Sox17 is indispensable for acquisition and maintenance of arterial identity - PubMed (original) (raw)

Fabrizio Orsenigo, Marco Francesco Morini, Mara Elena Pitulescu, Ganesh Bhat, Daniel Nyqvist, Ferruccio Breviario, Valentina Conti, Anais Briot, M Luisa Iruela-Arispe, Ralf H Adams, Elisabetta Dejana

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Sox17 is indispensable for acquisition and maintenance of arterial identity

Monica Corada et al. Nat Commun. 2013.

Abstract

The functional diversity of the arterial and venous endothelia is regulated through a complex system of signalling pathways and downstream transcription factors. Here we report that the transcription factor Sox17, which is known as a regulator of endoderm and hemopoietic differentiation, is selectively expressed in arteries, and not in veins, in the mouse embryo and in mouse postnatal retina and adult. Endothelial cell-specific inactivation of Sox17 in the mouse embryo is accompanied by a lack of arterial differentiation and vascular remodelling that results in embryo death in utero. In mouse postnatal retina, abrogation of Sox17 expression in endothelial cells leads to strong vascular hypersprouting, loss of arterial identity and large arteriovenous malformations. Mechanistically, Sox17 acts upstream of the Notch system and downstream of the canonical Wnt system. These data introduce Sox17 as a component of the complex signalling network that orchestrates arterial/venous specification.

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Figures

Figure 1

Figure 1. Selective expression of Sox17 in developing and mature arteries.

(a,b) Confocal analysis of whole-mount ephrinB2/β-gal reporter mice. The vasculature of E10.5 embryo head (a) and yolk sac (b) was stained with PECAM (red), _ephrinB2/_β-gal (green) and Sox17 (light blue). Nuclear staining of Sox17 is seen in the internal carotid artery (ICA, yellow arrowhead), which also expressed ephrinB2, while the veins (V, white arrows) are negative. Inset: PECAM staining of the whole embryo showing the area of the head that was analysed. (b) Sox17 expression (light blue) in the E10.5 yolk sac is restricted to large arteries (yellow arrowhead), which also expressed ephrinB2. (c–e) Analysis of the vasculature in whole-mount retina. P5, retina from control was stained with isolectin B4 (IB4, white), αSMA (green) and Sox17 (red). Sox17 is highly expressed in large and small arteries (A) while veins (V) are almost negative. Higher magnifications of the central (d) and marginal (e) areas of the retina are shown. Binarized images of Sox17 and isolectin B4 from boxed area in panel (e) were obtained by imposing a threshold of 40 and 78 a.u., respectively. Sox17-positive nuclei are highlighted in yellow and indicated by arrowheads (see Supplementary Fig. S1B for quantification). (f,g) Artery-specific Sox17 staining (red) of the adult mouse vasculature. VE-cadherin antibody (VEC, green) stained both arteries (A) and veins (V) in the different tissues and organs (as indicated). Sox17 was detected only in the arteries. In the intestine and diaphragm, the arteries were labelled by αSMA staining (green) (g). Scale bar, 100 μm.

Figure 2

Figure 2. Deletion of Sox17 alters vascular development.

(a,b) Isolectin B4 (IB4) staining of P5, and P9 retinas from control and Sox17 iECKO newborn mice shows marked defects in vascular development. Higher magnification of the angiogenic front (boxed areas) are shown and tip cells are indicated by yellow dots. (c,d) Quantification of vascular parameters in retinas from control and Sox17 iECKO mice. Binarized images of retinas (P9) from control and Sox17 iECKO (c, left and right, respectively), as an examples of the quantification performed in (d). The vascular front density was measured in the areas highlighted in red, while vascular progression was measured as the average distance covered by the growing vessels (see green arrows in c). The quantification of the tip cells per length, the vascular front density, the vascular progression and the number of filopodia per tip cell are shown in (d). Retinas from control and Sox17 iECKO (blue and red columns, respectively) at P5, P9 and P12 are shown. Data are means ±s.d. of at least five mice per group. (e) Detailed analysis of vascular sprouts stained with Isolectin B4 in control and Sox17 iECKO retinas. Tip cells nuclei are highlighted by yellow circles (see also Supplementary Fig. S2C and Methods for details) and filopodia protrusions by yellow dots (middle panels). Higher magnification of red boxed areas are shown on the right panels where tip cells nuclei and corresponding filopodia are colours coded. (f) High magnification of the angiogenic front show multidirectional hyper sprouting in the Sox17 iECKO vasculature. Tip cells growing above the vascular plane are indicated by arrows; filopodia are also indicated as yellow dots. Bottom panels show depth-coded stacks (see Methods for details). Tip cells indicated by arrows are located above vascular plexus (green). (g) Higher magnification of Isolectin B4 (IB4, white) and Dll4 (green) staining at the vascular front shows high expression of Dll4 in Sox17 iECKO hyper sprouting retinas (lower panels, yellow arrowheads). Control tip cells are shown in the upper panel. Scale bar, 500 μm (top panels a–c); Scale bar, 100 μm (bottom panels a,b,e); Scale bar, 25 μm (magnifications of e–g). *P<0.05; **P<0.01, two-tailed _t_-test assuming unequal variances.

