etsrp Is Required for <i>vegf</i> and <i>scl</i> Signaling

<p>(A–D) Etsrp is required for Vegf signaling as assayed for <i>flk1</i> expression at 26 hpf. (A) Control uninjected embryo, (B) <i>vegf</i> RNA-injected embryo, (C) 7.5 ng of <i>etsrp</i> MO2-injected embryo, (D) <i>vegf</i> RNA- and <i>etsrp</i> MO2-co-injected embryo. Note that <i>vegf</i> RNA induces strong <i>flk1</i> expression in (B) while <i>vegf</i> RNA and <i>etsrp</i> MO co-injection results in loss of <i>flk1</i> expression in (D), similar to the <i>etsrp</i> morphant phenotype in (C). (E,F) Etsrp expression analysis in Vegf morphants at 26 hpf. (E) Control uninjected embryo; (F) 10.5 ng of <i>vegf</i> MO-injected embryo. Note that <i>vegf</i> morphants have lost <i>etsrp</i> expression in the intersegmental vessels (arrowhead, E). (G–J) Scl knockdown affects <i>gata1</i> but not <i>etsrp</i> expression at the 15-somite stage. Dorsal view, anterior is to the left. (G,I) Control uninjected embryo; (H,J) 10 ng <i>scl</i> UTR-MO-injected embryo. (G,H) <i>gata1</i> expression; (I,J) <i>etsrp</i> expression. (K–N) Etsrp is required for <i>scl</i> signaling in <i>clo</i> mutants as analyzed for <i>flk1</i> expression at the 15-somite stage. (K) Control uninjected embryo; (L) 7.5 ng <i>etsrp</i> MO2-injected embryo; (M) <i>scl</i> RNA-injected embryo; (N) <i>scl</i> RNA- and <i>etsrp</i> MO2-co-injected embryo. Note that <i>scl</i> RNA causes ectopic <i>flk1</i> expression in (M) which is lost upon knockdown of Etsrp in (N).</p

Expression Pattern of <i>etsrp</i> as Analyzed by In Situ Hybridization

<p>Anterior is to the left except as noted. (A) five-somite stage. <i>etsrp</i> is expressed within lateral mesoderm in two distinct expression domains, in the anterior and posterior parts of an embryo. (B) 15-somite stage, lateral view. (C) dorsal view. (D) transverse section. <i>etsrp</i> is expressed in two bilateral stripes of presumptive angioblasts within the lateral mesoderm in the anterior and the trunk and posterior parts of an embryo (arrows, D). In addition, a stripe of <i>etsrp</i>-expressing cells is apparent at the midline and extends through the middle and posterior parts of an embryo (arrowhead, C and D). (E) 26 hpf stage. <i>etsrp</i> is expressed in vascular endothelial cells of the axial, head, and intersomitic vessels. Note a group of <i>etsrp</i>-expressing cells bilaterally located in the intermediate mesoderm (arrowhead). (F) Transverse section through the trunk region of a 30 hpf embryo. Arrowhead shows one of the <i>etsrp</i>-expressing cells located within the lateral/intermediate mesoderm. Expression of <i>etsrp</i> in the axial vessels is weak at this stage and not apparent in this section. nt, neural tube; n, notochord; y, yolk. (G) 36 hpf stage. <i>etsrp</i> is expressed in a subset of head vessels, the aortic arches (aa), the cardinal vein plexus (pl) region, posterior intersomitic vessels, and the dorsal longitudinal anastomotic vessel (dlav). Note a group of <i>etsrp</i>-expressing cells located in the endodermal region (arrowhead). (H) 52 hpf stage. <i>etsrp</i> expression is observed in a subset of head vessels, common cardinal vein, pectoral fin bud blood vessels (fb), cardinal vein plexus region, and weakly in posterior intersegmental vessels and the dorsal vessel.</p

Molecular Analysis of Early Vasculogenesis in <i>etsrp</i> Morphants

<p>(A, B, E, F, I, K) Uninjected control embryo; (C, D, G, H, J, L) 8–10 ng <i>etsrp</i> MO2-injected embryo. Anterior is to the left in all panels. (A–H) Embryos were flat mounted with their yolk removed. (A–D) <i>scl</i> expression; six-somite (A,C) and ten-somite (B,D) stages. Note that the anterior domain of <i>scl</i> expression (arrows) is reduced and the trunk domain (arrowheads) is missing in <i>etsrp</i> morphants. (E–H) <i>fli1</i> expression; six-somite (E,G) and ten-somite (F,H) stages. Note that the anterior domain of <i>fli1</i> expression (arrows) is missing in the <i>etsrp</i> morphants, while the posterior domain is not affected. Also note that the trunk domain of <i>fli1</i> expression (arrowheads, F,H) is missing at the ten-somite stage in <i>etsrp</i> morphants. (I–L) Etsrp knockdown blocks angioblast migration towards the midline as assayed by <i>etsrp</i> expression at the 16-somite (I,J) and 20-somite (K,L) stages. (I,K) Uninjected control embryo; (J,L) 7.5 ng <i>etsrp</i> MO2-injected embryo. Dorsal view, anterior is to the left. Note that the midline stripe of angioblasts (arrows) is missing in <i>etsrp</i> morphants. Also notice more intense <i>etsrp</i> expression in pre-migratory angioblasts (arrowheads) in <i>etsrp</i> morphants as compared to control embryos.</p

