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Vascular Notch proteins and Notch signaling in the peri-implantation mouse uterus.

Shawber CJ, Lin L, Gnarra M, Sauer MV, Papaioannou VE, Kitajewski JK, Douglas NC - (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Division of Reproductive Sciences, College of Physicians and Surgeons, Columbia University Medical Center, 630 West 168th St, New York, NY 10032 USA ; Department of Surgery, College of Physicians and Surgeons, Columbia University Medical Center, 630 West 168th St, New York, NY 10032 USA.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus

Location of Notch2 (N2) in relation to endothelial cells and mural cells in the peri-implantation uterus. H&E and double staining IF of E3.5 (A – D) and E6.5 (E – H) uterine sections. Ovals indicate areas of the uteri magnified (B1 – D1, F1 – H1). (A) H&E at E3.5 highlighting the luminal epithelium (arrows), and myometrium (myo), and endometrial stroma (st). (B – D) Notch2+ cells are observed throughout the stroma, myometrium and serosa (s), with low levels of Notch2 in endometrial glands (g). (B) Notch2 and CD31. Notch2+ cells in association with CD31+ ECs are scattered sparsely throughout the endometrium (arrrowhead in B1) and serosa (s). (C) Notch2 and PDGFR-β. Notch2 is not associated with PDGFR-β+ mural cells in the stroma and myometrium. (D) Notch2 and SMA. SMA+ smooth muscle cells are observed adjacent to Notch2+ cells in myometrium. Some uterine smooth muscle cells express both Notch2 and SMA. (E) H&E at E6.5 highlighting the embryo (e), inter-implantation sites (i), and myometrium (myo). (F – H) Notch2+ cells are observed throughout the myometrium. (F) Notch2 and CD31. Notch2 is not expressed in CD31+ decidual vessels. (G) Notch2 and PDGFR-β. Notch2 is not associated with PDGFR-β in the decidua or myometrium. (H) Notch2 and SMA staining. SMA+ smooth muscle cells are observed adjacent to Notch2+ cells in myometrium. Some uSMCs express both Notch2 and SMA. DAPI identifies all nuclei in IF images. am, anti-mesometrial; m, mesometrial. Bar in A and E =100 μm. Bar in B – D and F – H =500 μm. Bar in B1 – D1 and F1 – H1 = 50 μm.
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Fig7: Location of Notch2 (N2) in relation to endothelial cells and mural cells in the peri-implantation uterus. H&E and double staining IF of E3.5 (A – D) and E6.5 (E – H) uterine sections. Ovals indicate areas of the uteri magnified (B1 – D1, F1 – H1). (A) H&E at E3.5 highlighting the luminal epithelium (arrows), and myometrium (myo), and endometrial stroma (st). (B – D) Notch2+ cells are observed throughout the stroma, myometrium and serosa (s), with low levels of Notch2 in endometrial glands (g). (B) Notch2 and CD31. Notch2+ cells in association with CD31+ ECs are scattered sparsely throughout the endometrium (arrrowhead in B1) and serosa (s). (C) Notch2 and PDGFR-β. Notch2 is not associated with PDGFR-β+ mural cells in the stroma and myometrium. (D) Notch2 and SMA. SMA+ smooth muscle cells are observed adjacent to Notch2+ cells in myometrium. Some uterine smooth muscle cells express both Notch2 and SMA. (E) H&E at E6.5 highlighting the embryo (e), inter-implantation sites (i), and myometrium (myo). (F – H) Notch2+ cells are observed throughout the myometrium. (F) Notch2 and CD31. Notch2 is not expressed in CD31+ decidual vessels. (G) Notch2 and PDGFR-β. Notch2 is not associated with PDGFR-β in the decidua or myometrium. (H) Notch2 and SMA staining. SMA+ smooth muscle cells are observed adjacent to Notch2+ cells in myometrium. Some uSMCs express both Notch2 and SMA. DAPI identifies all nuclei in IF images. am, anti-mesometrial; m, mesometrial. Bar in A and E =100 μm. Bar in B – D and F – H =500 μm. Bar in B1 – D1 and F1 – H1 = 50 μm.

Mentions: To determine Notch2 and Notch3 expression patterns, E3.5 and E6.5 uteri were stained for Notch2 or Notch3 and EC marker, CD31 or mural cell markers, PDGFR-β and SMA. At E3.5, Notch2 expression is observed in a subset of CD31+ endometrial ECs (Fig. 7B, arrowhead). In contrast, Notch2 expression is not associated with CD31+ ECs in the E6.5 decidua (Fig. 7F). At both time points, Notch2 expression does not overlap with the vascular mural cell marker, PDGFR-β. In the myometrium, Notch2 is expressed in the SMA+ uSMCs of the inner circular and outer longitudinal fibers (Fig. 7D and H, yellow signal). The expression pattern of Notch2 in the decidua does not support a role for Notch2 in decidual angiogenesis.Fig. 7


Vascular Notch proteins and Notch signaling in the peri-implantation mouse uterus.