Figure 3

Figure 3. Altered arterial and venous differentiation in the absence of Sox17.

Analysis of the vasculature of retina, diaphragm and small intestine at P9. (a) Whole-mount immunofluorescence for Isolectin B4 (IB4, white) and endomucin (green) of control and Sox17 iECKO retinas. Endomucin is expressed in control veins (V). Arrowheads indicate endomucin-positive arteries (A) in Sox17 iECKO mouse. (b) Staining for VE-cadherin (VEC, white) and endomucin (green) of vessels of control and Sox17 iECKO mice diaphragm. Arrowheads indicate an artery endomucin positive in Sox17 iECKO mouse. Quantification of data in (a,b) is shown in panel f (see below). (c) Immunostaining for Isolectin B4 (IB4, white) and EphB4 (green) of control and Sox17 iECKO retinas. The arrowheads indicate the EphB4 staining in the arteries of Sox17 iECKO mouse. (d) Isolectin B4 (IB4, white) and Jag1 (green) staining of the retina vasculature in control and Sox17_iECKO_ mice. The arrowheads point to decreased Jag1 staining in Sox17 iECKO mouse. (e) Immunofluorescence for VE-cadherin (VEC, white) and Dll4 (green) staining in the vasculature of the intestine in control and Sox17 iECKO mice. Arterial Dll4 staining is strongly reduced (see arrowheads) in Sox17 iECKO mice. (f) Quantification of endomucin-positive arteries in control (blue) and Sox17 iECKO (red) retinas. Data are expressed as percentages of total vessels ±s.d. (_n_>20). (g) qRT–PCR analysis of control and Sox17 iECKO lungs derived endothelial cells (red columns) and cultured venous endothelial cells infected with Sox17 (blue columns). Modifications induced in the expression of arterial (CXCR4, ephrinB2) and venous markers (COUP-TFII) are reported. The RNA level obtained for control was set to 1 and the ratio for Sox17 iECKO versus control or venous endothelial cells infected with Sox17 versus control is shown for each gene. Standard deviation of control values did not exceed 10% of the means. Scale bar, 100 μm; **P<0.01; *P<0.05, two-tailed _t_-test assuming unequal variances. (h) Confocal analysis of cryosections stained with PECAM (green) and DAPI (blue) of control and Sox17 ECKO embryos (E10.5) revealed the presence of arteriovenous shunts (arrowheads) leading to a bypass circulation. Scale bar, 200 μm.

Figure 4

Figure 4. Impairment of mural cells coverage in the absence of Sox17.

(a–d) Analysis of the control and Sox17 iECKO retinal vasculature by whole-mount staining for Isolectin B4, αSMA, NG2 and Desmin. (a,b) Isolectin B4 (IB4, white) and αSMA (green) staining. Arrowheads indicate αSMA-negative arteries in Sox17 iECKO mice. Quantification of αSMA fluorescence intensity in arteries of control and Sox17 iECKO (blue and red, respectively) is shown in (b). Data are expressed as mean fluorescence ±s.d. (_n_>3 mice per group) *P<0.01, two-tailed _t_-test assuming unequal variances. (c) Isolectin B4 (IB4, white) and NG2 (green) staining. (d) Isolectin B4 (IB4, white) and Desmin (green) staining. Higher magnification of the insets is shown in the right panels. Arrowheads indicate regions of incomplete pericyte coverage in Sox17 iECKO mice. These cells appear poorly organized around the vessel wall leaving relatively large, uncovered areas. Scale bar, 25 μm in magnifications and 100 μm elsewhere.

Figure 5

Figure 5. Sox17 is upstream of Notch signalling.

(a) qRT–PCR analysis of freshly isolated endothelial cells from lungs of vehicle or DAPT-treated pups (P6). DAPT does not affect the expression of Sox17, while inhibits the expression of Hes, Hey1 and Hey2. Data are expressed as fold change versus vehicle and are means ±s.d. from cells obtained from at least four mice per group. The s.d. of the values obtained in vehicle-treated mice did not exceed 10% of the mean. (b) Retina whole-mount immunostaining for Isolectin B4 (IB4, green) and Sox17 (red) after vehicle (left) or DAPT (right) treated pups (P6). Inhibition of Notch signalling does not significantly change Sox17 expression (arrowheads). (c) Whole-mount immunofluorescence for Isolectin B4 (IB4, green), and Sox17 (red) of P6 control and Rbpj _iECKO_retinas. The genetic ablation of Rbpj does not change the expression of Sox17 (arrowheads). (d,e) qRT–PCR analysis of Rbpj iECKO P6 mouse lungs. The genetic ablation of Rbpj does not change the expression of Sox17 (d), while it inhibits the expression of Hes, Hey1, Dll4, Jag1, Notch1 and Notch4. Data are expressed as fold change versus controls and are means ±s.d. from at least three mice per group. The s.d. of the values obtained in control animals did not exceed 10% of the mean. (f) Whole-mount immunofluorescence for Isolectin B4 (IB4, green), and Sox17 (red) of P6 control and Dll4 _iECKO_retinas. The genetic ablation of Dll4 does not change the expression of Sox17 (right panels, arrowheads). (g) Isolectin B4 (IB4, green) and Sox17 (red) staining of P6 retinas from control (ROSANotch IC-CRE) and the endothelial-cell-specific gain-of-function mutant of Notch signaling (ROSANotch IC+CRE). Activation of Notch signaling does not significantly change Sox17 expression (arrowheads). The upregulation and ectopic expression of αSMA (white) and Dll4 (red) in veins is shown in (h), see arrowheads. Columns are means ±s.d. of triplicates from a representative experiment. *P<0.05; **P<0.01, two-tailed _t_-test assuming unequal variances. Scale bar, 100 μm.

