Peri-arterial specification of vascular mural cells from naïve mesenchyme requires Notch signaling

Abstract

Mural cells (MCs) are essential for blood vessel stability and function; however, the mechanisms that regulate MC development remain incompletely understood, in particular those involved in MC specification. Here, we investigated the first steps of MC formation in zebrafish using transgenic reporters. Using pdgfrb and abcc9 reporters, we show that the onset of expression of abcc9, a pericyte marker in adult mice and zebrafish, occurs almost coincidentally with an increment in pdgfrb expression in peri-arterial mesenchymal cells, suggesting that these transcriptional changes mark the specification of MC lineage cells from naïve pdgfrb^low mesenchymal cells. The emergence of peri-arterial pdgfrb^high MCs required Notch signaling. We found that pdgfrb-positive cells express notch2 in addition to notch3, and although depletion of notch2 or notch3 failed to block MC emergence, embryos depleted of both notch2 and notch3 lost mesoderm- as well as neural crest-derived pdgfrb^high MCs. Using reporters that read out Notch signaling and Notch2 receptor cleavage, we show that Notch activation in the mesenchyme precedes specification into pdgfrb^high MCs. Taken together, these results show that Notch signaling is necessary for peri-arterial MC specification.

Keywords: Mural cells; Notch; Pericytes; Vascular smooth muscle cells; Zebrafish.

Fig. 1.

MC marker expression during early developmental stages. (A) Confocal stack images of the brain vasculature of 96 hpf TgBAC(pdgfrb:GFP);Tg(fli1a:Myr-mCherry) (left), TgBAC(tagln:GFP);Tg(kdrl:DsRed2) (middle) or TgBAC(abcc9:GAL4FF);Tg(UAS:GFP);Tg(kdrl:DsRed2) (right) larvae. CtAs or the vessels in the cerebral base such as the BCA, PCS and BA are shown in the top rows, and the boxed regions are enlarged in the bottom rows. Note that pdgfrb- and abcc9 reporter-positive MCs were readily observed, whereas tagln reporter expression was faint in BCA and PCS (arrowheads) and not detected in CtAs. (B,C) Confocal stack images of the brain vasculature of 54 hpf TgBAC(abcc9:GAL4FF);Tg(UAS:GFP);Tg(kdrl:DsRed2) (B) or TgBAC(abcc9:GAL4FF);Tg(UAS:GFP);Tg(pdgfrb:mCherry) embryos (C). abcc9-positive cells were observed only around vessels (B) and these abcc9-positive cells were positive for pdgfrb:mCherry expression (C). Note that pdgfrb:mCherry expression was broadly detected in the cerebral base but only pdgfrb^high cells around these vessels expressed the abcc9 reporter. (D) Changes of pdgfrb^high MC number (top row), percentage of abcc9 reporter-positive (middle row) or percentage of tagln reporter-positive (bottom row) pdgfrb^high MCs at indicated time points and vessels. The number of pdgfrb^high MCs in ISV is shown per somite in one side of the trunk. (E) Time-lapse imaging of the trunk region of TgBAC(abcc9:GAL4FF);Tg(UAS:GFP);TgBAC(pdgfrb:mCherry) embryo at indicated time points. Boxed regions are enlarged to the bottom. Increase of pdgfrb:mCherry signal could be observed after 67.5 hpf (arrows). Soon after the increase of pdgfrb:mCherry signal, GFP expression was observed only in these pdgfrb^high cells (arrows). (F) Schematic time course of pdgfrb-positive cell emergence (left) (Ando et al., 2016) and the changes of pdgfrb^high-, abcc9-positive or tagln-positive MCs in brain vessels (BCA/PCS, CtAs), DA or ISV, based on D (right). Peri-arterial pdgfrb^low precursors can be detected by 24 hpf. Subsequently, the specification from those pdgfrb^low precursors into pdgfrb^high MCs takes place, and the expression of other MC markers is induced in pdgfrb^high MCs at the cerebral base and beneath the DA after 36 hpf, as well as in the ISV after 48 hpf. BA, basilar artery; CaDl, caudal division of the internal carotid artery; CVP, choroidal vascular plexus; FP, floor plate; HP, hypochord; NC, notochord; PHBC, primordial hindbrain channel. Scale bars: 50 µm in A,E; 100 µm in B; 30 µm in C; 20 µm in enlarged images in A,E.

Fig. 2.

