|
Fig. S6 Antisense morpholino targeting of H3.3 chaperones. a, Zebrafish have one predicted copy each of hira, daxx, and dek genes in their genomes. Antisense morpholino oligonucleotides were designed to inhibit splicing at specific exon/intron boundaries (green arrowheads) within hira (exon 1/intron 1–2), daxx (exon 3/intron 3–4), and dek (exon 3/intron 3–4) transcripts. Morpholinos were injected into one-cell-stage zebrafish embryos at 400 μM. b–d, Morpholino efficacy was demonstrated by PCR amplification between exons flanking targeted splice junctions from 10 hpf cDNA from 20 pooled embryos (position of primers shown as red arrows in a). Morpholino-treated samples exhibited a significant decrease in PCR product representing spliced transcript (b, hira, 96 bp; c, daxx, 143 bp; d, dek, 169 bp) and a concomitant increase in un-spliced PCR product (b, hira, 637 bp; d, dek, 247 bp) or an alternative spliced transcript (c, daxx, 169 bp: utilization of cryptic splice donor site 26 nucleotides into adjacent intron, predicted to result in frameshift and early termination). e–j, Wholemount in situ hybridization for sox10 at 11 hpf. Morpholinos against hira (f), daxx (h) and dek (j) have no effect on early sox10 expression within CNC cells when compared to uninjected controls (e, g, i) (n≥9 for each). k–p, 5 dpf larval head skeletons stained with Alcian blue (cartilage) and Alizarin red (bone and teeth). Craniofacial development is unaffected in surviving hira (l), daxx (n) and dek (p) morpholino-treated individuals and uninjected controls (k, m, o) (n≥35 for each). hira morpholino-injected embryos did exhibit a high level of death after 24 hpf but prior to 5 dpf (morpholino injected, 55.1%; uninjected, 0%) and a curved/kinked tail phenotype in surviving 5 dpf larvae (morpholino injected, 48.6%; uninjected, 0%). q–t, Compared to uninjected controls (q & s), embryos injected with a combination of all three morpholinos at 200 μM had no defects in CNC sox10 expression (r) (n≥9 for each) or development of the larval head skeleton (t) (n≥66 for each). un, uninjected; MO, morpholino. Scale bars = 250 μm.