Fig 5

(A) Head expression of all mef2 paralogs was quantified by RT-qPCR in full-sibling mef2ca mutant and wild-type embryos from low- and high-penetrance strains at 28 hpf. Expression of each paralog in mef2ca mutants was normalized to wild-type sibling expression levels (black line). Asterisks mark paralogs with expression levels significantly different in mef2ca mutants compared with mef2ca wild types (T-test at alpha = 0.05). Error bars are standard deviation. (B) Animals heterozygous for mef2ca from the low-penetrance strain were intercrossed and offspring treated with nonsense-mediated decay inhibitor (NMDi14) or vehicle control (DMSO) from 18–42 hpf. Treated animals were fixed and stained with Alcian Blue (cartilage) and Alizarin Red (bone) at 6 dpf. Genotyped skeletal preparations were scored for penetrance of mef2ca-associated phenotypes. (C) Intercrosses from low-penetrance heterozygotes were immunostained with anti-MEF2C antibody. Genotyped animals were imaged in whole mount. 9/9 homozygous wild types had strong signal in the pharyngeal arches, 0/9 homozygous mutants exhibited any signal in the pharyngeal arches. Scale bar: 50 μm. (D) Exonic structure of mef2ca with protein domains and the location of the mef2ca^b1086 mutation indicated. Primer pairs either span the mutant exon (F1/R1) or amplify across exon-exon boundary (F2/R1). (E) cDNA from wild-type and mutant siblings from low- and high-penetrance strains was used as a template for amplification across the mutant exon. These primers are predicted to amplify bands of indicated different sizes if the mutant exon is included versus excluded from mature transcripts. (F) cDNA from wild-type animals was collected before and after zygotic genome activation and used as a template for PCR amplification across an exon-exon junction. Reverse transcriptase (RT) negative control ensured that only cDNA and not genomic DNA was amplified under these conditions. (G) A homozygous mutant female was crossed with a heterozygous male sibling from the low-penetrance strain and offspring were fixed and stained with Alcian Blue (cartilage) Alizarin Red (bone) at 6 dpf. Genotyped maternal-zygotic mutants were scored for ectopic bone which develops between the opercle (op) and the branchiostegal ray (br) in mutants from the high-penetrance strain. Scale bar: 100 μm.