*2.2. Characterization of Notch Expression in the Decidual Vasculature*

To determine expression of Notch proteins in the decidual vasculature, implantation sites were double stained with antibodies to detect Notch1 and Notch4 with respect to ECs and mural cells. In the mesometrial region, CD31<sup>+</sup> capillary ECs at the mesometrial pole and in the central region express Notch1 and Notch4 (Figure 2A,B). In SpAs at the mesometrial pole, PDGFRβ <sup>+</sup> mural cells are adjacent to ECs that express both Notch1 and Notch4 (Figure 2A). In the anti-mesometrial region, Notch1 is expressed in CD31<sup>+</sup> ECs surrounded by Notch1 negative PDGFRβ <sup>+</sup> mural cells (Figure 2C). Notch4 expression was not detected in the anti-mesometrial region (data not shown).

Double staining for Jag1 or Dll4 and a vascular cell marker revealed that these Notch ligands are dynamically expressed in the decidual vasculature. At the mesometrial pole of the mesometrial region, CD31<sup>+</sup> ECs in capillaries and SpAs express both Jag1 and Dll4 (Figure 3A,D). A subset of the NG2<sup>+</sup> and PDGFRβ <sup>+</sup> mural cells in the SpAs also express Jag1, but not Dll4 (Figure 3A,D). In the central region of the mesometrial region, expression of Jag1 is not detected (Figure 3B), while Dll4 is expressed in a perinuclear, non-vascular pattern in decidual stromal cells (Figure 3E). In the anti-mesometrial region, Dll4 is expressed in CD31<sup>+</sup> ECs and Dll4<sup>+</sup> cells are associated with PDGFRβ <sup>+</sup> mural cells. Expression of Jag1 is scant in the anti-mesometrial region but is found in CD31<sup>+</sup> ECs and surrounding PDGFRβ <sup>+</sup> mural cells. Together, these data demonstrate that capillary ECs in the mesometrial region and anti-mesometrial region and SpA ECs co-express Notch1, as well as Jag1 and Dll4, suggesting these proteins mediate Notch signaling to regulate decidual angiogenesis and SpA remodeling.

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 5 of 19

**Figure 2.** Expression of receptors, Notch1 and Notch4, in the E7.5 decidual vasculature. High magnification images of sections double stained to detect expression of Notch1 or Notch4 and the endothelial cell marker, CD31 or mural cell marker, PDGFRββ. Merged images are outlined in yellow. (**A**) In the MP, Notch1 and Notch4 are expressed in CD31+ SpA ECs (white arrows). PDGFRβ+ mural cells are associated with both Notch1+ and Notch4+ ECs in the SpAs (white arrowheads). (**B**) In the CTR, CD31+ capillary ECs express Notch1 and Notch4 (white arrows). (**C**) In the AMR, CD31+ capillary ECs express Notch1 (white arrows) and PDGFRβ+ pericytes are associated with Notch1+ cells (white arrowhead). AMR = anti-mesometrial; CTR = central region; MP = mesometrial pole. Scale bars = 50 μm. **Figure 2.** Expression of receptors, Notch1 and Notch4, in the E7.5 decidual vasculature. High magnification images of sections double stained to detect expression of Notch1 or Notch4 and the endothelial cell marker, CD31 or mural cell marker, PDGFRββ. Merged images are outlined in yellow. (**A**) In the MP, Notch1 and Notch4 are expressed in CD31<sup>+</sup> SpA ECs (white arrows). PDGFRβ <sup>+</sup> mural cells are associated with both Notch1<sup>+</sup> and Notch4<sup>+</sup> ECs in the SpAs (white arrowheads). (**B**) In the CTR, CD31<sup>+</sup> capillary ECs express Notch1 and Notch4 (white arrows). (**C**) In the AMR, CD31<sup>+</sup> capillary ECs express Notch1 (white arrows) and PDGFRβ <sup>+</sup> pericytes are associated with Notch1<sup>+</sup> cells (white arrowhead). AMR = anti-mesometrial; CTR = central region; MP = mesometrial pole. Scale bars = 50 µm.

