Fig. 9. Msx1/2 are required for the survival of cranial neural crest cells. (A-F) Double-label immunostaining of apoptotic and proliferating cells. Immunostaining of phosphorylated histone H3 (Rhodamine, red) and TUNEL (FITC, green) were carried out on the same sections to detect cell proliferation and apoptosis in Msx1/2 mutants. Nuclei were counterstained with DAPI. Arrowheads indicate increased apoptotic neural crest-derived cells in the region of the trigeminal ganglion (B), the proximal portion of pharyngeal arch1 (maxillary prominence; D), and the distal tip of pharyngeal arch1 (mandiblular prominence; F) of Msx1/2 mutants at E9.5. Cell proliferation within these sites was not noticeably altered. (G-L) Nile Blue-stained E9.5 control (G,I,K) and Msx1/2 mutant (H,J,L) embryos; lateral (G-J) and dorsal (K,L) views. Increased cell death was detected in neural crest-derived craniofacial structures of the Msx1/2 mutant (arrows, arrowheads and open arrowheads in H and J) consistent with results of the TUNEL assay. We did not detect increased cell death in cardiac neural crest cells in mutant embryos (J). Note the lack of discernable change of cell death in the mutant hindbrain (L). Scale bar in L: 0.2 mm (for G-L).