VE-cadherin enables trophoblast endovascular invasion and spiral artery remodeling during placental development

  1. Derek C Sung
  2. Xiaowen Chen
  3. Mei Chen
  4. Jisheng Yang
  5. Susan Schultz
  6. Apoorva Babu
  7. Yitian Xu
  8. Siqi Gao
  9. TC Stevenson Keller IV
  10. Patricia Mericko-Ishizuka
  11. Michelle Lee
  12. Ying Yang
  13. Joshua P Scallan
  14. Mark L Kahn  Is a corresponding author
  1. Cardiovascular Institute, Department of Medicine, University of Pennsylvania, United States
  2. Department of Radiology, Hospital of the University of Pennsylvania, United States
  3. University Laboratory Animal Resources, University of Pennsylvania, United States
  4. Department of Molecular Pharmacology and Physiology, University of South Florida, United States
4 figures, 2 tables and 1 additional file

Figures

Figure 1 with 3 supplements
Deletion of VE-cadherin in trophoblasts disrupts cell migration resulting in placental and fetal growth restriction.

(A–D) Gross examination and quantification of E12.5 Control and CYP19A1(Tg)Cre; Cdh5fl/fl placentas and weights. Control n = 11, CYP19A1(Tg)Cre; Cdh5fl/fl n = 5. (E–H) Gross examination and …

Figure 1—source data 1

Excel file containing quantification for embryo weights, placenta weights, trophoblast density, trophoblast migration distance, and fetal labyrinth vasculature in Figure 1.

https://cdn.elifesciences.org/articles/77241/elife-77241-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Deletion of VE-cadherin in CYP19A1(Tg)Cre; Cdh5fl/fl placentas.

(A, B) Immunofluorescence staining and quantification of E12.5 Control and CYP19A1(Tg)Cre; Cdh5fl/fl placentas for VE-cadherin (green) and CK8 (red). Yellow arrowheads indicate VE-cadherin+

Figure 1—figure supplement 1—source data 1

Excel file containing quantification for VE-cadherin expression in trophoblasts, embryo weights, and placenta weights in Figure 1—figure supplement 1.

https://cdn.elifesciences.org/articles/77241/elife-77241-fig1-figsupp1-data1-v2.xlsx
Figure 1—figure supplement 2
Loss of trophoblast VE-cadherin causes defects in brain, liver, and heart development.

(A) Sagittal hematoxylin and eosin (H&E) sections from E12.5 Control and CYP19A1(Tg)Cre; Cdh5fl/fl embryos. Boxes show magnified regions of the brain, heart, and liver. Note the thinness of the …

Figure 1—figure supplement 2—source data 1

Excel file containing quantification for liver area and myocardial thickness in Figure 1—figure supplement 2.

https://cdn.elifesciences.org/articles/77241/elife-77241-fig1-figsupp2-data1-v2.xlsx
Figure 1—figure supplement 3
VE-cadherin expression is retained in the vasculature of affected organs in CYP19A1(Tg)Cre; Cdh5fl/fl embryos.

(A) Immunofluorescence staining of E12.5 Control and CYP19A1(Tg)Cre; Cdh5fl/fl brains for VE-cadherin (green) and Endomucin (red) at sites of hemorrhage. Images in gray scale are VE-cadherin alone. …

Figure 1—figure supplement 3—source data 1

Excel file containing quantification for VE-cadherin expression in various organs in Figure 1—figure supplement 3.

https://cdn.elifesciences.org/articles/77241/elife-77241-fig1-figsupp3-data1-v2.xlsx
VE-cadherin is required in trophoblasts to remodel spiral arteries (SAs) and to displace SA endothelium.

(A) Immunofluorescence staining of E12.5 Control and CYP19A1(Tg)Cre; Cdh5fl/fl placentas for Endomucin (green) and alpha-smooth muscle actin (αSMA) (red). White arrowheads indicate αSMA+ cells. …

Figure 2—source data 1

Excel file containing quantification for smooth muscle cells per spiral artery, spiral artery diameter, and trophoblast-endothelial cell displacement in Figure 2.

https://cdn.elifesciences.org/articles/77241/elife-77241-fig2-data1-v2.xlsx
Spiral artery remodeling defects result in placental insufficiency and fetal distress.

(A) Schematic of workflow for Doppler ultrasound of pregnant dams. The embryo, UA/UV (yellow arrow), and placenta (dotted white outline) are labeled. (B) Representative umbilical artery Doppler …

Figure 3—source data 1

Excel file containing quantification for ultrasound studies (resistance index, pulsatility index, fetal heart rate) in Figure 3.

https://cdn.elifesciences.org/articles/77241/elife-77241-fig3-data1-v2.xlsx
Figure 4 with 2 supplements
RNA-sequencing reveals defects in the decidual extracellular matrix and immune microenvironment.

