Integrated molecular analysis identifies a conserved pericyte gene signature in zebrafish

Abstract

Pericytes reside in capillary beds where they share a basement membrane with endothelial cells and regulate their function. However, little is known about embryonic pericyte development, in part, due to lack of specific molecular markers and genetic tools. Here, we applied single cell RNA-sequencing (scRNA-seq) of platelet derived growth factor beta (pdgfrb)-positive cells to molecularly characterize pericytes in zebrafish larvae. scRNA-seq revealed zebrafish cells expressing mouse pericyte gene orthologs, and comparison with bulk RNA-seq from wild-type and pdgfrb mutant larvae further refined a pericyte gene set. Subsequent integration with mouse pericyte scRNA-seq profiles revealed a core set of conserved pericyte genes. Using transgenic reporter lines, we validated pericyte expression of two genes identified in our analysis: NDUFA4 mitochondrial complex associated like 2a (ndufa4l2a), and potassium voltage-gated channel, Isk-related family, member 4 (kcne4). Both reporter lines exhibited pericyte expression in multiple anatomical locations, and kcne4 was also detected in a subset of vascular smooth muscle cells. Thus, our integrated molecular analysis revealed a molecular profile for zebrafish pericytes and allowed us to develop new tools to observe these cells in vivo.

Keywords: pdgfrb; Pericyte; Vascular development; Zebrafish.

Fig. 1.

scRNA-seq analysis of pdgfrb:egfp-positive cells. (A) UMAP plot of pdgfrb:egfp-positive cells from 5 dpf TgBAC(pdgfrb:egfp)^ncv34 larvae. Clusters with highest pdgfrb levels are shown. Pericyte cluster is circled. (B) Violin plot of top cluster 39-enriched genes (sorted by decreasing log₂ fold change relative to all other clusters, adjusted P<0.05) and genes with known pericyte-expressed orthologs (in red; log₂ fold change>0.75, adjusted P<0.05, Wilcoxon Rank Sum test). Pericyte cluster highlighted by rectangle. (C) Violin plot of smooth muscle gene expression in indicated clusters. (D) Venn diagram of genes enriched in pdgfrb:citrine-positive cells (compared with pdgfrb:citrine-negative cells, log₂ fold change>1, adjusted P<0.05), reduced in pdgfrb:citrine-positive pdgfrb^um148 mutant cells (compared with wild type; log₂ fold change<−1, adjusted P<0.05), and enriched in cluster 39 (log₂ fold change>0.75, adjusted P<0.05). (E) Venn diagram of genes enriched in mouse brain and lung pericytes and zebrafish pericytes. Only genes with log₂ fold change>0.75, adjusted P<0.05 in each respective individual analysis were considered. Violin plots show log₂-normalized expression levels across distribution of cells in indicated cluster.

Fig. 2.

The ndufa4l2a locus drives pericyte-specific expression. (A-D″) Confocal micrograph of 5 dpf TgBAC(ndufa4l2a:sfgfp)^um382;(abcc9:gal4ff)^ncv34;(uas:rfp)^nkuasrfp1a larvae subjected to QDot angiography (blue). (A) ndufa4l2a:sfgfp expression in pericytes (selected cells denoted by arrowheads) along branches emanating from the middle mesencephalic central artery (mmcta) and posterior mesencephalic central artery (pmcta), and along the metencephalic artery (mta). Positive cells also seen along central arteries (cta). ba, basilar artery. (A′,A″) abcc9:rfp expression (A′) and overlay (A″) for same embryo as A. Asterisks denote cells only expressing sfGFP. (B-B″) Magnified image of boxed area in ndufa4l2a:sfgfp and abcc9:rfp co-expression (arrows) in pericytes shown in A. (C,C′) ndufa4l2a:sfgfp (C) and abcc9:rfp (C′) expression in retinal pericytes. (C″) Overlay: arrowheads denote co-expressing pericytes. (D,D′) ndufa4l2a:sfgfp (D) and abcc9:rfp (D′) expression in pericytes along the intersegmental vessels (isv). (D″) Overlay: arrows denote co-expressing pericytes; asterisk indicates cells only expressing sfGFP. da, dorsal aorta; pcv, posterior cardinal vein. (A-C) Dorsal views, anterior is up. (D) Lateral view, dorsal is up, anterior is to the left. Scale bars: 30 μm (A); 10 μm (B); 50 μm (C,D).

Fig. 3.

The kcne4 locus drives expression in pericytes and selected vascular smooth muscle cells. (A-G) Confocal micrographs of 5 dpf TgBAC(kcne4:sfgfp)^um333 larvae subjected to angiography with QDots also bearing TgBAC(pdgfrb:gal4ff)^ncv24;(uas:rfp)^nkuasrfp1a (referred to as pdgfrb:rfp) (A,B) or Tg(acta2:mcherry)^ca8 (C-G). (A) kcne4:sfgfp in pericytes (selected cells denoted by arrowheads) on cranial vessels. (A′,A″) pdgfrb:rfp expression (A′) and overlay (A″) for same embryo as A. (B,B′) kcne4:sfgfp (B) and pdgrb:rfp (B′) in retinal pericytes. (B″) Overlay: arrowheads denote co-expressing pericytes. (C,C′) kcne4:sfgfp in pericytes (arrows) along the intersegmental vessels (isv) (C) and acta2:mcherry in vascular smooth muscle cells (VSMCs) (arrowheads with asterisk) on dorsal aorta (da) (C′). pcv, posterior cardinal vein. (C″) Overlay. Magnification of boxed area shown in E-E″. (D,D′) Circle of Willis. (D) Overlay image showing acta2:mcherry-positive VSMCs (arrows) and nearby cranial arteries with kcne4:sfgfp-positive pericytes (arrowheads). (D′) kcne4:sfgfp channel only, showing absence of VSMC expression. (E-E″) Magnification of boxed area in C″ showing single confocal section of the intestine. (E,E′) kcne4:sfgfp detected in pericytes on intestinal vessels (arrowheads) and scattered acta2:mcherry-positive smooth muscle cells (arrowhead denoted by asterisk). (E″) Overlay. (F-G″) Ventral view of outflow tract and associated vasculature. (F) Overlay showing kcne4:sfgfp expressed in pericytes on the hypobranchial artery (ha). Ventral aorta (va) is noted, as is cardiac ventricle (vent). (G-G″) Magnification of boxed area in F. Channels and overlay as indicated in each panel. Plain arrowheads denote kcne4:sfgfp expression in acta2-positive VSMCs. Arrowheads marked with an asterisk are sfGFP-positive cells located ventral to the ventral aorta (see Fig. S3D,E). (A-B,D) Dorsal views, anterior is up. (C,E) Lateral view, dorsal is up, anterior is to the left. (F,G) Ventral views, anterior is up. Scale bars: 50 μm (A-D,F); 25 μm (E,G).