A dominant H3.3 mutation results in losses of CNC–derived head skeleton and pigment cells.
a, b, fli1a:GFP-labeled arch ectomesenchyme (arrowheads) is greatly reduced, yet fli1a:GFP-positive endothelial cells (top) are unaffected, in both homozygous and heterozygous h3f3adb1092 mutants at 34 hpf (7/7 mutant; 0/10 wild-type). c, d, Homozygous h3f3adb1092/db1092 embryos specifically lack the CNC-derived head skeleton at 5 dpf (36/71 complete loss; 35/71 partial loss). Diagrams show the CNC-derived cartilage (blue) and bone and teeth (red), mesoderm-derived cartilage (green), pectoral fin cartilage (black), and eyes (yellow). e, f, h3f3adb1092/+ heterozygous larvae exhibit a wide range of craniofacial defects. In some cases, no defects are observed in the facial skeleton and heterozygotes are adult viable (not shown). In mild cases (e), dorsal cartilage and bone of the first and second arches are preferentially reduced, including the dorsal hyosymplectic cartilage and opercular bone of the second arch (arrow). In more severe cases (f), the cartilage and bone of the first arch and dorsal second arch are greatly reduced, with the anterior neurocranium and the posterior ceratobranchial cartilages being less affected. The frequency of skeletal phenotypes in h3f3adb1092/+ heterozygous larvae is highly variable between clutches. g, h, At 27 hpf, dct-positive melanophore precursors are selectively missing anterior to the ear (arrowheads) in both homozygous and heterozygous h3f3adb1092 embryos (5/5 mutant; 0/5 wild-type). i, j, At 27 hpf, cranial xdh-positive xanthophore precursors are mildly reduced in both homozygous and heterozygous h3f3adb1092 embryos (6/8 mutant; 0/4 wild-type). k, l, Wild-type and both homozygous and heterozygous h3f3adb1092 embryos have comparable numbers of foxd3-positive glial cells at 24 hpf (4 mutant; 5 wild-type). m-r, D123N h3f3a mRNA-injected but not wild-type h3f3a mRNA-injected embryos lack fli1a:GFP-positive ectomesenchyme (9/19 D123N; 0/12 wild-type), CNC-derived head skeleton (24/45 D123N; 0/21 wild-type) and cranial dct-positive melanophore precursors (anterior to the ear: arrowheads) (7/14 D123N; 0/11 wild-type). fli1a:GFP-positive blood vessels are unaffected (arrows). s, Except for the loss of the majority of the skull (no facial structures below the level of the eye: arrowheads) and mild heart edema (arrows), the overall morphologies of wild-type, homozygous and heterozygous h3f3adb1092, wild-type h3f3a-injected, and D123N h3f3a-injected larvae are indistinguishable at 5 dpf. Melanophores (black) and xanthophores (yellow) are also largely normal. Except for panels e and f, homozygous h3f3adb1092 examples are shown. Scale bars: a, b, m & n, 50 μm; c–l, o–s, 250 μm.