Fig. 4

The fin blistering of rafels mutants is caused by mutations in Frem1a, which shows partial functional redundancy with Frem1b.

(A–C) The rafels mutant displays mild blistering of the posterior fin at 48 hpf (B) compared to a sibling (A), and is phenocopied in wild-type embryos by injection of a morpholino targeting frem1a (C). Lower panels show dorsal views tail fin, providing a more striking image of the blistering in the mutant and morphant. (D,E) Expression of frem1a in the medial fin fold at 24 hpf (D) and pectoral fin at 48 hpf (D-inset). Expression of frem1b is weaker but can be seen in the tail region, in particular in the blood islands, at 24 hpf (E). (F) Genetic map (set out as in Figure 1A), showing the approximate location of the rafels locus on linkage group 7 between markers z9249 and z13880. Independent radiation hybrid mapping localised the frem1a gene to this region, with distances to the mapping markers given in grey. (G) Schematic of the zebrafish Frem1a protein with conserved domains as defined in Figure 2I. The purple circle depicts the C-type Lectin domain. The positions and natures of the molecular lesions of the rfl^tc280b, rfl^fr23 and the rfl^tr240 alleles are indicated. (H–L) Sequence chromatograms of the mutations in the frem1a cDNA of rfl^{tc280b/tc280b} (H), rfl^fr23/fr23 (I), and rfl^tr240/tr240 (J) (lower panels) compared to wild-type siblings (upper panels). The 13 nucleotide insertion in the rfl^tr240/tr240 allele is delineated by orange lines (J). Genomic sequencing of the intron32-exon33 boundary reveals generation of a novel splice site in the rfl^tr240/tr240 mutants (K; lower chromatogram), leading to aberrant splicing as depicted in (L) (red lines). (M–P) frem1b splice MO enhances the rafels phenotype. Lateral views of WT (M) or rfl^fr23/fr23 (N–P) embryos either uninjected (N) or injected with frem1b splice MO (M,O,P) photographed at 32 hpf (M–O) or 48 hpf (P). Whilst the frem1b MO does not generate a phenotype alone (M), it reveals a blistering phenotype in rafels mutants at 32 hpf (O), a time when a phenotype is not seen in uninjected mutants (N). The frem1b MO injected frem1a mutants often display degeneration of the fin at 48 hpf (P; compare to B).