*2.8. Vasculature Is Similar in Implantation Sites of Control and Jag1*∆*EC Pregnancies*

The recruitment and differentiation of mural cells, into pericytes and vascular smooth muscle cells, promotes vessel stabilization and is essential for vascular integrity [11,12,47,48]. We have shown that Jag1 is expressed in the ECs of SpAs at the mesometrial pole and in a subset of decidual mural cells (Figure 3A, [27]). To assess whether loss of *Jag1* in ECs impacts vascular density or mural cell content, implantation sites from *Jag1*∆*EC* mutant and *Cdh5-CreERT2* control pregnancies were stained for expression of CD31 and mural cell markers, PDGFRβ, NG2, and SMA (Figure S3). *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 12 of 19

Implantation sites stained for CD31 were quantified for percentage CD31<sup>+</sup> ECs in the mesometrial pole and central region in the mesometrial region and anti-mesometrial region. In all regions, CD31<sup>+</sup> expression is similar in *Jag1*∆*EC* mutant and control pregnancies, suggesting that loss of EC-specific *Jag1* does not impact decidual vascular density (Figure 9A–C). To assess whether loss of *Jag1* in ECs impacts mural cell content, implantation sites were analyzed for the expression of mural cell markers. No difference in the expression of NG2, PDGFRβ or SMA at the mesometrial pole of the mesometrial region (Figure 9D–F) was observed, suggesting that loss of endothelial *Jag1* does not impact mural cells in SpAs. Together, these data suggest that EC *Jag1* is not essential for decidual angiogenesis or maintenance of the SpA until E7.5. Implantation sites stained for CD31 were quantified for percentage CD31+ ECs in the mesometrial pole and central region in the mesometrial region and anti-mesometrial region. In all regions, CD31+ expression is similar in *Jag1*Δ*EC* mutant and control pregnancies, suggesting that loss of EC-specific *Jag1* does not impact decidual vascular density (Figure 9A–C). To assess whether loss of *Jag1* in ECs impacts mural cell content, implantation sites were analyzed for the expression of mural cell markers. No difference in the expression of NG2, PDGFRβ or SMA at the mesometrial pole of the mesometrial region (Figure 9D–F) was observed, suggesting that loss of endothelial *Jag1* does not impact mural cells in SpAs. Together, these data suggest that EC *Jag1* is not essential for decidual angiogenesis or maintenance of the SpA until E7.5.

**Figure 9.** EC-specific loss of *Jag1* does not impact endothelial and mural cell density. (**A**–**C**) Expression of CD31+ was quantified to determine blood vessel density in each region of the implantation site. (**A**) Density of CD31+ ECs was similar in *Cdh5-CreERT2* control and *Jag1ΔEC* pregnancies in the MP (A) and CTR (**B**) of the MR and in the AMR (**C**). (**D**–**F**) Implantation sites stained for mural markers, NG2, PDGFRβ or SMA are assessed for mural cell content at the MP. Expression of NG2 (**D**), PDGFRβ (**E**) and SMA (**F**) is similar in *Cdh5-CreERT2* control and *Jag1*Δ*EC* pregnancies in the MP. AMR = antimesometrial region; CTR = central region; MP = mesometrial pole; MR = mesometrial region. Data shown as median + IQR. **Figure 9.** EC-specific loss of *Jag1* does not impact endothelial and mural cell density. (**A**–**C**) Expression of CD31<sup>+</sup> was quantified to determine blood vessel density in each region of the implantation site. (**A**) Density of CD31<sup>+</sup> ECs was similar in *Cdh5-CreERT2* control and *Jag1*∆*EC* pregnancies in the MP (A) and CTR (**B**) of the MR and in the AMR (**C**). (**D**–**F**) Implantation sites stained for mural markers, NG2, PDGFRβ or SMA are assessed for mural cell content at the MP. Expression of NG2 (**D**), PDGFRβ (**E**) and SMA (**F**) is similar in *Cdh5-CreERT2* control and *Jag1*∆*EC* pregnancies in the MP. AMR = anti-mesometrial region; CTR = central region; MP = mesometrial pole; MR = mesometrial region. Data shown as median + IQR.

