Clarification of mural cell coverage of vascular endothelial cells by live imaging of zebrafish

Abstract

Mural cells (MCs) consisting of vascular smooth muscle cells and pericytes cover the endothelial cells (ECs) to regulate vascular stability and homeostasis. Here, we clarified the mechanism by which MCs develop and cover ECs by generating transgenic zebrafish lines that allow live imaging of MCs and by lineage tracing in vivo To cover cranial vessels, MCs derived from either neural crest cells or mesoderm emerged around the preformed EC tubes, proliferated and migrated along EC tubes. During their migration, the MCs moved forward by extending their processes along the inter-EC junctions, suggesting a role for inter-EC junctions as a scaffold for MC migration. In the trunk vasculature, MCs derived from mesoderm covered the ventral side of the dorsal aorta (DA), but not the posterior cardinal vein. Furthermore, the MCs migrating from the DA or emerging around intersegmental vessels (ISVs) preferentially covered arterial ISVs rather than venous ISVs, indicating that MCs mostly cover arteries during vascular development. Thus, live imaging and lineage tracing enabled us to clarify precisely how MCs cover the EC tubes and to identify the origins of MCs.

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

Fig. 1.

Generation of Tg zebrafish lines for live imaging of MCs. (A) A schematic structure of the BAC clone (CH1073-606I16) used to generate Tg zebrafish lines for live imaging of MCs. cDNA encoding either EGFP, mCherry or Gal4FF was inserted at the start codon of pdgfrb gene. (B) Confocal stack fluorescence image of trunk vasculature in a 96 hpf TgBAC(pdgfrb:Gal4FF);Tg(UAS:GFP);Tg(fli1a:Myr-mCherry) larva. Lateral view, anterior to the left. Merged image of pdgfrb:Gal4FF;UAS:GFP (green) and fli1a:Myr-mCherry (red). (C-F) Confocal images of hindbrain vasculature (C,D), hyaloid vessels (E) and anterior region of dorsal aorta (F) in the TgBAC(pdgfrb:EGFP);Tg(fli1a:Myr-mCherry) larvae at 60 hpf (C) and 80 hpf (D-F). Dorsal view, anterior to the left. Merged images of pdgfrb:EGFP (green) and fli1a:Myr-mCherry (red). In C, the boxed areas are enlarged to the right. (G) Confocal images of trunk vasculature in a 1 mpf TgBAC(pdgfrb:EGFP);Tg(fli1a:Myr-mCherry) juvenile. Cross-sectional views (200 μm thick) through the caudal region as depicted in Fig. S1H are shown. Upper left, pdgfrb:EGFP (green); upper center, fli1a:Myr-mCherry (red); upper right, merged image. The boxed areas labeled a and b are enlarged below. (H) Confocal images of blood vessels in the intercostal muscle of a 1 mpf TgBAC(pdgfrb:EGFP);Tg(fli1a:Myr-mCherry) juvenile. Pleural tissue as indicated by the box shown in g was cut out and immunostained with anti-α-SMA antibody to visualize VSMCs. The merged image of pdgfrb:EGFP (green) and fli1a:Myr-mCherry (red) is shown on the left (a). The boxed area in a is enlarged to the right: pdgfrb:EGFP (b), fli1a:Myr-mCherry (c), α-SMA (d), merge of pdgfrb:EGFP (green) and fli1a:Myr-mCherry (red) (e) and merge of pdgfrb:EGFP (green), fli1a:Myr-mCherry (red) and α-SMA (blue) (f). (g) Brightfield image of the thorax showing the region where the image shown in a was taken. BA, basilar artery; BCA, basal communicating artery; CCtA, cerebellar central artery; DA, dorsal aorta; LDA, lateral DA; HV, hyaloid vessel; PCS, posterior communicating segment; PHBC, primordial hindbrain channel. Scale bars: 20 μm (enlarged images in C and H; D-F); 50 μm (B,C); 100 μm (G,H).

Fig. 2.

