Developmental Biology

Glial betaPix is essential for blood vessel integrity in the zebrafish brain

ShihChing Chiu
Qinchao Zhou
Chenglu Xiao
Linlu Bai
Xiaojun Zhu author has email address
Wanqiu Ding author has email address
Jing-Wei Xiong author has email address

Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, and College of Future Technology, Peking University, Beijing, China
School of Basic Medical Sciences, The Second Affiliated Hospital, Institute of Biomedical Innovation, The MOE Basic Research and Innovation Center for the Targeted Therapeutics of Solid Tumors, Jiangxi Medical College, Nanchang University, Nanchang, China

https://doi.org/10.7554/eLife.106665.1

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Figures and data

Generation of betaPix conditional trap (betaPix^ct) allele by an homologous recombination (HDR)-mediated knock-in method.
(A) Schematic diagram illustrates the HDR-mediated Zwitch strategy for generating zebrafish knock-in allele at the betaPix locus.
(B) Genomic PCR analysis of the F₁ embryos confirming the right Zwitch insertions.
(C) Sanger sequencing confirming the junction of betaPix^ct (after HDR-mediated insertion) or betaPix^m (after Cre-mediated inversion) that are highlighted by RED lines in (A).
(D) Representative stereomicroscopy images of erythrocytes stained with o-dianisidine in betaPix^ct/ct and betaPix^m/m embryos at 48 hpf. Brain hemorrhages, indicated with arrows, in Cre mRNA-injected embryos (betaPix^m/m). Lateral views, anterior to the left.
(E) Quantification of hemorrhagic parameters in (D). Left panel showing hemorrhage percentages with each dot representing an individual experiment (n>100 each), and right panel showing hemorrhage areas with each dot representing one embryo.
(F) qRT-PCR analysis showing the expression of betaPix, RFP and alphaPix in betaPix^ct/ct, betaPix^ct/m, and betaPix^m/m embryos at 48 hpf. Each dot represents one embryo. cryaa, αA-crystallin; PA, polyadenylation signal; SA, splice acceptor; T2A, T2A self-cleaving peptide.

betaPix^m/m mutant have brain hemorrhages, central artery defects and abnormal glial structure that was partially rescued by Pak1 inhibitor IPA3 treatment.
(A) Left panel showing the maximum intensity projection of the glial structures in the hindbrain of betaPix^ct/ct and betaPix^m/m embryos at 48 hpf. Lateral view, anterior to left. Middle panel showing representative optical sections and right panel showing the higher magnifications of boxed area, presenting atypical glial structures with disoriented arrangements (yellow arrows) in betaPix^m/m embryos.
(B) Quantification of glial parameters in (A). Left panel showing the average glia length, and right panel showing glia length index normalized to individual head length, which each dot represents one embryo.
(C) Whole-mount RNA in situ hybridization revealed nestin and pax2a expression pattern in betaPix^ct/ct and betaPix^m/m embryos at 48 hpf. Dorsal view, anterior to the left.
(D) Optical sections of glial structure (green) and blood vessels (magenta) in the heads of siblings and CRISPR-mediated betaPix F₀ knockout embryos. Arteries in the hindbrain of betaPix KO mutants had developmental defects (white arrowheads), showing shorter distance between basilar artery (BA) and glial cell bodies.
(E) 3D reconstruction of the sox2-positive precursors (green) and vasculatures (magenta) in the heads of siblings and CRISPR-mediated betaPix F₀ knockout embryos at 48 hpf. Box areas are shown in higher magnifications at the middle panels, with optical sections shown in the right panels. Arrows indicate CtA with enlarged perivascular space.
(F) Representative stereomicroscopy images of o-dianisidine staining of betaPix^ct/ct and betaPix^m/m embryos at 48 hpf that were treated with DMSO or PAK inhibitor IPA3. Brain hemorrhages indicated with the arrows.
(G) Quantification of brain hemorrhagic parameters in (F).
(H) 3D reconstruction of the glial structure (green) and vasculature (magenta) in the heads of betaPix^ct/ct and betaPix^m/m embryos at 48 hpf treated with DMSO or IPA3. Lateral view, anterior to left. Defects in CtAs (white arrows) and glia (yellow arrows) shown in betaPix^m/m embryos treated with DMSO or IPA3.
(I) Quantification of CtA parameters in (H).
(J) Quantification of glia parameters in (H).

