Expression of pax1a and pax1b in the mature pouches. (A–D) Fluorescence in situ hybridization of pax1a and pax1b (green) in conjunction with the GFP immunohistochemistry (red) in wild-type Tg(sox17:GFP) reporter lines marking the pharyngeal endoderm and pouches. Pouches are marked as p1-p6. Arrowheads indicate the expression of pax1a and pax1b in the first pouch (p1). (A, C) At 36 hpf, transcripts for pax1a and pax1b are seen in sox17-positive pouches. (B, D) At 48 hpf, the expression of pax1a and pax1b continues in sox17-positive pouches. (A′–D′) Green channel only. Scale bar: 40 μm. Anterior is to the left.

Requirement of pax1a in hyomandibular cartilage development. (A–I) Craniofacial skeletal elements stained with Alcian Blue (cartilage) and Alizarin Red (bone) at 4 dpf, with the hyomandibular (HM) plate circled. (A, C, F, H) Ventral views of dissected facial cartilages. (B, D, E, G, I) Lateral views of dissected facial skeletal elements of the mandibular and hyoid arches, along with inserts showing the anterior and posterior regions of HM (circled areas). (A, B) All facial skeletal elements are present in wild types, including the triangular HM plate, with an arrowhead indicating the multi-layered cells in the anterior region of HM. Five CBs on one side are underlined. (C–E) In pax1a mutants, a specific defect is seen in the HM plate, with other skeletal elements unaffected. Arrowheads indicate mono-layered cells (D) and missing cells (E) in the anterior region of HM. (F, G) In pax1b mutants, all facial skeletal elements are normal, including the HM plate. The normal multi-layered cells in the anterior region of HM are marked with an arrowhead (H, I) In double mutants for pax1a and pax1b, the HM plate is defective, and most CB cartilages are missing. Arrowhead indicates the missing cells in the anterior region of HM. M, Meckel’s cartilage; PQ, Palatoquadrate cartilage; HS, Hyosymplectic cartilage; CH, Ceratohyal cartilage; CB, Ceratobranchial cartilage; OP, Opercle bone; A, anterior; P, posterior. Anterior is to the left. (J) The number of mutants analyzed and the penetrance for HM defects. (K) Quantification of the number of cells in HM. Data is represented on a boxplot. *** shows p < 0.001.

Rescue of the defects in the first pouch and hyomandibular cartilage in pax1 double mutants with an enforced expression of pax1a at 16 hpf. (A, C) Zn5 staining labels to visualize pouches at 37 hpf. The first pouches are marked with arrowheads. After the heat-shock treatment at 16 hpf for 40 min, while only two pouches (p1 and p) with abnormal shapes are seen in pax1 double mutants (A), the first pouch (p1) is pretty normal, with other pouches disorganized, in pax1 double mutants carrying Tg(hsp70I:Pax1a) transgene (C). p, pouch. Scale bar = 40 μM. (B, D) Unilateral dissections of the skeletons of the mandibular and hyoid arches stained with Alcian Blue (cartilage) and Alizarin Red (bone) at 5 dpf, along with inserts showing the zoomed-in HM plate (circled areas). (B) After the heat shock treatment in pax1 double mutants, a rod-shape HM is seen in the severe group, more cells appear in the rod-shape HM in the intermediate group compared to the severe group, and the foramen is seen, with adjacent mono-layered cells in the weak group. Arrowheads indicate the cells appearing in the HM plate in the intermediate group and the mono-layered cells adjacent to the foramen in the weak group. No wild-type HM plate is observed. (D) After the heat shock treatment in pax1 double mutants bearing Tg(hsp70I:Pax1a) transgene, HM defects in all three groups were observed, but with a different frequency than those observed in (B); wild-type HM plates are also observed. Arrowheads indicate the cells appearing in the HM plate in the intermediate group and the multi-layered cells adjacent to the foramen in the wild-type HM plate. (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/wild-type groups, respectively. **** indicates p < 0.0001. (F) Quantification of HM defects. The number of cells in HM is counted. Data is represented on a boxplot. **** indicates p < 0.0001. n, number of animals analyzed.

Rescue of the defects in the hyomandibular cartilage in pax1 double mutants with an enforced expression of pax1a at 36 hpf. (A, C) Zn5 immunohistochemistry to visualize pouches at 37 hpf. Arrowheads mark pouches. After the heat-shock treatment at 36 hpf for 40 min, pax1 double mutants display two pouches (p1 and p) with abnormal shapes (A), and pax1 double mutants carrying Tg(hsp70I:Pax1a) transgene show a distorted pouch (p), with the first pouch (p1) barely seen (C). Scale bar = 40 μM. (B, D) Lateral views of dissected facial skeletal elements of the mandibular and hyoid arches, along with 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:Pax1a) transgene. No wild-type HM plate is observed in (B, D). Cells appearing in the HM plate in the intermediate group and the mono-layered cells adjacent to the foramen in the weak group are indicated by arrowheads. (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, respectively. * 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 < 0.01. n, number of animals analyzed.

