Vascular Notch proteins and Notch signaling in the peri-implantation mouse uterus
© Shawber et al. 2015
Received: 29 July 2015
Accepted: 17 November 2015
Published: 1 December 2015
Angiogenesis is essential for uterine decidualization, the progesterone-mediated transformation of the uterus allowing embryo implantation and initiation of pregnancy. In the current study, we define the vasculature, expression of Notch proteins and Notch ligands, and Notch activity in both endothelial cells and vascular-associated mural cells of blood vessels in the pre-implantation endometrium and post-implantation decidua of the mouse uterus.
We used immunofluorescence to determine the expression of Notch in endothelial cells and mural cells by co-staining for the endothelial cell marker, CD31, the pan-mural cell marker, platelet-derived growth factor receptor beta (PDGFR-β), the pericyte markers, neural/glial antigen 2 (NG2) and desmin, or the smooth muscle cell marker, alpha smooth muscle actin (SMA). A fluorescein isothiocyanate-labeled dextran tracer, was used to identify functional peri-implantation vasculature. CBF:H2B-Venus Notch reporter transgenic mice were used to determine Notch activity.
Notch signaling is observed in endothelial cells and pericytes in the peri-implantation uterus. Prior to implantation, Notch1, Notch2 and Notch4 and Notch ligand, Delta-like 4 (Dll4) are expressed in capillary endothelial cells, while Notch3 is expressed in the pericytes. Jagged1 is expressed in both capillary endothelial cells and pericytes. After implantation, Notch1, Notch4 and Dll4 are expressed in endothelial cells of newly formed decidual capillaries. Jagged1 is expressed in endothelial cells of spiral arteries and a subset of decidual pericytes. Notch proteins are not expressed in lymphatic vessels or macrophages in the peri-implantation uterus.
We show Notch activity and distinct expression patterns for Notch proteins and ligands, suggesting unique roles for Notch1, Notch4, Dll4, and Jag1 during decidual angiogenesis and early placentation. These data set the stage for loss-of-function and gain-of-function studies that will determine the cell-type specific requirements for Notch proteins in decidual angiogenesis and placentation.
KeywordsNotch Dll4 Jagged1 Endothelial cells Pericytes Decidua Angiogenesis Implantation
Angiogenesis, the formation of new vessels from pre-existing vasculature, is critical in the uterine endometrium for embryo implantation, maintenance of early pregnancy, and development of the placenta. After fertilization, angiogenesis within the uterus occurs simultaneously with stromal cell decidualization, the rapid proliferation and differentiation of endometrial stromal fibroblasts into glycogen and lipid rich decidual cells [1–4]. In mice and humans, the uterine decidua supports early pregnancy prior to placenta development. The decidua serves as a scaffold for the newly formed decidual vascular plexus, as well as the maternal spiral arteries that are remodeled by embryo-derived trophoblasts during placenta formation. The decidual vascular plexus serves as the first exchange apparatus between the maternal circulation and the embryo and is necessary to maintain pregnancy prior to placenta formation [5–7]. In mice, inadequate decidual vascular development results in pregnancy failure by mid-gestation [7, 8]. In humans, inadequate decidual vascular development is associated with implantation failure, first trimester miscarriages, and abnormal placenta formation and function which leads to preeclampsia and intrauterine growth restriction [4, 9]. Ovarian estrogen and progesterone regulate decidua formation; however, the underlying molecular signaling pathways active in decidual angiogenesis have not as yet been fully characterized.
Sprouting angiogenesis is a multi-step process that begins with endothelial cells (ECs) sprouting out from mature vessels. ECs then migrate and proliferate to form a new sprout consisting of a tip cell at the front and neighboring stalk cells. New sprouts form capillary loops to create the vessel lumen and recruit vascular mural cells, which include pericytes and vascular smooth muscle cells (vSMCs), necessary for vessel stabilization . Well-known regulators of angiogenesis during development and adult life include the vascular endothelial growth factor (VEGF) and Notch signaling pathways. In mice and non-human primates, VEGF activates VEGF receptors (VEGFR) to mediate increased uterine vascular permeability and decidual angiogenesis required for embryo implantation [5, 6, 11]. We have shown that inhibition of VEGFR-2, blocks decidual angiogenesis observed at embryonic day 7.5 (E7.5) and results in embryonic lethality prior to E10.5 . Whereas continuous VEGFR-1 blockade significantly reduces decidual angiogenesis  and VEGFR-3 inhibition moderately reduces decidual angiogenesis , neither VEGFR-1 nor VEGFR-3 blockade has a notable negative effect on pregnancy prior to E10.5.
