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The zebrafish is a powerful model for studying vascular development, demonstrating remarkable conservation of this process with mammals. Here, we identify a zebrafish mutant, redhead ([rhd.sup.mi149]), that exhibits embryonic CNS hemorrhage with intact gross development of the vasculature and normal hemostatic function. We show that the rhd phenotype is caused by a hypomorphic mutation in p21-activated kinase 2a (pak2a). PAK2 is a kinase that acts downstream of the Rho-family GTPases CDC42 and RAC and has been implicated in angiogenesis, regulation of cytoskeletal structure, and endothelial cell migration and contractility among other functions. Correction of the Pak2a-deficient phenotype by Pak2a overexpression depends on kinase activity, implicating Pak2 signaling in the maintenance of vascular integrity. Rescue by an endothelial-specific transgene further suggests that the hemorrhage seen in Pak2a deficiency is the result of an autonomous endothelial cell defect. Reduced expression of another PAK2 ortholog, pak2b, in Pak2a-deficient embryos results in a more severe hemorrhagic phenotype, consistent with partially overlapping functions for these two orthologs. These data provide in vivo evidence for a critical function of Pak2 in vascular integrity and demonstrate a severe disease phenotype resulting from loss of Pak2 function. [beta]-pix | CNS | endothelial cell | p21-activated kinase | vasculature

Vlad tepes ([vlt.sup.m651]) is one of only five 'bloodless' zebrafish mutants isolated through large-scale chemical mutagenesis screening. It is characterized by a severe reduction in blood cell progenitors and few or no blood cells at the onset of circulation. We now report characterization of the mutant phenotype and the identification of the gene mutated in [vlt.sup.m651]. Embryos homozygous for the [vlt.sup.m651] mutation had normal expression of hematopoietic stem cell markers through 24 h postfertilization, as well as normal expression of myeloid and lymphoid markers. Analysis of erythroid development revealed variable expression of erythroid markers. Through positional and candidate gene cloning approaches we identified a nonsense mutation in the gata1 gene, 1015C [right arrow] T (Arg-339 [right arrow] Stop), in [vlt.sup.m651]. The nonsense mutation was located C-terminal to the two zinc fingers and resulted in a truncated protein that was unable to bind DNA or mediate GATA-specific transactivation. A BAC clone containing the zebrafish gata1 gene was able to rescue the bloodless phenotype in [vlt.sup.m651]. These results show that the [vlt.sup.m651] mutation is a previously uncharacterized gata1 allele in the zebrafish. The [vlt.sup.m651] mutation sheds new light on Gata1 structure and function in vivo, demonstrates that Gata1 plays an essential role in zebrafish hematopoiesis with significant conservation of function between mammals and zebrafish, and offers a powerful tool for future studies of the hematopoietic pathway.

Type IIB von Willebrand disease (vWD) is a hereditary disorder characterized by the presence of missense mutations in the glycoprotein IB binding region of the von Willebrand factor. Functional analysis of the most common type IIB missense mutation, arginine to tryptophan substitution at residue 543, reveal that this single amino acid substitution alone could account for the typical hematologic picture found in type IIB vWD. This mutation is sufficient to induce increased platelet adhesiveness characteristic of type IIB vWD and no other posttranslational defects are necessary to produce this effect.