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.

Genetic linkage of EphrinB2a with Pax1a in hyomandibular plate development. (A–F)In situ hybridization of efnb2a (green) in conjunction with the GFP immunohistochemistry (red). In wild types bearing Tg(sox10:GFP) transgene, efnb2a expression in the first pouch at 36 (A) and 40 hpf (C) is seen (arrows). In the first pouch of pax1a mutants, efnb2a expression at 36 hpf is unaffected [arrow in (B)] but is reduced at 40 hpf in 22 out of 32 animals [arrowhead in (D)]. In the first pouch of pax1b mutants, efnb2a expression at 40 hpf is unaffected [arrow in (E)]. In pax1a and pax1b double mutants, efnb2a expression in the first pouch at 40 hpf is abolished [arrowhead in (F)]. Scale bar = 20 μM. Anterior is to the left. (G) Relative expression levels of efnb2a mRNAs in wild types and pax1 mutants. Expression in wild types set at 1. Transcripts of efnb2a are downregulated in pax1a mutants at 40 hpf but not at 36 hpf. Expression of efnb2a mRNAs is further reduced in pax1a and pax1b double mutants at 40 hpf compared to pax1a mutants. Transcripts of efnb2a are barely affected in pax1b mutants at 40 hpf compared to wild types. ** and **** show p < 0.01 and p < 0.0001, respectively. n.s., not significant. (H, I) Confocal projections from live imaging of embryos bearing Tg(her5:mCherryCAAX) (red) and Tg(sox10:GFP) (green) transgenes at 34 hpf, followed by Alcian Blue staining (blue) in the same individuals at 4 dpf. In a wild-type individual, a bilayered normal first pouch is seen at 34 hpf [white arrow in (H)], with the triangular HM plate observed at 4 dpf [black arrow in (H)]. In an efnb2a mutant individual, the first pouch was hypoplastic at 34 hpf [white arrowhead in (I)], with the HM plate defective at 4 dpf [black arrowhead in (I)]. (J–M)In situ hybridization for pax1a (green). Compared to wild types, pax1a expression in the first pouch was unaffected in efnb2a mutants at 36 and 40 hpf [arrows in (K, M)]. Scale bar = 20 μM. Anterior is to the left. n, number of animals analyzed.

Requirement of Pax1a-EphrinB2a in the first pouch for hyomandibular plate development. (A–C) Unilateral dissections of the skeletons of the mandibular and hyoid arches stained with Alcian Blue (cartilage) and Alizarin Red (bone) at 4 dpf. HM plates are circled. The severe, intermediate, and weak defects in HM are seen in both pax1 double mutants (A) and their siblings expressing Pax1a (B) or EfnB2a (C) in sox17-positive endoderm. Anterior is to the left. (D, E) Quantification of the frequency of each group between pax1 double mutants and their siblings carrying Tg(sox17:Gal4VP16) and Tg(UAS:Pax1a)(D) or Tg(sox17:Gal4VP16) and Tg(UAS:EfnB2a) transgenes (E). The frequency of each group is counted. Data is represented on a pie chart. Black, yellow, and green represent the severe, intermediate, and weak/wild-type groups. *** and * indicate p < 0.001 and p < 0.05, respectively. (F, G) Quantification of the number of cells in HM. The number of cells in HM is counted. Data is represented on a boxplot. **** and *** show p < 0.0001 and p < 0.001, respectively. n, number of animals analyzed.

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.

A model for the role of Pax1a in hyomandibular plate development. In wild types, Pax1a is necessary for the morphogenesis of the first pouch that forms at 18 hpf, with the loss of Pax1a leading to a distorted first pouch. After pouch formation, pax1a expression in the first pouch continues by 48 hpf, which is required to maintain efnb2a expression there. After the condensation of the skeletogenic mesenchyme in the hyoid arch at 36 hpf, Efnb2a in the first pouch would provide a survival signal to the barx1-positive osteochondral progenitors in the dorsal region of the hyoid arch that differentiate into chondrocytes contributing to the anterior plate of hyomandibular cartilage at 48 hpf. In pax1a mutants, reduced expression of efnb2a in the first pouch at 40 hpf would be insufficient to promote the survival of barx1-positive osteochondral progenitors in the dorsal region of the hyomandibular arch. Consequently, in pax1a mutants, osteochondral progenitors contributing to the anterior plate of hyomandibular cartilage die, and the anterior HM plate is defective in the larval head. p1, first pouch; MA, mandibular arch; HA, hyoid arch; HS, hyosymplectic cartilage; HM, hyomandibular cartilage.