Expression of <i>meis1.1,</i> but not <i>cdx4</i>, is impaired in <i>hoxd4a</i> morphants at the shield stage.

<p>Expression of <i>cdx4</i> (A,B), <i>hoxd4a</i> (C–E) and <i>meis1.1</i> (F–H) in control (A,C,F), <i>hoxd4a</i> morphants (B,D,G) and rescuants injected with <i>hoxd4a</i> mRNA (E,H) observed at the shield stage. Asterisks denote the ventral-most mesoderm fated to give rise to hemangioblast in addition to unipotential hematopoietic and angiogenic progenitors. Ratios indicate the fraction of embryos showing the presented phenotype. Scale bars equal 100 µm. (I) qRT-PCR was used to quantitate relative mRNA levels in controls, morphants and rescuants as indicated. Error bars present the standard error. Decreased expression of <i>hoxd4a</i> and <i>meis1.1</i> in <i>hoxd4a</i> morphants is significantly lower than both controls and rescuants to p≤0.02.</p

<i>hoxd4a</i> knockdown disrupts primitive hematopoiesis and is highly specific.

<p><i>In situ</i> hybridization revealing expression at 13 hpf of erythroid lineage markers <i>gata1</i> (A,C,E) and <i>β embryonic globin 1</i> (<i>hbbe1</i>) (B,D,F). (A,B) Normal expression of <i>gata1</i> and <i>hbbe1</i> in the LPM. (C,D) Expression of <i>gata1</i> and <i>hbbe1</i> is strongly down-regulated in <i>hoxd4a</i> morphants, but rescued by co-injection of capped mRNA for <i>hoxd4a</i> (E,F). All embryos have been flat-mounted and are shown in dorsal view. Anterior is to the left. Ratios in the bottom left corner of all panels indicate the number of embryos showing the presented phenotype. ctrl, embryos injected with a non-specific morpholino. MO, embryos injected with the anti-<i>hoxd4a</i> morpholino. <i>hoxd4a</i> mRNA, embryos simultaneously injected with the anti-<i>hoxd4a</i> MO plus capped mRNA for <i>hoxd4a</i>. Scale bars equal 100 µm. All images are at the same magnification.</p

Expression pattern of <i>hoxd4a</i> and phenotype of <i>hoxd4a</i> morphants.

<p>(A–D) Detection of <i>hoxd4a</i> transcripts in early zebrafish embryos. Animal pole is to the top. (A) 1 cell. (B) 3 hpf. (C) 50% epiboly. (D) 75% epiboly. (E) Dorsal view of flat-mounted embryo at 12 hpf (5–6 somites) doubly stained for expression of <i>hoxd4a</i> and <i>krox20a</i>. The arrow indicates the anterior expression border of <i>hoxd4a</i>, while asterisks denote expression of <i>krox20a</i> in r3 and r5. Anterior is to the left. The rostral-most portion of the embryo is not captured in the image. (F) Dorsal view of a flat-mounted embryo at 26–28 hpf (>26 somites) showing <i>hoxd4a</i> expression in hindbrain, branchial arches (white arrowhead, left side only) and pectoral fin field (black arrowhead, right side only). The white arrow marks the <i>hoxd4a</i> anterior expression border in the hindbrain at the boundary between r6 and r7. Asterisks denote <i>krox20a</i> expression in r3 and r5. (G–H) Lateral views of 26–28 hpf embryos showing <i>hoxd4a</i> expression in the central nervous system (arrows) of control embryos (G) and reduced expression in <i>hoxd4a</i> morphants (H). (I–K) <i>hoxd4a</i> expression in the caudal half at 26–28 hpf as shown in lateral views, anterior to the left. <i>hoxd4a</i> expression seen in the PBI of control embryos (I, white arrow) is greatly reduced in <i>hoxd4a</i> morphants (J). Expression is rescued following co-injection with capped <i>hoxd4a</i> mRNA (K). (L–N) Results of <i>In situ</i> hybridization for <i>hoxd4a</i> at 48 hpf showing expression in the AGM and caudal vein plexus (site of future CHT) of control injected embryos (L, black arrows), greatly reduced expression in <i>hoxd4a</i> morphants (M), and rescued expression in embryos simultaneously injected with capped mRNA for <i>hoxd4a</i> (N). (O–T) Hemoglobin within RBCs revealed by o-dianisidine staining. Ventral views at 48 hpf (O,P) and 72 hpf (Q,R) show greatly reduced levels of hemoglobin in the ducts of Cuvier in <i>hoxd4a</i> morphants (P and R) vs controls (O and Q). Co-injection with capped mRNA for <i>hoxd4a</i> results in rescued RBC production (S). (T) O-dianisidine staining of the caudal half of a control larva (upper panel) and <i>hoxd4a</i> morphant (middle panel) showing overall reduction in hemoglobin levels at 72 hpf in morphants, and rescue by co-injection of capped <i>hoxd4a</i> mRNA (lower panel). (U,V) Lateral views of trunk regions of Tg(gata1:dsRed) embryos at 26 hpf, anterior to the left. The expression of dsRed within proerythroblasts is readily detected in the ICM (arrow) and PBI (arrowhead) of control-injected embryos (U) but not <i>hoxd4a</i> morphants (V). Scale bars = 100 µm. Ratios indicate the number of embryos showing the presented phenotype. (W) qRT-PCR shows an overall 3-fold reduction of <i>hoxd4a</i> expression in morphants at 26–28 hpf. Error bars = standard error. p = 0.02. (X) Quantitation by flow cytometry of dsRed-positive cells in Tg(gata1:dsRed) embryos at 48 hpf showing that morphants display an 88% reduction in RBCs relative to control-injected embryos. Error bars = standard deviation. p<0.0002.</p