Live imaging of MC coverage in cranial vessels. (A-C) Time-lapse confocal images of the hindbrain vasculature in TgBAC(pdgfrb:EGFP);Tg(fli1a:Myr-mCherry) larvae at 35-42 hfp (A), 59-63 hpf (B) and 65-71 hpf (C). Upper, pdgfrb:EGFP; lower, merged images of pdgfrb:EGFP (green) and fli1a:Myr-mCherry (red). (A) Dorsal view, anterior to the left. The central panels are enlarged and subsequent time-lapse images of the boxed areas in the leftmost column. The transverse sectional views of the areas indicated by dashed lines on the 42 hpf image are shown in the rightmost column. Top, pdgfrb:EGFP (green); middle (rightmost column only), fli1a:Myr-mCherry (red); bottom, merged image. Note that the cells located in the vicinity of the BA (arrowheads) gradually emitted strong EGFP signal and tightly contacted ECs. (B) 3D-rendered confocal images of EGFP-positive cells (green) migrating along the CCtAs. Lateral view, dorsal to the top and anterior to the front. Note that EGFP-positive cells located around the BA (arrowheads) dorsally migrated along the CCtAs. (C) Dorsal view, anterior to the left. Arrowheads with numbers indicate individual EGFP-positive cells spreading on the CCtAs. Note that EGFP-positive cells spreading on the CCtA (1 and 2) divided into two daughter cells (1-1/1-2 and 2-1/2-2). (D) Schematic of how CCtAs become covered by MCs. MCs develop around the BA and migrate towards the CCtAs. During their migration, the MCs actively proliferate to cover the CCtAs. (E) Confocal images of hindbrain vasculature of pdgfrb wild-type (WT/WT), heterozygous (sa16389/WT) and homozygous (sa16389/sa16389) larvae in the TgBAC(pdgfrb:EGFP);Tg(fli1a:Myr-mCherry) background at 5 dpf. Dorsal view, anterior to the left. The vessels in the cerebral base, such as BCA, PCA and BA (BCA/PCS/BA), and the CtA, are shown in the left and right columns, respectively. Upper, pdgfrb:EGFP; lower, merged images of pdgfrb:EGFP (green) and fli1a:Myr-mCherry (red). Arrowheads indicate MCs emerged around the BCA, PCS and BA. Scale bars: 20 μm (transverse sectional image in A); 50 μm (A,B,C,E).

Fig. 3.

Migration of MCs along inter-EC junctions. (A) Time-lapse confocal images of an MC migrating along the CtA in the hindbrain of a TgBAC(pdgfrb:mCherry);Tg(fli1a:pecam1-EGFP) larva. 3D-rendered confocal images at 62 hpf (leftmost column) and their subsequent time-lapse images with the elapsed time (min) at the top right. Top, pdgfrb:mCherry (green); middle, fli1a:pecam1-EGFP (magenta); bottom, merged images. Arrowheads and asterisk indicate the tip of the MC process and the cell body, respectively. Note that the MC extended a process along the Pecam1-EGFP-labeled inter-EC junctions. (B) Alignment of MC processes along inter-EC junctions, as observed in A, expressed as a percentage of the total length (n=28). Bar and circles indicate the average and the values of individual processes, respectively. (C) Time-lapse confocal images of an MC migrating along the CtA, as in A. 3D-rendered confocal images at 72 hpf (leftmost column) and their subsequent time-lapse images with the elapsed time (min) at the top right. Note that the MC moved forward by sequentially relocating its cell body (asterisks) to the punctate structures formed within the preceding processes (arrows). CVP, choroidal vascular plexus. (D) Schematic of how MCs migrate along the EC tube to cover the CtA. Red lines indicate the inter-EC junctions, green cells represent MCs. Scale bars: 10 μm (A,C).

Fig. 4.