VEGF and Notch signaling pathways interact to coordinate developmental and postnatal angiogenesis [13–15], angiogenesis in tumors , and angiogenesis modeled in vitro [17–21]. Notch proteins (Notch1, Notch2, Notch3, and Notch4) are single-pass transmembrane receptors that interact with membrane-bound ligands of the Delta-like (Dll) (Dll1, Dll3, Dll4) and Jagged (Jag1 and Jag2) families in adjacent cells [22, 23]. In mice, Notch1 and Notch4 are expressed in endothelium of the developing vasculature [24–26] and Notch3 is expressed in mural cells, pericytes and vSMCs [26–28]. In tissues, such as the developing postnatal retina Notch ligand, Dll1 and Dll4 are expressed in ECs, while Jag1 is expressed in both ECs and vascular mural cells [23, 29]. Genetic studies demonstrate that Notch proteins and ligands are essential for embryonic vascular development [30–32] and maturation of vSMCs in mice [33, 34] and humans [35, 36].
Given the interactions between the Notch and VEGF signaling pathways in vascular development, Notch signaling likely functions in mammalian decidual angiogenesis to coordinate EC VEGFR signaling. A role for Dll4 in vascular development and differentiation in the decidua has recently been shown. Dll4 mediates decidual angiogenesis through induction of a tip/stalk phenotype in decidual ECs, suggesting a requirement for Notch signaling for proper decidual vascular development . However, a comprehensive analysis of the expression of Notch proteins and ligands in decidual angiogenesis has yet to be described. The goal of this study is to define the expression of Notch proteins and Notch ligands in the peri-implantation uterus as a framework for genetic studies that will identify cell-type specific requirements for Notch signaling in decidual angiogenesis and placenta formation. Herein, we characterize the distribution of blood and lymphatic vessels, vascular associated mural cells, and macrophages in the pre- and post-implantation mouse uterus and use a fluorescein isothiocyanate (FITC)-labeled dextran tracer to identify the functional peri-implantation vasculature. We determine the expression of Notch proteins, Notch1-4, Notch ligands, Dll4 and Jag1, and Notch activity with respect to ECs and mural cells in the pre- and post-implantation mouse uterus. Our data provide strong support for a role for Notch signaling in decidual angiogenesis and pericyte/EC interactions.
The Columbia University Institutional Animal Care and Use Committee approved protocols used in animal studies. All mice were maintained on a C57BL/6 background. For assessment of wild type expression patterns, we used C57BL/6J virgin female mice and males of proven fertility (The Jackson Laboratory). The CBF:H2B-Venus transgenic mouse strain that expresses human histone H2B fused to yellow fluorescent protein (YFP) Venus in response to Notch/CSL transcriptional activation was used to determine Notch activity . Mice were bred; noon on the day a mating plug was observed was designated embryonic day (E) 0.5. Pieces of uteri and implantation sites from pregnant females at E3.5 and E6.5, respectively, were embedded in Tissue-Tek® O.C.T.™ Compound (Sakura Fine Technical Co, Ltd, Tokyo, Japan), snap-frozen on dry ice in ethanol and stored at −80 °C in methylbutane (Sigma-Aldrich).
Histology, immunohistochemistry (IHC) and immuofluorescence (IF)
Antibodies for analysis of the pregnant mouse uterus
Jagged1 extracellular domain
Dr. Kitajewski’s laboratory
Anti-goat-IgG Alexa-Fluor 594
Anti-rabbit-IgG Alexa-Fluor 594
Anti-rabbit-IgG Alexa-Fluor 488
Anti-rat-IgG Alexa-Fluor 594
Anti-rat-IgG Alexa-Fluor 488
Mice were given tail vein injections of 200 μl (25 mg/mL) of FITC-conjugated 40 kDa dextran (Invitrogen D-1820) at E3.5 and 10 kDa dextran (Invitrogen D-1845) at E6.5 [41–43]. After 10 minutes, animals were euthanized. Uteri and implantation sites were dissected in cold phosphate buffered saline, fixed in Carnoy’s solution and embedded in paraffin wax. Sections (7 μm) were deparaffinized, rehydrated and mounted in Vectashield medium containing DAPI or stained for Notch1 prior to mounting. Dextran was administered to 3 mice at each stage. Specific staining was performed at least 3 times and 5 different uterine sections or implantation sites were analyzed at each stage.
IHC and H&E staining were examined with a Nikon MICROPHOT-FXA microscope and images were captured using NIS-Elements D3.10 software. Fluorescent images were captured using a Nikon A1 scanning confocal microscope on an Eclipse Ti microscope stand (Nikon Instruments, Melville, NY). Standard lasers and filters were used to image DAPI, AlexaFluor 488, and TRITC. Maximum intensity projections are shown.
