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Mural lymphatic endothelial cells regulate meningeal angiogenesis in the zebrafish

Abstract

Mural cells of the vertebrate brain maintain vascular integrity and function, play roles in stroke and are involved in maintenance of neural stem cells. However, the origins, diversity and roles of mural cells remain to be fully understood. Using transgenic zebrafish, we identified a population of isolated mural lymphatic endothelial cells surrounding meningeal blood vessels. These meningeal mural lymphatic endothelial cells (muLECs) express lymphatic endothelial cell markers and form by sprouting from blood vessels. In larvae, muLECs develop from a lymphatic endothelial loop in the midbrain into a dispersed, nonlumenized mural lineage. muLEC development requires normal signaling through the Vegfc–Vegfd–Ccbe1–Vegfr3 pathway. Mature muLECs produce vascular growth factors and accumulate low-density lipoproteins from the bloodstream. We find that muLECs are essential for normal meningeal vascularization. Together, these data identify an unexpected lymphatic lineage and developmental mechanism necessary for establishing normal meningeal blood vasculature.

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Figure 1: Mural lymphatic endothelial cells are present at the adult meninx.
Figure 2: muLECs and meningeal lymphatics are present in the zebrafish larval brain.
Figure 3: muLECs form by sprouting lymphangiogenesis and disperse throughout the larval meninx.
Figure 4: muLECs are immediately adjacent to endothelium and take up LDL.
Figure 5: Mural lymphatic endothelial cells are transcriptionally distinct from other mural cell types and produce endothelial growth factors.
Figure 6: muLECs are correlated with meningeal blood endothelial cell numbers.
Figure 7: Asymmetric muLEC ablation leads to asymmetric meningeal angiogenesis.

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Acknowledgements

We thank K. Georgas for design of graphics in the manuscript and thank V. Nink, G. Osborne, H. Chen, D. Paul, J. Springfield and G. Baillie for technical assistance. We thank V. Oorschot and G. Ramm (Ramaciotti Centre for Structural-Cryo Electron Microscopy, Monash Univeristy) for excellent support with electron microscopy. B.M.H. was supported by an NHMRC/NHF Fellowship (1083811); M.F. was supported by an NHMRC Career Development Fellowship (1111169). The Australian Regenerative Medicine Institute is supported by funds from the State Government of Victoria and the Australian Federal Government.

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Authors and Affiliations

Authors

Contributions

N.I.B. conceived, designed, performed experiments, analyzed data and wrote the manuscript. K.K., C.P.-T., B.W.L., S.P., A.K.L., W.W., S.B., M.R.-G., N.M. and M.F. performed experiments and provided unpublished reagents. I.V., D.G.H., C.A.W., C.S. and S.J.B. performed computational experiments and data analysis. J.K. designed and performed experiments. B.M.H. conceived and designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Benjamin M Hogan.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 lyve1-expressing cells in the adult zebrafish meninx co-express the lymphatic marker genes prox1a and fli1a

(a) Confocal images of adult zebrafish brain in Tg(-5.2lyve1b:DsRed)nz101; TgBAC(prox1a:KalTA4 uq3bh;10xUAS:Venus) animals showing cells that co-express the lymphatic markers prox1a and lyve1. Expression of prox1a is mosaic due to the Gal4 system. Full quantification of marker co-expression can be found in Supplementary Figure 1e. Scale bar represents 100μm.

(b) Confocal images of adult zebrafish brain in Tg(-5.2lyve1b:DsRed)nz101; Tg(fli1a:EGFP)y1 showing co-expression (arrows) of the lymphatic markers fli1a and lyve1. Full quantification of marker co-expression can be found in Supplementary Figure 1f. Scale bar represents 100μm.

(c) Quantification of muLEC density on the surface of the brain (meningeal surfaces n=6 adult brains. Error bars represent mean +/− sem, Statistical testing N/A.)

(d) Quantification of the number of lyve1-positive meningeal cells (over the surface of the tectum, or adjacent to the central arteries (cTA)) compared with non-meningeal lyve1-positive cells from cross sections of the adult zebrafish brain. n=4 adult brain cross sections, p<0.0001, from two-tailed student t-test (t=8.926 df=6).

(e) Quantification of co-expression of cells from Tg(prox1a:KalTA4uq3bh;10xUAS:Venus) and lyve1 (Tg(-5.2lyve1b:DsRed)nz101) and the macrophage marker mpeg1 (Tg(mpeg1:mcherry)). 215 cells from n=4 larvae (lyve1) and 255 cells from n=5 larvae (mpeg1) were scored from confocal Z-stack images. Error bars represent mean +/− sem, Statistical testing N/A.