Figure 6

Figure 6. Sox17 affects Notch signaling.

(a) qRT–PCR analysis of control and Sox17_iECKO_ lungs derived endothelial cells (P6) shows that several members of the Notch signaling pathway (Hes, Dll4 and Notch4) are downregulated by the absence of Sox17. Data are expressed as fold change versus controls and are means ±s.d. from at least three mice per group. The s.d. of the values obtained in control animals did not exceed 10% of the mean. (b) Vein endothelial cells which do not express Sox17 (see Supplementary Fig. S3A,B) were infected with lentiviral vectors coding for GFP, Sox17, Sox18RaOP or both Sox17 and Sox18RaOP in combination. Immunofluorescence analysis of confluent monolayers stained for Sox17 (green) and NICD (red). Nuclear NICD staining is significantly increased by Sox17 expression (white asterisks), while the Sox18RaOP mutant is inactive. The Sox18RaOP mutant reduced the effects of Sox17 on NICD nuclear staining when it is expressed in combination. Scale bar, 100 μm. Quantification of nuclear fluorescence intensity is reported in (c). Data are means ±s.d. of three experiments. (d) qRT–PCR analysis of Sox17 infected venous endothelial cells (see above). Sox17 expression (red columns) upregulates several members of the Notch signaling pathway, as Hey1, Dll4, Dll1 and Notch4, and arterial markers, such as ephrinB2. Conversely, venous markers, EphB4 and COUP-TFII are downregulated. DAPT treatment (magenta columns) prevent the Sox17-induced upregulation of Hey1 and Dll4. Data are means ±s.d. of four experiments. (e–g) ChIP analysis of Sox17 interaction with the promoters of Notch4, Notch 1, Dll1 and Dll4. The positions of the putative Sox17-binding sites for all the genes analysed are indicated. Chromatin from cultured endothelial cells was immunoprecipitated with Sox17 antibody and qRT–PCR on the indicated regions was carried out. The levels of DNA are normalized to input. (e) ChIP analysis of venous cells infected with GFP (blue) or Sox17 (red); (f) ChIP analysis of arterial cells from lungs expressing endogenous levels of Sox17 (violet); (g) ChIP analysis of arterial cells of embryonic origins expressing endogenous levels of Sox17 (green). Columns are means ±s.d. of triplicates from a representative experiment. *P<0.05; **P<0.01, two-tailed _t_-test assuming unequal variances.

Figure 7

Figure 7. Canonical Wnt signaling up-regulates Sox17 expression.

(a,b) qRT–PCR analysis of Sox17 expression in endothelial cells derived from β_-_catenin gain-of-function (GOF) embryos (a) and yolk sac vasculature (b). Data are means ±s.d. of endothelial cells from at least four embryos. (c,d) Whole-mount Sox17 (light blue) and VE-cadherin (VEC, red) staining of E9.5 β_-_catenin gain-of-function embryos and yolk sacs. The vasculature of the β_-_catenin gain-of-function mice embryos was severely affected, as reported previously. Nuclear staining of Sox17 is specifically in arteries of control embryos (c,d; left panels). In contrast, in β_-_catenin gain-of-function, Sox17 nuclear staining is expressed in most types of vessels (arrowheads). Bottom panels of (c) shows higher magnifications of the boxed areas (white arrow indicates control vein). (e) Confocal images of whole-mount control, β_-_catenin loss-of-function (LOF) and gain-of-function P6 retinas stained with isolectin B4 (IB4, green) and Sox17 (red). Sox17 expression in the small arteries was reduced in LOF while in GOF pups was increased (see arrowheads). FireLUT (pixels are false coloured according to fluorescent intensity, ranging from dark blue, 0 to white 255) visualization for boxed areas is shown in bottom panels. (f) Confocal images of PECAM (green) and Sox17 (red) stained whole-mounts P5 brains. Sox17 is strongly reduced in β_-_catenin loss-of-function (LOF). Scale bar, 100 μm. *P<0.05; **P<0.01, two-tailed _t_-test assuming unequal variances.

Figure 8

Figure 8. Role of Sox 17 on vascular development.

Schematic representation of the suggested interaction among the different signaling pathways that regulate arterial/venous specification. Wnt is upstream of Sox17 that, in turn, activates Notch signaling and arterial differentiation. Endothelial-specific inactivation of Sox17 prevents Notch signaling and acquisition of arterial identity by endothelial cells.

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