(A,B) Whole mount ISH for gfp in Tg(Bre:gfp) embryos, lateral views, anterior to the left. (A) Wildtype and (B) following injection with 25 ng/embryo BMP4/7 into the arch region at 20 hpf. (C) Histogram quantifying Mc length (µM) in controls, dntcf3+ embryos, and dntcf3+ embryos injected with either 50 ng of BMP4/7 or 5 ng EDN1. (D) Histograms quantifying numbers of dntcf3+ embryos, alone or injected with BMP4/7 or EDN1 proteins, with reductions in Ch (left) and joint fusions between Mc and Pq (right). Numbers of embryos with rescue of Ch or Mc-Pq were averaged from three independent experiments (minimum of 10 embryos per protein injection experiment) and samples compared using a paired student t-test. (E, G, I) Whole mounted, alcian-stained 4 dpf larvae; control (E), dntcf3+ (G), and dntcf3+ injected with 25 ug BMP4/7 (I), lateral views, anterior to the left. Arrowhead in G indicates Ch loss. Black lines in G and I indicate Mc length and asterisks indicate Mc-Pq joint fusion. (F, H, J–L) Dissected, flat-mounted alcian-stained cartilages at 4 dpf, ventral views, anterior to the left; control with unilateral injection of 50 ng BMP4/7 (F), dntcf3+ (H), dntcf3+ with 25 ng BMP4/7 (J), control with 5 ng EDN1 (K) and dntcf3+ with 5 ng EDN1 (L). Arrowhead in F indicates duplicate Mc. Black lines in J indicate Mc length. Arrowheads in H indicate Sy and Ch. Asterisks in H, J and L indicate Mc-Pq joint fusions. **P<0.001. Abbreviations: Ch, ceratohyal; mc, Meckel′s; mc′, duplicated Meckel′s; Sy, symplectic. Scale bar: 100 µm.
