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Lymphatic vessels arise from specialized angioblasts within a venous niche

Abstract

How cells acquire their fate is a fundamental question in developmental and regenerative biology. Multipotent progenitors undergo cell-fate restriction in response to cues from the microenvironment, the nature of which is poorly understood. In the case of the lymphatic system, venous cells from the cardinal vein are thought to generate lymphatic vessels through trans-differentiation. Here we show that in zebrafish, lymphatic progenitors arise from a previously uncharacterized niche of specialized angioblasts within the cardinal vein, which also generates arterial and venous fates. We further identify Wnt5b as a novel lymphatic inductive signal and show that it also promotes the ‘angioblast-to-lymphatic’ transition in human embryonic stem cells, suggesting that this process is evolutionarily conserved. Our results uncover a novel mechanism of lymphatic specification, and provide the first characterization of the lymphatic inductive niche. More broadly, our findings highlight the cardinal vein as a heterogeneous structure, analogous to the haematopoietic niche in the aortic floor.

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Figure 1: Lymphatic progenitors originate in the vPCV.
Figure 2: vPCV cells are specialized angioblasts.
Figure 3: LEC specification is induced in the vPCV angioblasts.
Figure 4: Wnt5b is necessary and sufficient for LEC specification.
Figure 5: Wnt5b induces lymphatic specification in zebrafish and hESCs.
Figure 6: Wnt5b induces LEC specification through activation of the canonical pathway.

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Primary accessions

Gene Expression Omnibus

Data deposits

RNA-Seq raw data and processed values have been submitted to the NCBI Gene Expression Omnibus (GEO) under the accession number GSE65751.

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Acknowledgements

The authors would like to thank B. Cohen, N. Strasser, R. Solomon and F. Bochner for technical assistance, N. Stettner and A. Harmelin for animal care, G. Beck and E. Ainbinder for assistance with hESC experiments, E. Winter for RNA-Seq analyses, F. Argenton for providing the Tg(7xTCF-Xla.Siam:nlsmCherry)ia5 transgenic line, G. Weidinger for the Tg(hsp70l:wnt5b-GFP)w33 line, E. Ober for the TgBAC(prox1a:KalT4-UAS:uncTagRFP)nim5 line, S. Schulte-Merker for the Tg(flt4BAC:mCitrine)hu7135 line, S. Sumanas for the Tg(etv2:GFP)ci1 line, M. Affolter and H. G. Belting for the Tg(fli1:gal4ubs3;uasKaederk8) line, A. Inbal for the pCS2-axin plasmid, B. Weinstein for the pME-nr2f2 plasmid and the cas mutants, M. Beltrame for the pCMV sox18 plasmid, and E. Tzahor, E. Zelzer, M. Neeman and B. Shilo for critical reading of the manuscript. The authors are grateful to all the members of the Yaniv laboratory for discussion, technical assistance and continuous support. This work was supported in part by Marie Curie Actions-International Reintegration grants FP7-PEOPLE-2009-RG 256393 (to K.Y.), Minerva Foundation 711128 (to K.Y.), German-Israeli Foundation Young Investigator Program 1967/2009 (to K.Y.), Israel Cancer Research Foundation Postdoctoral Fellowship (to G.M.), Lymphatic Research and Education Network postdoctoral fellowship (to G.M.), Northrine Westphalia Return fellowship (to W.H.), US National Institutes of Health (NIH) R01 HL122599 (to N.D.L.), JSPS Postdoctoral Fellowships for Research Abroad (to M.S.), ERC 310927 (to I.Y.). K.Y. is supported by the Karen Siem Fellowship for Women in Science; the Willner Family Center for Vascular Biology; the estate of Paul Ourieff; the Carolito Stiftung; Lois Rosen, Los Angeles, CA; and the Adelis Foundation. K.Y. is the incumbent of the Louis and Ida Rich Career Development Chair.

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Authors

Contributions

J.N. and G.M. designed and conducted experiments, analysed data, and co-wrote the manuscript; Y.S. designed and conducted experiments on human ESCs and analysed data; T.L, L.A., O.M., A.J.-V. and M.S. conducted experiments and data analyses; I.A.-D. and V.K. conducted in vitro experiments, N.S. and T.H. conducted RNA-Seq experiments and data analyses; R.H. assisted with animal care and genotyping; L.G.-B. and J.W.A. generated transgenic lines; G.A. managed the fish facility; S.B-D. performed bioinformatics analyses; O.G. assisted with image processing analyses; P.S.C. provided the Tg(lyve1:EGFP)nz150 and Tg(lyve1:dsRed2)nz101 transgenic lines. W.H. and N.D.L. designed and supervised part of the experiments; I.Y. designed and supervised RNA-Seq experiments; J.H.H. supervised part of the hESCs experiments; K.Y. initiated and directed the study, designed experiments, analysed data and co-wrote the paper with input from all authors.

