Constitutively active Notch1 signaling promotes endothelial‑mesenchymal transition in a conditional transgenic mouse model - PubMed (original) (raw)
Constitutively active Notch1 signaling promotes endothelial‑mesenchymal transition in a conditional transgenic mouse model
Ju Liu et al. Int J Mol Med. 2014 Sep.
Abstract
Endothelial-mesenchymal transition (EndoMT) is a process in which endothelial cells lose their cell-type‑specific characteristics and gain a mesenchymal cell phenotype. The Notch signaling pathway is crucial in the regulation of EndoMT; however, its roles have not been fully studied in vivo. In a previous study, we reported the generation of transgenic mice with a floxed β-geo/stop signal between a CMV promoter and the constitutively active intracellular domain of Notch1 (IC-Notch1) linked with a human placental alkaline phosphatase (hPLAP) reporter (ZAP-IC-Notch1). In this study, we examined the results of activating IC-Notch1 in endothelial cells. ZAP-IC‑Notch1 mice were crossed with Tie2-Cre mice to activate IC-Notch1 expression specifically in endothelial cells. The ZAP-IC-Notch1/Tie2-Cre double transgenic embryos died at E9.5-10.5 with disruption of vasculature and enlargement of myocardium. VE-cadherin expression was decreased and EphrinB2 expression was increased in the heart of these embryos. Mesenchymal cell marker α-smooth muscle actin (SMA) was expressed in IC-Notch1‑expressing endothelial cells. In addition, upregulation of Snail, the key effector in mediating EndoMT, was identified in the cardiac cushion of the double transgenic murine embryo heart. The results of the present study demonstrate that constitutively active Notch signaling promotes EndoMT and differentially regulates endothelial/mesenchymal cell markers during cardiac development.
Figures
Figure 1
Strategy for Cre-conditional IC-Notch1 expression. The floxed β geo/stop signal was placed between a CMV promoter and the constitutively active intracellular domain of Notch1 (IC-Notch1). The reporter coding sequence of human placental alkaline phosphatase (hPLAP) fused with the internal ribosomal entry site (IRES) was placed downstream of the IC-Notch1 cDNA to allow the co-expression of IC-Notch1 and hPLAP from the same transcript. In the endothelial cells of double transgenic embryos, Tie2-Cre transgene expresses Cre recombinase, which eradicates the β geo/stop signaling sequence. IC-Notch1 expression is then driven by the CMV promoter and detected through visualization of hPLAP.
Figure 2
Constitutively active Notch1 signaling in endothelial cells causes defects in cardiovascular development. (A and C) E9.5 wild-type embryo (A) with and (C) without yolk sac. (B and D) E9.5 ZAP-IC-Notch1/Tie2-Cre double transgenic embryo showed (C) disorganized vasculature in the yolk sac and (D) an enlarged heart with hemorrhage in the embryo. The rectangular box refers to the enlarged heart. (E and F) C and D after whole-mount alkaline phosphatase staining.
Figure 3
Endothelial-specific expression of intracellular domain of Notch1 (IC-Notch1) disrupts embryonic vasculature. Platelet endothelial cell adhesion molecule-1 (PECAM-1) immunostaining of sagittal sections of (A) normal E9.5 embryo and (C) yolk sac as well as (B) ZAP-IC-Notch1/Tie2-Cre double transgenic embryo and (D) yolk sac. DA, dorsal aorta; S, somite.
Figure 4
Intracellular domain of Notch1 (IC-Notch1) expression in endocardium promotes endothelial-mesenchymal transition (EndoMT) in the embryo heart. (A and B) Alkaline phosphatase staining of sections of E9.5 wild-type (A) and ZAP-IC-Notch1/Tie2-Cre double transgenic embryo heart (B). Thin arrow refers to positively stained cells on endocardium, thick arrows refer to positive cells in myocardium. (C) Alkaline phosphatase staining of a blood vessel on a section of a ZAP-IC-Notch1/Tie2-Cre double transgenic embryo. Thin arrow refers to positively stained endothelial cells on the blood vessel. (D) α-smooth muscle actin (SMA) immunostaining on the serial section of C. Thick arrow refers to positively stained endothelial cells on the blood vessel.
Figure 5
Expression of endothelial-mesenchymal transition (EndoMT)-related proteins in the ZAP-IC-Notch1/Tie2-Cre embryo heart. (A) Semi-quantitative PCR of EndoMT-related genes of RNA extracted from wild-type (WT) and ZAP-IC-Notch1/Tie2-Cre mouse embryo hearts. (B) Representative immunoblots of EndoMT-related proteins examined in protein extracts from WT and ZAP-IC-Notch1/Tie2-Cre mouse embryo hearts.
Figure 6
Intracellular domain of Notch1 (IC-Notch1) promotes Snail and EprinB2 expression. (A) Schematic image of endothelial-mesenchymal cell transition. (B-E) Sagittal section of E9.5 embryo heart ventricle: (B) Snail immunostain on wild-type and (C) ZAP-IC-Notch1/Tie2-Cre embryo; (D) EprinB2 immunostain on wild-type and (E) ZAP-IC-Notch1/Tie2-Cre embryo. Dark arrow refers to endocardium, single arrow refers to mesenchymal cells. A, atrium; V, ventricle.
References
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