(f) Quantification of co-expression of cells in Tg(-5.2lyve1b:DsRed)nz101 and Tg(fli1a:EGFP)y1, TgBAC(pdgfrβ:EGFP)uq15bh, TgBAC(acta2:EGFP)uq17 and Tg(nkx2.2a:EGFP)vu16Tg. 100 cells from n=5 larvae were scored from confocal Z-stack images. Error bars represent mean +/− sem, Statistical testing N/A.

(g) Quantification of the distance from the nucleus of muLECs to the center of the parenchyma (defined as the centre of the unlabeled space surrounded by a vascular loop) shows that muLECs are more closely associated with blood endothelial cells than the space between vessels. 77 muLEC nuclei from n=3 larvae, p<0.0001, from two-tailed student t-test (t=15.64 df=130). Error bars represent mean +/− sem.

(h) Quantification of the distance from the nucleus of a given muLEC to the nearest vessel branch point, compared with the distance from the nucleus of a given muLEC to the midpoint between branches of the closest vessel. muLECs are quantitatively closer to vessel branch points than vessel midpoints. 77 branch and 77 midpoints points from n=3 larvae, p<0.0001, from two-tailed student t-test (t=4.74 df=151). Error bars represent mean +/− sem.

Supplementary Figure 2 High-molecular-weight dye can be taken up by meningeal lymphatics but not by muLECs.

(a) Dorsal confocal image of 7mm Tg(-5.2lyve1b:DsRed)nz101 zebrafish larvae following injection of FITC-labeled high molecular weight (2000kDa) dextran. Arrowheads indicate the presence of the dye in lymphatic vessels, but the dye is absent from muLECs. Images representative of data obtained from injection of n=6 larvae. Scale bar represents 100μm.

(b) Magnification of boxed region in (a) showing muLECs do not absorb high MW dextran. Images representative of data obtained from injection of n=6 larvae. Scale bar represents 100μm.

(c) Dorsal confocal image of a Tg(-5.2lyve1b:DsRed)nz101; Tg(fli1a:EGFP)y1 zebrafish larvae. Image representative of n=5 larvae. Scale bar represents 100μm.

(d) Magnification of boxed region in (c) showing muLECs co-express lyve1 and fli1a (arrowheads). Image representative of n=6 larvae. Full quantification of marker co-expression can be found in Supplementary Figure 1f. Scale bar represents 100μm.

(e) muLECs are closely associated with blood vessels in Tg(-5.2lyve1b:DsRed)nz101 larvae. Image representative of n=6 larvae. Scale bar represents 100μm.

(f) Confocal image of a cross-section of adult (20mm stage, 3 month old) Tg(-5.2lyve1b:DsRed)nz101; TgBAC(pdgfrβ:EGFP)uq15bh brain showing pdgfrβ expressing pericytes are associated with blood vessels throughout the adult CNS. Scale bar represents 100μm. Full quantification of marker co-expression can be found in Supplementary Figure 1f. Image representative of data obtained from injection of n=3 male and female 20mm zebrafish adults.

Supplementary Figure 3 muLECs do not express markers of neuronal and/or oligodendrocyte cells and smooth muscle cells.

(a-d) Confocal images of (a,b) 7mm stage Tg(-5.2lyve1b:DsRed)nz101;Tg(nkx2.2a:EGFP)vu16Tg and (c,d) 7mm Tg(-5.2lyve1b:DsRed)nz101;TgBAC(acta2:EGFP)uq17 zebrafish larvae showing a lack of co-expression of these markers (quantified in Supplementary Figure 1f) corresponding to neuronal/oligodendrocyte and smooth muscle respectively. Smooth muscle cells are present around the dorsal aorta in the lateral view of the trunk (arrow in d). Scale bars represent 100μm.

Supplementary Figure 4 Cells of the putative lymphatic loop express lymphatic markers and are separate from the blood vasculature.

(a) Confocal images of a 5dpf Tg(-5.2lyve1b:DsRed)nz101; Tg(kdrl:EGFP)s843 zebrafish midbrain showing that cells of the lymphatic loop do not co-express lyve1 and the blood vessel marker kdrl. n=5 embryos. Full quantification of marker co-expression can be found in Supplementary Figure 1f.