Corresponding author

Correspondence to K. Yaniv.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Mesoderm-derived angioblasts generate LECs through asymmetric cell division.

a, Snapshots from a time-lapse sequence of a Tg (fli1:nEGFP)y7 zebrafish embryo, showing the origin of a PAC cell (yellow) in the vPCV (nimaged embryos = 7). b, vPCV (left panel), and dPCV (right panel) Kaede photoconverted cells at 48 hpf. c, Kaede-photoswitched ‘medial’ (left panel) and ‘early lateral’ (right panel) angioblasts. d, Snapshots from a time-lapse sequence of a plcg1 mutant, showing the origin of a PAC cell (green) in the vPCV (nimaged embryos = 3). e, Quantification of symmetric and asymmetric division events in the vPCV and dPCV of double Tg(flt1_9a_cFos:GFP; lyve1:dsRed2 nz101) embryos (nimaged embryos = 6). Scale bars, 30 μm.

Extended Data Figure 2 Analysis of cell division in the zebrafish axial vessels.

a, Phospho-histone H3 staining shows no difference in the number of proliferative endothelial cells among the DA, dPCV and vPCV (n24 hpf embryos = 17, n26 hpf embryos = 16, n28 hpf embryos = 16, n30 hpf embryos = 16). b, Ectopic induction of Wnt5b in Tg(hsp70l:wnt5b; fli1:EGFP) does not result in enhanced proliferation of endothelial cells (26 hpf; ncontrol embryos = 15, nhsp70:wnt5b embryos = 8, 28 hpf; ncontrol embryos = 14, nhsp70:wnt5b embryos = 8, 30 hpf; ncontrol embryos = 14, nhsp70:wnt5b embryos = 10). Scale bar, 60 μm. Error bars, mean ± s.e.m.

Extended Data Figure 3 Fate map analysis of vPCV cells.

a, Schematic representation of the subintestinal plexus at 72 hpf. Subintestinal vein (SIV, green), interconnecting SI vessels (purple), supraintestinal artery (SIA, pink), posterior cardinal vein (PCV, blue), dorsal aorta (DA, red). b, Quantification of the number of intersegmental arteries (ISA) and intersegmental veins (ISV) in the first four segments of Tg(flt1_9a_cFos:GFP; lyve1:dsRed2) double transgenic embryos (nembryos = 41). IS# denotes the position of intersegmental vessel. c, Confocal images of Tg(lyve1:dsRed2) (left panel) and Tg(flt1_9a_cFos:GFP; lyve1:dsRed2) (right panel) embryos showing lyve1:dsred2+ endothelial cells in PACs, venous intersegmental vessels (ISVs), PCV and SIV and flt1_9a:GFP+ endothelial cells in the SIA. d, flt1_9a:GFP+ vPCV angioblast (light-blue arrowhead), divides asymmetrically (curved arrow) to generate cells that populate the SIV (31.5 hpf, white arrowhead), and the SIA (53.5 hpf, white arrowhead). Scale bar, 30 µm. Error bars, mean ± s.e.m.

Extended Data Figure 4 Transcriptional profiling of vPCV angioblasts.

a, Experimental setup used for RNA sequencing analysis of FACS isolated vPCV and dPCV cells. b, FACS isolation of green vs red (photoconverted) endothelial cells from Tg(fli1:gal4;uasKaede) embryos following photoswitching of dorsal or ventral PCV (nindependent experiments = 4). c, qRT–PCR analysis of selected candidates shows enrichment in ventral vs dorsal PCV cells (nindependent experiments = 2). d, Gene Ontology enrichment in vPCV vs dPCV cells (results represent 2 out of 4 independent biological repeats). Error bars, geometrical mean ± s.e.g.m.

