pak2a Mutations Cause Cerebral Hemorrhage in Redhead Zebrafish.

Abstract

Zebrafish are a powerful vertebrate model system for the study of human disease as they share many molecular pathways with mammals. Of note, nearly all of the mammalian coagulation factors are also highly conserved in fish. As part of a whole genome ENU mutagenesis screen, we identified a mutant zebrafish which displayed intraventricular hemorrhage between 2–3 days post fertilization (dpf), which we named redhead. Using an F2 intercross strategy, we mapped this recessive mutant to a 100 kilobase interval on chromosome 2 and identified a splice site mutation in the gene for an ortholog of the p21-activated kinase Pak2. This IVS9 T+2A mutation renders greater than 90% of transcripts nonfunctional, resulting in a hypomorphic allele. Surveying zebrafish expressed sequence tag databases, we identified two Pak2 orthologs in zebrafish, designated Pak2a and Pak2b, with the redhead mutation in pak2a. Central nervous system (CNS) hemorrhage in redhead embryos was rescued by injection of wild type pak2a mRNA. Morpholino knockdown of pak2a in wild type fish phenocopies the redhead mutant, but with an increase in penetrance and severity, including hydrocephalus and pericardial edema secondary to severe hemorrhage. Injection of either pak2a or pak2b mRNA was able to rescue this phenotype. In addition, pak2b knockdown worsened the bleeding phenotype in redhead embryos, with no effect on wild type fish. These results suggest a partial overlap in function between Pak2a and Pak2b. Confocal microscopy was performed at 2.5 dpf in pak2a knockdown embryos transgenic for Gata1-dsRED and Flk1-eGFP, distinctively labeling erythrocytes and endothelial cells, respectively. The sites and magnitude of hemorrhage were variable between individual embryos, but there were no obvious abnormalities in vessel patterning. In summary, we have identified a critical role for Pak2 in maintaining CNS vessel integrity in zebrafish, without apparent effects on other vascular beds. Further analysis of this signaling pathway in the vessel wall may provide novel insight into the mechanisms underlying vascular heterogeneity in mammals, and the tissue specific function of Pak2 in the CNS vasculature.

Genome editing of factor X in zebrafish reveals unexpected tolerance of severe defects in the common pathway

“…26,30 Multiple vascular mutants have been described in zebrafish with intracranial hemorrhage as a primary phenotype secondary to various mechanisms, including defects in vascular development, patterning, integrity, and remodeling. [27][28][29] Our analysis did not reveal any such vascular phenotypes, suggesting that the consequences of F10 loss are solely defects in hemostasis. This implies that thrombin generation is not a key player in zebrafish vasculogenesis or operates through non-F10-dependent pathways.…”

“…15 Thus, we have turned to the zebrafish model (Danio rerio) with advantages that include high fecundity, external and transparent development, ease of manipulation and maintenance, accessibility of genetic techniques and imaging, as well as extensive conservation of the hemostatic and vascular systems as we and others have demonstrated. [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] In this article, we report targeted mutagenesis of zebrafish f10 through genome editing with transcription activator-like effector nucleases (TALENs). We find that loss of F10 results in late-onset lethality by 4 months of age secondary to intracranial and intra-abdominal hemorrhage, without any evidence of a vascular defect.…”

Gateway compatible vectors for analysis of gene function in the zebrafish

“…In this case, we have identified an endothelial enhancer for the purpose of generating a useful reagent to drive gene expression in blood vessels. Accordingly, these Gateway compatible fli1ep constructs have been used to rapidly generate constructs for endothelial autonomous rescue experiments in the zebrafish redhead mutant (Buchner et al,2007). We have also constructed Gateway compatible Tol2 vectors containing the fli1ep fragment for generating N‐terminal fusions with 6xmyc, Egfp, and mCherry (data not shown and Table 1).…”

“…This cassette was transferred to pTol2000 (kindly provided by Koichi Kawakami) that had been digested with Xho I and treated with Klenow. A description of the construction of the fli1ep containing vectors can be found elsewhere (Buchner et al,2007). In all cases, plasmids containing Destination cassettes were grown in ccdB ‐tolerant bacterial strains (Invitrogen) and maintained in the presence of both ampicillin and chloramphenicol.…”

Keeping your vascular integrity: What can we learn from fish?

“…(21) Positional cloning showed that the redhead mutation is located in the Pak2a gene, which is a downstream signaling partner of bPix. (22) In addition to BIRC2, the role of another IAP, survivin, was recently studied by knockdown of its expression in zebrafish. Downregulation of survivin expression resulted in perturbation of angiogenesis in vessels of the trunk, the eyes and the intestines, at least in part by an increase in endothelial apoptosis.…”