Fig. 4

sof^tm272a is an allele of lamb2. (A) sof^tm272a was meiotically mapped to a 2.3 cM region on chromosome 23, delineated by the SSLP markers z13424 and z31657. The non-recombinant marker z31489 was found to reside in an intron of lamb2l. The markers used have been placed on a physical map of chromosome 23, with recombination frequencies shown (number of recombinants/number of meioses). The numbers below the line represent the genetic (top) and physical (bottom) location of markers (Zv7; http://www.ensembl.org). The arrow above the genes indicates transcriptional orientation. (B) The zebrafish lamb2 and lamb2l genes maintain a syntenic relationship with USP19 in human. LAMB2 regions of human chromosome 3 and zebrafish chromosome 23 are displayed, with distances between genes shown (not to scale). Distances were taken from the Ensembl database and Durkin et al. (Durkin et al., 1999). (C) Genomic sequence electropherograms of the mutated region in wild-type, heterozygote and homozygote sof embryos. A single T to C transition was found in exon 3 of lamb2 (asterisk), resulting in the substitution of a proline for a leucine at amino acid 38 of the unprocessed form of Lamb2. The translation is shown below each electropherogram. (D) Domains of Lamb2. The sof^tm272a mutation is in the LN domain, shown in red. Other domains represented are LF domain (blue), EGF-like domain (grey circles) and coiled-coiled domain (grey bar) (Aumailley et al., 2005). (E) Amino acid sequence alignment of the N-terminal region of laminin β2 from zebrafish (Dr), human (Hs), chicken (Gg) and the sole Caenorhabditis elegans β-laminin, LAM-1 (Ce), as well as zebrafish laminin β1, β4 and β2-like. The mutation affects a completely conserved leucine, 21 amino acids from the predicted secreted N-terminal of zebrafish Lamb2. Asterisks denote universally conserved residues. The signal peptide cleavage sites of each sequence were predicted using SignalP3.0 (Bendtsen et al., 2004). (F) Injection of a lamb2 antisense MO precisely phenocopies the loss of birefringency seen in sof homozygous mutants. Top, uninjected wild-type embryo; middle, lamb2 MO3-injected embryo; bottom, sof mutant embryo.