(b) Confocal images of lyve1/prox1a positive cells of the lymphatic loop at 5dpf in Tg(-5.2lyve1b:DsRed)nz101; Tg(prox1a:KalTA4uq3bh;10xUAS:Venus) zebrafish. n=3 embryos. Full quantification of marker co-expression can be found in Supplementary Figure 1f.

(c) Confocal images of a 5dpf zebrafish head showing cells of the lymphatic loop co-express the lymphatic markers lyve1 and fli1a in Tg(-5.2lyve1b:DsRed)nz101; Tg(fli1a:EGFP) (arrowheads). n=5 embryos. Full quantification of marker co-expression can be found in Supplementary Figure 1f.

(d) Confocal images of 5dpf Tg(-5.2lyve1b:DsRed)nz101 embryo injected with dextran into the blood vasculature. The lyve1 positive cells do not contain dextran demonstrating they are separate from the blood vasculature. Scale bars represent 100μm. Representative of analysis from n=4 embryos.

Supplementary Figure 5 Kaede photoconversion demonstrates that the lymphatic loop gives rise to the meningeal muLEC population.

(a-b) Confocal images of Tg(prox1a:KalTA4)uq3bh; Tg(10xUAS:Kaede)s1999t photoconverted at 10dpf and reimaged at 15dpf (a) and photoconverted at 20dpf and reimaged at 25dpf (b). Scale bars represent 50μm. For the 10-15 dpf stages n=34 cells from 7 larvae were examined and for the 20-25dpf stages n=29 cells from 3 larvae were analysed. Positive lineage tracing was confirmed in all cases. (c) Quantification of the average increase in photo-converted cell number over 5 days (proliferation rate, cell number 5 days after conversion/0 days after conversion) data combined for 5-20 dpf experiments, n=19 larvae overall. Error bars represent mean +/− sem, Statistical testing N/A.

Supplementary Figure 6 Still images of a Tg(-5.2lyve1b:DsRed)nz101;Tg(fli1a:nEGFP)y7 zebrafish embryo from Supplementary Movie 2.

(a-k) Images are representative of n= 5 movies.

(a) Tilted view showing the labeled endothelial nucleus in the choroidal vascular plexus (arrowhead).

(b,c) First cell (grey render, arrow) exits the choroidal plexus vessel and migrates dorsally along another pre-existing vessel.

(d-f) Second cell (blue render, arrow) exits vessel and migrates dorsally along pre-exiting vessel.

(g-i) First cell divides (arrowhead) and one daughter cell divides again (arrow).

(i,j) Second cell (blue) divides (arrowhead) and continues to migrate (k, arrowhead). Scale bar represents 50μm.

Supplementary Figure 7 muLEC sprouting is dependent upon Vegfc, Vegfd, Ccbe1 and Vegfr3 signaling.

(a-d) Dorsal confocal images of 5dpf Tg(-5.2lyve1b:DsRed)nz101; Tg(fli1a:nEGFP)y7 embryos showing the lymphatic loop that forms in wildtype embryos (a, arrows, representative image based on analysis of n=7 embryos) is reduced in vegfchu5055 (b, arrows, n=6 embryos) and vegfduq7bh (c, arrows, n=6 embryos) single mutants, but is absent in vegfchu5055/vegfduq7bh double mutants (d, asterisk, n=9 embryos). Scale bars represent 100μm.

(e) Quantification of the number of muLECs present in a single putative lymphatic loop at 5dpf in wildtype (n=7 embryos), vegfchu5055 (n=6 embryos), vegfduq7bh (n=6 embryos) single mutants and vegfchu5055/vegfduq7bh double mutants (n=9 embryos). Error bars represent mean +/− sem; **** p<0.0001, from one way ANOVA (F (3, 25) = 166.6).

(f-i) At 5 dpf, the lyve1/prox1a positive cells in Tg(-5.2lyve1b:DsRed)nz101; Tg(prox1a:KalTA4, 4xUAS:uncTagRFP)nim5; Tg(10xUAS:Venus) have formed a lymphatic loop in wildtype embryos (f, n=6 embryos), however in vegfchu5055 (g, n=6 embryos) and vegfd (h, n=6 embryos) mutants, the development of the lymphatic loop is reduced (arrows). In vegfchu5055 / vegfduq7bh double mutant embryos (i, n=6 embryos) the lymphatic loop is completely absent (asterisks). Scale bars represent 100μm.

(j-l) The lyve1 lymphatic loop structure (control, j) is absent in vegfr3 morphant (n=15 embryos) (k) and (l) ccbe1 morphant (n=15 embryos) embryos at 5dpf (asterisks).

