pak2a mutations cause cerebral hemorrhage in redhead zebrafish

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.␤-pix ͉ CNS ͉ endothelial cell ͉ p21-activated kinase ͉ vasculature

Genome-Wide Analysis of the Zebrafish ETS Family Identifies Three Genes Required for Hemangioblast Differentiation or Angiogenesis

“…The hemorrhage phenotype in the erg morphants is very similar to the hemorrhaged mutants recently identified in zebrafish, such as bubblehead/␤pix and redhead/pak2a. 41,42 It was suggested that ␤Pix-Pak2a signaling pathways regulate cerebral vascular stability, possibly via the p38 mitogen-activated protein kinase pathway. 41 Nevertheless, it would be intriguing to know whether expression of VE-cadherin was affected in these mutants, as in the erg and fli1 morphants described here.…”

“…41,42 It was suggested that ␤Pix-Pak2a signaling pathways regulate cerebral vascular stability, possibly via the p38 mitogen-activated protein kinase pathway. 41 Nevertheless, it would be intriguing to know whether expression of VE-cadherin was affected in these mutants, as in the erg and fli1 morphants described here. As a first test for effects on FGF or VEGF signaling, 2 pathways known to be important in angiogenesis, we asked whether activated forms of Erg or Fli1 could induce expression of the ligands (supplemental Figure VI, D through I).…”

The integrin coactivator Kindlin-2 plays a critical role in angiogenesis in mice and zebrafish

“…21,22 To study the effect of morpholino knockdown on vascular development, we used live imaging of a special marker zebrafish line that expresses GFP in all vascular ECs (GFP fused to the VEGF receptor promoter) and DsRed in all blood cells (DsRed fused to GATA-1 promoter). 27 Injection of a control morpholino into 1-2 cell stage embryos resulted in normal vascular development at 24 and 48 hpf ( Figure 4A). Injection of kindlin-2 morpholinos resulted in severe disturbances in embryonic vascular formation ( Figure 4B-C).…”

Regulatory Pathways Affecting Vascular Stabilization via VE-Cadherin Dynamics: Insights from Zebrafish (Danio Rerio)

“…21 The mutation in this strain is shown to be a splice site error in the p21-activated kinase 2a (pak2a) gene, which otherwise encodes a member of the pak2a gene family. 21 The PAK gene family are kinases that act downstream of the Rho-family GTPases, CDC42 and RAC, and regulate actin organization at the membrane-cytosol interface (Figure 2).…”

“…21 The mutation in this strain is shown to be a splice site error in the p21-activated kinase 2a (pak2a) gene, which otherwise encodes a member of the pak2a gene family. 21 The PAK gene family are kinases that act downstream of the Rho-family GTPases, CDC42 and RAC, and regulate actin organization at the membrane-cytosol interface (Figure 2). 22 The PAK gene family has been shown to regulate a multitude of relevant biologic processes in vitro, namely, cell motility, cytoskeletal rearrangements, and angiogenesis.…”