Extended Data Figure 5 Endoderm-derived Wnt5b is required for lymphatic development.

a, PAC-containing segments in WT (arrows) and cas mutants (asterisks). b, In situ hybridization at 20 hpf showing expression of wnt5b mRNA (blue arrowhead) in the endoderm of WT embryos. c, PAC-containing segments in uninjected (UI) (arrows) and wnt5b MO-injected embryos (asterisks). d, ppt mutants injected with wnt5b MO (subdose) display significant reduction in PAC number (nUI embryos = 38, nwnt5b-MO embryos sub = 38, nppt-UI embryos = 34, nppt, wnt5b MO sub-embryos = 34; *P = 1.2 × 10−5). e, wnt5b morphants exhibit marked reduction in the number of thoracic duct-containing segments (asterisks) as compared to uninjected (UI) siblings (arrows) (nUI-embryos = 38, nwnt5b MO-embryos = 32; *P = 4.5 × 10−30). f, The number of flt1+ vPCV progenitors is not affected in wnt5b morphants (nUI-embryos = 31, nwnt5b-MO embryos = 31). Scale bars, a, c, 60 μm; b, e, f, 30 μm. Error bars, mean ± s.e.m.

Extended Data Figure 6 Wnt5b is not required for sprouting from the PCV.

a, Phenotypic analysis of Wnt5b overexpression in Tg(hsp70l:wnt5b-GFP; lyve1:dsRed2) embryos, following 25–30 min heat shock (HS), at 23, 25 and 27 hpf (23 hpf embryos nHS-25 min = 18, nHS-30 min = 14, nHS-40 min = 15, 25 hpf embryos nHS-25 min = 14; nHS-30 min = 17, nHS-40 min = 20, 27 hpf embryos nHS-25 min = 19, nHS-30 min = 17, nHS-40 min = 10). b, The number of vISVs vs aISVs is unaltered in wnt5b morphants as compared to Control MO-injected siblings (nControl MO-embryos = 43, nwnt5b MO-embryos = 41). c, flt1_9a+ vPCV cells are detected in the supraintestinal artery (SIA) and subintestinal vein (SIV) of wnt5b MO-injected embryos (nCtrl MO = 16, nwnt5b MO = 16). Scale bars, 60 μm. Error bars, mean ± s.e.m.

Extended Data Figure 7 Wnt5b induces the “angioblast-to-lymphatic” specification.

a, Selected frames from a time-lapse sequence of a Tg(fli1:gal4;uasKaede) embryo injected with wnt5b MO. Photoconverted vPCV cell (white arrow) divides normally (arrows at 48 hpf point to 2 daughter cells), but does not engage in dorsal migration to generate PACs. b, In situ hybridization of Ctrl MO-, and wnt5b MO-injected zebrafish at 30 hpf, with lyve1, sox18, nr2f2 and cdh5 probes, showing specific decrease in lymphatic marker expression in the floor of the PCV (white arrowheads) of wnt5b morphants. The pan-endothelial marker cdh5, as well as the arterial expression of sox18, remain unchanged in wnt5b morphants. c, vegfc and ccbe1 mRNA levels remain unaltered in sox32 and wnt5b morphants. d, Immunostaining of Prox1 shows marked increase in protein levels following ectopic activation of Wnt5b in Tg(hsp70l:wnt5b; fli1:EGFP) embryos (co-localization channel is shown in yellow, white arrowheads). e, qRT–PCR analysis of FLT4 and CDH5 in hESCs treated with WNT5B (nindependent-experiments = 3; *P = 0.03 by one sample t-test). Scale bars, 60 μm. Error bars, geometrical mean ± s.e.g.m.

Extended Data Figure 8 Wnt5b induces LEC specification through activation of canonical pathway.

a, PAC-containing segments (arrows) in wnt5b MO-injected mbl mutants (nwnt5bMO = 42, nmbl;wnt5bMO = 52; *P = 3.4 × 10−10). b, apc mutants (nWT = 18, napc = 19; *P = 0.0006), c, axin1 mRNA-injected embryos (nUI = 33, naxin-mRNA = 46; *P = 1.73 × 10−14), and d, IWR-1 treated embryos (nDMSO = 55, nIWR = 54; *P = 1.05 × 10−21). Scale bars, 60 μm. Error bars, mean ± s.e.m.