Sequence Analysis of the Zebrafish Ets1-Related Protein Etsrp

<p>(A) Shows alignment of Etsrp and its closest human and zebrafish homologs Ets1 proteins. Etsrp and hEts1 share 88% homology within the ETS DNA-binding domain (underlined in red), and a very limited homology within the rest of the sequence. Identical and similar amino acids are shaded in grey. (B) The homology tree of Etsrp and its closest human, mouse, and zebrafish homologs. Length of horizontal branches is directly proportional to the evolutionary distance between the proteins. Zebrafish Ets1 and Ets2 protein sequences have been predicted using the available EST sequences TC282499 and TC270146 (<a href="http://www.tigr.org" target="_blank">http://www.tigr.org</a>). GeneWorks 2.5 has been used to build the alignment and the homology tree. (C) Chromosomal location of the zebrafish <i>etsrp,</i> mouse, and human <i>ets1</i> genes. In all cases, they are positioned next to a <i>fli1</i> homolog.</p

etsrp RNA Overexpression Induces Ectopic Expression of Vascular Endothelial Markers

<p>Dorsal view, anterior to the left in all panels except for (E,F) which are lateral views. (A, C, E, and G) Control uninjected embryo; (B, D, F, and H) 100 pg of <i>etsrp</i> RNA-injected embryo. (A,B) <i>scl</i> expression at the eight-somite stage; (C,D) <i>flk1</i> expression at the nine-somite stage. Note the strong ectopic induction of <i>scl</i> and <i>fli1</i> upon overexpression of <i>etsrp</i> RNA. (E,F) Live <i>flk1</i>-GFP embryo at the 14-somite stage; fluorescent and transmitted light images were overlayed. Note the very strong ectopic induction of GFP expression in different tissues including neuroectoderm (arrow, F) upon <i>etsrp</i> RNA overexpression. Fluorescence in the control uninjected <i>flk1</i>-GFP embryo in (E) is not detectable under the same exposure. (G,H) <i>gata1</i> expression at the 16-somite stage. Note that <i>gata1</i> expression is not affected upon <i>etsrp</i> overexpression. (I–L) <i>etsrp</i> RNA induces <i>flk1</i> expression in <i>clo</i> mutant embryos as analyzed using <i>flk1</i> probe at the ten- to 12-somite stages. (I) wt (or <i>clo+/−</i>) embryo, (J) wt (or <i>clo+/−</i>) embryo injected with 100 pg of <i>etsrp</i> RNA, (K) <i>clo−/−</i> embryo, (L) <i>clo−/−</i>embryo injected with 100 pg of <i>etsrp</i> RNA. Note that in a <i>clo+/−</i> (or wt) embryo <i>etsrp</i> RNA induces ectopic <i>flk1</i> (arrow, J) in addition to the endogenous <i>flk1</i> expression (arrowheads, J) while <i>clo-/− etsrp</i> RNA-injected embryo shows only ectopic <i>flk1</i> (arrows, L).</p

VEGF signaling inhibits <i>stab2</i> expression, while Notch inhibition has no effect on <i>stab2</i> expression.

<p>(A–F) Vegf overexpressing embryos display downregulated <i>stab2</i> expression evident by in situ hybridization analysis at the 20-somite (A,B), 24-somite (C,D) and 24 hpf (E,F) stages. Note that in wt embryos <i>stab2</i> is expressed in both the DA and the PCV at the 20–24-somite stage while its expression is primarily restricted to the PCV at 24 hpf. (G,H) Vegf morphants display an expansion of <i>stab2</i> expression into the DA at 24 hpf when expression is normally restricted to the PCV in wt embryos. (I–N) <i>Stab2</i> expression at 24 hpf is not affected by inhibition of Notch signaling either by DAPT treatment (K,L) or in <i>mindbomb</i> genetic mutants (M,N) while <i>flt4</i> expression is expanded in DAPT treated embryos (I,J). Arrows indicate DA and arrowheads indicate PCV in all panels. Lateral view, anterior is to the left, trunk and tail region is shown.</p

Molecular Analysis of Vascular Endothelial and Hematopoietic Markers in <i>etsrp</i> Morphants

