Brain pericyte number is consistently lower and does not recover in foxf2a mutant larvae.

(A) Zebrafish brains were imaged using endothelial (red; Tg(kdrl:mCherry)) and pericyte (light blue; Tg(pdgfrβ:Gal4, UAS:GFP)) transgenic lines (arrows: brain pericytes). (A’-A’’) Brain pericyte soma (white arrows) and processes (yellow arrows) are closely associated with the endothelium. (B) Serially imaged wildtype and foxf2a mutant brains at 3, 5, 7 and 10 dpf. (C) Total brain pericyte numbers at 3, 5, 7 and 10 dpf. (D) Individual brain pericyte trajectories of serially imaged embryos over the same period. (E) Dorsal images of embryos for the indicated genotypes from a foxf2a heterozygous incross at 75 hpf. (F) Total brain pericytes at 75 hpf. Statistical analysis was conducted using multiple Mann-Whitney tests (C) and one-way ANOVA with Tukey’s test (F). Scale bars, 50 µm (A-B, E).

Foxf2 affects embryonic pericyte numbers, but not endothelial cell pattern.

(A-B) Expression of foxf2 in a wildtype brain at 72 hpf using Hybridization Chain Reaction (HCR) is co-expressed with pericyte markers. (A) foxf2a is co-expressed with pericyte marker ndufa4l2a (arrows show overlapping expression). (B) foxf2b is co-expressed with pericyte marker pdgfrβ but also lowly expressed in the endothelium (kdrl) (arrow shows overlapping expression with endothelium). (C) Endothelium used to generate the total blood vessel network length. (D) Total vessel network length from Vessel Metrics software. (E) Scatter plot of hindbrain CtA diameters. (F) Scatter plot of pericyte density and pericyte coverage (G). Statistical analysis was conducted using one-way ANOVA with Tukey’s test. Scale bars, 10 µm (A), 50 µm (C).

foxf2a mutants show strong brain vascular defects as adults.

(A-B) 3D projections of iDISCO-cleared immunostained whole wildtype and foxf2a-/- brains at 3 mpf, viewed ventrally. (C-D) Wildtype and foxf2a mutant 2 brain regions, viewed dorsally (arrows = defects in coverage). (E) 3D projections of CUBIC-cleared whole wildtype and foxf2a-/- brains at 11 mpf with Tg(pdgfrβ:Gal4, UAS:GFP, kdrl:mCherry) viewed ventrally. (F) CUBIC-cleared wildtype and foxf2a mutant midbrain at 11 mpf (arrows: individual pericyte soma). C = caudal, D = dorsal, R = rostral, V = ventral. Scale bars, 500 μm (A-B), 200 μm (C-D), 700 μm (E), 50 μm (F).

foxf2a mutants show unusual pdgfrβ-expressing cells and blood vessels in the adult brain.

Immunolabeled sections in equivalent regions of wildtype and foxf2a-/- mutant brains at 11 mpf. (A) Region of the brain with an inset of pdgfrβ-expressing mural cells, likely vSMCs (arrows: large calibre vessel). (B) Region of the brain with an inset of pericytes (arrows: individual cell bodies. Scale bars, 50 µm (A-B).

foxf2a mutant brain pericytes show increased soma size and process length.

(A) Wildtype and foxf2a-/- mutant brain pericytes at 3 and 10 dpf with tracings of individual pericytes (indicated by arrows). (B) Brain pericyte soma area at 3 and 10 dpf. (C) Multispectral Zebrabow labelling reveals pericyte-process interactions in the larval brain. (arrows: pericyte interaction points). (D) Total process length per pericyte at 3 and 10 dpf. (E) Varying pericyte-pericyte interactions at 10 dpf (arrows: interaction points). (F) Number of each type of interaction at 10 dpf. (G) Length of overlap when process interaction occurs.Statistical analysis was conducted using multiple Mann-Whitney tests in B, a one-way ANOVA with Tukey’s test at 3 dpf and a Kruskal-Wallis test with Dunn’s multiple comparisons test at 10 dpf in D. Scale bars, 25 µm (A), 20 µm (C), 5 µm (E).

foxf2a mutant pericytes degenerate.