Extended Data Figure 9 Involvement of Tcf transcription factors in LEC specification.

a, PAC number remains unchanged in TNP-470 treated Tg(fli1:EGFP) embryos as compared to DMSO (control) (nDMSO = 19, nTNP-470 = 38). b, c, Quantification of PAC-containing segments in the trunk of UI, tcf7, lef1 and tcf4 MO-injected embryos (nUI-embryos = 59, ntcf7-MO embryos = 33, nlef1-MO embryos = 16, ntcf4-MO embryos = 25; *P = 4.53 × 10−25, **P = 9.62 × 10−8, ***P = 9.12 × 10−9). d, Photoswitching of vPCV cells in tcf7 MO-injected Tg(fli1:gal4;uasKaede) embryos (white arrowheads) at 24 hpf. At 48 hpf photoconverted, red vPCV cells (arrowheads) remain in the PCV and do not generate PACs. Scale bars, 30 μm. Error bars, mean ± s.e.m.

Extended Data Figure 10 Wnt5b-dependent activation of β-catenin/TCF in vPCV angioblasts.

a, Selected frames from a time-lapse sequence showing β-catenin/TCF activity in a single vPCV angioblast (light-blue arrowhead), which generates PACs (white arrowhead) through asymmetric cell division (n = 2). b, Confocal images of the trunks of Tg(7xTCFXla.Siam:nlsmCherry; fli1:EGFP) double transgenic zebrafish injected with wnt5b MO, showing decreased β-catenin/TCF activation in vPCV cells (quantified in c) (nUI-embryos = 18, nwnt5b-embryos = 17; *P = 4 × 10−5). Purple signal depicts co-localization of cytoplasmic EGFP and nuclear mCherry. Scale bars, 30 μm. Error bars, mean ± s.e.m.

Supplementary information

LEC progenitors originate in the floor of the PCV

This video shows time-lapse images of the trunk of a Tg(fli:EGFP) zebrafish between 24hpf-58hpf. Shown are two combined panels: the original images are on the left. On the right, a selected LEC progenitor was colored off-line in green to facilitate its visualization. Note its initial location at the ventral PCV (vPCV). (MP4 12837 kb)

LEC progenitors originate in the floor of the PCV in plcg1 mutant

This video shows time-lapseimages of the trunk of a plcg1 mutant, between 24hpf-50hpf. Shown are two combined panels: the original images are on the left. On the right, a selected LEC progenitor was colored off-line in green to facilitate its visualization. Note its initial location at the vPCV (green). Following asymmetric division, a daughter cell (blue), migrates dorsally to generate a PAC sprout. (MP4 15543 kb)

vPCV cells generate LECs through asymmetric cell division

This video shows time-lapse images of a photoswitched vPCV cell in the trunk of Tg(fli1:gal4;uasKaede) embryo between 25hpf-48hpf. Light-blue arrowhead points to a vPCV angioblast; white arrowhead points to daughter cell that generates PAC. The first frame was acquired before photoswitching. (MP4 6974 kb)

LECs arise from a pool of specialized angioblasts

This video shows time-lapse images of the trunk of Tg(flt1_9a_cFos:GFP; lyve1:dsRed) double ransgenic embryo between 30hpf-48hpf. Light-blue arrowheads point to flt1_9a:GFP+ vPCV angioblast; white arrowheads point to flt1_9a:GFP+ daughter cells that generate PACs, downregulate flt1_9a:GFP expression and upregulate lyve1:dsRed expression. (MP4 3896 kb)

PACs arise from prox1a-expressing LEC progenitors

This video shows time-lapse images of the trunk of Tg(fli1:EGFP; prox1a:KalT4-UAS:uncTagRFP) double transgenic embryo between 23-55 hpf. Cells showing co-localization were pseudo-coloredin yellow. The first cells expressing Prox1a are visible at ~22 hpf in the vPCV. Later on these cells divide and generate progeny that translocates dorsally and buds from the PCV to generate PACs. (MP4 7925 kb)

LEC progenitors do not generate PACs in wnt5b-MO injected embryo

This video shows time-lapse images of the trunk of a g(fli1a:nEGFP; fli1:dsRed) double transgenic embryo injected with wnt5b MO between 28hpf-44hpf. Shown are two combined panels: the original images are on the left.On the right panel, vPCV (colored) cells do not engage in dorsal migration to generate PACs, but undergo normal cell division. (MP4 5355 kb)

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Nicenboim, J., Malkinson, G., Lupo, T. et al. Lymphatic vessels arise from specialized angioblasts within a venous niche. Nature 522, 56–61 (2015). https://doi.org/10.1038/nature14425

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