Fig. 5

Injection of wild-type H3.3 RNA and reduction of mutant H3.3 levels both rescue craniofacial skeletal development in h3f3a^db1092 mutants.

a, b, Results from scoring the severity of craniofacial losses in 5 dpf larval head skeletons from h3f3a^db1092 siblings injected with 450 ng/µl mRNA encoding wild-type H3.3 or a control kikGR fluorescent protein. The scoring system ranges from Grade 0 (wild-type phenotype) to Grade 5 (complete loss of CNC derivatives); see Materials and Methods for more detail. H3.3-mRNA-injected h3f3a^db1092 homozygotes (a) and heterozygotes (b) exhibited a decrease in the severity of h3f3a^db1092 craniofacial phenotypes over kikGR-RNA-injected controls (significant by Fisher′s exact test: homozygotes, p = 7.7E-05; heterozygotes, p = 1.0E-04). c, An antisense morpholino oligonucleotide was designed to inhibit splicing at the exon 3/intron 3–4 boundary (green arrowhead) of the h3f3a transcript. Morpholinos were injected into one-cell-stage h3f3a^db1092 embryos at 400 µM. d, Morpholino efficacy was demonstrated by PCR amplification between exons flanking the targeted splice junction from 10 hpf cDNA from 20 pooled embryos (position of primers shown as red arrows in c). Compared to the sample from uninjected (un) embryos, the morpholino-treated sample (MO) exhibited a partial decrease in PCR product representing spliced transcript (295 bp) and a concomitant increase in un-spliced PCR product (390 bp). e, f, Compared to uninjected siblings, both morpholino-injected h3f3a^db1092 homozygotes (e) and heterozygotes (f) exhibited a decrease in the severity of h3f3a^db1092 craniofacial phenotypes (significant by Fisher′s exact test: homozygotes, p = 2.0E-04; heterozygotes, p = 6.3E-06). (g) Craniofacial development in wild-type embryos was unaffected by morpholino injection.