Fig. 2

nft ^y72 is a genetically null allele of reck. (A) WT zebrafish Reck (top) is 955 aa long and features the same domains and motifs found in mammalian RECK (Takahashi et al., 1998). N-terminal signal peptide (SP; brown), cysteine knot motifs (CK1-5; blue), epidermal growth factor-like repeats (EGF1-2; black), fibronectin-like type I module (Fnl1; green), Kazal motifs (K1-3; red), C-terminal GPI transferase cleaveage site (G; pink). Mutant Reck^y72 (bottom) harbors a missense amino acid change in an evolutionarily conserved Cys residue within CK4. (B-F) Providing WT reck mRNA to reck ^y72 mutants restores formation of both CtAs (B-D) and DRG (E,F). Embryos with unilateral CtA rescue {B,C; endothelium, green [Tg(fli1a:eGFP)^y1]} or bilateral DRG rescue (F; yellow circles, red HuC immunofluorescence; only one side shown). Quantification of CtA rescue expressed as the percentage of embryos (from incrossing heterozygous mutant carriers) with reck-like CtA deficits (D). (G-L) Exogenous co-expression of epitope-tagged zebrafish Reck (WT Reck or mutant Reck^y72) with cytosolic EGFP in cultured mammalian cells. (G-J) Immunofluorescence-based detection of surface 3×FLAG-Reck (red) and EGFP fluorescence (green) in non-permeabilized 293T cells. 3×FLAG-Reck^y72 fails to reach the cell surface (J). (K,L) WT and mutant Reck^y72 expressed in COS7 cells show similar abundance. (K) Western blot of total cell lysates from cells expressing WT or mutant 3xFLAG-Reck. (L) Densitometry-based quantification total cell lysates from cells expressing WT Reck or mutant Reck^y72 zebrafish proteins tagged with 3×FLAG or 2×HA. Protein levels normalized with EGFP and GAPDH. Error bars: s.e.m. Scale bars: 50µm in B-C, 100µm in E,F and 10µm in G-J. See also Figs S2, S3, Movies 1-14 and Table S1.