Zebrafish hoxd4a Acts Upstream of meis1.1 to Direct Vasculogenesis, Angiogenesis and Hematopoiesis

10.1371/journal.pone.0058857PLoS ONE83

<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

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

<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

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