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