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.

Fig. 1.

Cerebral hemorrhage in rhd embryos. (A) Three rhd embryos at 3 dpf in lateral views. Arrows indicate area of hemorrhage. (B) Injection of a pak2a MO causes cerebral hemorrhage similar to that seen in rhd. Negative control and pak2a MO-injected embryo at 3 dpf are shown. (C) pak2a RT-PCR of RNA isolated from embryos injected with pak2a MO show aberrant transcripts.

Fig. 2.

Identification of a pak2a splice site mutation in rhd zebrafish. (A) Genetic map of the rhd nonrecombinant interval. Recombination between two markers is indicated by an X. rhd is located between D2Umi14 and D2Umi17 on zebrafish chromosome 2. (B) Seven genes were identified within the rhd nonrecombinant interval. Arrows indicate the direction of transcription. (C) Schematic showing the four aberrant splice isoforms detected in rhd (numbered 1–4). (D and E) RT-PCR analysis of adult WT, rhd/+, and rhd/rhd fish reveals altered splice patterns in golph4-like (D) and pak2a (E). M indicates 1-kb Plus DNA ladder (Invitrogen). (F) Domain structure of Pak2 (13) illustrating the predicted truncation in rhd caused by skipping exon 9. (G) Fluorescent RT-PCR was performed to determine the relative level of full-length pak2a transcript versus the exon 9-skipped isoform in rhd zebrafish. Standard deviation is shown. (H) Diagram of the three pak2a exon 9 cryptic splice donor sites identified in rhd. Numbers 2–4 correspond to the splice forms shown in C. Uppercase letters represent the WT exonic sequence, whereas lowercase letters represent the WT intronic sequence. n, number of clones among eight that were sequenced.

Fig. 3.

In situ hybridization with pak2a and pak2b. (A–D) Lateral views with heads to the left of 36-hpf embryos hybridized with pak2a antisense (A), pak2a sense (B), pak2b antisense (C), or pak2b sense (D) probes. (E–J) In situ hybridizations of cryosectioned 48-hpf embryos. Staining with NBT/BCIP is shown by a brownish-pink color. (E–G) pak2a antisense. (H–J) pak2b antisense. The areas shown by a rectangle in F and I are enlarged in G and J, respectively. The asterisks in G and J label endothelial cells. b, brain; ep, epidermis; en, endoderm; da, dorsal aorta; sc, spinal cord; e, erythrocyte.

Fig. 4.

Pak2b deficiency modifies the severity of hemorrhage in rhd. Representative pak2b MO-injected embryos of each pak2a genotype are shown at 3 dpf with black arrows showing hemorrhage and the gray arrow indicating pericardial edema.

Fig. 5.

Normal vascular patterning in Pak2a-deficient animals. (A and B) Confocal imaging of 2-dpf Tg(flk1:EGFP);Tg(gata1:dsred) homozygous double transgenic animals injected with either control MO (A) or pak2a MO (B). Animals injected with pak2a MO display severe hemorrhage, but their vascular pattern does not differ significantly from control MO-injected animals. (C and D) Confocal imaging of ≈2-dpf Tg(flki-:EGFP) transgenic embryos injected with pak2a MO. Shown is an embryo that had CNS hemorrhage and was injected with quantum dots (2 μm). C is at time point 0, and D is after 30 min, when leakage has occurred. All images are dorsal views, head to the right. (Scale bar, 200 μm.)