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Furin is required for basement membrane anchorage, ectodomain shedding and proper basement membrane localisation of Frem2 and Fras1 proteins. (A,B) Lateral views of the medial fin of a sturgeon mutant at 48 hpf (B) showing mild blistering reminiscent of pinfin and blasen mutants compared to a sibling (A). (C,D) In situ hybridisation analysis of furina demonstrates broad expression in the embryo at 24 hpf (C) with clearly increased levels in the fin fold (D). (E) A consensus Furin cleavage site (red box) is conserved in Fras1 immediately N-terminal to the transmembrane domain across vertebrates. (F–I) furina and frem2a interact dose-dependently in zebrafish. Embryos injected with sub-phenotypic doses of morpholinos targeting frem2a (G) or furina (H) have medial fins as uninjected control embryos (F) at 48 hpf. Combined injection of the two MOs at these doses robustly induces single or multiple blisters of the fins (I). (J) Chemical inhibition of Furin function (far right lanes in all panels) reduces secretion of N-terminally 3xHA tagged Fras1 protein (upper left panel) and of N-terminally 3xMyc tagged Frem2 protein (upper right panel) from 293F cells into the medium. Proteins were detected by Western Blotting with an anti-HA antibody (for the Fras1 construct) or an anti-myc antibody (for the Frem2 construct). Cellular expression levels were not affected by addition of the inhibitor (lower panels). Due to the large size of the proteins and the small size of the cleaved C-terminus, the differences in sizes between the cellular and secreted proteins are indistinguishable. (K,L) Transverse sections of the posterior medial fin of 42 hpf pifte262/te262 embryos at 42 hpf, after transplantation of GFP-positive cells from either a WT (K) or a sturgeon mutant (L) donor at 6 hpf. The sections were immunostained for Fras1 (red), p63 (pink) and GFP (green), and nuclear DNA was counterstained with DAPI (blue). Fras1 protein from WT cells can be found in the basement membrane several cell diameters proximal of its source (white bar in K). In contrast, Fras1 from stu mutant cells lacking FurinA remains restricted to the basal surface of the donor cell (arrowhead, L). (M–P) Transverse sections through tail of embryos at 24 hpf (M) or 30 hpf (N–P) showing localisation of fras1 mRNA by in situ hybridisation (M,N,P: blue precipitate) compared to Fras1 protein (O: red precipitate, P: brown precipitate). Fras1 protein is present in the basement membrane along the entire fin fold (O,P), whereas fras1 mRNA is largely restricted to apical cells of the fin fold (M,N,P). Apical restriction is more pronounced at 30 hpf (N), while at 24 hpf, the fras1 expression domain appears to extend further proximally (M). (Q,R) Cell tracing analysis after transplantation of GFP-labelled, tg(bactin::hras-egfp) transgenic presumptive epidermal cells into non-transgenic hosts. Comparison of labelled cells in the same chimeric embryos at 24 hpf (Q) and 48 hpf (R) revealed that proximal clones had approximately doubled their cell number, whilst distal cells had not proliferated, but had increased their surface area by acquiring a flat elongate shape.
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