Dll4 assembles the umbilical cord and placental vasculature
Derek C. Sung, Hana A. Ahanger, Sweta Narayan, Jesse A. Pace, Mei Chen, Jisheng Yang, Siqi Gao, T.C.S. Keller IV, Jenna Bockman, Xiaowen Chen, Erica Nguyen, Alan T. Tang, Patricia Mericko-Ishizuka, Ivan Maillard, Mark L. Kahn
Derek C. Sung, Hana A. Ahanger, Sweta Narayan, Jesse A. Pace, Mei Chen, Jisheng Yang, Siqi Gao, T.C.S. Keller IV, Jenna Bockman, Xiaowen Chen, Erica Nguyen, Alan T. Tang, Patricia Mericko-Ishizuka, Ivan Maillard, Mark L. Kahn
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Research Article Development Reproductive biology Vascular biology

Dll4 assembles the umbilical cord and placental vasculature

Abstract

Proper development of the umbilical cord and placental vasculature is essential for embryonic development. While the allantois is known give rise to endothelial cells (ECs) within the placenta, whether the allantois gives rise to ECs in the umbilical cord is debated. Furthermore, a lack of genetic tools to study placental vascular development independent of the embryo proper has hindered robust investigation into the primary cause of vascular defects from early studies utilizing global KOs. In this study, we delineate the contribution of the allantois to the umbilical vessels and utilize a mouse genetic tool previously developed by our lab to revisit the role of Notch signaling during placental development. We show that the allantois has mosaic contribution to the umbilical endothelium with higher contributions closer to the placenta. Allantoic deletion of Dll4 disrupts umbilical cord and placental vascular formation with secondary defects in the heart. Lastly, we identify Unc5b downstream of Notch signaling that restricts EC migration while promoting chemokine signaling for vascular smooth muscle cell (vSMC) recruitment to arteries. These findings identify a genetic tool for investigating placental vascular development and give insights into the ontogeny and mechanisms of placental vascular and umbilical cord development.

Authors

Derek C. Sung, Hana A. Ahanger, Sweta Narayan, Jesse A. Pace, Mei Chen, Jisheng Yang, Siqi Gao, T.C.S. Keller IV, Jenna Bockman, Xiaowen Chen, Erica Nguyen, Alan T. Tang, Patricia Mericko-Ishizuka, Ivan Maillard, Mark L. Kahn

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Figure 5

UNC5B acts downstream of DLL4 to restrict endothelial migration by stabilizing cell-cell junctions.

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UNC5B acts downstream of DLL4 to restrict endothelial migration by stabi...
(A) Heatmap of significantly (Padj < 0.05) upregulated and downregulated genes comparing HUAECs treated with siDLL4 relative to siCTRL (n = 3 independent experiments per condition). (B) Expression of specific NOTCH, arterial, and venous genes in siDLL4-treated HUAECs relative to siCTRL. All genes are significant with an adjusted P < 0.05. (C) Immunofluorescence staining of arteries from E10.5 control and Hoxa13Cre;Dll4fl/fl placentas for αSMA (green), CD31 (red), and UNC5B (gray) and quantification of mean fluorescence intensity (n = 3–5 placentas per genotype, each data point represents the average of at least 3 vessels per placenta). Scale bars: 25 μm. (D) Heatmap of significantly (Padj < 0.05) upregulated and downregulated genes comparing HUAECs treated with siUNC5B relative to siCTRL (n = 3 independent experiments per condition). (E) Expression of specific NOTCH, arterial, and venous genes in siUNC5B-treated HUAECs relative to siCTRL. All genes are significant with an adjusted P < 0.05 unless denoted with n.s. (not significant). (F) Gene ontology (GO) analysis (biological process) of the 940 upregulated genes in siUNC5B-treated HUAECs related to cell migration and cell adhesion. Dotted line indicates the threshold for significance. (G) Phalloidin (F-actin, magenta) stained HUAECs treated with siCTRL, siDLL4, or siUNC5B following 24 hours of wound closure (n = 6 independent experiments per condition). Dotted lines demarcate leading edge. Scale bars: 200 μm. (H) Quantification of percent wound closure in siCTRL, siDLL4, and siUNC5B HUAECs after 24 hours. (I) Immunofluorescence staining of HUAECs treated with siCTRL, siDLL4, or siUNC5B for β-catenin (green), VE-cadherin (red), and F-actin (blue). White arrowheads point to continuous cell-cell junctions in siCTRL cells that are more frequently disrupted in siDLL4 and siUNC5B cells. Scale bars: 50 μm. (J) Quantification of mean fluorescence intensity of β-catenin and VE-cadherin (n= 4 independent experiments per condition). Data represent mean ± SD. A 1-way ANOVA with post hoc Tukey HSD was performed for statistical analysis.