Volume 18, Issue 4, Pages 569-578 (April 2010) Tel1/ETV6 Specifies Blood Stem Cells through the Agency of VEGF Signaling  Aldo Ciau-Uitz, Philip Pinheiro, Rajeev Gupta, Tariq Enver, Roger Patient  Developmental Cell  Volume 18, Issue 4, Pages 569-578 (April 2010) DOI: 10.1016/j.devcel.2010.02.009 Copyright © 2010 Elsevier Inc. Terms and Conditions

Developmental Cell 2010 18, 569-578DOI: (10.1016/j.devcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions

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 40× 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. Developmental Cell 2010 18, 569-578DOI: (10.1016/j.devcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions

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. Developmental Cell 2010 18, 569-578DOI: (10.1016/j.devcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions

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. Developmental Cell 2010 18, 569-578DOI: (10.1016/j.devcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions

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. Developmental Cell 2010 18, 569-578DOI: (10.1016/j.devcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions

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. Developmental Cell 2010 18, 569-578DOI: (10.1016/j.devcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions

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. Developmental Cell 2010 18, 569-578DOI: (10.1016/j.devcel.2010.02.009) Copyright © 2010 Elsevier Inc. Terms and Conditions