**1. Introduction**

During early pregnancy, from embryo implantation to placentation, formation of new capillaries and physiologic remodeling of uterine spiral arteries (SpAs), which are distal branches of the maternal uterine arteries, are essential for normal embryonic growth and development [1–3]. Abnormal uterine vascular development early in pregnancy can set off a "ripple effect", resulting in poor placentation, reduced placental function and adverse downstream effects, such as abnormal growth and development of the embryo and pregnancy failure by mid-gestation [1,4]. Thus, understanding the pathways that regulate pre-placentation vascular development in the uterus will help to better elucidate the pathways that impact maintenance and success of pregnancy at later stages. poor placentation, reduced placental function and adverse downstream effects, such as abnormal growth and development of the embryo and pregnancy failure by mid-gestation [1,4]. Thus, understanding the pathways that regulate pre-placentation vascular development in the uterus will help to better elucidate the pathways that impact maintenance and success of pregnancy at later stages.

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 2 of 19

Abnormal uterine vascular development early in pregnancy can set off a "ripple effect", resulting in

In mice, uterine vascular changes are initiated by embryo implantation. Embryo implantation, which occurs by embryonic day (E) 4.5, triggers uterine stromal cell decidualization and concomitant decidual angiogenesis [1,2,5–8]. Blood vessels grow into the uterus on the mesometrial side creating a mesometrial-anti-mesometrial polarity of the implantation site and dividing the implantation site into two regions, mesometrial region and anti-mesometrial region [1,7] (Figure 1A–C). The mesometrial region can be further divided into the mesometrial pole and central region. In the central region of the mesometrial decidua, sprouting angiogenesis is most pronounced at E4.5 and remodeling angiogenesis occurs from E6.5–E8.5 [7]. In the anti-mesometrial region, angiogenesis begins at E4.5 and initiates endothelial cell (EC) proliferation, which peaks by E6.5, and results in a greater than 2-fold increase in vascular density at E8.5, as compared to E4.5 [7]. From E4.5–E7.5, decidual angiogenesis creates a rich, new capillary network that supports the growing pregnancy prior to placentation. In mice, uterine vascular changes are initiated by embryo implantation. Embryo implantation, which occurs by embryonic day (E) 4.5, triggers uterine stromal cell decidualization and concomitant decidual angiogenesis [1,2,5–8]. Blood vessels grow into the uterus on the mesometrial side creating a mesometrial-anti-mesometrial polarity of the implantation site and dividing the implantation site into two regions, mesometrial region and anti-mesometrial region [1,7] (Figure 1A–C). The mesometrial region can be further divided into the mesometrial pole and central region. In the central region of the mesometrial decidua, sprouting angiogenesis is most pronounced at E4.5 and remodeling angiogenesis occurs from E6.5–E8.5 [7]. In the anti-mesometrial region, angiogenesis begins at E4.5 and initiates endothelial cell (EC) proliferation, which peaks by E6.5, and results in a greater than 2-fold increase in vascular density at E8.5, as compared to E4.5 [7]. From E4.5–E7.5, decidual angiogenesis creates a rich, new capillary network that supports the growing pregnancy prior to placentation.

**Figure 1.** Characterization of the E7.5 uterine vasculature in wildtype mice. (**A**) Schematic representation of an E7.5 implantation site (created with BioRender.com). The implantation site is **Figure 1.** Characterization of the E7.5 uterine vasculature in wildtype mice. (**A**) Schematic representation of an E7.5 implantation site (created with BioRender.com). The implantation site is divided (represented

