Fig. 1

Twist1 genes are required for ectomesenchyme specification in zebrafish.

(A–D) Whole mount in situ hybridizations of sox10 expression at 18 hpf show ectopic expression in the arches (numbered and second arch indicated by black arrow) of twist1a/1b-MO (n = 16/16) and sox10:Gal4VP16; UAS:dnTwist1b (n = 4/4) embryos compared to un-injected (n = 0/14) and sox10:Gal4VP16 only (n = 0/9) controls. White arrowheads indicate otic expression. (E–H) Whole mount in situs at 24 hpf show reduction of fli1a expression in the arch ectomesenchyme (numbered) of twist1a/1b-MO (n = 12/12) and sox10:Gal4VP16; UAS:dnTwist1b (n = 5/5) embryos compared to un-injected (n = 0/13) and sox10:Gal4VP16 only (n = 0/8) controls. Insets in E and F highlight arch ectomesenchyme which is reduced in twist1a/1b-MO embryos. Vascular expression of fli1a (red arrowheads) is unaffected. (I–L) Whole mount in situs at 18 hpf show a slight reduction of dlx2a in sox10:Gal4VP16; UAS:dnTwist1b embryos (n = 4/4) but not un-injected (n = 0/6), twist1a/1b-MO (n = 0/8), and sox10:Gal4VP16 only (n = 0/6) embryos. (M-P) Skeletal staining at 5 dpf shows severe loss of CNCC-derived head skeleton in twist1a/1b-MO embryos (n = 21/21) and primarily jaw reductions in sox10:Gal4VP16; UAS:dnTwist1b embryos (n = 9/9) compared to no defects in un-injected (n = 0/24) and sox10:Gal4VP16 only (n = 0/16) controls. Whereas only small remnants remain of the CNCC-derived skeleton (arrows), the mesoderm-derived otic capsule cartilage (arrowheads) and posterior neurocranium are less affected in twist1a/1b-MO embryos. (Q–T) In situs for dct expression at 28 hpf show normal melanophore precursors in un-injected (n = 14), twist1a/1b-MO (n = 12), sox10:Gal4VP16 only (n = 8), and sox10:Gal4VP16; UAS:dnTwist1b (n = 8) embryos. (U–X) In situs for xdh expression at 28 hpf show normal xanthophore precursors in un-injected (n = 7), twist1a/1b-MO (n = 5), sox10:Gal4VP16 only (n = 9), and sox10:Gal4VP16; UAS:dnTwist1b (n = 8) embryos. (Y-AB) In situs for foxd3 expression at 48 hpf reveal largely normal patterns of glia in un-injected (n = 9), twist1a/1b-MO (n = 8), sox10:Gal4VP16 only (n = 9), and sox10:Gal4VP16; UAS:dnTwist1b (n = 8) embryos. Scale bar = 50 μm.