(m) Quantification of the number of muLECs present in a single putative lymphatic loop at 5dpf in uninjected controls (n=15 embryos), vegfr3 morphant (n=15 embryos) and ccbe1 morphant (n=15 embryos). **** p<0.0001, from two-tailed student t-test (t=33.62 df=28). Scale bars represent 100μm.

(n,o) Dorsal confocal images from 24 mm Tg(-5.2lyve1b:DsRed)nz101 vegfchu5055 (n) and vegfduq7bh (o) mutants showing the presence of either ligand is sufficient for development of the muLECs. Representative image based on analysis of n=4 adult brains. Scale bars represent 200μm.

Supplementary Figure 8 Mature muLECs display a distinctive ultrastructure and take up LDL.

(a,b) Schematic diagram indicating the region used for immuno-electron microscopy.

(c) Low magnification immuno-electron micrograph image showing overview of imaged region in Fig. 4a,b. Scale bar represents 1μm.

(d) Higher magnification of image shown in (c). Scale bar represents 1μm. * = prominent vacuoles or inclusion bodies consistent with confocal imaging in Figure 1 and panel g below, L=Lumen, EC= endothelial cell, BM=basement membrane.

(e) High magnification of muLEC cell showing positive immunostaining by anti-gfp antibody. Scale bar represents 1μm.

(f) High molecular weight dextran is not observed in muLECs (asterisks) 3 hours post injection into the blood stream of Tg(-5.2lyve1b:DsRed)nz101 larvae. n=63 muLECs were analysed in detail by scoring confocal z-stacks from n= 3 larvae.

(g) Alexa 488-labelled acetylated LDL is observed in the endothelial cells and the inclusion bodies (arrows) of 61 +/− 11 % of muLECs 3 hours post injection into the blood vasculature. Scale bars represent 20μm. n=72 muLECs were analysed in detail by scoring confocal z-stacks from 3 embryos.

(h,i) Three hours post injection of high molecular weight dextran, there is no obvious vascular leakage in wildtype (h) or vegfchu5055 / vegfduq7bh double mutant larvae (i) which lack muLECs. Scale bar represents 50μm. n=3 larvae were analysed in detail by scoring confocal z-stacks.

Supplementary Figure 9 muLECs can be FAC-sorted and separated from blood endothelial cells and pericytes.

(a) Representative plots of FAC sorted cells from adult brains in unlabeled wildtype and Tg(-5.2lyve1b:DsRed)nz101 positive samples.

(b) Representative plots of FAC sorted EGFP positive cells in unlabeled wildtype and Tg(-5.2lyve1b:DsRed)nz101; Tg(kdrl:EGFP)s843 positive samples.

(c) Representative plots of FAC sorted EGFP positive cells in unlabeled wildtype and Tg(-5.2lyve1b:DsRed)nz101; TgBAC(pdgfrβ:EGFP)uq15bh positive samples.

(d) Scree plot of variance (eigenvalue versus component number) and principle component comparisons from Fig. 5b shown in 2-dimensional plots (PC1 vs PC2, PC2 vs PC3 and PC1 vs PC3), clear separation of muLECs from control samples is observed.

(e) Representative plots of FAC sorted EGFP positive cells in wildtype and Tg(pdgfrβBAC:EGFP)uq15bh; Tg(kdrl:mCherry)s843 positive samples.

Supplementary Figure 10 vegfchu5055vegfduq7bh double-mutant larvae have reduced meningeal BEC nuclei compared to wild-type larvae.

(a) Dorsal confocal image (a) and hyperstack (a’) of z-stacks from the Tg(-5.2lyve1b:DsRed)nz101;Tg(fli1a:nEGFP)y7 signal with darker shades representing z-stack slices closer to the objective, showing vegfchu5055/vegfduq7bh double mutant larvae (a, representative images of n=8 larvae analysed), which have reduced meningeal BEC number when compared to size matched (5.7mm) wildtype siblings (b, n=7 larvae). Scale bar represents 100μm.

Supplementary Figure 11 Visual confirmation of muLEC ablation and mock ablation.

(a) Single z-slices from a dorsal confocal image of a 10dpf Tg(-5.2lyve1b:DsRed)nz101;Tg(fli1a:nEGFP)y7 larvae before and after targeted ablation of muLECs. Ablation can be confirmed by direct observation of cell death, with “bubbles” which disperse post ablation. Scale bar represents 50μm. Representative images of n=27 larvae analysed.

