Fig. 2. Isolation and molecular characterization of a kit mutant D. albolineatus and comparison with D. rerio. (A-D) Melanophore phenotypes of embryos at 60 hpf. (A) Wild-type D. rerio. (B) Wild-type D. albolineatus have fewer melanophores overall compared with D. rerio. (C) Homozygous kit^b5 mutant D. rerio exhibit fewer melanophores, especially at sites distant from their origin in the neural crest (e.g. covering the yolk, arrow). An ectopic patch of melanophores (arrowhead) occurs posterior to the otocyst. (D) Homozygous wp.a14e1 D. albolineatus have fewer peripheral melanophores (arrow) and ectopic post-otic melanophores (arrowhead), although total melanophore numbers are greatly diminished compared with kit mutant D. rerio. (E) RT-PCR for amplicons (A1-A3) from D. albolineatus kit cDNAs. A1, lower transcript abundance in wp.a14e1 compared with wild type. A2, reveals the predicted 652 bp product in wild type but a 296 bp product in wp.a14e1. A3, failure to amplify a 234 bp product demonstrates absence of wild-type kit transcript in wp.a14e1. ß-actin, loading control. (F) Schematics of D. albolineatus kit cDNAs and locations of amplicons A1-A3. Green, predicted signal sequence and transmembrane domain. Red, predicted kinase domains. In kit^wp.a14e1 two exons are deleted, resulting in a frameshift with novel amino acids (orange) and a premature stop codon. Sequence analyses confirm that D. albolineatus kit is orthologous to D. rerio kit (kita), rather than a second kit locus identified in D. rerio, kitb (see Discussion) (Mellgren and Johnson, 2005). (G) Genomic structure of kit in wild type and wp.a14e1 mutant D. albolineatus. Shown are exons 4-6 and intervening introns (i4-i6). The region deleted in wp.a14e1 is shown in brown, and a novel inserted sequence is shown in orange. GenBank accession number for D. albolineatus kit: EF035010.