Characterization of blood vessels in the pre-implantation murine uterus
At E3.5 prior to embryo implantation, CD31+ ECs in the endometrial stroma, myometrium and serosa are closely associated with NG2+ and PDGFR-β+ mural cells (Fig. 1C, D, yellow signal). Magnified areas of the endometrial stroma are representative of the stroma, myometrium and serosa (Fig. 1, C1, D1). Endometrial CD31+ vessels are not associated with SMA+ cells (Fig. 1E). SMA weakly labels glandular epithelia. SMA expression is seen throughout the myometrium and serosa, likely staining vSMCs, as well uterine smooth muscle cells (uSMCs) that also express NG2 and desmin (Fig. 1C, E, data not shown). Desmin expression is observed throughout the endometrium, stroma, myometrium and serosa and does not appear to be a specific marker of uterine vascular mural cells (data not shown).
The close association of NG2+, PDGFR-β+, and SMA− pericytes with CD31+ ECs suggests that these vessels are functional capillaries. Intravenous injection of FITC-conjugated dextran shows dextran in vascular structures throughout the pre-implantation endometrial stroma (Fig. 1F). We conclude that functional capillaries are present a day prior to embryo implantation. These capillaries are evident as pericyte-covered CD31+ vessels within the stroma.
Characterization of blood vessels in the post-implantation murine uterus
Characterization of lymphatic vessels and macrophages in the peri-implantation uterus
CD11b+ macrophages and F4/80+ macrophages are abundant throughout the myometrium and serosa at E3.5 and E6.5 (Fig. 4). These CD11b+ cells may be undifferentiated monocytes, as well as neutrophils or dendritic cells that also express this marker. Macrophages are abundant in the endometrial stroma before implantation (Fig. 4B, C), while their density is reduced throughout the decidua after implantation (Fig. 4E, F). The staining patterns suggest that different populations of macrophages exist within the peri-implantation uterus: a CD11b+ subset and a F4/80+ subset at both E3.5 and E6.5 and a LYVE1+ and F4/80+ double positive subset in the myometrium at E6.5.
Notch1 expression in the peri-implantation uterus
Notch4 expression in the peri-implantation uterus
Notch2 and Notch3 are predominantly expressed in SMCs in the peri-implantation uterus
Angiogenic Notch ligand expression in the peri-implantation uterus
Notch activity in ECs and pericytes in the peri-implantation uterus
Transformation of the pre-implantation E3.5 uterine endometrium to the post-implantation E6.5 uterine decidua requires angiogenesis and vascular remodeling to increase vascular permeability, and immune/inflammatory changes. This vascularization process assures successful embryo implantation and placenta formation [1, 2]. Herein we show that prior to implantation, most vessels in the uterine endometrium are capillaries, defined as CD31+ ECs covered by NG2+/PDGFR-β+ pericytes and lacking SMA expression. Macrophages are abundant throughout the endometrium, myometrium and serosa, whereas lymphatic vessels are restricted to the myometrium and serosa. After implantation, we found patent vessels lateral to the embryo running between the inter-implantation sites and the embryo. NG2+ and PDGFR-β+ pericytes are most abundant on newly formed decidual capillaries in the anti-mesometrial decidua. It is plausible that vessels in the mesometrial decidua, the site of vascular remodeling during placenta formation, may not require extensive pericyte coverage. Alternatively, a different population of pericytes, that does not express NG2, but may express desmin, could support the mesometrial vessels that do not express NG2. However, desmin is also expressed in the stromal cells and decidual cells and thus it is not possible to discriminate between desmin+ pericytes and stromal cells.
Summary of Notch activity and expression in the peri-implantation mouse uterus
Notch1 and Notch1/Notch4 mutant embryos have lethal angiogenic defects, with more severe phenotypes in Notch1/Notch4 double mutants . Endothelium-specific activation of Notch1 or Notch4 and disruption of Notch signaling within the vasculature [24, 31, 32] also results in embryonic lethality by mid-gestation with defects in fetal angiogenesis. These mutants reveal the importance of Notch1 and Notch4 in formation of fetal-derived embryonic and placental components. Notch1 has been previously been reported to be expressed in mouse and primate maternal uterine stromal cells during decidualization [53, 54]. The contribution of Notch1 and Notch4 signaling to maternal-derived placental vascular components or the pre-placental decidual vasculature has not as yet been investigated. We show that Notch1 is expressed in capillaries and excluded from macrophages in the peri-implantation uterus. Notch1+ ECs are uniformly distributed throughout the decidua, while Notch4 expression in ECs is more abundant in the mesometrial decidua. The partially overlapping expression patterns for Notch1 and Notch4 suggests that Notch1 and Notch4 may have some functionally redundancy in decidual angiogenesis. Further, active Notch signaling in both decidual ECs, which express Notch1 and Notch4, and pericytes suggests that the Notch pathway has a fundamental role in decidual angiogenesis.
