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, 104 (35), 13990-5

A betaPix Pak2a Signaling Pathway Regulates Cerebral Vascular Stability in Zebrafish

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A betaPix Pak2a Signaling Pathway Regulates Cerebral Vascular Stability in Zebrafish

Jing Liu et al. Proc Natl Acad Sci U S A.

Abstract

The vasculature tailors to the needs of different tissues and organs. Molecular, structural, and functional specializations are observed in different vascular beds, but few genetic models give insight into how these differences arise. We identify a unique cerebrovascular mutation in the zebrafish affecting the integrity of blood vessels supplying the brain. The zebrafish bubblehead (bbh) mutant exhibits hydrocephalus and severe cranial hemorrhage during early embryogenesis, whereas blood vessels in other regions of the embryo appear intact. Here we show that hemorrhages are associated with poor cerebral endothelial-mesenchymal contacts and an immature vascular pattern in the head. Positional cloning of bbh reveals a hypomorphic mutation in betaPix, a binding partner for the p21-activated kinase (Pak) and a guanine nucleotide exchange factor for Rac and Cdc42. betaPix is broadly expressed during embryonic development and is enriched in the brain and in large blood vessels. By knockdown of specific betaPix splice variants, we show that they play unique roles in embryonic vascular stabilization or hydrocephalus. Finally, we show that Pak2a signaling is downstream of betaPix. These data identify an essential in vivo role for betaPix and Pak2a during embryonic development and illuminate a previously unrecognized pathway specifically involved in cerebrovascular stabilization.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
bubblehead zebrafish develop multiple hemorrhages in the brain because of a mutation in βPix. (A–F) Isolectin staining of wild type [whole-mount (A), head (B), top view(C)] and bbhm292 mutant embryos with three separate hemorrhages (arrows in D–F). (G and H) Brain hemorrhages and hydrocephalus (*) are also visible in live mutant zebrafish. (G) Wild type. (H) bbhm292 mutant. (I and J) An overlay of isolectin staining on vascular pattern as visualized with flk:GFP localizes hemorrhages near the middle cerebral vein (MCeV) and primordial midbrain and hindbrain channels (PMBC and PHBC, respectively). All embryos are 52–56 hpf. (K and L) Mapping revealed linkage to chromosome 1 at the βPix locus in the bbhm292 and bbhfn40 alleles (K) and a splice site mutation in βPix (L). (M) Four splice variants of βPix were identified, including two variants with alternative 5′ exons and an alternatively spliced internal exon (exon 19). βPix is truncated in the pleckstrin homology domain in bbhm292 due to mutation of a splice donor site. (N) Amplification of βPix cDNA with primers surrounding exon 14 shows a reduction in wild type and increased mutant transcripts in bbhm292. (O) Bbhfn40a mutants have strongly reduced expression of βPix by quantitative PCR.
Fig. 2.
Fig. 2.
βPix splice variants have unique expression and knockdown phenotypes. (A–E) Pan-βPix morphants, βPix-B-specific morphants, or Pak2a morphants (A–C and E) show hemorrhages in the brain with occasional hydrocephalus, whereas exon19 morphants (D) show hydrocephalus at high frequency at 52 hpf. (F and F′) A pan-βPix probe shows expression in the whole brain, in large blood vessels in the trunk, and the major cerebral blood vessels at 31 hpf. (G, G′, H, H′, J, and K) At 36 hpf, βPix-A and -B are expressed in the brain. Only βPix-B is expressed in the blood vessels in the trunk. (I, I′, L, and M) At 48 hpf, Pak2a is highly expressed in the mesenchymal cells around the major cerebral vessels and in the major blood vessels in the trunk. DA, dorsal aorta; V, posterior cardinal vein; He, hemorrhage; MC, mesenchymal cells.
Fig. 3.
Fig. 3.
Altered blood vessel pattern in bbhm292 mutants. Transgenic Flk:GFP marks developing blood vessels in wild-type and bbhm292 mutants. Although indistinguishable at 48 hpf, the extensive angiogenesis occurring between 48 and 54 hpf in wild-type embryos is lacking in mutants, with a partial recovery by 77 hpf.
Fig. 4.
Fig. 4.
Ultrastructural defects in endothelial cell–mesenchymal contacts in bbhm292 mutants. (A and A′) Normal cerebral blood vessels are closely surrounded by mesenchymal cells (A) and have numerous and extensive tight junctions between adjacent endothelial cells (arrow in A′). (B) In bbhm292 mutants, there is poor or no contact of endothelial cells with surrounding substratum, and bbhm292 mutant endothelial cells have a tortuous stretched abluminal surface. (A′ and B′) Enlargements of Insets in A and B, respectively, showing the structure of the vessel wall. (B, C′, and D) Endothelial cells are often stretched (C and D); however, tight junctions between the dysmorphic endothelial cells are maintained (arrows in B′ and D). L, lumen; *, intercellular space. (Scale bars: 1 μm.)

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