Live imaging of MC coverage of axial vessels in the trunk. (A) Time-lapse confocal images of an axial vessel in the trunk of TgBAC(pdgfrb:EGFP);Tg(fli1a:Myr-mCherry) embryos (38-48 hpf). Upper, pdgfrb:EGFP; lower, merged images of pdgfrb:EGFP (green) and fli1a:Myr-mCherry (red). Arrowheads with numbers indicate individual EGFP-positive cells emerging at the ventral part of the DA. Arrow indicates hypochord (HP). (B) Time-lapse confocal images of a trunk axial vessel in TgBAC(pdgfrb:Gal4FF);Tg(UAS:NLS-mCherry,mVenus-geminin) embryo (60-67.5 hpf). Top, NLS-mCherry (red); middle, mVenus-geminin (the cells in the S/G2/M phase of the cell cycle) (green); bottom, merged images. Arrowheads with numbers indicate individual mCherry/mVenus double-positive cells located in the ventral part of DA. Note that mCherry/mVenus double-positive cells (1 and 2) divided into two daughter cells (1-1/1-2 and 2-1/2-2), which subsequently lost mVenus fluorescence. Arrow indicates hypochord. Lateral view, anterior to the left. Scale bars: 50 μm.

Fig. 5.

Live imaging of MC coverage of ISVs. (A,B) The number of EGFP-positive cells covering mCherry-labeled ISVs (A) and the percentage of ISVs covered by more than one EGFP-positive cell (B) in the left side of TgBAC(pdgfrb:EGFP);Tg(fli1a:Myr-mCherry) embryos or larvae at the stages indicated below. (n≥8). (C) Time-lapse confocal images of the trunk vasculature in a TgBAC(pdgfrb:EGFP);Tg(fli1a:Myr-mCherry) embryo (57.5-75 hpf). Upper, pdgfrb:EGFP; lower, merged image of pdgfrb:EGFP (green) and fli1a:Myr-mCherry (red). Arrowheads indicate an EGFP-positive cell that initially located in the ventral part of DA and subsequently migrated towards the ISV. (D) Time-lapse confocal images of the trunk vasculature in a TgBAC(pdgfrb:EGFP);Tg(fli1a:Myr-mCherry) embryo (63-85.5 hpf), as in C. Arrowheads indicate EGFP-positive cells covering the ISVs. Note that the cells around the ISVs gradually emitted a stronger EGFP signal. (E) Confocal images of trunk vasculature in a TgBAC(pdgfrb:EGFP);Tg(fli1a:Myr-mCherry) larva at 120 hpf. Upper, pdgfrb:EGFP; lower, merged image of pdgfrb:EGFP (green) and fli1a:Myr-mCherry (red). Yellow and blue arrowheads indicate the EGFP-positive cells covering arterial ISVs (aISVs) and those covering venous ISVs (vISVs), respectively. ‘a’ and ‘v’ in the merged image indicate aISVs and vISVs, respectively. (F) Percentage of aISVs/vISVs covered by more than one EGFP-positive cell, as observed in E (n≥12). (G) The number of EGFP-positive cells covering aISVs/vISVs, as observed in E (n≥12). (H) Percentage of EGFP-positive cells covering the upper and lower half of aISVs/vISVs, as observed in E (n=12). (I) Confocal images of trunk vasculature in a TgBAC(pdgfrb:EGFP);Tg(flt1:mCherry) larva at 100 hpf. Fluorescence signal derived from flt1:mCherry labels arterial ECs (Bussmann et al., 2010). The merged image of pdgfrb:EGFP (green) and flt1:mCherry (red) is shown at the top. The boxed areas labeled 1 and 2 are enlarged at the bottom, showing the merged image of pdgfrb:EGFP and flt1:mCherry (left), pdgfrb:EGFP (center) and flt1:mCherry (right). Yellow and blue arrowheads indicate the EGFP-positive cells covering aISVs and those covering vISVs, respectively. Note that MCs adhered to the flt1:mCherry-positive arterial ECs within vISVs. (J) Percentage of EGFP-positive cells adhering to arterial ECs (aEC) and those adhering to venous ECs (vECs) within vISVs, as observed in I (n=10). (K) Confocal images of trunk vasculature of the pdgfrb heterozygous (sa16389/WT) and homozygous (sa16389/sa16389) larvae in the TgBAC(pdgfrb:EGFP);Tg(fli1a:Myr-mCherry) background at 100 hpf are shown, as in E. In A,B,F,G and J, bars and circles indicate averages and individual values, respectively. ***P<0.001. Lateral view, anterior to the left. DLAV, dorsal longitudinal anastomotic vessel; ISV, intersegmental vessel; DA, dorsal aorta; PCV, posterior cardinal vein; aISV, arterial ISV; vISV, venous ISV. Scale bars: 20 μm (C, enlarged images in I); 50 μm (D,E,I,K).

