Fig. 2

Positional cloning of pku300 from the sco^te382 locus in zebrafish. (A) The sco^te382 locus was mapped on linkage group 22 by bulk segregation analysis with microsatellite markers. Note that the sco^te382 locus, which contains calr, trh1, pku300 and cherp genes, was determined between markers 6 and 9 by genotyping 1632 informative meiosis using SLP and SNP methods. A G3935T missense mutation of fbn2b is reported to be responsible for the sco^te382 locus (Mellman et al., 2012), but fbn2b is located outside of the genetic interval in our mapping cross DNA panels. fbn2b is about 570kb away from pku300. (B) pku300 consists of 6 exons and encodes a 540-amino-acid protein. Note that a C946T mutation in exon 3 of pku300 led to a premature stop code (TAG) in the sco^te382 allele (arrowhead). (C) Sequencing traces of wild-type and homozygous sco^te382 mutant embryos. Note a single base pair change from C to T in the sco^te382 allele (arrowhead). (D) Pku300 protein was predicted to contain three extracellular IgG domains, a transmembrane domain and a short intracellular tail. (E–G) pku300 was broadly expressed, including in the heart (arrowhead) during early embryogenesis. Staged wild-type 24 (E), 48 (F) and 48hpf (G) hearts were subjected to in situ hybridization with anti-sense pku300 riboprobe. (H) pku300 was expressed in day-2 Tg(myl7:eGFP) embryonic hearts by RT-PCR. (I) pku300 was reduced in cloche (clo) mutant embryos that have no endocardial cells. RNA was prepared from siblings (sib) and clo mutant embryos (clo) from heterozygous cloche^m39 crosses. (J) Pku300-mCherry fusion proteins were transiently expressed in the heart of a Tg(kdrl:EGFP) transgenic embryo. Note the red Pku300-mCherry fusion protein localization on cell membranes (arrowhead), which was not overlapped with cytoplasmic localization of EGFP. Pku300-mCherry was driven by the zebrafish kdrl promoter. a, atrium; v, ventricle. Scale bar: 30μm.