Double staining for Jag1 or Dll4 and a vascular cell marker revealed that these Notch ligands are dynamically expressed in the decidual vasculature. At the mesometrial pole of the mesometrial region, CD31+ ECs in capillaries and SpAs express both Jag1 and Dll4 (Figure 3A,D). A subset of the NG2+ and PDGFRβ+ mural cells in the SpAs also express Jag1, but not Dll4 (Figure 3A,D). In the central region of the mesometrial region, expression of Jag1 is not detected (Figure 3B), while Dll4 is expressed in a perinuclear, non-vascular pattern in decidual stromal cells (Figure 3E). In the antimesometrial region, Dll4 is expressed in CD31+ ECs and Dll4+ cells are associated with PDGFRβ<sup>+</sup> mural cells. Expression of Jag1 is scant in the anti-mesometrial region but is found in CD31+ ECs and surrounding PDGFRβ+ mural cells. Together, these data demonstrate that capillary ECs in the mesometrial region and anti-mesometrial region and SpA ECs co-express Notch1, as well as Jag1 and remodeling.

**Figure 3.** Expression of Notch ligands, Jag1 and Dll4, in the E7.5 decidual vasculature. High magnification images of sections double stained to detect expression of Jag1 or Dll4 and the endothelial cell marker, CD31 or mural cell marker, PDGFRβ. (**A**) In MP SpAs, Jag1 is expressed in CD31+ ECs (white arrow) and in NG2+ and PDGFRβ+ mural cells (white arrows). Some Jag1+ ECs are closely associated with PDGFRβ+/Jag1 mural cells (white arrowheads). (**B**) Expression of Jag1 is not detected in the central region. (**C**) In the AMR, expression of Jag1 is sparse. Jag1 is expressed in CD31+ ECs and PDGFRβ+ mural cells (white arrows). Jag1+ cells are closely associated with PDGFRβ+ mural cells (white arrowheads). (**D**) In MP SpAs, Dll4 is expressed in CD31+ ECs (white arrows). NG2+ and PDGFRβ+ mural cells are closely associated with Dll4+ cells (white arrowheads). (**E**) In the CTR, Dll4 is expressed in a punctate pattern (white arrowheads). (**F**) In the AMR, Dll4 is expressed in CD31+ ECs (white arrows). Dll4+ cells are associated with PDGFRβ+ mural cells (white arowheads). AMR = antimesometrial; CTR = central region; MP = mesometrial pole. Scale bars = 50 μm. **Figure 3.** Expression of Notch ligands, Jag1 and Dll4, in the E7.5 decidual vasculature. High magnification images of sections double stained to detect expression of Jag1 or Dll4 and the endothelial cell marker, CD31 or mural cell marker, PDGFRβ. (**A**) In MP SpAs, Jag1 is expressed in CD31<sup>+</sup> ECs (white arrow) and in NG2<sup>+</sup> and PDGFRβ <sup>+</sup> mural cells (white arrows). Some Jag1<sup>+</sup> ECs are closely associated with PDGFRβ <sup>+</sup>/Jag1<sup>−</sup> mural cells (white arrowheads). (**B**) Expression of Jag1 is not detected in the central region. (**C**) In the AMR, expression of Jag1 is sparse. Jag1 is expressed in CD31<sup>+</sup> ECs and PDGFRβ <sup>+</sup> mural cells (white arrows). Jag1<sup>+</sup> cells are closely associated with PDGFRβ + mural cells (white arrowheads). (**D**) In MP SpAs, Dll4 is expressed in CD31<sup>+</sup> ECs (white arrows). NG2<sup>+</sup> and PDGFRβ <sup>+</sup> mural cells are closely associated with Dll4<sup>+</sup> cells (white arrowheads). (**E**) In the CTR, Dll4 is expressed in a punctate pattern (white arrowheads). (**F**) In the AMR, Dll4 is expressed in CD31<sup>+</sup> ECs (white arrows). Dll4<sup>+</sup> cells are associated with PDGFRβ <sup>+</sup> mural cells (white arowheads). AMR = anti-mesometrial; CTR = central region; MP = mesometrial pole. Scale bars = 50 µm.
