Name: analysis of cardiac chamber development during mouse embryogenesis using whole mount epifluorescence
Uploaded: 2019-04-17T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence at JoVE.com

This Work was supported by NIH R03 HL140264 (Y.-J. N) and Gilead Sciences Research Scholar Program (Y.-J. N).

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
<ol>
	<li>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.</li>
	<li>Check the dams for vaginal plugs every morning. Noon on ...

<ol>
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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...

Chamber formation during heart development is a complex process transitioning through several morphologically distinct embryonic stages1,2. 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 numerous previous studies on heart development, our understanding of this complex process remains limited.
In situ hybridization, immunohistochemistry, and beta-galactosidase staining using LacZ reporter mice have been widely used to study chamber formation during mouse heart development by labeling cardiac specific or chamber specific structural genes or proteins (e.g., Nppa, Coup-TFII, Irx4, MLC-2a and MLC-2v)3,4,5,6,7,8,9,10. However, these experiments using mouse embryos require significant time and expertise, because several different experimental steps have to be performed sequentially11. Here, we describe a simple whole mount epifluorescent microscopy method to visualize the developing ventricles using embryos dissected from MLC-2v-tdTomato reporter knock-in mice12. The advantage of this method compared to previously used methods is to avoid complex experimental steps which may often create experimental variations. The main purpose of this protocol is to describe how to dissect mouse embryos and developing hearts and to examine each stage of mouse cardiac chamber development without tedious histochemical experiments. This method can be easily applied to assess heart development using various other transgenic mouse lines labeling early cardiac markers (e.g., Mesp1Cre: Rosa26EYFP13, Isl1Cre: Rosa26EYFP13, Hcn4H2BGFP14, Hcn4Cre: Rosa mT/mG14, Nkx2-5Cre: Rosa mT/mG14, Hcn4-eGFP15, Isl1Cre: Rosa mT/mG14, Nkx2.5Cre: Rosa26tdTomato15, and TgMef2c-AHF-GFP16 mice).

<table border="0" cellpadding="0" cellspacing="0" style="width:647px;" width="647">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">dissecting microscope</td>
			<td style="width:83px;">Leica</td>
			<td style="width:119px;">MZ125</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DNA ladder (100 bp)</td>
			<td style="width:83px;">Promega</td>
			<td style="width:119px;">G2101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">epifluorescence dissecting microscope</td>
			<td style="width:83px;">Leica</td>
			<td style="width:119px;">M165 FC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">GoTaq Green master Mix</td>
			<td style="width:83px;">Promega</td>
			<td style="width:119px;">M712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">PCR machine (master cycler)</td>
			<td style="width:83px;">Eppendorf</td>
			<td style="width:119px;">6336000023</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of cardiac chamber development during mouse embryogenesis using whole mount epifluorescence

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.

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...

Watch this Scientific Journal Video about Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence at JoVE.com

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

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.

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

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