En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Summary

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

Abstract

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.

Introduction

The heart is the first functional organ of a mammalian embryo. Around embryonic day (E) 7.5 in mice, bilateral precardiac mesoderm cells form the cardiac crescent in the ventral side¹.The cardiac crescent contains two populations of precardiac cells that include progenitors of the myocardium and the endocardium². Around E8.0, the cardiac precursors fuse in the midline, forming the primitive heart tube consisting of two epithelial tissues, the outer myocardium, and the inner endocardium, which is a specialized endothelium separated by an extracellular matrix named cardiac jelly. Later, at E8.5, the heart tube undergoes ri....

Protocol

Animal studies were approved by the Centro Nacional de Investigaciones Cardiovasculares (CNIC) Animal Experimentation Ethics Committee and by the Community of Madrid (ref. PROEX 155.7/20). All animal procedures conformed to EU Directive 2010/63EU and Recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enacted in Spanish law under Real Decreto 1201/2005.

1. Obtention of AVC and/or OFTs (adapted from Xiao et al.²⁵)

1. Set up crosses in the afternoon between female mTmG and male VE-Cadherin-Cre-ERT/+

Results

The data generated using this protocol show that it is possible to perform en face imaging of the endocardium of the AVC. The first objective was to analyze the cell shape of the endocardium during the formation of the valves at a cellular resolution (Figure 1). In order to highlight individual endocardial cells at E9.5, we used two transgenic mouse strains. (1) ROSA^mT/mG is a two-color fluorescent Cre reporter allele (tdTomato/mT and EGFP/mG) w.......

Discussion

The endocardium is an epithelial monolayer that covers the entire inner surface of the embryonic heart tube. During valve development, endocardial cells at the prospective valvular regions undergo EMT, thus endocardial cells transform and rearrange their cytoskeleton to delaminate from the endocardium toward the cardiac jelly. We and others have obtained relevant data about valve development in mouse embryos by analyzing transversal sections of E8.5 and E9.5 embryonic hearts, where the endocardium is shown as a row of ce.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-OH-Tamoxifen	Sigma Aldrich	H-6278
16 % Paraformaldheyde	Electron Microscopy Sciences	157-10	Dilute to 4% in water
anti-GFP	Aves Labs	FGP-1010
anti-VECadherin	BD Biosciences	555289
Goat anti-Chicken, Alexa Fluor 488	Thermo Fisher Scientific	A-11039
Goat Anti-Mouse Alexa Fluor 647	Jackson ImmunoResearch	115-605-174
DAPI	AppliChem	A4099,0005
Slides Superfrost PLUS	VWR	631-0108	25 mm x 75 mm x 1.0 mm
Triton X-100	Sigma Aldrich	X100-100ML
Tween 20	A4974,0500	AppliChem
Vectashield Mounting Medium	Vector Laboratories	H-1000-10