### **3. Discussion 3. Discussion**

In this report, we focused on the role the endothelial Jag1 in decidual vasculature in the postimplantation, pre-placentation mouse uterus. When *Jag1* was deleted from the endothelium at E4.5 and pregnancies were assessed at E7.5, the start of placentation, we found that *Jag1∆EC* mutants In this report, we focused on the role the endothelial Jag1 in decidual vasculature in the post-implantation, pre-placentation mouse uterus. When *Jag1* was deleted from the endothelium at E4.5 and pregnancies were assessed at E7.5, the start of placentation, we found that *Jag1*∆*EC* mutants

ECs in the anti-mesometrium, suggesting that the increased Notch signaling activity was restricted to capillary ECs throughout the implantation site. In the anti-mesometrial decidua, increased expression of Notch1 and Notch signaling in angiogenic capillary ECs, leads to decreased endothelial VEGFR2 expression and EC proliferation. Despite aberrant Dll4 expression and Notch signaling

maintain pregnancies with no change in embryo development. Loss of *Jag1* in SpA and capillary ECs

maintain pregnancies with no change in embryo development. Loss of *Jag1* in SpA and capillary ECs resulted in a loss of Jag1 expression, but increased expression of Notch effectors, *Hey2* and *Nrarp,* consistent with a loss of Jag1 leading to increased Notch signaling activity. This correlated with an increase in Dll4 expression in capillary ECs, but not SpA ECs, in the mesometrium and in capillary ECs in the anti-mesometrium, suggesting that the increased Notch signaling activity was restricted to capillary ECs throughout the implantation site. In the anti-mesometrial decidua, increased expression of Notch1 and Notch signaling in angiogenic capillary ECs, leads to decreased endothelial VEGFR2 expression and EC proliferation. Despite aberrant Dll4 expression and Notch signaling activity, decidual vasculature development up to E7.5 in *Jag1*∆*EC* mutants was not altered in the time window we evaluated.

Angiogenesis occurs in the setting of both physiologic and pathologic growth and is largely controlled by pro-angiogenic signaling pathways, such as the VEGF and Notch pathways [16,19,49–51]. In the post-implantation, pre-placentation pregnant mouse uterus, physiologic angiogenesis is a dynamic process that involves sprouting and remodeling angiogenesis in the decidua that is regulated by VEGF/VEGFR2 [5,7,52] and Dll4/Notch signaling [53]. At E7.5, we found that the central region of the mesometrial decidua contains newer, immature, capillary networks with few, if any, associated mural cells, as there was little to no expression of NG2, PDGFRβ or SMA. In contrast, the ECs associated with NG2+, PDGFRβ <sup>+</sup> pericytes in the anti-mesometrial region are consistent with the remodeling phase of angiogenesis [11,54]. Together, our findings agree with previous literature in suggesting that the central region and anti-mesometrial region are regions of early and active angiogenesis at E7.5.

Our evaluation of Notch proteins and ligands at E7.5 supports and extends our previous findings in the pre-placentation decidua [27,39]. We find Notch1 and Notch4 in the ECs of angiogenic capillaries in the central region, whereas, Notch1, Dll4 and Jag1, but not Notch4, are expressed in the ECs of remodeling capillaries in the anti-mesometrial region. Co-expression of Jag1 and Dll4 in sites of neovascularization and angiogenesis suggests that both these ligands could function in anti-mesometrial region ECs during decidual angiogenesis. In contrast, in the central region, Dll4 expression was detected in stromal, but not endothelial cells, whereas Jag1 expression was not detected in any central region cell-type. Further studies are needed to identify other Notch ligands, such as Jag2 or Dll1, that support angiogenesis and Notch signaling activity in the central region [27,39].

In mouse retinal vascular development [17], in skin wound healing [55] and in coronary plexus formation [56], EC-specific Jag1 has been shown to antagonize Dll4/Notch signaling. Disruption of endothelial *Jag1* leads to disinhibition of Dll4/Notch signaling, followed by increase in Dll4 expression and Notch activation, decreased *VEGFR2* and vascular density, and decreased EC proliferation [17,55]. Further, in wound angiogenesis, it has been proposed that a feedforward signaling mechanism maintains increased Dll4 expression [55]. We observed that loss of EC-specific *Jag1* increases Dll4 and Notch1 expression, as well as increases Notch signaling activity in areas of the decidua with remodeling angiogenesis. Consistent with increased Dll4/Notch signaling, we observed a decrease in VEGFR2 expression and EC proliferation, suggesting that Jag1 antagonizes Dll4/Notch signaling in angiogenic decidual capillaries. Our analysis does not distinguish the type of cellular interaction, *cis*or *trans*-, that underlies Notch signaling activation in angiogenic decidual capillaries. Further studies are needed to determine if loss of a Jag1/Notch *trans*-interaction promotes Dll4/Notch *cis*-activation or *trans*-activation in anti-mesometrial decidual angiogenesis.

