controls (Fig. 7D,E). Unlike in Alk5/Wnt1-Cre mutant embryos, increased apoptosis of NC-derived cells is not responsible for the observed defects in the OFT septation in corresponding Alk2 mutants (Fig. 7C,E).
Figure 7  Aberrant apoptosis of NCCs in Alk5/Wnt1-Cre mutants. TUNEL (A-H) and Cleaved Caspase-3 (I, J) staining at E11.0 demonstrates a notable increase in apoptosis in Alk5/Wnt1-Cre mutants (B, H, J) on the aortic sac level when compared to controls (A, G, I) or Alk2/Wnt1-Cre mutants (C) (frontal sections), while sections on the OFT level do not demonstrate differences between controls (D) and Alk5 (E) or Alk2 (F) mutants. G,H, TUNEL staining of lacZ-stained embryos demonstrates that apoptotic cells are of neural crest origin. G, control; H, mutant. AS, aortic sac, arrows point to clusters of apoptotic cells surrounding the aortic sac. To conclude, our results suggest that in Alk5/Wnt1-Cre mutants a noticeable increase in apoptosis coincides with the abnormal patterning of the PAAs and the aortic sac, and with the failed AP-septation. These data support a specific role for ALK5 signaling, either directly or indirectly, in CNCC survival, since a similar apoptosis of NC-derived cells is not seen in Tgfbr2/Wnt1-Cre mutants [8,9].