divided (represented by the dashed line) into two regions, the anti-mesometrial region (AMR) and the mesometrial region (MR), which is further divided into the mesometrial pole (MP) and central by the dashed line) into two regions, the anti-mesometrial region (AMR) and the mesometrial region (MR), which is further divided into the mesometrial pole (MP) and central region (CTR). Labeled are spiral arteries (white ellipses) in the MP, capillaries (white rectangles) in the CTR, capillaries (red rectangles) in the AMR and the embryo (e). (**B**) H&E of an implantation site at E7.5 showing the embryo and central parts of the decidua. (**C**) An E7.5 implantation site double stained for the endothelial cell marker, CD31 and pericyte marker, NG2. Ellipses identify NG2<sup>+</sup> SpAs at the MP. Rectangles identify CD31<sup>+</sup> capillaries in the central region (white rectangle) and the AMR (red rectangle). (**D**–**F**) High magnification images of the vessels of the MR at the MP and CTR and AMR. Sections were double stained to detect expression of CD31 and mural cell markers, NG2, PDGFRβ or SMA. Merged images are outlined in yellow. CD31<sup>+</sup> endothelial cells (ECs) are present throughout the implantation site in all three regions. (**D**) The vasculature in the MP is composed of SpAs seen as CD31<sup>+</sup> ECs covered by NG2+, PDGFRβ <sup>+</sup> and SMA<sup>+</sup> mural cells (white arrowheads) and capillaries comprised of ECs that are not associated with NG2, PDGFRβ or SMA (yellow rectangles). NG2 and PDGFRβ are co-expressed (white arrow). (**E**) In the CTR, expression of NG2 is sparse and expression of PDGFRβ was not detected. Few CD31<sup>+</sup> capillaries are associated with NG2<sup>+</sup> mural cells (white arrowhead). (**F**) In the AMR, capillaries are comprised of CD31<sup>+</sup> ECs closely associated with NG2+/PDGFRβ <sup>+</sup> mural cells (white arrowheads). Scale bars = 200 µm in (**B**,**C**) and 50 µm in (**D**–**F**).

Placenta formation begins at E8.5 and is complete by mid-gestation, or E10.5 [1,2]. Transformation of the maternal SpAs, initiates at E6.5 and is the key vascular change occurring from E8.5–E10.5. Embryo-derived trophoblasts migrate from the implantation site to the SpAs and mediate SpA remodeling [3,9,10]. SpAs, like most arteries, are comprised of two key cell types, ECs, which make up the inner lining of the vessel, and mural cells, which include pericytes and vascular smooth muscle cells, that surround the endothelium and are essential for maintenance of vascular integrity [11–13]. During the process of SpA remodeling, trophoblasts intercalate the vessels, replacing both ECs and vascular smooth muscle cells, and transform constricted artery-like structures into dilated and enlarged vein-like SpAs, which will support adequate blood flow through to the placenta [1,3,14,15]. Prior to mid-gestation, E10.5, two distinct vascular events, decidual angiogenesis and SpA remodeling, are essential for normal placentation and placental function.

The Vascular Endothelial Growth Factor (VEGF) and Notch signaling pathways are interconnected and together have been shown to be critical for vascular development of the retina, embryo and ovary [16–19]. VEGF signaling is essential for vascular proliferation and expansion in decidual angiogenesis and is necessary to support early pregnancy [5,7]. Experimental mouse models with targeted deletions of Notch signaling pathway members possess a variety of vascular defects that cause embryonic lethality by mid-gestation [20–26], but loss of Notch signaling in decidual angiogenesis and vascular remodeling in early pregnancy has not been fully elucidated.

Notch proteins are expressed in ECs and mural cells. Vascular ECs express receptors, Notch1 and Notch4, and ligands delta-like (Dll) 1, Dll4, Jagged1 (Jag1) and Jag2 [23,27–31]. Mural cells have been previously shown to express Notch1, Notch3 and Jag1 [26,27,32–34]. Ligand binding of Notch triggers release of the Notch intracellular domain (NICD), which translocates into the nucleus and promotes transcription of downstream effectors of Notch signaling, including members of the Hairy/Enhancer of Split related with YRPW motif (*Hey*) families and Notch-regulated ankyrin repeat-containing protein (*Nrarp*) [29,35,36]. Notch activation can be elicited by ligands expressed within the same cell (*cis*-activation) as the Notch receptor or on an adjacent cell (*trans*-activation) [37]. Alternatively, blockade of an inhibitory *cis*-interaction between receptor and ligand can drive Notch activation [38].

We have previously shown that Dll4 is expressed in the ECs of SpAs, while Jag1 is expressed in both ECs and pericytes of SpAs [27]. Consistent with ligand expression, Notch signaling is active in ECs in decidual capillaries and SpAs [27,39]. Herein, we investigated the role of endothelial Jag1/Notch signaling in the formation and maintenance of the maternal decidual vasculature in early mouse pregnancy. Mice carrying an EC-specific, tamoxifen inducible Cre recombinase, *Cdh5-CreERT2* [40] and a *Jagged1flox*/*flox* alleles [41] were used to achieve cell-type specific deletion of *Jag1* during decidual angiogenesis. In order to assess the decidual vasculature after embryo implantation and prior to placentation, we evaluated pregnancies at E7.5, a stage of pregnancy when decidual angiogenesis is nearing completion and decidual vascular density is greatest.
