Grem2 promotes the dorsal and intermediate facial skeleton. (A) Structure of the grem2 gene and the Grem2:GFP fusion construct. grem2-MO #1 recognizes the ATG start site in Exon 2 and grem2-MO #2 recognizes the 5′ UTR. (B,C) Grem2:GFP fluorescence in uninjected (B) or grem2-MO-#1-injected (C) embryos at 9 hpf. (D,E) Confocal sections of 30 hpf BRE:GFP transgenic embryos. Compared with uninjected controls (D, n=5), injection of grem2-MO-#1 (E, n=8) increased BRE:GFP throughout the mandibular (1) and hyoid (2) arches. (F-H) Confocal sections of in situ hybridizations for hand2 (green) and dlx3b (red). Relative to the first pouch (white outlines), dlx3b and hand2 were mildly expanded in grem2-MO embryos (G, n=45/52) and lost in hsp70I:Gal4; UAS:Grem2 embryos subjected to a 16-17 hpf heat-shock (H, n=16/16). Arrow indicates expanded hand2 expression. (I-M) Ventral views of 5 dpf facial skeletons from control (I), grem2-MO-#1-injected (J), UAS:Grem2 (K), edn1–/– mutant (L) and an edn1–/– mutant injected with grem2-MO-#1 (M). Schematics show skeletal regions derived from dorsal (green), intermediate (red) and ventral (blue) arch domains, with bones lightly shaded. Elements of undefined morphology or derived from the maxillary domain or more posterior arches are grey. Scale bar: 50 μm.