FIGURE 6

FIGURE 6

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.