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A somitic Wnt16/Notch pathway specifies haematopoietic stem cells

Abstract

Haematopoietic stem cells (HSCs) are a self-renewing population of cells that continuously replenish all blood and immune cells during the lifetime of an individual1,2. HSCs are used clinically to treat a wide array of diseases, including acute leukaemias and congenital blood disorders, but obtaining suitable numbers of cells and finding immune-compatible donors remain serious problems. These difficulties have led to an interest in the conversion of embryonic stem cells or induced pluripotent stem cells into HSCs, which is not possible using current methodologies. To accomplish this goal, it is critical to understand the native mechanisms involved in the specification of HSCs during embryonic development. Here we demonstrate in zebrafish that Wnt16 controls a novel genetic regulatory network required for HSC specification. Non-canonical signalling by Wnt16 is required for somitic expression of the Notch ligands deltaC (dlc) and deltaD (dld), and these ligands are, in turn, required for the establishment of definitive haematopoiesis. Notch signalling downstream of Dlc and Dld is earlier than, and distinct from, known cell-autonomous requirements for Notch, strongly suggesting that novel Notch-dependent relay signal(s) induce the first HSCs in parallel to other established pathways. Our results demonstrate that somite-specific gene expression is required for the production of haemogenic endothelium.

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Figure 1: Wnt16 is required for the specification of HSCs.
Figure 2: The wnt16 loss-of-function phenotype is specific.
Figure 3: Wnt16 acts upstream of Notch ligands Dlc and Dld.
Figure 4: Non-cell-autonomous requirement for Notch in HSC specification.

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Acknowledgements

The authors wish to thank L. Zon, K. Poss, D. Kimelman, M. Lardelli, B. Appel, C. Burns, J. Campos-Ortega, D. Ransom, N. Trede, J. Lewis, M. Pack, S. Holley, C. Moens, B. Paw, R. Karlström and J. Waxman for probe constructs. L. Zon, R. Dorsky, S. Lin and S. Holley provided transgenic and mutant zebrafish lines. C. Weaver, K. Willert, K. J. P. Griffin, J. Bertrand, D. Stachura and Y. Lee provided critical evaluation of the manuscript. This research was funded by an AHA Postdoctoral Fellowship 0725086Y to W.K.C., an AHA Predoctoral Founders Affiliate Fellowship 0815732D to J.C.M., NIH R01-HL093467 to N.L. and NIH R01-DK074482, CIRM New Investigator Award, and March of Dimes 6-FY09-508 to D.T.

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Authors

Contributions

W.K.C. and D.T. designed all experiments. Whole-mount immunofluorescence, double fluorescence in situs, and Kaede-based fate mapping was performed by A.D.K. K.G.O. cloned and subcloned multiple constructs. J.C.M and N.L. generated Notch reporter lines. All other experiments were performed by W.K.C. The manuscript was written by W.K.C. and edited by N.L. and D.T., with critical input as described in the Acknowledgments.

Corresponding author

Correspondence to David Traver.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-15 with legends and Supplementary Tables 1-9. (PDF 3823 kb)

Supplementary Movie 1

The movie shows timelapse imaging of GFP+ HSCs in the trunk region of untreated cd41:GFP transgenic animals captured at 1 frame per 3 minutes from 50 hpf to 75 hpf, documenting the behaviour of HSCs in this time period. (MOV 8601 kb)

Supplementary Movie 2

The movie shows timelapse imaging of lack of GFP+ HSCs in the trunk region of W16MO-injected cd41:GFP transgenic animals captured at 1 frame per 3 minutes from 50 hpf to 75 hpf, documenting decreased HSCs in this time period. (MOV 6967 kb)

Supplementary Movie 3

The movie shows timelapse imaging of GFP+ thymic immigrants in the head region of untreated cd41:GFP transgenic animals captured at 1 frame per 3 minutes from 50 hpf to 75 hpf, documenting the transition of HSCs to nascent T cells in this time period. (MOV 9791 kb)

Supplementary Movie 4

The movie shows timelapse imaging of lack of GFP+ thymic immigrants in the head region of W16MO-injected cd41:GFP transgenic animals captured at 1 frame per 3 minutes from 50 hpf to 75 hpf, documenting decreased transition of HSCs to nascent T cells in this time period. (MOV 6550 kb)

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Clements, W., Kim, A., Ong, K. et al. A somitic Wnt16/Notch pathway specifies haematopoietic stem cells. Nature 474, 220–224 (2011). https://doi.org/10.1038/nature10107

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