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

Stab2-HA pathway functions in parallel to the Vegf pathway.

<p>The proposed model shows that Stab2 interacts with HA to induce Erk phosphorylation, acting to upregulate arterial fate and suppressing venous fate in the arterial progenitors. This pathway may function in parallel to the Vegf pathway. Additionally, Vegf acts in a negative feedback loop to repress <i>stab2</i> expression, although the mechanism by which this occurs is unknown (indicated by dashed line).</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

Stab2 and Has2 combinatorial knockdown results in a synergistic inhibitory effect on ISV formation.

<p>(A–C) A subphenotypic 1.25 ng dose of Stab2 (B) and 8 ng dose of Has2 (C) morpholinos do not affect vascular development when injected alone. (D) In contrast, Stab2 and Has2 MO co-injection results in 51 percent of embryos that lack ISVs suggesting that Stab2 and HA genetically interact. Arrows indicate ISVs. (E) Graph shows percentages of embryos displaying reduced or absent ISVs. Lateral view of 24 hpf embryos, anterior is to the left.</p

Stab2 morphants display a lack of intersegmental vessels.

<p>(A–H) In situ hybridization analysis shows a lack of ISVs in Stab2 morphants as observed by endothelial marker <i>fli1a</i> (A and B), <i>kdrl</i> (C and D), <i>esam</i> (E and F), and <i>she</i> (G and H) expression at 24 hpf, as compared to uninjected controls. Arrows indicate ISVs in control embryos and missing ISVs in morphants. Arrowheads indicate stronger PCV expression of <i>kdrl</i> and <i>esam</i> in morphant embryos (D and F). Lateral view, anterior is to the left; trunk and tail region is shown. Morphants were injected with a cocktail containing Stab2 MO1 and MO2, as well as p53 MO. ISVs: intersegmental vessels; PCV: posterior cardinal vein.</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

Stab2 knockdown affects Erk phosphorylation.

<p>(A–D) Phosphorylated Erk expression is observed in the DA of uninjected control embryos by whole mount immunostaining (A,B), while a majority of Stab2 morphants display a reduction in arterial P-Erk expression (C,D). (E) Percentages of embryos displaying normal or decreased arterial P-Erk expression. Arrows indicate P-Erk expression in the DA (A,C). Experiments performed in a Tg(<i>fli1a</i>:GFP) line. Green staining: GFP, Blue Staining: DAPI, Red staining: P-Erk. Lateral view, anterior is to the left. All embryos are at the 20-somite stage. Morphants were injected with a cocktail containing Stab2 MO1, MO2 and p53 MO.</p

Stab2 morphants display expanded expression of certain arterial and venous markers.

<p>(A,B and E,F) As analyzed by in situ hybridization, <i>aqp8a</i> (A,B) and <i>cldn5b</i> (E,F) expression is restricted to the DA in wild type uninjected embryos while it is expanded into the PCV in Stab2 morphants at 24 hpf. (I,J) Arterial <i>grl</i> expression is not affected in Stab2 morphants. (C,D, G,H, and K,L) Expression of venous specific markers <i>flt4</i> (C,D), <i>mrc1</i> (G,H), and <i>stab1l</i> (K,L) is expanded into the area of the DA in Stab2 morphants. (M,N) Venous expression of <i>stab2</i> itself is also expanded into the area of the DA at 24 hpf in Stab2 morphants. Arrows indicate DA and arrowheads indicate PCV. Lateral view, anterior is to the left, trunk and tail region is shown. DA: dorsal aorta; PCV: posterior cardinal vein. All embryos are 24 hpf. Morphants were injected with a cocktail containing Stab2 MO1, MO2 and p53 MO.</p

Myocardium and BMP Signaling Are Required for Endocardial Differentiation

Endocardial and myocardial progenitors originate in distinct regions of the anterior lateral plate mesoderm and migrate to the midline where they coalesce to form the cardiac tube. Endocardial progenitors acquire a molecular identity distinct from other vascular endothelial cells and initiate expression of specific genes such as nfatc1. Yet the molecular pathways and tissue interactions involved in establishing endocardial identity are poorly understood. The endocardium develops in tight association with cardiomyocytes. To test for a potential role of the myocardium in endocardial morphogenesis, we used two different zebrafish models deficient in cardiomyocytes: the hand2 mutant and a myocardial-specific genetic ablation method. We show that in hand2 mutants endocardial progenitors migrate to the midline but fail to assemble into a cardiac cone and do not express markers of differentiated endocardium. Endocardial differentiation defects were rescued by myocardial but not endocardial-specific expression of hand2. In metronidazole-treated myl7:nitroreductase embryos, myocardial cells were targeted for apoptosis, which resulted in the loss of endocardial nfatc1expression. However, endocardial cells were present and retained expression of general vascular endothelial markers. We further identified bone morphogenetic protein (BMP) as a candidate myocardium-derived signal required for endocardial differentiation. Chemical and genetic inhibition of BMP signaling at the tailbud stage resulted in severe inhibition of endocardial differentiation while there was little effect on myocardial development. Heat-shock-induced bmp2b expression rescued endocardial nfatc1 expression in hand2 mutants and in myocardium-depleted embryos. Our results indicate that the myocardium is crucial for endocardial morphogenesis and differentiation, and identify BMP as a signal involved in endocardial differentiation

Myocardium and BMP Signaling Are Required for Endocardial Differentiation

Endocardial and myocardial progenitors originate in distinct regions of the anterior lateral plate mesoderm and migrate to the midline where they coalesce to form the cardiac tube. Endocardial progenitors acquire a molecular identity distinct from other vascular endothelial cells and initiate expression of specific genes such as nfatc1. Yet the molecular pathways and tissue interactions involved in establishing endocardial identity are poorly understood. The endocardium develops in tight association with cardiomyocytes. To test for a potential role of the myocardium in endocardial morphogenesis, we used two different zebrafish models deficient in cardiomyocytes: the hand2 mutant and a myocardial-specific genetic ablation method. We show that in hand2 mutants endocardial progenitors migrate to the midline but fail to assemble into a cardiac cone and do not express markers of differentiated endocardium. Endocardial differentiation defects were rescued by myocardial but not endocardial-specific expression of hand2. In metronidazole-treated myl7:nitroreductase embryos, myocardial cells were targeted for apoptosis, which resulted in the loss of endocardial nfatc1expression. However, endocardial cells were present and retained expression of general vascular endothelial markers. We further identified bone morphogenetic protein (BMP) as a candidate myocardium-derived signal required for endocardial differentiation. Chemical and genetic inhibition of BMP signaling at the tailbud stage resulted in severe inhibition of endocardial differentiation while there was little effect on myocardial development. Heat-shock-induced bmp2b expression rescued endocardial nfatc1 expression in hand2 mutants and in myocardium-depleted embryos. Our results indicate that the myocardium is crucial for endocardial morphogenesis and differentiation, and identify BMP as a signal involved in endocardial differentiation