Requirement of Pax1a-EphrinB2a after pouch formation in hyomandibular plate development. (A, C) Zn5 immunohistochemistry to visualize pouches at 40 hpf. Arrowheads mark the first pouch (p1). After the heat-shock treatment at 39 hpf for 40 min, the first pouch is hypoplastic in pax1 double mutants carrying Tg(hsp70I:EfnB2a) transgene, which is similar to the hypoplasia first pouch in pax1 double mutants. Scale bar = 40 μM. (B, D) Lateral views of dissected facial skeletal elements of the mandibular and hyoid arches and inserts showing the zoomed-in HM plate (circled areas). After the heat shock treatment, the severe, intermediate, and weak defects in HM are seen in both pax1 double mutants and those bearing Tg(hsp70I:EfnB2a) transgene. No wild-type HM plate is observed in (B, D). Arrowheads indicate cells appearing in the HM plate in the intermediate group and the mono-layered cells adjacent to the foramen in the weak group. (E) Quantification of the frequency of each group between (B, D). The frequency of each group is counted. Data is represented on a pie chart. Black, yellow, and green represent the severe, intermediate, and weak groups. * indicates p < 0.05. (F) Quantification of HM defects. The number of cells in HM is counted. Data is represented on a boxplot. *** indicates p < 0.001. n, number of animals analyzed.
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