Identification of zebrafish vhl and loss-of-function alleles. (A) The zebrafish VHL ortholog vhl encodes a single 175-amino-acid protein that is 52% identical and 70% similar to human pVHL. ClustalW alignment shows high conservation of the pVHL Elongin C and HIF-α binding domains (HIF-1α core hydroxyproline Pro⁵⁶⁴ binding sites are highlighted in yellow). The VHLp30 isoform-specific acidic domain (residues 1-53) is not present. By screening the Hubrecht target-selected ENU-mutagenized library, we identified 2 different zebrafish lines carrying inactivating germline vhl mutations within the HIF-α binding domain (Q23X and C31X; highlighted in red). (B) Whole-mount in situ hybridizations show ubiquitous vhl expression in wild-type embryos at 14 and 24 hpf. Original magnification ×5. (C) Vhl protein (∼ 19 kDa) is not detected in 6.5 dpf vhl mutant zebrafish. One embryo equivalent is loaded per lane. β-Actin is used as a loading control. Wt, wild-type; sib, sibling.