30 hpf scRNAseq (see Excel sheet).

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.</div

Nkx3.1 function is required to regulate brain pericyte numbers.

Lateral view of control nkx3.1+/− (A) and nkx3.1−/− MZ (B) mutants showing brain hemorrhage (arrowhead) at 52 hpf, and quantification (C; N = 3, proportions of hemorrhage). (D) nkx3.1−/− have decreased CtA vessel diameter in comparison to nkx3.1+/− hets. Dorsal images of nkx3.1+/− hets (E) and nkx3.1−/− (F) embryos expressing Tg(pdgfrβ:GFP) and Tg(kdrl:mCherry) showing fewer brain pericytes (arrows) in mutants at 75 hpf. Quantification of (G) decreased pericyte number and (H), decreased pericyte density (defined as the number of pericytes divided by the length of the vessel network) in mutants. In comparison to control wild-type embryos (I, Tg(hsp70l:tBFP), nkx3.1 gain-of-function (GOF) embryos expressing Tg(hsp70l:tBFP-2a-nkx3.1)) show more pericytes (J, arrowheads) (n = 20 control and 19 GOF)) as quantified (K). Pericyte density is also increased (L). Dorsal views of embryos labelled with TgBAC(nkx3.1:Gal4) and Tg(UAS:NTR-mCherry) under fluorescence (M, N) or under brightfield (O, P) that are untreated (M, O) or treated with metronidazole (N, P) to ablate nkx3.1-expressing cells. Ablated embryos show brain hemorrhage (P, arrowhead) at 48 hpf (P). (Q) Quantification of pericyte number in ablated embryos. Statistical significance was calculated using the Student t test (n = 5 wild type and 11 nkx3.1 mutants). Scale bars are 50 μm. The data underlying this figure can be found in S3 Table.</p

<i>nkx3</i>.<i>1</i> expression patterns and cell behavior.

All images are captured dorsally and the anterior (A) and posterior (P) axis is marked. (A-C) Expression of nkx3.1 by HCR in situ hybridization. (A) At 16 hpf, nkx3.1 is expressed in the hindbrain and anterior trunk. Arrowheads mark nkx3.1 expression. (B) At 30 hpf, nkx3.1 is expressed in the posterior head and trunk. (C) At 48 hpf, nkx3.1 expression is not detectable. (D-E’) nkx3.1NTR-mcherry cells (red) are adjacent to endothelium (green; Tg(flk:GFP)) in brain vessels at 4 dpf. Pericytes in midbrain (arrowheads, E, F) and hindbrain (arrowheads, E’, F’) are denoted in dual channel (E, E’) and single-channel pericyte (F, F’) images. (G) Brain pericytes labelled by TgBAC(pdgfrβ:GFP) coexpress nkx3.1NTR-mcherry 75 hpf. (H) Enlargement of an individual brain pericyte marked by a square in G. (I) Quantification brain pericytes coexpressing nkx3.1 at 75 hpf (N = 3 experiments and 30 embryos). (J-M) Single images from time-lapse of nkx3.1NTR-mcherry cells in the midbrain. White and yellow arrowheads track individual cells that migrate and divide with time. Scale bar in all images is 50 μm.</p

<i>cxcl12b</i> is regulated by <i>nkx3</i>.<i>1</i>; loss of Cxcr4 signaling reduces brain pericytes.

All embryos were imaged dorsally in the head region. cxcl12b mRNA expression is reduced in the embryonic head of nkx3.1−/− mutants (B) as compared to wild-type controls (A) at 30 hpf as quantified by fluorescent intensity (C) of regions marked by dotted lines in A and B. (n = 10 wild-type and 10 mutant embryos). Inhibition of Cxcr4 from 24–75 hpf using AMD 3100 leads to reduced brain pericytes in treated (E) vs. untreated (D) embryos (at 75 hpf (n = 10)). Pericytes (red cells, white arrows) are labeled by TgBAC(pdgfrβ:Gal4); Tg(UAS:NTR-mCherry). Brain vessels in D and E are labeled by Tg(flk:GFP; green). Quantification of (F) pericyte number and (G) pericyte density. (n = 10 wild type and 10 mutants). Statistical significance was calculated using the Student t test. Scale bar is 50 μm. The data underlying this figure can be found in S3 Table.</p

Zygotic <i>nkx3.1</i> mutants have wild-type pericyte numbers at 75 hpf.

(A, B) Dorsal views of embryonic brain of zygotic nkx3.1 mutants showing no change in brain pericyte numbers as compared to wild type. Pericytes (green, arrowheads) are labelled with TgBAC(pdgfrβ:GFP) and vessels (red) are labelled with Tg(kdrl:mCherry). (C) Quantitation of pericyte numbers shows no significance using one-way ANOVA with Tukey’s test. (n = 8 wild types, 17 heterozygotes, and 10 mutants). Scale bar is 50 μm. The data underlying this figure can be found in S3 Table. (PDF)</p

Expression analysis of <i>nkx3.1</i> and <i>cxcl12b</i> using HCR.

(A) Dorsal view of the posterior head region of 16 hpf embryo showing expression overlap (white bracket) between cxcl12b (green) and nkx3.1 (red). (B) Lateral view of the posterior head region of 24 hpf embryo showing expression overlap (white bracket) between cxcl12b and nkx3.1. A-Anterior, P-Posterior, D-Dorsal, V-Ventral. Scale bar is 50 μm. (PDF)</p

Featureplots of canonical pericyte markers in the nkx3.1-positive cell scRNAseq data.

Featureplots of canonical pericyte markers in the nkx3.1-positive cell scRNAseq data.</p

Central artery vessel network length is unchanged across multiple experimental manipulations.

Vessel network length of the hindbrain CtAs was measured using VesselMetrics. Genotypes and treatments are labelled. No treatment or mutant significantly alters the endothelial vessel length. Statistics used a Student t test. The data underlying this figure can be found in S3 Table. (PDF)</p

No overlap between <i>cxcl12b</i> and <i>cxcr4a</i> mRNA at 36 hpf.

Dorsal view of the ventral head region of 36 hpf embryo showing no expression overlap between cxcl12b (green) and cxcr4a (red). A-Anterior, P-Posterior, Scale bar is 20 μm. (PDF)</p

Python Script for velocity analysis.

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.</div