(b) Second example of single z-slices from a dorsal confocal image of 10dpf Tg(-5.2lyve1b:DsRed)nz101;Tg(fli1a:nEGFP)y7 larvae before and after ablation of muLECs. Ablation can be confirmed by direct observation of cell death. The analysis in Fig. 7 used the same approach to verify cell ablations for n=27 embryos in total.

(c) Example of single z-slices from a dorsal confocal image of 10dpf Tg(-5.2lyve1b:DsRed)nz101;Tg(fli1a:nEGFP)y7 larvae before and after mock ablation in which cells adjacent to the muLECs are targeted. Ablation confirmed by direct observation and this approach was used in Fig. 7 for analysis of n=15 larvae. This is the same larvae as in Fig. 7g,h. Scale bar represents 50μm.

(d) Second example of single z-slices from a dorsal confocal image of 10dpf Tg(-5.2lyve1b:DsRed)nz101;Tg(fli1a:nEGFP)y7 larvae before and after mock ablation in which cells adjacent to the muLECs are targeted. This is the same larvae as above and in Fig. 7g,h.

(e and f) 10 dpf Tg(-5.2lyve1b:DsRed)nz101;Tg(fli1a:nEGFP)y7 larvae before (e) and after (f) mock ablation. Representative of n=15 larvae analysed. Scale bar represents 100μm.

Supplementary Figure 12 Schematic summary of muLEC development and function

a. Mural lymphatic endothelial cells (muLECs) emerge from the choroidal vascular plexus (CVP) at 54 hpf (a) and sprout to the periphery of the midbrain at 96 hpf (a’).

b. The resulting lymphatic vascular loop in the midbrain (MB) begins to undergo a transition to a mesenchymal morphology between 5 (b’) and 10 dpf (b’’)

c - d. muLECs (blue) are present on the midbrain, fore brain (FB) and hind brain (HB) in 7 mm larvae (c) and expand over the adult brain (d). c’ and d’ provide lateral views and indicate the meningeal lymphatics (green, c’) and muLEC location relative to blood vessels (red) (d’).

e and f. Three dimensional (e) and cross sectional schematic representation of the blood vasculature and mural cells at the zebrafish meninx. muLECs (blue) secrete vascular growth factors and take up LDL from the bloodstream. PVM= perivascular macrophage.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 (PDF 3704 kb)

Supplementary Methods Checklist (PDF 466 kb)

3D reconstruction of Tg(prox1a:KalTA4 uq3bh;10xUAS:Venus) Z-stacks from Fig. 2a.

The meningeal lymphatic vessels and muLECs surround the outer curvature of the brain in a 7mm larvae. Representative of n=6 larvae analysed. (AVI 1819 kb)

Time lapse imaging of muLECs sprouting from the choroidal vascular plexus.

Left side: Confocal time lapse imaging overview of a Tg(-5.2lyve1b:DsRed)nz101; Tg(fli1a:nEGFP)y7 zebrafish embryo from 30 hpf to 4 dpf. At approximately 54 hpf, cells migrated dorsally from a vein running parallel to the primary head sinus and subsequently give rise to the lymphatic loop observed at 4dpf. Representative of n=5 movies of individual embryos analysed. Right side: Zoomed and rendered version of left hand movie, showing confocal time lapse imaging of a Tg(-5.2lyve1b:DsRed)nz101; Tg(fli1a:nEGFP)y7 zebrafish embryo from 30 hpf to 4 dpf. Rendered nuclei (grey and blue) highlight cells sprouting at approximately 54 hpf, from a vein running parallel to the primary head sinus, subsequently dividing and up-regulating lyve1. Representative of n=5 movies of individual embryos analysed. (MOV 19546 kb)

3D reconstruction showing processing used in Imaris to confirm the correct identification of meningeal BECs as represented in Fig. 6 and Fig.7.

In this example, the deconvoluted z-stack was surface rendered (white) to detect the EGFP signal from Tg(-5.2lyve1b:DsRed)nz101; Tg(fli1a:nEGFP)y7 14 dpf mock ablated larvae. The EGFP signal corresponding to the meningeal BECs was rendered magenta on the left side and green on the right side. The white render corresponding to the total EGFP signal was removed to leave the meningeal BECs. The channel for DsRed corresponding to the muLECs was added (grey) followed by the surface render of the signal. (AVI 2740 kb)

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Bower, N., Koltowska, K., Pichol-Thievend, C. et al. Mural lymphatic endothelial cells regulate meningeal angiogenesis in the zebrafish. Nat Neurosci 20, 774–783 (2017). https://doi.org/10.1038/nn.4558

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