Fig. 8

H3.3 function is required tissue- and cell-autonomously for CNC development.

a, Wild-type cells were transplanted unilaterally into the CNC precursor domain of h3f3a^{db1092/db1092} homozygous mutants at 6 hpf. b, Compared to the non-recipient control side (bottom), expression of the early CNC marker snai2 is restored in the recipient side at 11hpf (top) (n = 17/29 with rescue). c–e, fli1a:GFP (green) marks CNC ectomesenchyme of the pharyngeal arches at 30 hpf and facial skeletal elements at 5 dpf. The red fluorescent dye, Alexa568, marks transplanted wild-type cells, whereas both donor and host cells harbor the fli1a:GFP transgene. When transplanted into an h3f3a^{db1092/db1092} homozygous host, wild-type Alexa568+ CNC precursors contribute to pharyngeal arch ectomesenchyme and rescue arch size (c) and form wild-type cartilage and bone (e). In contrast, the non-recipient control side (d) has reduced pharyngeal arch ectomesenchyme. Whereas wild-type donor cells appear yellow due to red Alexa568 and green fli1a:GFP, mutant host cells have only fli1a:GFP and hence appear green. Rescue of arch size was observed in 8/11 cases. f, Alcian staining at 5 dpf shows that cartilage is restored to half the face in an h3f3a^{db1092/db1092} larvae that received an unilateral wild-type CNC precursor transplant. Compare the recipient side (left) to the control side that forms little facial cartilage (right). Skeletal rescue was observed in 21/30 cases. g, Individual cells of 32-cell stage sox10:GFP embryos were injected with mRNA encoding mCherry-tagged versions of wild-type or D123N H3.3 to generate mosaic mCherry-H3.3 expression at later stages. h, Soon after the appearance of GFP-labeled CNC at approximately 11 hpf, mosaic embryos were assessed for incorporation of mCherry-H3.3-expressing cells (red) into the sox10:GFP-positive CNC domain (green). i/i′/i′′ and j/j′/j′′, Confocal images from sox10:GFP embryos with mosaic expression of wild-type (i/i′/i′′) and D123N (j/j′/j′′) versions of mCherry-H3.3. Cells doubly-positive for wild-type mCherry-H3.3 (red) and GFP (green) (arrowheads) were observed within the CNC domain (7/14 cells over 4 embryos), whereas mutant D123N mCherry-H3.3 cells within the CNC domain failed to up-regulate sox10:GFP (arrowheads) (0/18 cells over 4 embryos). Only cells with strong mCherry-H3.3 were used in the analysis. hs: hyosymplectic cartilage, pq: palatoquadrate cartilage, ch: ceratohyal cartilage, op: opercular bone. Scale bars: b, f, 250 μm; c–e, 50 μm; i and j,10 μm.