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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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.

Author information

Author notes

    • J. Nicenboim
    •  & G. Malkinson

    These authors contributed equally to this work.

Affiliations

  1. Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel

    • J. Nicenboim
    • , G. Malkinson
    • , T. Lupo
    • , L. Asaf
    • , Y. Sela
    • , O. Mayseless
    • , L. Gibbs-Bar
    • , A. Jerafi-Vider
    • , I. Avraham-Davidi
    • , R. Hofi
    • , G. Almog
    •  & K. Yaniv
  2. Faculty of Biology, Technion – Israel Institute of Technology, Haifa 32000, Israel

    • N. Senderovich
    • , T. Hashimshony
    •  & I. Yanai
  3. Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA

    • M. Shin
    •  & N. D. Lawson
  4. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

    • V. Krupalnik
    •  & J. H. Hanna
  5. School of Medical Sciences, University of Auckland, Auckland 1142, New Zealand

    • J. W. Astin
    •  & P. S. Crosier
  6. Biological Services Unit, Weizmann Institute of Science, Rehovot 76100, Israel

    • O. Golani
    •  & S. Ben-Dor
  7. University of Muenster, 48149 Muenster, Germany

    • W. Herzog
  8. Max Plank Institute for Molecular Biomedicine, 48149 Muenster, Germany

    • W. Herzog

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to K. Yaniv.

Extended data

Supplementary information

Videos

  1. 1.

    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).

  2. 2.

    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.

  3. 3.

    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.

  4. 4.

    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.

  5. 5.

    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.

  6. 6.

    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.

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DOI

https://doi.org/10.1038/nature14425

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