The emergence of pdgfrb^high MCs was inhibited by DAPT treatment during the specification period. (A,B) Confocal stack images of pdgfrb^high MCs at 72 hpf in brain vessels (A) or trunk vessels (B) after treatment with vehicle-DMSO (control), 50 µM SB431542 or 100 μM DAPT from 24 hpf through 72 hpf. E3 medium-containing inhibitors were replaced with fresh ones every 12 h. (C,D) Brain vessels (C) and trunk vessels (D) of 72 hpf TgBAC(pdgfrb:GFP);Tg(fli1a:Myr-mCherry) larvae injected with 6 ng alk1 MO. Note that alk1 MO induced abnormal vasculature in the cerebral base or caudal vein but did not inhibit the emergence of pdgfrb^high MCs. (E) Trunk vessels of TgBAC(pdgfrb:GFP);Tg(fli1a:Myr-mCherry) larvae treated with vehicle-DMSO or 100 µM DAPT at the time point indicated in the top schematic. Boxed regions are enlarged to the right. (F) Confocal stack images of TgBAC(pdgfrb:GFP);Tg(kdrl:Hsa.HRAS-mCherry) larvae treated with either 1% DMSO, 25 μM DAPT or 5 μM LY411575 at the time point indicated in the schematic on the right. Arrows and arrowheads indicate pdgfrb^high MCs beneath DA and in BSA/PCS/BA/ISVs, respectively. Quantitative results are shown on the right of each representative image. Bars and circles in the graph indicate averages and each value of observed larvae, respectively. ****P<0.0001, ***P<0.001 and **P<0.01, significant difference between control and indicated group. BA, basilar artery; CA, caudal artery; CV, caudal vein; HP, hypochord; n.s., not significant; PCS, posterior communicating segment. Scale bars: 30 µm in A,C; 50 µm in B,D,E; 20 µm in enlarged images in B,D,E; 15 µm in F.

Fig. 3.

Both Notch2 and Notch3 are essential for pdgfrb^high MC emergence. (A,B) Confocal stack images of the brain (A) or trunk vessels (B) of 78 hpf TgBAC(pdgfrb:GFP);Tg(fli1a:Myr-mCherry) larvae injected with 10 ng control MO, 10 ng notch2 MO or 5 ng each of notch2 and notch3 MOs. (C) The percentage of aISV with pdgfrb^high MCs, as observed in B (n≥11). (D) Confocal stack images of the brain vessels of 57 hpf TgBAC(abcc9:GAL4FF);Tg(UAS:GFP);Tg(kdrl:DsRed2) embryos injected with 10 ng control MO or 5 ng each of notch2 and notch3 MOs. (E,G) Confocal images of brain (E) and trunk (G) vasculature of the wild-type (WT) siblings or mutants carrying both heterozygous notch2 and notch3 mutations (N2-Het/N3-Het), homozygous notch2 mutation (N2-null/N3-WT), both homozygous notch2 and heterozygous notch3 mutations (N2-null/N3-Het), homozygous notch3 mutation (N2-WT/N3-null) or both heterozygous notch2 and homozygous notch3 mutations (N2-Het/N3-null) with the TgBAC(pdgfrb:GFP);Tg(fli1a:Myr-mCherry) background at 78 hpf. (F,H,I) The number of pdgfrb^high MCs in BCA and PCS in notch2 and notch3 mutants, as shown in panel E (F), the percentage of the DA with MC coverage (MC-covered/total DA length in observed areas were measured using Imaris software) (H) or the percentage of embryos with pdgfrb^high MCs in aISV (I) in notch2 and notch3 mutants, as shown in G. The total number of observed larvae are shown on the top of each bar in I. (J) Confocal stack image of the trunk vessels of 72 hpf TgBAC(pdgfrb:GFP);Tg(fli1a:Myr-mCherry) larvae injected with control MO or low dose (0.75 ng each) of notch2 and notch3 MO combined with subsequent treatment with DMSO or low dose (5 µM) of DAPT from 36 to 72 hpf as shown in the scheme to the top right of J. (K,L) The percentage of aISV with pdgfrb^high MCs (K) and pdgfrb^high MCs number in aISV on one side of the observed trunk (L), as observed in J. Data are mean±s.e.m. (n≥4). (M) Confocal stack image of the hyaloid vessels of 78 hpf TgBAC(pdgfrb:GFP);Tg(fli1a:Myr-mCherry) larvae injected with 10 ng control MO (top) or 5 ng each of notch2 and notch3 MO (bottom). Boxed regions are enlarged to the bottom or right. Arrows and arrowheads indicate pdgfrb^high MCs beneath the DA and in ISV/BCA/PCS, respectively. *P<0.05, **P<0.01, ***P<0.001, significant difference between two groups. n.s., not significant. In F and H, significant difference between WT and indicated mutant group. HP, hypochord; NC, notochord; PCV, posterior cardinal vein. Scale bars: 50 µm; 20 µm in enlarged images.

Fig. 4.