(A) Schematic of bulk RNA-sequencing (RNA-seq) workflow on deciduas from Control (WT) and CYP19A1(Tg)Cre; Cdh5fl/fl (KO) E12.5 placentas. (B) Heatmap of the top 100 differentially regulated genes …

Figure 4—figure supplement 1
Disrupting trophoblast migration results in decidual extracellular matrix defects.

(A) Mmp15 mRNA gene expression in control and CYP19A1(Tg)Cre; Cdh5fl/fl placentas from RNA-sequencing (RNA-seq). (B) Immunofluorescence staining for Endomucin (green), CK8 (red), and MMP15 (gray) in …

Figure 4—figure supplement 1—source data 1

Excel file containing quantification for Mmp15 gene expression, laminin expression in the decidua, and vinculin expression in trophoblasts in Figure 4—figure supplement 1.

https://cdn.elifesciences.org/articles/77241/elife-77241-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Persistent uterine natural killer (uNK) cells at the junctional zone-decidual interface of Cdh5 knockout placentas.

(A) Immunofluorescence staining for DBA (a lectin that specifically binds uterine natural killer (uNK) cells, gray), vimentin (green), and cleaved caspase-3 (red).

Dotted line demarcates the border of the junctional zone and decidua. Scale bars = 50 μm. (B, C) Quantification of DBA+ cell density and cleaved caspase-3+ cell density in the decidua. (D) Gene ontology (GO) analysis of upregulated biological process terms related to NK cells. Statistical analysis was performed using two-tailed, unpaired Welch’s t-test. Data are shown as means ± SD.

Figure 4—figure supplement 2—source data 1

Excel file containing quantification for uterine natural killer cell density and apoptotic cell density in Figure 4—figure supplement 2.

https://cdn.elifesciences.org/articles/77241/elife-77241-fig4-figsupp2-data1-v2.xlsx

Tables

Table 1
Decreased survival of CYP19A1(Tg)Cre; Cdh5fl/fl mutants in late gestation.

Cdh5fl/fl male mice were crossed with CYP19A1(Tg)Cre; Cdh5fl/+ females to generate litters with mixed genotypes. The expected percentage is listed under the genotype label. The observed number of …

CYP19A1(Tg)Cre; Cdh5fl/fl (25%)CYP19A1(Tg)Cre; Cdh5fl/+ (25%)Cdh5fl/fl (25%)Cdh5fl/+ (25%)Total (100%)Fisher Exact Test
E10.5-E12.513* (26%)13 (26%)12 (24%)12 (24%)50 (100%)P=1.0000
E14.5-E16.51 (2.9%)13 (38.2%)12 (35.3%)8 (23.5%)34 (100%)P=0.0033
P211 (2.2%)11 (24.4%)15 (33.3%)18 (40.0%)45 (100%)P=0.0333
  1. *

    One embryo at E12.5 was dead.

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculcus)CYP19A1(Tg)-CreWenzel and Leone, 2007
Genetic reagent (Mus musculcus)Cdh5 floxYang et al., 2019
AntibodyAnti-Endomucin (goat polyclonal)R&DAF4666IF(1:400)
AntibodyAnti-Endomucin (rat monoclonal)Abcamab106100IF(1:300)
AntibodyAnti-TER119 (rat monoclonal)Abcamab91113IF(1:300)
AntibodyAnti-VE-cadherin (goat polycloncal)R&DAF1002IF(1:200)
AntibodyAnti-CK8 (rabbit monoclonal)Abcamab53280IF(1:300)
AntibodyAnti-CK8 (rat monoclonal)DSHBTROMA-1IF(1:400)
AntibodyAnti-αSMA-Cy3 (mouse monoclonal)SigmaC6198IF(1:300)
AntibodyAnti-Cleaved Caspase-3 (rabbit polyclonal)Millipore SigmaAB3623IF(1:100)
AntibodyAnti-MMP15 (rabbit polyclonal)Thermo Fisher ScientificPA5-13184IF(1:200)
AntibodyAnti-Laminin (rabbit polyclonal)SigmaL9393IF(1:200)
AntibodyAnti-Vimentin (goat polyclonal)R&DAF2105IF(1:300)
AntibodyAnti-Vinculin (mouse monoclonal)SigmaV9131IF(1:200)
Commercial assay or kitDirect-zol RNA Miniprep KitsZymo ResearchR2053
Software, algorithmImageJNIH, Bethesda, MD, USARRID:SCR_003070
Software, algorithmGraphPad PrismGraphPadRRID:SCR_002798
Software, algorithmPicard v2.17.11PicardRRID:SCR_006525
OtherDBA-BiotinVector LabsB-1035(1:500)

Additional files

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