Loss of <i>hoxd4a</i> function impairs development of the vasculature.

<p>(A–D) Fluorescent images of the trunk and tail regions of Tg(<i>fli1</i>:EGFP) embryos at 48 hpf. The panels present merged bright field and fluorescent images (A,C) or fluorescent images only (B,D) The normal pattern of the vasculature (A,B) is severely disrupted in <i>hoxd4a</i> morphants (C,D) Dorsal extremities of ISV sprouts that fail to contact the DLAV are marked by white dots (D). The caudal vein plexus of control embryos (E, arrowheads) is replaced by a disorganized mass of endothelial tissue in <i>hoxd4a</i> morphants (F, arrowheads). (G–I) Alkaline phosphatase staining at 72 hpf revealing the vasculature in (G) control-injected larvae, (H) <i>hoxd4a</i> morphants, and (I) rescued larvae co-injected with capped mRNA for <i>hoxd4a</i>. Dorsal aorta (DA), posterior cardinal vein (PCV), inter-segmental vessels (ISV), caudal artery (CA), dorsal longitudinal anastomotic vessel (DLAV), caudal vein (CV) and vertebral artery (VTA). All images show lateral views, with anterior to the left and dorsal on top. Scale bars equal 100 µm.</p

<i>hoxd4a</i> expression is required for transient and definitive hematopoiesis.

<p><i>In situ</i> hybridization on 28 hpf (A–D) and 48 hpf (E–H) embryos showing expression of <i>runx1</i> (A,C,E,G) and <i>cmyb</i> (B,D,F,H) in presumptive HSCs arising in the PBI (arrows in A and B) and AGM (arrows in E and F). Expression of both genes was severely reduced in <i>hoxd4a</i> morphants (C,D and G,H). Ratios in the bottom right corner of images indicate the fraction of embryos showing the presented phenotype. ctrl, embryos injected with a non-specific morpholino. MO, embryos injected with the anti-<i>hoxd4a</i> morpholino. <i>hoxd4a</i> mRNA, embryos simultaneously injected with the anti-<i>hoxd4a</i> MO plus capped mRNA for <i>hoxd4a</i>. Scale bars equal 100 µm. All images are at the same magnification. (I) qRT-PCR confirms the strong depletion of hematopoietic gene expression in <i>hoxd4a</i> morphants at 26–28 hpf, and restored expression following co-injection with capped mRNA for <i>hoxd4a</i>. Samples were normalized to β-actin. Error bars indicate standard error. By comparison to controls and rescuants, the gene expression levels of all morphants were statistically different to p≤0.02 except for <i>gata1</i> control vs morphant (p = 0.04) and <i>hbbe1</i> rescuant vs morphant (p = 0.09).</p

<i>scl1</i> and <i>lmo2</i> act downstream of <i>hoxd4a</i> to direct formation of the hemangioblast.

<p>All images are of <i>hoxd4a</i> morphants at 13 hpf. <i>In situ</i> hybridization was performed to detect expression of <i>scl1</i> and <i>lmo2</i> (A–D), <i>gata1</i> and <i>hbbe1</i> (E–H) and <i>fli1</i> and <i>flk1</i> (I–L). To test for rescue of gene expression, embryos were co-injected with capped mRNA for either <i>scl1</i> or <i>fli1</i> as indicated on the left. Ratios indicate the fraction of embryos showing the presented phenotype. Scale bars equal 100 µm.</p

<i>hoxd4a</i> is required for hemangioblast formation.