At E7.5, we did not observe a change in vascular density in the anti-mesometrial or central regions. Given that Jag1 is not expressed in central region ECs, the lack of change in Dll4 expression or vascular density in the central region, was not unexpected. In the anti-mesometrial region, where increased expression of EC Dll4 is observed, we predicted an increase in vascular density, as was previously reported with loss of EC-specific *Jag1* [17]. Had we found a change in vascular density, perhaps we would also have seen a change in the progression or survival of the pregnancy. Here, we show that the embryo can tolerate disturbances in the Jag1/Notch signaling by utilizing other members of the signaling pathway. Thus, while loss of Dll4/Notch signaling disrupts decidual angiogenesis [53], loss

of endothelial Jag1, which leads to increased Dll4/Notch signaling, does not appear to be essential for early uterine angiogenesis prior to placentation.

SpAs in mice and humans are the distal branches of the uterine artery and are essential for bringing nutrients to the maternal fetal interface before development of the placenta. These arteries undergo important phases of circumferential remodeling, in part triggered by uNK cells, between E6.5 and E10.5 [3,9]. SpA remodeling involves the initial thickening of vascular smooth muscle lined vessel walls between E6.5 and E7.5 followed by their gradual thinning from E8.5 to E9.5. Trophoblast invasion of the SpAs mediates loss of smooth muscle coverage and the breakdown of extracellular matrix, resulting in thin walled vessels with wide lumens that allow for low resistance, high capacity maternal blood flow through the placenta to meet the demands of the growing embryo [3,9,57]. We show that at E7.5, the resident SpAs are still closely associated with mural cells that express NG2, PDGFRβ and SMA, which is consistent with mature vessels in the mesometrial pole that are not undergoing angiogenesis and have not, as yet, been remodeled. SpA ECs express Notch1 and Notch4 and Notch ligands, Jag1 and Dll4. In the pre-implantation uterus, we have previously shown that both Notch3 and Jag1 are expressed in mural cells in vessels in the stroma [27]. Whereas Jag1 is expressed in mural cells in vessels in the decidua, we did not find expression of Notch3 in post-implantation decidual vasculature at E6.5 [27] or E7.5 (data not shown). Thus, endothelial Jag1 and/or Dll4 could activate Notch1 and Notch4 in adjacent ECs to promote Notch signaling in SpA ECs, whereas SpA mural cells which do not express Notch proteins cannot be activated by endothelial Jag1 [39]. We found that loss of EC-specific *Jag1* does not result in increased Dll4 in SpAs in the mesometrial pole, suggesting no compensation or disinhibition of Dll4 with Jag1 loss, as was seen in the anti-mesometrial region capillaries. We also found that loss of EC-specific *Jag1* does not change vascular density or mural cell content in the SpAs. Together, our findings suggest Jag1/Notch signaling in SpA ECs is not essential for maintenance of decidual SpAs from implantation to E7.5. The impact of loss of EC-specific *Jag1* in SpAs may become evident later in gestation, nearing the completion of SpA transformation in the placenta.

Taken together, while we saw no vascular phenotype at E7.5 with loss of EC-specific *Jag1*, our data show that Jag1 and Dll4 mediate Notch signaling activity during decidual angiogenesis. Increased Dll4 in decidual capillary ECs is associated with increased Notch signaling, consistent with loss of inhibitory function of EC Jag1, which has been previously described with EC-specific loss of *Jag1* in retinal angiogenesis and wound healing [17,55]. Thus, our data identify another vascular bed, the newly formed uterine decidual capillaries, in which Jag1 functions as an antagonist of EC Dll4/Notch signaling during angiogenesis.