<p>(A, C, E, G, I, K, M, O, Q, S) Uninjected control embryo; (D, H, J, L, P, R, T) 8 ng <i>etsrp</i> MO2-injected embryo; (B, F, N) 12 ng <i>etsrp</i> MO1+MO2 (1:1) mix-injected embryos for the maximum knockdown. All embryos are at 24 hpf, except as noted. Scale bar, 0.2 mm. (A,B) <i>flk1</i> expression; (C,D) <i>admr</i> expression; (E,F) <i>cdh5</i> expression; (G,H) <i>dusp5</i> expression; (I,J) <i>flt4</i> expression; (K,L) <i>crl</i> expression. Note that the vascular expression of the markers in (A–L) is almost absent in <i>etsrp</i> morphants. (M,N) <i>fli1</i> expression; (O,P) <i>etsrp</i> expression. Note the more intense <i>etsrp</i> expression and reduced <i>fli1</i> expression in angioblasts which remain dispersed and fail to coalesce into blood vessels in <i>etsrp</i> morphants. Inset, (P) DIC image of scattered angioblasts in <i>etsrp</i> morphants. (Q,R) <i>scl</i> expression, 22 hpf. Note that <i>scl</i> expression appears unaffected at this stage except for more intense staining in a subset of head vessels in <i>etsrp</i> morphants (arrowhead). (S,T) <i>gata1</i> expression, 22 somites. Note that no significant difference in hematopoietic <i>gata1</i> expression is observed between the control embryos and <i>etsrp</i> morphants.</p

MO Knockdown of Etsrp Protein Function Disrupts Blood Vessel Formation in the Zebrafish Embryos

<p>(A,B) Morphological analysis of live <i>etsrp</i> morphants at 34 hpf. (A) Uninjected control embryo. (B) 5 ng of <i>etsrp</i> MO1-injected embryo. Notice that red blood cells are scattered throughout the circulatory system in the control uninjected embryo while they accumulate at their formation site within the intermediate cell mass (arrow, B) in the <i>etsrp</i> morphant. (C–E) o-dianisidine staining of heme in the red blood cells of uninjected control (C), 5 ng of <i>etsrp</i> MO1-injected (D) and 5 ng of <i>etsrp</i> MO2-injected (E) embryos at 34 hpf. While many circulating blood cells are apparent within the common cardinal vein before entering the heart in the control embryo (arrow, C), they stay at their formation site within the ICM region in <i>etsrp</i> morphants (arrows). (F–I) Microangiography analysis of the circulatory system by injecting fluorescein-labeled dextran into the sinus venosus of <i>etsrp</i> morphants at 55 hpf. (F) Control uninjected, (G) 1 ng of <i>etsrp</i> MO1-injected (H) 2.5 ng of <i>etsrp</i> MO1-injected (I) 5 ng of <i>etsrp</i> MO1-injected embryos. Note that the embryo in (G) has lost circulation in the posterior vessels, the embryo in (H) has lost circulation in most vessels, and the embryo in (I) has no circulation at all. (J–M) Analysis of blood vessels in live <i>flk1</i>-GFP transgenic embryos at 26 hpf. (J) Control uninjected, (K) 1 ng of <i>etsrp</i> MO1-injected, (L) 2.5 ng of <i>etsrp</i> MO1-injected, (M) 15 ng of <i>etsrp</i> MO1+MO2 (1:1) mix-injected embryos. Note the gaps in formation of intersegmental vessels in (K) (arrowhead), the missing (arrowhead) and abnormally branched, (arrow) intersegmental vessels in (L), and the nearly completely eliminated <i>flk1</i> expression from axial vessels (arrow) in (M).</p

Stab2 morphants display ISV defects at 22–28 hpf.

<p>Tg(<i>kdrl</i>:GFP) expression in wild type uninjected embryos (A,C, and E) and Stab2 morphant embryos (B, D, and F) at 22 hpf (A,B), 24 hpf (C,D) and 28 hpf (E,F). Morphant embryos display absent (B and D) or reduced (F) ISVs at all three stages. Lateral view shown, anterior is to the left. Morphants were injected with a cocktail containing Stab2 MO1, MO2 and p53 MO.</p

Downregulation of venous markers by PI3K inhibition is restored in Stab2 morphants.

<p>Both wild type and Stab2 morphant embryos were treated with either 1% DMSO or 20 µM LY294002 and stained for <i>stab1l</i> and <i>flt4</i> expression by in situ hybridization analysis. (A,B, E,F) Wild type embryos display a reduction in intensity of staining of either <i>stab1l</i> (A,B) or <i>flt4</i> (E,F) upon LY294002 treatment. Note that because venous <i>stab1l</i> and <i>flt4</i> expression is reduced, arterial and venous expressions appear of similar intensity. (C,G) DMSO treated Stab2 morphants display an expansion of <i>stab1l</i> (C) and <i>flt4</i> (G) expression into the area of the DA. (D,H) LY294002 treated Stab2 morphants display expanded arterial <i>stab1l</i> (D) and <i>flt4</i> (H) expression and the intensity of venous expression are restored to wild type levels. Arrows indicate DA and arrowheads indicated PCV. Lateral view of 24 hpf embryos, anterior is to the left, trunk and tail region is shown. Morphants were injected with a cocktail containing Stab2 MO1, MO2, and p53 MO.</p