Ciau-Uitz A et al. (2010), Tel1/ETV6 specifies blood stem cells through th...

XB-ART-48503

Dev Cell 2010 Apr 20;184:569-78. doi: 10.1016/j.devcel.2010.02.009.

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Tel1/ETV6 specifies blood stem cells through the agency of VEGF signaling.

Ciau-Uitz A , Pinheiro P , Gupta R , Enver T , Patient R .

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The regulation of stem cell ontogeny is poorly understood. We show that the leukemia-associated Ets transcription factor, Tel1/ETV6, specifies the first hematopoietic stem cells (HSCs) in the dorsal aorta (DA). In contrast, Tel1/ETV6 has little effect on embryonic blood formation, further distinguishing the programming of the long- and short-term blood populations. Consistent with the notion of concordance of arterial and HSC programs, we show that Tel1/ETV6 is also required for the specification of the DA as an artery. We further show that Tel1/ETV6 acts by regulating the transcription of VegfA in both the lateral plate mesoderm and also in the somites. Exogenous VEGFA rescues Tel1/ETV6 morphants, and depletion of VEGFA or its receptor, Flk1, largely phenocopies Tel1/ETV6 depletion. Few such links between intrinsic and extrinsic programming of stem cells have been reported previously. Our data place Tel1/ETV6 at the apex of the genetic regulatory cascade leading to HSC production.

