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 6

UNC5B controls endothelial chemokine signaling to promote smooth muscle cell migration.

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UNC5B controls endothelial chemokine signaling to promote smooth muscle ...
(A and B) Volcano plots of siDLL4- and siUNC5B-treated HUAECs compared with siCTRL restricted to genes with Padj < 0.05 and log2(Fold Change) < –0.5 and > 0.5. (C) Venn diagrams demonstrating number of shared upregulated and downregulated differentially expressed genes (DEGs) between siDLL4- and siUNC5B-treated HUAECs. (D) Gene ontology (GO) analysis (molecular function) of the 70 shared downregulated DEGs. Dotted line indicates the threshold for significance. (E) Expression of genes contained within chemokine-related GO terms for siDLL4- and siUNC5B-treated HUAECs relative to siCTRL (dotted line) (n = 3 independent experiments per condition). All genes are significant with an adjusted P < 0.05. (F and G) Immunofluorescence staining and RNA-FISH of arteries from E10.5 control and Hoxa13Cre;Dll4fl/fl placentas for αSMA (green), CD31 (red), and Cxcl12 (F) or Cxcr4 (G) (gray, RNA) and quantification of mean fluorescence intensity (n = 6–9 placentas per genotype, each data point represents the average of at least 3 vessels per placenta). Yellow arrowheads point to Cxcl12-expressing vSMCs. White arrowheads point to Cxcl12- and Cxcr4-expressing ECs. Scale bars: 25 μm. (H) Schematic of workflow for conditioned media (CM) experiments (n = 4 independent experiments per condition). HUAEC, human umbilical artery endothelial cell; HUASMC, human umbilical artery smooth muscle cell; HUAEC-CM, HUAEC-conditioned media. (I) Phalloidin staining and fluorescence of HUASMC migration experiments. Phalloidin was pseudo colored to red for t=0 hrs and cyan for t=24 hrs then overlayed. Dotted red line shows cell edge at t=0 hrs, and dotted cyan line shows cell edge at t=24 hrs. Scale bars: 50 μm. (J) Quantification of HUASMC migration cultured in siCTRL HUAEC-CM or siUNC5B HUAEC-CM after 24 hours. Data represent mean ± SD. An unpaired t test was performed for statistical analysis.