Fig. 5

Identification of the llk gene. (A) Genetic map for the llk locus. The llk locus mapped to linkage group (LG) 7. The RDA marker, EcoRI-46 is located at 0 cM map-distance from the llk locus (0 per 2054 meioses). (B) Genomic structure of the zebrafish scrb1. The scrb1 gene is encoded by 45 exons spanning 120 kb in the genome. EcoRI-46 is located in the first intron of the gene. Each of the two mutant alleles, lkl^rw16 and llk^rw468, carries a nucleotide substitution in exons 11 and 17, respectively. (C) Schematic drawing for the putative cDNA encoding 45 exons shown in B. Six alternatively used exons, 16, 28, 31, 34, 40 and 43 are shown as red boxes. Exon numbers and first ATG site are indicated above. Regions encoding LRR domain and four PDZ domains are indicated below. (D) RT-PCR analyses were performed to identify the predominant gene product. Primers were designed in the flanking exons encompassing each exon of interest. Total RNA was extracted from 1.5, 10, 18, 24, 36 and 48-hpf embryos. Arrowheads in each panel indicate the predominant RT-PCR product expressed during migration of the nVII motor neurons at 18-24 hpf. The predominant RT-PCR products contain exon 16, but no other exons (exons 28, 31, 34 and 43). The lesser RT-PCR products contain exons 28, 31, 34 and 43, but not exon 16 (indicated by arrows). 100 bp-interval molecular markers (bp) are shown in each panel. (E) Sequence diagrams of the mutation sites for the llk^rw16 and llk^rw468 alleles compared to the wild-type allele. (F) Schematic drawings of the wild-type (zScrb1) and mutant Scrb1 proteins (Scrb^rw16 and Scrb^rw468). Percentage identity of the amino acid sequences (%) to the mouse Scrb (mScrb1) is shown for each domain. The allele llk^rw16 carries a mis-sense amino acid substitution in the first PDZ domain, while llk^rw468 carries a stop codon in the LRR domain. (G-M) Lateral views of wild-type embryos stained with the scrb1 RNA probe in the 8-cell stage (G), dome-stage (H), 18 hpf (I), 22 hpf (J) and 48 hpf (K) embryos. (L,M) scrb1 mRNA expression in the brain at 20 hpf (L, dorsal view) and (M) cross section at r5 (indicated by the broken line in L). M′ shows the cross section at r5 of the 24 hpf-Isl1-GFP embryo stained with anti-acetylated α-tubulin antibody. Arrowheads indicate medial longitudinal fascicles (MLF); nc indicates notochord. Scale bars: 50 µm.