Glial-specific betaPix knockouts recapture global betaPix mutant phenotypes.
(A) Representative stereomicroscopy images of erythrocytes stained with o-dianisidine in betaPix^ct/ct siblings and gfap:GFP-Cre; betaPix^ct/ct mutant embryos treated with DMSO or IPA3 at 48 hpf. Brain hemorrhages indicated with arrows in glial-specific betaPix knockouts. Lateral view with anterior to the left.
(B) 3D reconstruction of the vasculatures (magenta) in the heads at 48 hpf, showing lateral view with anterior to the left. The box areas are shown in higher magnifications of brain vasculatures at the right panels. CtA defects (yellow arrows) were evident in gfap:GFP-Cre; betaPix^ct/ct mutant embryos.
(C) Quantification of brain hemorrhages (A) and CtA parameters (B). Left panel showing hemorrhage percentages with each dot representing an individual experiment (n>100 each). Middle panel showing hemorrhage areas with each dot representing one embryo. Right panel showing CtA length index normalized to individual head length, with each dot representing one embryo.

Single cell transcriptome reveals that a subcluster of glial progenitor and stathmin family members are associated with betaPix mutation.
(A) Experimental strategy for single cell RNA sequencing of embryonic heads from wild-type siblings and betaPix CRISPR mutants at 1 dpf and 2 dpf.
(B) UMAP visualization and clustering of cells labeled by cell type. Four samples were aggregated and analyzed together.
(C) Proportions of 24 cell clusters were differentially distributed among four sample groups. ctrl_1d, PBS-injected siblings at 1 dpf; ko_1d, betaPix CRISPR mutants at 1 dpf; ctrl_2d, PBS-injected siblings at 2 dpf; ko_2d, betaPix CRISPR mutants at 2 dpf.
(D) Enriched GO terms for differentially expressed genes for progenitor sub-cluster comparing ko_2d to ctrl_2d groups.
(E) Violin plots showing the expression of the stathmin family genes by all cells among four sample groups (left panel) or by progenitor sub-cluster among four sample groups (right panel).
(F) qRT-PCR analysis showing expression of betaPix, stmn1a, stmn1b, stmn4l and ppfia3 in glia of FACS-sorted betaPix^ct/ct siblings and betaPix^m/m mutants at 48 hpf. Each dot represents cells sorted from one embryo.
(G) qRT-PCR analysis showing expression of betaPix, stmn1a, stmn1b, stmn4l and ppfia3 in glia of FACS-sorted betaPix^ct/ct siblings and gfap:GFP-Cre; betaPix^ct/ct mutants at 48 hpf. Each dot represents cells sorted from one embryo.
(H) Whole-mount RNA in situ hybridization revealing down-regulation of stmn1a, stmn1b and stmn4l in betaPix^ct/ct siblings and gfap:GFP-Cre; betaPix^ct/ct mutant embryos at 48 hpf.

Stathmin acts downstream of betaPix in glial migration via regulating tubulin polymerization.
(A) Whole-mount RNA in situ hybridization showing that stmn1a, stmn1b and stmn4l expression in gfap:GFP-Cre; betaPix^ct/ct embryos were partially rescued by Pak1 inhibitor IPA3 treatment at 48 hpf. Dorsal views, anterior to the left.
(B) qRT-PCR analysis showing that stmn1a and stmn4l expression were rescued in gfap:GFP-Cre; betaPix^ct/ct mutants by IPA3 treatment at 48 hpf. Each dot represents one embryo.
(C) Representative stereomicroscopy images of erythrocytes stained with o-dianisidine in siblings and stmn1a/1b/4l CRISPR mutants at 48 hpf. Brain hemorrhages, indicated with arrows, appeared in stmn1a/1b/4l mutants. Lateral views with anterior to the left.
(D) Quantification of hemorrhagic parameters in (C). Left panel showing hemorrhage percentages with each dot representing an individual experiment (n>100 each), and right panel showing hemorrhage areas with each dot representing one embryo.
(E) 3D reconstruction of glial structure (green) and vasculature (magenta) in the heads at 48 hpf. Lateral view with anterior to the left. CtA defects (white arrows) appeared in stmn1a/1b/4l mutants.
(F) Quantification of CtA and glia parameters in (E). Length index normalized to individual head length, with each dot representing one embryo.
(G) Representative stereomicroscopy images of erythrocytes stained with o-dianisidine in bbh^fn40a and bbh^fn40a; Tg(gfap:GFP-stmn1b) embryos at 48 hpf. Brain hemorrhages, indicated with arrows, decreased in bbh^fn40a mutants with glia-specific overexpression of stmn1b, compared with bbh^fn40a mutant siblings. Lateral views with anterior to the left.
(H) Quantification of hemorrhagic parameters of (G). Left panel showing hemorrhage percentages with each dot representing an individual experiment (n>100 each), and right panel showing hemorrhage area with each dot representing one embryo.
(I) 3D reconstruction of the gfap:GFP-stmn1b overexpression (green) and vasculature (magenta) in the heads of bbh^fn40a siblings and bbh^fn40a; Tg(gfap:GFP-stmn1b) mutants at 48 hpf. Lateral view with anterior to left. White arrows indicate CtA defects.
(J) Quantification of CtA and glia parameters in (I). Length index normalized to individual head length, with each dot representing one embryo.
(K) Representative stereomicroscopy images of U251 cells at 0 and 24 hours after wounding. U251 cells were transfected with negative control siRNA or betaPIX siRNA separately, in combination with pcDNA3.1 vector, betaPIX overexpression plasmid, and STMN1 overexpression plasmid. The wound edges are highlighted by dashed lines, with arrow lines indicating the wound width.
(L) Quantification of wound closure in (K), showing *P<0.05, ***P<0.005 compared to negative control siRNA with empty vector transfection. ^##P<0.01, ^###P<0.005 compared to betaPix knockdown with empty vector transfection.
(M) Representative immunofluorescence image of alpha-tubulin and DAPI signals in U251 cells. U251 cells were transfected with negative control siRNA or betaPIX siRNA separately, in combination with pcDNA3.1 vector, betaPIX overexpression plasmid, and STMN1 overexpression plasmid. Box areas are shown in higher magnifications. Arrows indicate protrusions at the cell periphery.
(N) Quantification of cell percentages with protrusions in (M). ***P<0.005 compared to negative control siRNA with empty vector transfection. ^#P<0.05, ^##P<0.01 compared to betaPix knockdown with empty vector transfection.

