Positional cloning of chuzhoi demonstrates mutation of Ptk7. (A) Genome wide scan shows linkage of chuzhoi to Chromosome 17; percentage of mice typing as homozygous BALB/c is shown for each marker. (B) Genetic map with additional markers; numbers on left show Mb position along the chromosome, vertical arrow demarcates chuzhoi critical region. (C) Sequence and translation of Ptk7 cDNA, revealing nine base pair insertion in chuzhoi, encoding three additional amino acids (highlighted in blue). (D) Genomic sequence from Ptk7 revealing a single nucleotide substitution in chuzhoi (circled). (E) Comparison with consensus splice sequence reveals chuzhoi mutation (circled) creates a novel splice acceptor site 9 bp upstream to the wild-type site, with greater identity to the consensus sequence (asterisks; identity 14/15 for chuzhoi, 10/15 for wild-type). Lower case, intron; upper case, exon; y, pyrimidine; n, any nucleotide; bold, determines intron subtype. (F) Schematic diagram of Ptk7 protein structure with seven immunoglobulin (IG)-like domains, a single transmembrane region (TM) and an intracellular kinase homology domain. The insertion in chuzhoi occurs between the fifth and sixth IG domains. (G) Western blot analysis on membrane (m) or soluble (s) protein fractions from E10.5 embryos (left blot), and total cell lysates from E8.5 embryos (right blot) demonstrating changes in Ptk7 protein expression in chuzhoi mutants; β-tubulin was used as a loading control. (H) Quantitation of expression of Ptk7 isoforms in wild-type, heterozygous and mutant chuzhoi E10.5 embryos, normalised to β-tubulin, and expressed as the abundance relative to that of the wild-type upper isoform.