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Species referenced: Xenopus
Genes referenced: atm dll4 efnb2 etv6 flt1 flt4 gata2 gfi1 kcnh2 kdr lmo2 notch1 notch4 notch4.2 odc1 runx1 tal1 tek vegfa
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	Figure 1. Tel1 Is Expressed in the Embryonic DA and Is Required for the Emergence of HSCs (A) In situ hybridization on transverse sections showing expression of Tel1 in the ventral wall of the DA and emerging HSCs (red arrows). (B) Scl staining on transverse sections shows that intra-aortic hematopoietic clusters containing HSCs do not emerge in Tel1 morphants. All sections shown are at 40Ã— magnification, with dorsal to the top. g, ganglia; n, notochord; PCV, posterior cardinal vein; Pd, pronephric duct. Stages of development are as indicated. See also Figures S1 and S2, and Table S1.
	Figure 2. Establishment of the HSC Program and Arterial Specification of the DA Is Abolished in Tel1-Depleted Embryos (A) In situ hybridization analysis showing that expression of hematopoietic genes in the ventral wall of the DA (arrows) is absent in Tel1 morphants. In contrast, expression in embryonic erythrocytes (Runx1, Scl, and Lmo2, arrowheads) and myeloid cells (SpiB and Gfi1, arrowheads) is still observed. (B) Expression analysis showing that the DA (arrows) of Tel1 morphants does not express the arterial affiliated genes, Notch4, Dll4, and EphrinB2, nor the Notch target, Gata2. (C) Endothelial expression of Erg1 reveals the presence of DA precursors (arrows) coalescing in the midline of Tel1 morphants. All embryos were hybridized as whole mounts and are shown in lateral view, with anterior to the left and dorsal to the top. Stages of development are as indicated. The numbers of embryos represented by each panel, out of the number analyzed, are indicated in the top right corner. See also Figure S1 and Table S1.
	Figure 3. Tel1 Is Required for the Programming of Adult Hemangioblasts in the Lpm (A) Expression analysis showing that Tel1 is expressed in the DLP (arrows) at the time adult hemangioblasts are specified. (B) Analysis of hematopoietic expression in the DLP of Tel1 morphants. Tel1 is dispensable for Gata2 and Lmo2 expression, but required for Scl expression in the DLP (arrows). Arrowheads indicate expression in embryonic erythrocytes located in the ventral blood island. (C) Analysis of endothelial expression in the DLP of Tel1 morphants. Tel1 is required for DLP expression of Flt1 and Flt4, but not Flk1 (arrows). All embryos were hybridized as whole mounts and are shown in lateral view, with anterior to the left and dorsal to the top. Stages of development are as indicated. The numbers of embryos represented by each panel, out of the number analyzed, are indicated in the top right corner. See also Figure S1 and Table S1.
	Figure 4. Tel1 Regulates VegfA Expression in the DLP and Somites, and VEGFA Rescues the Programming of Hemangioblasts, the DA, and HSCs in Tel1 Morphants (A) In situ hybridization analysis showing that Tel1 and VegfA expression overlap in several tissues, including the DLP and somites. BA, branchial arches; s, somites. (B) Analysis of VegfA expression in Tel1-depleted embryos. A general reduction of VegfA expression is observed in Tel1 morphants, with the greatest reductions in the DLP (arrows) and the somites. (C) qPCR analysis of the levels of VegfA mRNA in wild-type and Tel1 morphant DLP (top) and somite (bottom) explants (see Experimental Procedures). Explants were taken at stage 27, and RNA was extracted and assayed for VegfA expression normalized to ODC. Error bars indicate the SEM. (D) Expression analysis showing that VegfA mRNA (2 ng) injection into Tel1 morphants rescues hematopoietic (Scl) and endothelial (Flt1 and Flt4) gene expression in the DLP (arrows). Exogenous VegfA also rescues expression of Dll4 in the DA (arrows) of morphants and, in addition, induces ectopic expression of Dll4 in other blood vessels (arrowheads). A total of 54% of morphants recover Runx1 expression in the ventral wall of the DA (arrows). (E) Expression analysis on stage 43 sectioned embryos shows that exogenous VegfA mRNA rescues expression of Scl (red arrows) in the ventral wall of the DA of Tel1 morphants. n, notochord; PCV, posterior cardinal vein; Pd, proneprhic duct. Embryos hybridized as whole mounts are shown in lateral view, with anterior to the left and dorsal to the top. Sections are in transverse orientation and shown with dorsal to the top. Stages of development are as indicated. The numbers of embryos represented by each panel, out of the number analyzed, are indicated in the top right corner. See also Figure S1 and Tables S1 and S2.
	Figure 5. Depletion of VEGFA or Its Receptor, Flk1, Phenocopies the Tel1 Hemangioblast Phenotype (A) Injection of VegfA MO indicates that Gata2 and Lmo2 expression in the DLP is independent of VEGFA signaling, while Scl expression is dependent (arrows). Although a decrease in Gata2 expression is observed in the ventral blood island, VEGFA depletion appears to have little effect on developing embryonic erythrocytes (arrowheads). (B) Expression analysis in VegfA morphants showing that VEGFA is required for the expression of Flt1 and Flt4, but not Flk1, in the DLP (arrows). (C) Expression analysis in VegfA morphants showing that VEGFA is required for the formation of the DA (arrows). DA precursors located in the DLP do not migrate to the midline when VegfA is depleted. (D) Expression analysis in Flk1 morphants showing that Flk1 is required for SCL expression in the DLP, but that expression of Gata2 and Lmo2 is independent (arrows). (E) Expression analysis in Flk1 morphants showing that VEGFA is required for the expression of Flt1 and Flt4, but not Flk1 in the DLP (arrows). (F) Flk1 is required for the formation of the DA (arrows). DA precursors located in the DLP do not migrate to the midline when Flk1 is depleted. All embryos were hybridized as whole mounts and are shown in lateral view, with anterior to the left and dorsal to the top. Stages of development are as indicated. The numbers of embryos represented by each panel, out of the number analyzed, are indicated in the top right corner. See also Table S1.
	Figure 6. Tel1-Dependent VEGFA in the DLP and in the Somites Drives HSC Development (A) Schematic depiction of experimental design. Wild-type or MO-injected two cell embryos were cultured to stage 22 when DLPs and/or somites were explanted and cultured separately or together until sibling embryos had reached stage 27/28. RNA was extracted from the test DLPs and assayed for Scl expression normalized to ODC. Error bars indicate the SEM. (B) Induction of Scl expression in the DLP is tissue autonomous. DLPs at stage 22 (sample 1), expressing no Scl, express substantial quantities when cultured to stage 27/28 (sample 2). (C) Loss of Scl expression in the DLP of Tel1 morphants (sample 3) can be rescued by coculture with wild-type somitic tissue (sample 5), but not with wild-type DLP tissue (sample 4) or somitic tissue injected with either VegfA MO (sample 6) or Tel1 MO (sample 7). (D) Tissue-autonomous expression of Scl is dependent on VEGFA. The robust expression in wild-type DLPs (sample 2) is abolished by VegfA MO injection (sample 8). (E) Model illustrating the contribution of Tel1 and VEGFA to HSC development. Correct programming of adult hemangioblasts, the earliest precursors of HSCs, requires VEGFA signaling. Endogenous (adult hemangioblast) and exogenous (somites) VEGFA, controlled by Tel1, is required. Although hemangioblasts are not correctly programmed in the absence of Tel1, DA precursors are capable of migrating to the midline, where VEGFA is still produced in the hypochord (see Figure 4B). VEGFA signaling in the midline also appears to be sufficient for the endothelial differentiation and lumenization of a vessel in the position of the DA. Critically, though, the DA is not specified as an artery, hematopoiesis fails, and HSCs are not produced. Genes downregulated and cell types not specified in Tel1 morphants are indicated in red. See also Figure S1 and Table S2.
	Figure S1, related to Figures 1-4 and 6. Morpholino design and general phenotype of Tel1 morphants (A) Two antisense MO oligonucleotides were designed to block the translation of X. laevis Tel1. MO1 (pink) targets a sequence overlapping with the translation initiation codon whereas MO2 (red) targets a sequence within the 5ï¿½ UTR. The targets of both MO1 and MO2 are conserved between the two pseudoallelic forms of Tel1 found in the X. laevis genome. (B) Structure of Tel1-GFP fusion construct used to test the activity of Tel1 MO1. (C) Embryo injected with the Tel1-GFP fusion mRNA shows GFP expression (arrow). Image shows a lateral view of a stage 22 embryo, anterior to the left and dorsal to the top. (D) Embryo initially injected with Tel1-GFP mRNA and subsequently injected with Tel1 MO1 shows no GFP expression. Photo shows a lateral view of a stage 22 embryo, anterior to the left and dorsal to the top. (E) In situ hybridisation at stage 39 showing that injection of either Tel1 MO1 or Tel1 MO2 abolishes Runx1 expression in the ventral wall of the DA (red arrows). Images show lateral views with anterior to the left and dorsal to the top. Numbers of embryos represented by the image, out of the numbers analysed, are indicated in the top right corner. (F) qPCR analysis on isolated stage 35 VBIs showing no significant difference in ï¡T4- globin expression between control and Tel1-depleted embryos. (G) Embryos stained for Tie2 at stage 34 show that the development of the vasculature is affected in Tel1 morphants. Embryos exhibit a poorly developed vitelline vessel network (VitV) and an absence of aortic arches (AoArch). In contrast, although Tie2 expression levels are reduced, development of the posterior cardinal vein (PCV) and the DA are not disrupted. Embryos are shown in lateral view with anterior to the left and dorsal to the top.
	tal1 (T-cell acute lymphocytic leukemia 1) gene expression in X. laevis embryo, NF stage 43, assayed via in situ hybridization, transverse section through dorstal aorta (DA), showing positive staining in clusters of hematopoietic stem cells (HSC).