Fig. 6

Differential mef2ca^b1086 transcript expression, transposon DNA methylation and functional consequences. (A) mef2ca^b1086 mutant transcripts were detected by in situ hybridization in the low and high penetrance strains. Images are single focal planes, lateral views, anterior is towards the left and dorsal is upward. Scale bar: 100 µm. (B) mef2ca^b1086 mutants from the mild (low penetrance) strain were injected with mef2ca^b1086 mRNA, or mef2ca^b1086 mutants from the severe (high penetrance) strain were injected with mef2ca morpholino (MO) and scored for penetrance of ectopic bone. mef2ca^b631 heterozygotes were incrossed, or crossed to mef2ca^b1086 heterozygotes from either the mild or severe strain and mef2ca mutant offspring were scored for penetrance of ectopic bone. (E) Full sibling mef2ca^b631 heterozygous adults were crossed in single-pair matings and their mef2ca^b631 homozygous mutant offspring were stained for cartilage and bone and scored at 6 dpf for penetrance of the ligament-to-bone fate-switch phenotype as in Fig. 5. The offspring penetrance score was assigned to the parents, which are color coded as indicated. (D) DNA methylation analysis of 6 dpf mef2ca^b1086 mutant larvae from the low and high penetrance strains. Bisulfite sequencing revealed the overall percentage of CpG methylation of the transposon at the mef2ca locus from four animals from each strain. Bisulfite sequencing results from a representative animal from each strain are shown in which six transposon clones from each animal, each with seven CpG sites, were sequenced. Filled circles represent methylated CpG sites. (E) Model for transposon DNA methylation and interaction with the mef2ca locus.