Homozygous null mutants of Jag1 and Dll4 die during embryogenesis due to vascular defects [26, 55–58]. The umbilical artery and placental blood vessels are decreased in size in Dll4 heterozygous mutant embryos . The requirement for Dll4-mediated signaling in decidual angiogenesis was recently investigated . Peri-implantation administration of a Dll4 blocking antibody resulted in increased, but non-productive decidual angiogenesis that compromised pregnancies by E9.5, with very few embryos surviving to E13.5 . In this model, Dll4-mediated signaling was blocked during decidualization, however it is unclear whether subsequent placentation was affected, leading to the abnormal embryonic development observed. Our finding of Dll4 in capillaries in the anti-mesometrial uterine decidua supports the finding that Dll4 signaling is required for proper decidual angiogenesis. Jag1 is expressed in decidual ECs of maternal spiral arteries and in pericytes. The different expression patterns of Dll4 and Jag1 in the decidual vasculature suggest unique functions for these Notch ligands.
During pregnancy, the lymphatic vasculature is believed to play a role in regulating the fluid balance between the maternal and fetal compartments and in maintaining maternal tolerance of the semi-allogeneic fetus [45, 60, 61]. There is general agreement that the myometrium and serosa of both humans and mice contain lymphatic vessels [60, 62, 63]. Whereas LYVE1+ lymphatics have not been detected in the endometrium of the non-pregnant human uterus [60, 64], podoplanin+ lymphatics are abundant in the endometrium basalis, the region directly adjacent to the myometrium and sparse in the endometrium functionalis, the region that is shed during menses . LYVE1+ lymphatics are prominent in the decidua during all trimesters of human pregnancy, but have not been detected in the murine decidua . Similarly, we do not observe LYVE1+ lymphatic vessels in the peri-implantation murine decidua. Further investigation with additional lymphatic markers is necessary to confirm the difference between humans and mice.
Pregnancy is associated with an influx of macrophages into the uterus [65, 66]. Macrophages are proposed to function in coordinating the maternal immune response, in apoptosis and tissue remodeling at the maternal-fetal interface, as well as in promoting angiogenesis [67, 68]. Macrophages are recruited into the endometrium during the peri-implantation period such that decidual macrophages are the second most abundant immune cell population at the implantation site, comprising 20-30 % of immune cells in the uterine decidua [69, 70]. Whereas CD11b is expressed by myeloid cells other than macrophages, F4/80 is expressed by mature macrophages [46, 50]. F4/80 is commonly used to identify macrophages in the female reproductive tract [12, 71–73]. We recently showed that VEGFR-1+ ECs are often in direct contact with CD11b+ monocytes/macrophages and F4/80+ macrophages in the peri-implantation uterus . Blockade of VEGFR-1 significantly decreases both the number macrophages in the decidua of E7.5 pregnant mice, likely as an indirect result of the decrease in VEGFR-1 function in ECs . Here we show F4/80+ macrophages in direct contact with Notch1+ vessels prior to implantation. Available data suggest that macrophages do not have an obligatory role in uterine decidualization and embryo implantation [12, 71]. However, macrophages are implicated in trophoblast invasion and spiral artery remodeling during placental development [74, 75].
Taken together, our findings support unique roles for Notch1, Notch4 and angiogenic ligands, Dll4 and Jag1 in decidual angiogenesis. Abnormal decidual angiogenesis can result in spontaneous miscarriages early in pregnancy or have adverse “ripple effects” throughout pregnancy, leading to abnormal placentation, fetal growth restriction, and/or preeclampsia . Proper trophoblast invasion of maternal spiral arteries in the uterine decidua is integral to placenta formation. Notch proteins are implicated in EC-trophoblast interactions during this vascular remodeling in the uterine decidua . Our data set the stage for genetic studies to evaluate the requirement for Notch signaling in decidual angiogenesis and early placentation.
neural/glial antigen 2
platelet-derived growth factor receptor beta
alpha smooth muscle actin
uterine smooth muscle cells
vascular smooth muscle cells
vascular endothelial growth factor receptor
yellow fluorescent protein
Images were collected in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant P30 CA013696. The confocal microscope was purchased with NIH grant S10 RR025686. The authors thank Theresa Swayne, Ph.D. for her technical assistance with confocal microscopy.
Support was provided by NIH/NCI grant R01 CA136673 (C.J.S.), NIH/NICHD grant 5R37 HD033082 (V.E.P.), NIH/NHLBI grant R01 HL112626 (J.K.K.), the Reproductive Scientist Development Program/NIH/NICHD grant K12 HD000849 (N.C.D.) and the Robert Wood Johnson Foundation/Amos Medical Faculty Development Program (N.C.D.).
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