Zfhx3/4 acts downstream of betaPix to regulate vascular integrity development.
(A) Whole-mount RNA in situ hybridization revealing that vegfaa decreased in bbh^fn40a mutants compared with siblings at 36 hpf. Lateral views with anterior to the left. Arrows indicate vegfaa expression in the CtAs.
(B) qRT-PCR analysis revealing that ZFHX3, ZFHX4 and VEGFA decreased in U251 cells transfected with betaPIX siRNA, which were rescued by betaPIX overexpression.
(C) Whole-mount RNA in situ hybridization showing that vegfaa decreased in CRISPR-mediated zfhx3/4 F₀ knockout embryos at 36 hpf. Lateral views with anterior to the left. Arrows indicate vegfaa expression in the CtAs.
(D) Representative stereomicroscopy images of erythrocytes stained with o-dianisidine in siblings and CRISPR-mediated zfhx3/4 F₀ knockout embryos at 48 hpf. Arrows indicated brain hemorrhages in the knockout brains. Lateral views with anterior to the left.
(E) Quantification of hemorrhagic parameters in (D). Left panel showing hemorrhage percentages with each dot representing an individual experiment (n>100 each), and right panel showing hemorrhage areas with each dot representing one embryo.
(F) 3D reconstruction of the glial structure (green) and vasculature (magenta) in the hindbrains of siblings and CRISPR-mediated zfhx3/4 F₀ knockouts at 48 hpf. Lateral view with anterior to the left. White arrows indicate CtA defects in zfhx3/4 knockout embryos.
(G) Quantification of CtA and glia length parameters of (F). Length index normalized to individual head length, with each dot representing one embryo.
(H) Representative stereomicroscopy images of erythrocytes stained with o-dianisidine in CRISPR-mediated betaPix F0 knockout embryos with or without zfhx4 mRNA injection at 48 hpf. Arrows indicate brain hemorrhages. Lateral views with anterior to the left.
(I) Quantification of hemorrhagic parameters in (H). Left panel showing hemorrhage percentages with each dot representing an individual experiment (n>100 each), and right panel showing hemorrhage area with each dot representing one embryo.
(J) 3D reconstruction of the glial structure (green) and vasculature (magenta) in CRISPR-mediated betaPix F₀ knockout embryos with or without zfhx4 mRNA injection at 48 hpf. White arrows indicate CtAs and yellow arrows indicate glia.
(K) Quantification of CtA and glia length parameters in (J). Length index normalized to individual head length, with each dot representing one embryo.
(L) Whole-mount RNA in situ hybridization revealed that zfhx4 and vegfaa decreased in CRISPR-mediated betaPix F₀ knockout embryos at 36 hpf, which were rescued by zfhx4 mRNA injection. Lateral views with anterior to the left. Arrows indicate hindbrain regions.
(M) Dot plots of several angiogenesis-associated genes expression in endothelial cell cluster. Dot size indicates the percentage of cells with gene expression, and dot color represents the average gene expression level.

Working Model on the function of glial betaPix in zebrafish vascular integrity development of the hindbrain.
betaPix is enriched in glia and regulates the PAK1-Stathmin axis on microtublin stabilization, thus fine-tuning glial cell migration and their interactions with vanscular endothelial cells; and in paralelle, betaPix may also regulate Zfhx3/4-Vegfaa signaling in glia, which then modulates angiogenesis during cerebral vessel development and maturation. Deletion of betaPix affects glial cell migration and interaction with cerebral endothelial cells. MT, Microtubule.

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