Fig. 2

Mutation in a splice donor (SD) site of the lamb1a gene results in a truncated protein in s804 mutants and impairs expression of the multimeric protein laminin 1 in the basement membrane of lamb1a^s804 mutants. (A) The lamb1a ORF in zebrafish (exons are numbered). Functional domains of the corresponding protein are represented by colored boxes. Gray blocks represent the cloning strategy. (B) G-to-T replacement in the SD site for exon 24 of lamb1a in s804 mutants (6/6). (C) Alternative splicing in lamb1a^s804 mutants. Mutation of the SD site for exon 24 results in excision of exon 24 (i), maintenance of intron 24 (I24, ii) or splicing of exon 23 into a cryptic site within exon 24 (iii). These alternative splice events result in a frame-shift and premature stop signals (asterisks) that disrupt the C-terminal coiled-coil domains (blue boxes). (D) PCR amplification of block C from wild-type cDNA produces a single band of 1.5 kb. In s804 mutants, alternative products are detected. (E) Injection of a lamb1a splice-blocking MO recapitulates the s804 mutant phenotype. Scale bars: 20 μm. (F-H′) Transverse sections of wild-type (F,G,H) and lamb1a^s804 mutant (F′,G′,H′) embryos at 16 ss (F-G′) and 30 hpf (H,H2). Anterior (F,F′) and posterior (G,G′) level sections. Lam1 (red), Tg(XIEef1a:GFP) (green). L, left; R, right. Scale bars: 20 μm. Neural tube (nt, broken line), notochord (n), somite (s) and gut endoderm (e). Dotted line identifies marked atrophy of the notochord.