Wnt9a Is Required for the Aortic Amplification of Nascent Hematopoietic Stem Cells - PubMed (original) (raw)

. 2016 Nov 1;17(6):1595-1606.

doi: 10.1016/j.celrep.2016.10.027.

Jenna Richter 2, Raquel Espín Palazón 1, Claire Pouget 1, Brianna Lonquich 1, Sara Wirth 1, Kathrin Sabine Grassme 3, Wiebke Herzog 4, Matthew R Swift 5, Brant M Weinstein 6, David Traver 7, Karl Willert 8

Affiliations

Wnt9a Is Required for the Aortic Amplification of Nascent Hematopoietic Stem Cells

Stephanie Grainger et al. Cell Rep. 2016.

Abstract

All mature blood cell types in the adult animal arise from hematopoietic stem and progenitor cells (HSPCs). However, the developmental cues regulating HSPC ontogeny are incompletely understood. In particular, the details surrounding a requirement for Wnt/β-catenin signaling in the development of mature HSPCs are controversial and difficult to consolidate. Using zebrafish, we demonstrate that Wnt signaling is required to direct an amplification of HSPCs in the aorta. Wnt9a is specifically required for this process and cannot be replaced by Wnt9b or Wnt3a. This proliferative event occurs independently of initial HSPC fate specification, and the Wnt9a input is required prior to aorta formation. HSPC arterial amplification occurs prior to seeding of secondary hematopoietic tissues and proceeds, in part, through the cell cycle regulator myca (c-myc). Our results support a general paradigm, in which early signaling events, including Wnt, direct later HSPC developmental processes.

Keywords: HSCs; HSPCs; Myc; Wnt; Wnt9a; aorta; hematopoietic stem and progenitor cells; hematopoietic stem cells; proliferation.

Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1.

Figure 1.. Wnt Signaling Is Required Transiently Prior to 20 hpf for HSPC Development

(A) kdrl:mCherry; cmyb:eGFP fish were treated with IWP-L6 or LiCl to inhibit and activate Wnt signaling, respectively (van de Water et al., 2001; Wang et al., 2013), from 10 hpf to 36 hpf and imaged at 36 hpf. A, aorta; V, vein. Scale bar, 30 mm. (B) Quantitation of HSPCs per millimeter of aorta. (C) Schematic of heat shock regimen. (D) hsp:dntcf fish were heat shocked every hour from 13 hpf to 24 hpf, fixed at 40 hpf, and analyzed for cmyb expression by WISH. Scale bar, 100 mm. (E) Quantitation of cmyb+ cells from (D). (F) Schematic of experimental layout. (G) hsp:dntcf fish were heat shocked at 16.5 hpf, pools were fixed every hour from 23 to 36 hpf, and they were analyzed for cmyb expression by WISH. Scale bar, 100 μm. (H) Quantitation of _cmyb_+ cells from (G). (I) HSPCs are specified as endothelial cells ingress toward the midline. Endothelial cells receive specification cues, at least in part, from somatic cells. At 26 hpf, HSPCs start to emerge from the floor of the dorsal aorta. A, aorta; V, vein. **p < 0.01; ***p < 0.001; n.s., not significant. Error bars represent SD. See also Figure S1.

Figure 2.

Figure 2.. Wnt Cue Is Required in Cells of the HE

(A) Schematic of transgenic elements used for endothelial specific expression of dntcf in endothelial cells. (B) WISH for cmyb at 40 hpf in control and in fli1a:Gal4; UAS:dntcf fish. (C) Quantitation of _cmyb_+ cells from (B). (D) Schematic of transgenic elements used for endothelial specific expression of dntcf in the HE. (E) WISH for cmyb at 40 hpf in control and in gata2b:Gal4; UAS:dntcf fish. (F) Quantification of _cmyb_+ cells from (E). ***p < 0.001. Scale bars, 100 μm. Error bars represent SD. See also Figure S2.

Figure 3.

Figure 3.. Wnt9a Is Required for HSPC Formation

(A and B) WISH for wnt9a at 16.5 hpf, 19 hpf, and 24 hpf (A). Scale bars, 0.2 mm; representative images of n = 15. Imaged area is shown in (B). (C and D) Representative images of kdrl:mCherry; cmyb:eGFP injected as listed (C). HSPCs per millimeter of aorta are quantified in (D). Scale bars, 30 mm. (E) WISH for cmyb in 4 days post fertilization (dpf) fish treated with 0.1 ng or 1 ng of wnt9a MO. Scale bars, 0.2 mm. (F) Wnt9a mutants were generated by injection of guide RNA targeting the last portion of the first exon. (G and H) Expression of cmyb was examined by WISH in 36 hpf WT, wnt9a _+/_D and wnt9a_D/D_- zebrafish (G) and quantified in (H). Scale bars, 100 mm. *p < 0.05; ***p < 0.001; n.s., not significant. Error bars represent SD. See also Figure S3.

Figure 4.

Figure 4.. HSPCs Expand in the Aorta

(A) The heart tubes of gata2b:GFP fish were injected with 1 nL of 10 mM EdU at 26 hpf, and fixed and stained for cells that had divided as well as GFP at 33 hpf (n = 10). (B) Representative confocal image of _gata2b_+ cells that have undergone cell division in the aorta (arrowheads). (C–E). gata2b:GFP;kdrl:mCherry fish were treated with 10 mM 5ˊ fluorouracil (5FU) from 24 to 35 hpf (C), confocal imaged at 36 hpf (D), and gata2b+ cells quantified (E). (F and G) G1 arrested (mCherry+) endothelial cells (GFP+) from wnt9a mor-phant and control fish (n = 100 embryos per condition) were collected by FACS at 28 hpf (F) and compared by qPCR for gata2b (G). Scale bars, 30 μm. Error bars represent SD. See also Figure S4.

Figure 5.

Figure 5.. Wnt9a Affects Endothelial gata2b+ G1-S Cell Cycle Progression through myca

(A) Endothelial cells (mCherry+) were collected by FACS from hs:dntcf:IRES-GFP; kdrl:mCherry fish at 30 hpf after heat shock at 16 hpf (n = 100 embryos per condition). (B) Wnt inhibited (dntcf;GFP+) cells were com-pared to control (GFP ) cells by qPCR after being sorted by FACS. (C and D) AB* fish were injected with wnt9a MO, myca mRNA, or both; phldb4:Gal4 fish were injected with wnt9a MO, UAS;myca plasmid, and transposase mRNA, fixed at 40 hpf, analyzed by WISH for cmyb (C), and quantified in (D). Scale bar, 100 μm. *p < 0.05; ***p < 0.001; n.s., not significant. Error bars represent SD. See also Figure S5.

Figure 6.

Figure 6.. Early Wnt9a Cue Is Required for Later Hematopoietic Stem Cell Amplification

Prior to 20 hpf, ingressing cells of the posterior lateral mesoderm travel beneath the somites. Inductive cues direct from the somite instruct the fate of these cells, some of which are destined to become HE, and later, HSPCs. Wnt9a is expressed in the somite at this stage. By 26 hpf, the aorta has formed and HSPCs have begun to emerge. In the absence of Wnt9a, this early emergence is unaffected, indicating the HSPC fate specification has occurred properly. By 31 hpf, normal HSPCs undergo an expansion event, whereas those in Wnt9a-deficient animals do not.

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