Depletion of both notch2 and notch3 hardly affected Notch-Rbpj-mediated transcriptional activity in ECs. (A) Confocal stack images of the trunk vasculature of the wild-type (WT) sibling or notch2/notch3 mutants with the Tg(fli1a:H2B-GFP);Tg(TP1:H2B-mCherry) background at 74 hpf. (B) The percentage of ECs in observed trunk region with TP1:H2B-mCherry expression, as observed in A. Total number of EC nuclei marked by fli1a:H2B-GFP and the number of EC nuclei positive for both fli1a:H2B-GFP and TP1:H2B-mCherry were counted using Imaris software. Bars and circles indicate averages and each value of observed larvae, respectively. (C) Confocal images of trunk vasculature of the 50 hpf Tg(fli1a:H2B-GFP);Tg(TP1:H2B-mCherry) embryos injected with 10 ng control MO (left), 5 ng each of notch2 and notch3 MOs (middle, N2/N3 MOs) or 10 ng of dll4 MO (right). (D) The expression of deltaC or ephrin-B2a mRNA, as detected by whole-mount in situ hybridization, in 48 hpf embryos injected with 10 ng control MO or 5 ng each of notch2 and notch3 MOs. Numbers indicate embryos with the similar expression as shown in each image/total number of embryos. Two independent experiments were performed for each marker with at least seven embryos per experiment. In A and C, arrowheads indicate ECs with TP1:H2B-mCherry expression; boxed regions are enlarged to the right. Scale bars: 50 µm; 10 µm in enlarged images. DLAV, dorsal longitudinal anastomotic vessels; PCV, posterior cardinal vein.

Fig. 5.

Time-lapse imaging of TP1 reporter expression in MCs. (A) Time-lapse imaging of the trunk region (ISV or DA) of Tg(TP1:GFP);TgBAC(pdgfrb:GAL4FF);Tg(4xUAS:RFP) every 12 min (see also Movie 1). Increase of RFP signal was observed in the cells positive for GFP expression (Notch activation) (arrows). Representative images at indicated time points and fluorescence changes (a1-a3) showing % of the peak value of pdgfrb or TP1 reporter fluorescence intensity after the subtraction of each fluorescent intensity of the first flame. Quantifications a1 (dorsal side) and a2 (ventral side) correspond to one of the ISV-MCs in embryo 1 (see Movie 1), and a3 corresponds to an ISV-MC in embryo 3 (images not shown). Red dots, pdgfrb reporter; green dots, TP1 reporter. Polynomial fitting curves for the pdgfrb (red line) and TP1 (green line) were assigned using the polyfit function in MATLAB with the degree of 7. Scale bars: 15 μm.

Fig. 6.

Notch activation in pdgfrb^high MCs. (A) Schematic structure of the BAC clone (CH211-39M8) which was used to generate TgBAC(notch2:Notch2-GAL4FF) for visualizing Notch2-activated cells. A cDNA encoding GAL4FF was inserted at the C terminus of the notch2 gene. Notch2 intracellular region (N2ICD) fused with GAL4FF is proteolytically released and translocated to the nucleus upon Notch2 activation, therefore, Notch2 activated cells can be monitored by the expression of proteins such as GFP driven by the UAS/GAL4FF system. (B) Confocal images of the DA in TgBAC(notch2:Notch2-GAL4FF);Tg(UAS:GFP);TgBAC(pdgfrb:mCherry) larvae at 76 hpf. Left, stack images. Right, single slice images from stack images on the left. Arrows indicate pdgfrb^high MCs beneath the DA with Notch2 activation. The fluorescence intensity profile of pdgfrb:mCherry (red line) and UAS:GFP (green line) along the line in the single slice images (B1-B3) are shown on the right, indicating the colocalization of mCherry and GFP expression. The x-axes show distance from the beginning of the line. (C) Confocal stack images of the trunk vessels of TgBAC(notch2:Notch2-GAL4FF);Tg(UAS:GFP);TgBAC(pdgfrb:mCherry) larvae at 76 hpf. Vascular structure was visualized by injection of Qtracker 705 vascular labels (aqua) into the circulation. Boxed regions depicting pdgfrb^high MCs in aISV are enlarged to the right. Arrows indicate the Notch2-activated pdgfrb^high MCs. Notch2-activity-positive MCs in ISVs/DA at 74 hpf were seen in 31.6% (12/38) of embryos shown. (D) Time-lapse imaging of the trunk region (ISV) of TgBAC(notch2:Notch2-GAL4FF);Tg(UAS:GFP);TgBAC(pdgfrb:mCherry) embryo at the indicated time points. Note that Notch2 activation precedes the increase of pdgfrb expression. GFP signal was absent at 50 hpf, but induced in pdgfrb^low cells after 72 hpf. Subsequently, pdgfrb:mCherry expression was increased in Notch2-activated pdgfrb^low cells (arrows). Representative fluorescence changes (% of the peak value of pdgfrb- or Notch2-activity reporter fluorescence) are shown on the right (D1, ISV-MC; D2, DA-MC). Red dots, pdgfrb reporter. Green dots, Notch2-activity reporter. Polynomial fitting curves for the pdgfrb (red line) and Notch2-activity reporter (green line) were assigned using the polyfit function in MATLAB with the degree of 7. The x-axes show the relative time. (E) Comparison of the time showing the highest value of Notch2 activity and the pdgfrb reporter in fitting curve. The data are mean±s.e.m. (n=6). Note that the time showing the highest GFP fluorescence preceded that of mCherry fluorescence by more than 8 h on average, indicating that Notch2 is activated before the onset of pdgfrb increase. HP, hypochord; N2ECD, Notch2 extracellular region; PCV, posterior cardinal vein. Scale bars: 20 µm in B,D: 50 µm in C: 10 µm in enlarged images in C.