(A) foxf2a-/- mutant pericyte at 10 and 13 dpf with the degenerating process and cell body (arrows: individual pericyte). (B) Bar graph with process blebbing phenotype penetrance in wildtype and mutant brains (n = total samples examined). (C) Timelapse of a foxf2a-/- mutant midbrain from 4-5 dpf (arrows: individual pericyte). (D) Inset of mutant pericyte undergoing degeneration (arrows: blebbing). Scale bars, 20 µm (A, C).

foxf2a mutants do not have impaired regeneration of brain pericytes after genetic ablation.

Zebrafish brains were imaged using endothelial (red; Tg(kdrl:GFP)) and pericyte (light blue; Tg(pdgfrβ:Gal4, UAS:NTR-mCherry)) transgenic lines. (A) Wildtype and mutant brains at 3 dpf in control (DMSO) and treated (MTZ) groups. (B) Total brain pericytes at 3 dpf. (C) Wildtype and mutant brains at 10 dpf. (D) Total brain pericytes at 10 dpf. Statistical analysis was conducted using a one-way ANOVA (B) or Kruskal-Wallis test with Dunn’s multiple comparisons test (D). Scale bars, 50 μm (A, C).

Model of foxf2a mutant brain pericyte defects over the lifespan.

Wildtype pericytes develop normally in the embryo and establish extensive, continuous coverage over vessels by adulthood. foxf2a mutant pericytes exhibit abnormal morphology during development that changes over the lifespan, leaving mutant vasculature severely affected.

foxf2a mutants exhibit regional loss.

Serial imaging of a foxf2a-/- mutant brain at 3, 5, 7 and 10 dpf (arrows: brain pericytes; boxes: regional loss). Scale bars, 50 µm.

foxf2 knockouts have severe pericyte deficiency during development.

(A) Serial imaging of wildtype and foxf2a-/-;foxf2b-/- double mutant brains at 3, 5, 7 and 10 dpf (arrows: brain pericytes). (B) Scatter plot of total brain pericyte numbers at 3, 5, 7 and 10 dpf. (C) Individual mutant trajectories over the same period. Statistical analysis was conducted using multiple unpaired t-tests with Welch corrections. Scale bars, 50 µm (A).

Expression of foxf2a and foxf2b in single-cell sequencing data from Daniocell.

foxf2a is expressed strongly in mural cells, pericytes and smooth muscle (vascular and visceral), with lower expression of foxf2b in the same cell types. Available at: https://daniocell.nichd.nih.gov/.

foxf2a mutant adult brains have normal size as compared to wildtypes.

(A) 3-month-old and 11-month-old foxf2a-/- mutant and wildtype brains were dissected and imaged dorsally under Brightfield. (B) Standard length measured from snout to base of the tail. (C) Brain length was measured from the tip of the forebrain to the end of the cerebellum. (D) Widest portion of midbrain measured. (E) Ratio of brain length relative to standard length. Statistical analysis was conducted using two-way ANOVAs with Šídák’s multiple comparison test. Scale bars, 50 μm (A).

foxf2a mutants show strong brain vascular defects in early adulthood.

(A-B) 3D projections of iDISCO-cleared immunostained whole wildtype and foxf2a-/-brains at 3 mpf, viewed ventrally (arrows = defects in coverage). R = rostral, C = caudal, D = dorsal, V = ventral. Scale bars, 500 μm (A-B).

Pericyte heterogeneity in the adult zebrafish brain.

Immunolabeling for mural cell transgenes (kdrl:mCherry and pdgfrβ:Gal4, UAS:GFP) on zebrafish brain vibratome sections. Vascular smooth muscle cells (vSMC), and pericyte (ensheathing, mesh and thin strand) subtypes are present in the adult zebrafish brain. Scale bars, 5 µm.

Abnormal blood vessels become apparent in adult foxf2a mutant brains.

Immunolabeled sections in equivalent regions of wildtype and foxf2a-/- mutant brains at 11 mpf. Large aneurysm-like structure with downregulated kdrl compared to the matched wildtype region. Scale bars, 50 µm (A-B).