Fig. 6.

Lineage tracing for identification of MC origins. (A) Schematic of the protocol used for lineage-tracing analysis. TgBAC(pdgfrb:Gal4FF);Tg(UAS:loxP-mCherry (mC)-loxP-mVenus (mV)) zebrafish (pdgfrb reporter) express mCherry in the pdgfrb-positive cells (top). In the pdgfrb reporter fish crossed with Tg(sox10:Cre) or Tg(tbx6:Cre,myl7:EGFP) line, the sox10-positive neural crest-derived MCs or tbx6-positive mesoderm-derived MCs, respectively, are labeled with mVenus expression because of Cre-mediated excision of mCherry gene flanked by loxP (bottom). (B) Lateral view of a TgBAC(pdgfrb:Gal4FF);Tg(UAS:loxP-mC-loxP-mV);Tg(sox10:Cre) larva at 5 dpf. Upper, mVenus; lower, brightfield image. (C) Confocal images of head (left column) and trunk (center and right columns) regions in a TgBAC(pdgfrb:Gal4FF);Tg(UAS:loxP-mC-loxP-mV);Tg(tbx6:Cre,myl7:EGFP) larva at 5 dpf. Upper, mVenus; middle, merged images of mVenus (green) and mCherry (red); lower, differential interference contrast (DIC) images. (D) Confocal images of head regions in a 5 dpf TgBAC(pdgfrb:Gal4FF);Tg(UAS:loxP-mC-loxP-mV) larva crossed with Tg(sox10:Cre) (left three columns) or Tg(tbx6:Cre,myl7:EGFP) (right three columns) fish lines. Dorsal view, anterior to the left. Images of CCtA, BCA/PCS and HV are shown in the left, center and right columns, respectively. Upper, mVenus; lower, the merged images of mVenus (green) and mCherry (red). Note that mVenus-labeled cells indicate sox10-positive neural crest-derived MCs (left three columns) or tbx6-positive mesoderm-derived MCs (right three columns). Scale bars: 50 μm (CCtA, HV); 20 μm (BCA/PCS). (E) Confocal images of pharyngeal regions in 5 dpf TgBAC(pdgfrb:Gal4FF);Tg(UAS:loxP-mC-loxP-mV);Tg(IF:NLS-mCherry,fli1a:iRFP) larvae crossed with Tg(sox10:Cre) (left three columns) or Tg(tbx6:Cre,myl7:EGFP) (right three columns) fish lines. The larvae expressing iRFP670 under the control of fli1a promoter were identified by intestinal fatty acid binding protein (IF; also known as fabp2) promoter-driven expression of NLS-mCherry in the intestine. Ventral view, anterior to the left. Boxed areas showing hypobranchial artery (HA) (a,c) and aortic arches (AA) (b,d) are enlarged to the right of the original images. Top row, mVenus; second row, mCherry; third row, fli1a:iRFP (iRFP); bottom row, merged images of mVenus (green), mCherry (red) and iRFP (white). Scale bars: 50 μm. Asterisks in C and E indicate heart visualized by myl7:EGFP. mC, mCherry; mV, mVenus; i, iris; j, jaw; p, pharyngeal arch.