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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

Characterization of lymphatic vessels and macrophages in the peri-implantation uterus. H&E and double staining IF of E3.5 (A – C) and E6.5 (D – F) uterine sections. Ovals indicate areas of the uteri magnified (E1 and F1). (A) H&E at E3.5 highlighting the luminal epithelium (arrows), inner circular and outer longitudinal myometrium [dashed bracket (myo)], endometrial stroma (st) and serosa (s). (B, C) LYVE1+ lymphatics are observed throughout the myometrium and serosa, but are not detected in the endometrial stroma. CD11b+ macrophages (B) and F4/80+ macrophages (C) are abundant throughout the endometrium, myometrium, and serosa, but their distribution patterns differ. (D) H&E at E6.5 highlighting the embryo (e), inter-implantation sites (i) and myometrium (myo). (E, F) LYVE1+ lymphatics are observed throughout the myometrium at E6.5. The LYVE1 positive staining in the decidua does not have a vascular pattern (E1, F1). CD11b+ macrophages (E) and F4/80+ macrophages (F) are abundant throughout the myometrium and serosa, but scattered throughout the decidua at E6.5. LYVE1+ F4/80+ macrophages are detected in the myometrium at E6.5 (F, white arrowheads). DAPI identifies all nuclei in IF images. am, anti-mesometrial; m-mesometrial. Bar in A – D =100 μm. Bar in E and F = 500 μm. Bar in E1 and F1 = 50 μm.
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Fig4: Characterization of lymphatic vessels and macrophages in the peri-implantation uterus. H&E and double staining IF of E3.5 (A – C) and E6.5 (D – F) uterine sections. Ovals indicate areas of the uteri magnified (E1 and F1). (A) H&E at E3.5 highlighting the luminal epithelium (arrows), inner circular and outer longitudinal myometrium [dashed bracket (myo)], endometrial stroma (st) and serosa (s). (B, C) LYVE1+ lymphatics are observed throughout the myometrium and serosa, but are not detected in the endometrial stroma. CD11b+ macrophages (B) and F4/80+ macrophages (C) are abundant throughout the endometrium, myometrium, and serosa, but their distribution patterns differ. (D) H&E at E6.5 highlighting the embryo (e), inter-implantation sites (i) and myometrium (myo). (E, F) LYVE1+ lymphatics are observed throughout the myometrium at E6.5. The LYVE1 positive staining in the decidua does not have a vascular pattern (E1, F1). CD11b+ macrophages (E) and F4/80+ macrophages (F) are abundant throughout the myometrium and serosa, but scattered throughout the decidua at E6.5. LYVE1+ F4/80+ macrophages are detected in the myometrium at E6.5 (F, white arrowheads). DAPI identifies all nuclei in IF images. am, anti-mesometrial; m-mesometrial. Bar in A – D =100 μm. Bar in E and F = 500 μm. Bar in E1 and F1 = 50 μm.

Mentions: We used lymphatic endothelial hyaluronan receptor-1 (LYVE1) to detect lymphatic vessels [45] and CD11b [46, 47] and F4/80 [48–50] to detect macrophages. At E3.5, LYVE1+ lymphatic vessels are restricted to the myometrial and serosal layers of the uterus (Fig. 4B and C) and excluded from the endometrium and decidua. At E6.5, LYVE1+ cells are scattered throughout the decidua, without any apparent vascular pattern and likely do not represent lymphatic ECs (Fig. 4E, F), as LYVE1 is also expressed by a subset of macrophages [51]. F4/80 and LYVE1 double positive cells are observed in the myometrium at E6.5, but not at E3.5, suggesting that these cells are recruited subsequent to embryo implantation (Fig. 4F, arrowheads). The decidual LYVE1+ cells are neither CD11b, nor F4/80 positive indicating that they are not macrophages.Fig. 4


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)

Characterization of lymphatic vessels and macrophages in the peri-implantation uterus. H&E and double staining IF of E3.5 (A – C) and E6.5 (D – F) uterine sections. Ovals indicate areas of the uteri magnified (E1 and F1). (A) H&E at E3.5 highlighting the luminal epithelium (arrows), inner circular and outer longitudinal myometrium [dashed bracket (myo)], endometrial stroma (st) and serosa (s). (B, C) LYVE1+ lymphatics are observed throughout the myometrium and serosa, but are not detected in the endometrial stroma. CD11b+ macrophages (B) and F4/80+ macrophages (C) are abundant throughout the endometrium, myometrium, and serosa, but their distribution patterns differ. (D) H&E at E6.5 highlighting the embryo (e), inter-implantation sites (i) and myometrium (myo). (E, F) LYVE1+ lymphatics are observed throughout the myometrium at E6.5. The LYVE1 positive staining in the decidua does not have a vascular pattern (E1, F1). CD11b+ macrophages (E) and F4/80+ macrophages (F) are abundant throughout the myometrium and serosa, but scattered throughout the decidua at E6.5. LYVE1+ F4/80+ macrophages are detected in the myometrium at E6.5 (F, white arrowheads). DAPI identifies all nuclei in IF images. am, anti-mesometrial; m-mesometrial. Bar in A – D =100 μm. Bar in E and F = 500 μm. Bar in E1 and F1 = 50 μm.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig4: Characterization of lymphatic vessels and macrophages in the peri-implantation uterus. H&E and double staining IF of E3.5 (A – C) and E6.5 (D – F) uterine sections. Ovals indicate areas of the uteri magnified (E1 and F1). (A) H&E at E3.5 highlighting the luminal epithelium (arrows), inner circular and outer longitudinal myometrium [dashed bracket (myo)], endometrial stroma (st) and serosa (s). (B, C) LYVE1+ lymphatics are observed throughout the myometrium and serosa, but are not detected in the endometrial stroma. CD11b+ macrophages (B) and F4/80+ macrophages (C) are abundant throughout the endometrium, myometrium, and serosa, but their distribution patterns differ. (D) H&E at E6.5 highlighting the embryo (e), inter-implantation sites (i) and myometrium (myo). (E, F) LYVE1+ lymphatics are observed throughout the myometrium at E6.5. The LYVE1 positive staining in the decidua does not have a vascular pattern (E1, F1). CD11b+ macrophages (E) and F4/80+ macrophages (F) are abundant throughout the myometrium and serosa, but scattered throughout the decidua at E6.5. LYVE1+ F4/80+ macrophages are detected in the myometrium at E6.5 (F, white arrowheads). DAPI identifies all nuclei in IF images. am, anti-mesometrial; m-mesometrial. Bar in A – D =100 μm. Bar in E and F = 500 μm. Bar in E1 and F1 = 50 μm.
Mentions: We used lymphatic endothelial hyaluronan receptor-1 (LYVE1) to detect lymphatic vessels [45] and CD11b [46, 47] and F4/80 [48–50] to detect macrophages. At E3.5, LYVE1+ lymphatic vessels are restricted to the myometrial and serosal layers of the uterus (Fig. 4B and C) and excluded from the endometrium and decidua. At E6.5, LYVE1+ cells are scattered throughout the decidua, without any apparent vascular pattern and likely do not represent lymphatic ECs (Fig. 4E, F), as LYVE1 is also expressed by a subset of macrophages [51]. F4/80 and LYVE1 double positive cells are observed in the myometrium at E6.5, but not at E3.5, suggesting that these cells are recruited subsequent to embryo implantation (Fig. 4F, arrowheads). The decidual LYVE1+ cells are neither CD11b, nor F4/80 positive indicating that they are not macrophages.Fig. 4

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