Loss of skeletogenic mesenchyme in the anterior region of developing hyomandibular plate in pax1a mutants. (A, B) Fluorescent in situ hybridization for barx1 (green) at 36 hpf shows almost identical condensations of osteochondral progenitors in the dorsal region of the hyoid arch of wild types and pax1a mutants (arrows). (C, D) Double in situ hybridization for barx1 (green) and sox9a (red) at 48 hpf. (C) In wild types, chondrocytes partially co-expressing barx1 and sox9a and barx1-expressing mesenchymal cells are observed in the anterior region of the developing HM (arrow). (D) In 11 of 53 pax1a mutants, those chondrocytes and mesenchymal cells are not seen in the anterior region of the developing HM (arrowhead). Scale bar = 40 μM. Anterior is to the left. Dorsal is at the top. (E–H) BrdU staining (red) visualizes proliferating cells relative to Tg(sox10:GFP) expressing mesenchymal cells (green) in wild-type siblings and pax1a mutants. The dorsal regions of the hyoid arches are boxed. The upper and lower boundaries of the box are set along the dorsal and ventral ends of the first pouch. The anterior and posterior boundaries are defined between the first pouch anteriorly and the core mesoderm posteriorly located at the center of the hyoid arch. (I) Quantification of the number of proliferating cells in the dorsal region of the hyoid arch [boxed in (E–H)]. Data is represented on a scatter plot. n.s., not significant. (J–M) Lysotracker Red staining (red) labels dying cells relative to Tg(sox10:GFP) expressing mesenchymal cells (green) in wild-type siblings and pax1a mutants. The dorsal regions of the hyoid arches are boxed, which are set with the same definition as in (E–H). Scale bar = 20 μM. Anterior is to the left. Dorsal is at the top. (N) Quantification of the number of dying cells in the dorsal region of the hyoid arch [boxed in (E–H)]. Data is represented on a scatter plot. * shows p < 0.05. n.s., not significant. n, number of animals analyzed.
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