Forced down-regulation of Jag1 in endothelial cells removes Notch pathway cis inhibition by HoxA3 and initiates EHT.

<p><b>A)</b> Jag1 RNA expression level in Bend3 cells infected with empty vector (Con) or Jag1 shRNA (Jag1 KD). <b>B)</b> Western blot using specific antibody against Jag1 and GAPDH in Bend3 cells infected with empty vector (pGIPZ) or Jag1 shRNA (Plko.1 Jag1). <b>C)</b> Frequency of Flk1+/VE-cadherin+ cells obtained from day 6 EBs, transduced with empty vector (CON) or with shRNA-Jag1-GFP (JKD) and co-cultured on OP9 for 5 days in Control (Con) or HoxA3 overexpression. <b>D)</b> Gene expression levels of Notch pathway components Hes1, Hey1, Hey2 from purified control (white bar) or HoxA3-overexpressing cells (black bars), transduced with empty vector (CON) or with shRNA-Jag1-GFP (JKD) and co-cultured on OP9-DLL1 for 5 days. Graphs in panel D show one representative experiment with triplicate measurements. *: p<0.05; **: p<0.01; ***: p<0.001. Statistical analysis is reported on <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#pone.0186818.s011" target="_blank">S6 Table</a></b>.</p

Proposed model.

<p><b>A)</b> Illustration of ligand <i>cis</i> inhibition in the hemogenic endothelium. HoxA3 dependent Jag1 overexpression in <i>cis</i> interacts with Notch receptors, inhibiting Notch ligand in <i>trans</i> to interact with the receptor. When HoxA3 is withdrawal the pathway is activated in <i>trans</i> and Runx1 can be induced in the hemogenic endothelium. <b>B)</b> Effect of HoxA3 on Notch pathway. HoxA3 upregulates Jag1 to inhibit the Notch pathway. The pathway in turn will promote downregulation of endothelial specific transcripts, and initiate the EHT.</p

HoxA3 up-regulates the Notch ligand Jag1 but does not activate the Notch pathway.

<p><b>A)</b> Experimental procedure (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#sec002" target="_blank">methods</a> for details). <b>B)</b> Gene expression levels of the Notch pathway components in purified endothelial cells (Flk1⁺/VE-Cadherin⁺) derived from day 6 control EBs (white bars) or upon 6 hours of HoxA3 up-regulation (black bars). <b>C)</b> Western blot analysis using Jag1 specific antibody in purified endothelial cells (Flk1⁺/VE-Cadherin⁺) derived from day 6 control EBs (white bars) or upon 2 days of HoxA3 up-regulation (black bars) <b>D)</b> Experimental procedure (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#sec002" target="_blank">methods</a> for details). <b>E)</b> Immunofluorescence staining for Jag1 (red), VE-Cadherin (green) and Hoechst (blue) showing adherent endothelial clusters growing in Control (Con) or HoxA3 overexpression (HoxA3), derived from endothelial cells (Flk1⁺/VE-cadherin⁺) and co-cultured for 5 days on OP9 cells. Bar: 100 μM. <b>F)</b> Gene expression levels of Notch ligands Jag1/Jag2/Dll1/Dll3/Dll4, Notch receptors Notch1 to Notch4, cofactors Lfng (lunatic fringe) Mfng (manic fringe) and target genes Hes1/Hey2/Hey1 on purified control cells (white bars) or cells overexpressing HoxA3 (black bars) derived from endothelial cells (Flk1⁺/VE-cadherin⁺) co-cultured for 5 days on OP9 cells. *: p<0.05. Detailed statistical analysis is reported in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#pone.0186818.s007" target="_blank">S2 Table</a></b>.</p

Notch signaling in <i>trans</i> does not rescue HoxA3 mediated inhibition of Notch.

<p><b>A)</b> Experimental procedure <b>B)</b> Representative flow cytometric profile of endothelial surface markers Flk-1/Ve-Cadherin and hematopoietic surface markers c-Kit/CD41, and c-Kit/CD45 obtained from 200,000 EB-derived Flk1⁺/VE-cadherin⁺ cells and co-cultured on OP9 control (CON) or OP9 overexpressing Dll1 (OP9-Dll1) for 5 days in Control or HoxA3-overexpressing HE cells. <b>C)</b> Quantification of frequencies of hematopoietic surface markers (CD41, CD45) of the same cell as in <b>B</b>. <b>D)</b> Gene expression levels of the Notch pathway target genes (Hes1, Hey1, Hey2) and hematopoietic gene markers (PU.1, Runx1, Gata1) on control (white bar) or HoxA3-overexpressing (black bars) HE cells co-cultured on OP9 controls (CON) or OP9-DLL1 for 5 days (Flk1⁺/VE-cadherin⁺ and CD41⁺/c-Kit⁺ cells were pulled together). <b>E)</b> Iimmunofluorescence staining for activated Notch1 (NICD-red), VE-Cadherin (VECad-green) and Hoechst (blue) showing adherent endothelial clusters growing in Control (Con) or HoxA3 overexpression (HoxA3), derived from endothelial cells (Flk1⁺/VE-cadherin⁺) co-cultured on OP9-DLL1 cells *: p<0.05. Statistical analysis is reported on <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#pone.0186818.s010" target="_blank">S5 Table</a></b>.</p

Repression of EHT by HoxA3 is not affected by inhibition Notch pathway.

<p><b>A)</b> Experimental procedure, Endothelial Derived Cells (ENDO). <b>B)</b> Representative flow cytometric profile of endothelial surface markers Flk-1/Ve-Cadherin and hematopoietic surface markers c-Kit/CD41, and c-Kit/CD45 on 200,000 EB-derived Flk1⁺/VE-cadherin⁺ cells without or with HoxA3 overexpression and co-cultured on OP9 for 5 days in the presence or absence of the Notch inhibitor DAPT (20 μM). <b>C)</b> Frequency of endothelial surface markers Flk-1⁺/Ve-Cadherin⁺ in EB-derived cells. *: p<0.05; **: p<0.01. Two way ANOVA analyses of Flk-1⁺/Ve-Cadherin⁺, CD41⁺ and CD45⁺ frequencies are reported on <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186818#pone.0186818.s008" target="_blank">S3 Table</a></b>.</p