pak2a mutations cause cerebral hemorrhage in redhead zebrafish

doi:10.1073/pnas.0700947104

pak2a mutations cause cerebral hemorrhage in redhead zebrafish

*Howard Hughes Medical Institute and Life Sciences Institute,
^‡Department of Internal Medicine,
^¶Department of Pediatrics,
^‖Department of Molecular, Cellular, and Developmental Biology,
^‡‡Molecular and Behavioral Neuroscience Institute and Department of Biological Chemistry, and
^¶¶Division of Hematology/Oncology and Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI 48109;
^§Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892;
^††Department of Biological Sciences, University of North Texas, Denton, TX 76203; and
**Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605

Edited by Napoleone Ferrara, Genentech, Inc., South San Francisco, CA, and approved July 19, 2007 (received for review February 1, 2007)

Abstract

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^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.

Footnotes

^§§To whom correspondence should be addressed at:
University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216.
E-mail: ginsburg{at}umich.edu
Author contributions: D.A.B., F.S., J.A.S., D. Ginsburg, and S.E.L. designed research; D.A.B., F.S., J.S.Y., M.K., J.A.S., L.K.B., B.M., S.K., and A.W.H. performed research; M.K., J.D.A., P.J., D. Goldman, N.D.L., and B.M.W. contributed new reagents/analytic tools; D.A.B., F.S., M.K., J.A.S., P.J., P.A.R., B.M.W., D. Ginsburg, and S.E.L. analyzed data; and D.A.B., J.A.S., B.M.W., D. Ginsburg, and S.E.L. wrote the paper.
↵ ^†Present address: Department of Genetics, Case Western Reserve University, Cleveland, OH 44106.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0700947104/DC1.
Abbreviations:

dpf,

days postfertilization;

hpf,

hours postfertilization;

PAK,

p21-activated kinase;

CCM,

cerebral cavernous malformation;

MO,

morpholino.