Fig. 2. low^ts213 is a mutation in a zebrafish tfap2a. (A) The tfap2a gene is closely linked to the low^ts213 locus. Both map to the long arm of linkage group 24 between SSCP markers z23011 and z3399. (B) low^ts213 creates a stop codon in the conserved DNA-binding/dimerization domain of tfap2a, resulting in a truncated protein. (C) PCR products of 424 bp were amplified from exon 5 of tfap2a from preparations of individual 3 dpf low and sibling larvae. Digests by BlpI show co-segregation of the restriction site with the low phenotype producing bands of sizes 208 bp and 217 bp. (D) Unilateral rescue of melanophore development at 26 hpf by injection of a PAC containing tfap2a into one blastomere at the two-cell stage (arrows indicate rescued melanocytes). (E) Genomic organization of tfap2a and the locations of morpholino antisense oligonucleotides directed against splice acceptor sites. (F,G) Splicing defects in tfap2a transcripts following injection of splice-directed morpholinos. tfap2a was amplified from pools of 3.1mo (lanes 1-4) and 5.1mo (lanes 5-8) morpholino injected and control uninjected (lanes 9-12) animals using primers tfap2a-3f and tfap2a-3r directed to exons 3 and 7 (lanes 1,2,4-6,9,10) and primers tfap2a-4f and tfap2a-3r directed to exons 4 and 7 (lanes 3,4,7,8,11,12). Uninjected animals (lanes 9-12) showed PCR products of sizes 730 bp and 420 bp, in comparison with morpholino-injected animals in which additional PCR products were observed (black arrows) because of aberrant splicing (F). (G) These produce a phenotype similar to low^ts213. Scale bar: 100 µm.

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