Shawber CJ, Lin L, Gnarra M, Sauer MV, Papaioannou VE, Kitajewski JK, Douglas NC - (2015)

Location of Notch2 (N2) in relation to endothelial cells and mural cells in the peri-implantation uterus. H&E and double staining IF of E3.5 (A – D) and E6.5 (E – H) uterine sections. Ovals indicate areas of the uteri magnified (B1 – D1, F1 – H1). (A) H&E at E3.5 highlighting the luminal epithelium (arrows), and myometrium (myo), and endometrial stroma (st). (B – D) Notch2+ cells are observed throughout the stroma, myometrium and serosa (s), with low levels of Notch2 in endometrial glands (g). (B) Notch2 and CD31. Notch2+ cells in association with CD31+ ECs are scattered sparsely throughout the endometrium (arrrowhead in B1) and serosa (s). (C) Notch2 and PDGFR-β. Notch2 is not associated with PDGFR-β+ mural cells in the stroma and myometrium. (D) Notch2 and SMA. SMA+ smooth muscle cells are observed adjacent to Notch2+ cells in myometrium. Some uterine smooth muscle cells express both Notch2 and SMA. (E) H&E at E6.5 highlighting the embryo (e), inter-implantation sites (i), and myometrium (myo). (F – H) Notch2+ cells are observed throughout the myometrium. (F) Notch2 and CD31. Notch2 is not expressed in CD31+ decidual vessels. (G) Notch2 and PDGFR-β. Notch2 is not associated with PDGFR-β in the decidua or myometrium. (H) Notch2 and SMA staining. SMA+ smooth muscle cells are observed adjacent to Notch2+ cells in myometrium. Some uSMCs express both Notch2 and SMA. DAPI identifies all nuclei in IF images. am, anti-mesometrial; m, mesometrial. Bar in A and E =100 μm. Bar in B – D and F – H =500 μm. Bar in B1 – D1 and F1 – H1 = 50 μm.
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Related In: Results  -  Collection

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Fig7: Location of Notch2 (N2) in relation to endothelial cells and mural cells in the peri-implantation uterus. H&E and double staining IF of E3.5 (A – D) and E6.5 (E – H) uterine sections. Ovals indicate areas of the uteri magnified (B1 – D1, F1 – H1). (A) H&E at E3.5 highlighting the luminal epithelium (arrows), and myometrium (myo), and endometrial stroma (st). (B – D) Notch2+ cells are observed throughout the stroma, myometrium and serosa (s), with low levels of Notch2 in endometrial glands (g). (B) Notch2 and CD31. Notch2+ cells in association with CD31+ ECs are scattered sparsely throughout the endometrium (arrrowhead in B1) and serosa (s). (C) Notch2 and PDGFR-β. Notch2 is not associated with PDGFR-β+ mural cells in the stroma and myometrium. (D) Notch2 and SMA. SMA+ smooth muscle cells are observed adjacent to Notch2+ cells in myometrium. Some uterine smooth muscle cells express both Notch2 and SMA. (E) H&E at E6.5 highlighting the embryo (e), inter-implantation sites (i), and myometrium (myo). (F – H) Notch2+ cells are observed throughout the myometrium. (F) Notch2 and CD31. Notch2 is not expressed in CD31+ decidual vessels. (G) Notch2 and PDGFR-β. Notch2 is not associated with PDGFR-β in the decidua or myometrium. (H) Notch2 and SMA staining. SMA+ smooth muscle cells are observed adjacent to Notch2+ cells in myometrium. Some uSMCs express both Notch2 and SMA. DAPI identifies all nuclei in IF images. am, anti-mesometrial; m, mesometrial. Bar in A and E =100 μm. Bar in B – D and F – H =500 μm. Bar in B1 – D1 and F1 – H1 = 50 μm.
Mentions: To determine Notch2 and Notch3 expression patterns, E3.5 and E6.5 uteri were stained for Notch2 or Notch3 and EC marker, CD31 or mural cell markers, PDGFR-β and SMA. At E3.5, Notch2 expression is observed in a subset of CD31+ endometrial ECs (Fig. 7B, arrowhead). In contrast, Notch2 expression is not associated with CD31+ ECs in the E6.5 decidua (Fig. 7F). At both time points, Notch2 expression does not overlap with the vascular mural cell marker, PDGFR-β. In the myometrium, Notch2 is expressed in the SMA+ uSMCs of the inner circular and outer longitudinal fibers (Fig. 7D and H, yellow signal). The expression pattern of Notch2 in the decidua does not support a role for Notch2 in decidual angiogenesis.Fig. 7

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Division of Reproductive Sciences, College of Physicians and Surgeons, Columbia University Medical Center, 630 West 168th St, New York, NY 10032 USA ; Department of Surgery, College of Physicians and Surgeons, Columbia University Medical Center, 630 West 168th St, New York, NY 10032 USA.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus