Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

Summary

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

Abstract

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

Introduction

Chamber formation during heart development is a complex process transitioning through several morphologically distinct embryonic stages^1,2. The crescent shape of cardiac progenitor population cells forms a linear heart tube and then undergoes elongation and looping to form the spiral shape of the developing heart. After its septation process, the developing heart is transformed into the four-chambered heart. Interruption of any of these processes results in developmental heart defects. Thus, it is important to understand the molecular mechanisms underlying chamber formation during heart development. Despite nu....

Protocol

All animal procedures were performed with the approval of the Vanderbilt University Medical Center Institutional Animal Care and Use Committee.

1. Mouse embryo collection and dissection

1. Mate 8-10 week old female MLC-2v-tdTomato^+/- mice with 8-10 week old male MLC-2v-tdTomato^+/- mice to obtain MLC-2v-tdTomato^+/+, MLC-2v-tdTomato^+/- and MLC-2v-tdTomato^-/- embryos.
2. Check the dams for vaginal plugs every morning. Noon on the day of vaginal plug detection is considered as E0.5.
   NOTE: Vaginal examination for detecting a vaginal plug should be performed in the morn....

Results

During heart development, MLC-2v is considered to be the earliest marker for ventricular chamber specification¹⁷. As depicted in Figure 1, we dissected out mouse whole embryos and embryonic hearts from MLC-2v-tdTomato reporter knock-in mice and examined MLC-2v-tdTomato reporter expression during heart development. In MLC-2v-tdTomato reporter knock-in mice, constitutive tdTomato expression in the developing heart is visualized via epifl.......

Discussion

The method described here is relatively simple to examine ventricular chamber development, without performing labor-intensive experiments to label ventricular or cardiac-specific structural genes or proteins. Thus, this method minimizes technical variabilities that were often found in immunostained heart sections.

There are two critical steps for successfully performing this method including precise estimation of the embryonic age of mice and dissection of embryonic hearts. We practically esti.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
dissecting microscope	Leica	MZ125
DNA ladder (100 bp)	Promega	G2101
epifluorescence dissecting microscope	Leica	M165 FC
GoTaq Green master Mix	Promega	M712
PCR machine (master cycler)	Eppendorf	6336000023