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Fig. 3

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ZDB-IMAGE-100504-13
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Figures for Carney et al., 2010
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Fig. 3 The fin blistering of the blasen mutant is caused by mutation of Frem2a.

(A–C) Injection of a morpholino targeting the translation site of frem2a mRNA into WT embryos phenocopies the bla fin blisters; lateral views of posterior medial fin, 48 hpf; (A) uninjected control; (B) blata90/ta90 mutant; (C) frem2a morphant. (D) Genetic map (set out as in Figure 1A), showing the approximate location of the blasen locus on linkage group 10 between markers z9328 and z7504, with genes within this interval shown below, including the strong candidate frem2a (red arrow). (E,F) Lateral views of embryos at 24 hpf (E) and 48 hpf (F) stained by in situ hybridisation for frem2a, revealing expression in the medial and pectoral fin fold (arrow, F). (G) Schematic of the zebrafish Frem2a protein with conserved domains as in Figure 2I, and the position and nature of the blata90 mutation indicated. (H) Sequence chromatograms of frem2a cDNA from blata90/ta90 mutants (lower panel) and heterozygous siblings (upper panel) showing the missense mutation depicted in (G). (I) Protein sequence alignment of Fras and Frem proteins showing the absolute conservation of the arginine residue mutated in blata90 (red asterisks) across different vertebrate classes. Sequences from the top are: zebrafish blata90/ta90 mutant Frem2a; zebrafish wild-type Frem2a; zebrafish Frem2b; zebrafish Frem3; human FREM2; mouse Frem2; zebrafish Fras1; fugu Fras1; human FRAS1; mouse Fras1; zebrafish Frem1a; human FREM1; mouse Frem1. (J) Segregation linkage analysis: the blata90 mutation generates a BccI site, which is present in both alleles of all bla homozygotes (lanes 7–48), whilst wild-type siblings (lanes 1–6) show at least one copy of the uncut allele; lane 49, 100 bp ladder.

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