<p>(A,B) Normal expression at 13 hpf of posterior hemangioblast markers <i>scl1</i> (A) and <i>lmo2</i> (B) in the PLM. (C,D) Expression of these markers is greatly reduced in <i>hoxd4a</i> morphants, but is rescued by co-injection of capped mRNA for <i>hoxd4a</i> (E,F). Ratios indicate the fraction of embryos showing the presented phenotype. Anterior is to the left. A, C and E are dorsal views of flat-mounted specimens while B, D and F are lateral views. Scale bars equal 100 µm. A is at a lower magnification than C and E.</p

<i>cdx4</i>, <i>meis1.1</i> and Hox gene expression in <i>hoxd4a</i> morphants, and a genetic pathway for specification of hemangioblasts and unipotential stem cells.

<p>(A) qRT-PCR showing decreased expression of <i>meis1.1</i> and many, but not all, <i>hox</i> genes in <i>hoxd4a</i> morphants at 26–28 hpf. By contrast, <i>cdx4</i> levels are unchanged. Samples were normalized to <i>β-actin</i>. Error bars indicate standard error. All pairs marked with an asterisk meet statistical significance (p≤0.02). (B) Based on the results presented here and those in the literature, we propose a pathway in which <i>hoxd4a</i> and <i>meis1.1</i> occupy sequential steps downstream of <i>cloche</i> and <i>cdx1/4</i> in a genetic programme leading to the specification of hemangioblasts and unipotential angiogenic and hematopoietic stem cells. The effects of <i>hoxd4a</i> knockdown may be magnified through positive cross-regulatory interactions with <i>meis1.1</i>. The observed effects on the expression of multiple Hox genes could be due to the direct action of <i>hoxd4a</i> and <i>meis1.1</i>. Non-exclusively, <i>cdx1</i> and <i>cdx4</i> may act in conjunction with <i>hoxd4a</i> and <i>meis1.1</i> in a feed-forward type of mechanism to regulate one or more of these same Hox genes with widespread consequences for vasculogenesis, angiogenesis and hematopoiesis at all levels.</p

<i>meis1.1</i> is down regulated in <i>hoxd4a</i> morphants and <i>meis1.1</i> mRNA rescues hematopoietic and vasculogenic gene expression in <i>hoxd4a</i> morphants.

<p>Expression of <i>meis1.1</i> in control (A), morphant (B) and <i>hoxd4a</i>-rescuant (C) embryos at 26–28 hpf. (D–I) At 13 hpf, normal expression of <i>gata1</i>, <i>scl1</i> and <i>fli1</i> (D–F) is reduced in <i>hoxd4a</i> morphants (G–I), but rescued upon co-injection with capped mRNA for <i>meis1.1</i> (J–L). (M,N) Rescue of vascular patterning in <i>hoxd4a</i> morphants by co-injection with capped mRNA for <i>meis1.1</i> as visualized in Tg(fli1:EGFP) transgenics at 48 hpf (M) and by alkaline phosphatase staining at 72 hpf (N). Scale bars equal 100 µm.</p

Knockdown of <i>hoxd4a</i> disrupts the endothelial programme in zebrafish embryos.

<p><i>In situ</i> hybridization at 13 hpf revealing expression of pan-endothelial markers <i>fli1</i> and <i>flk1</i> in control-injected embryos (A,B), <i>hoxd4a</i> morphants (C,D), and rescued embryos co-injected with anti-<i>hoxd4a</i> MO and capped mRNA for <i>hoxd4a</i> (E,F). (G,I) Expression of the marker of arterial identity <i>efnb2a</i> in controls (G) and <i>hoxd4a</i> morphants (I). (H,J) Expression of the endothelial inducer <i>vegf</i> in controls (H) and <i>hoxd4a</i> morphants (J). (K,L) Expression of the venous marker <i>ephb4a</i> in controls (K) and <i>hoxd4a</i> morphants (L). Ratios indicate the fraction of embryos showing the presented phenotype. All images show dorsal views with anterior to the left except H, J, K and L which are lateral views with anterior to the left. Scale bars equal 100 µm. (M) qRT-PCR results showing depletion at 26–28 hpf of angioblast and vascular gene expression in <i>hoxd4a</i> morphants and rescue by co-injection of capped mRNA for <i>hoxd4a</i>. Samples were normalized to β-actin. Error bars indicate standard error. By comparison to controls and rescuants, the gene expression levels of all morphants were statistically different to p≤0.02 except for <i>lmo2</i> control vs morphant (p = 0.04) and <i>lmo2</i> rescuant vs morphant (p = 0.05).</p