Name: analysis of cardiac chamber development during mouse embryogenesis using whole mount epifluorescence
Uploaded: 2019-04-17T15:00:00.000+00:00
Duration: 6 min 27 s
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 the day of vaginal plug detection is considered as E0.5. 
	NOTE: Vaginal examination for detecting a vaginal plug should be performed in the morning (within 8-24 h after sexual activity), since a vaginal plug can be lost throughout the day.</li>
	<li>Euthanize the pregnant dams at different days post coitum (e.g., E8.5, E10.5, and E12.5) using CO2 inhalation followed by cervical dislocation.</li>
	<li>Lay the mice supine and spray 70% ethanol on the abdomen of mice to avoid mouse hair contamination during dissection.</li>
	<li>Open the abdominal cavity by incision of both the skin and abdominal wall using sharp surgical scissors and a forcep.</li>
	<li>Locate bilateral uterine horns in the dorsal part of the abdominal cavity.</li>
	<li>Separate the entire uterus by carefully cutting above the oviducts on both sides using sharp surgical scissors and a forcep.</li>
	<li>Place the entire dissected uterus in a 10 cm Petri dish with ice-cold PBS and carefully separate each amniotic sac along the uterine horn using sharp surgical scissors and a forcep.</li>
	<li>Transfer each embryo into individual wells of 6 well plate filled with ice-cold PBS using a transfer pipette.</li>
	<li>Under a dissecting microscope, open up an amniotic sac and expose each embryo by cutting off the umbilical cord using sharp surgical scissors and a forcep in an individual well of a 6 well plate with ice-cold PBS.</li>
	<li>Trim out extra-embryonic tissues as much as possible without damaging the embryo using sharp surgical scissors and a forcep. 
	NOTE: Whole mount epifluorescent imaging of whole mouse embryo is usually performed before dissecting the developing heart as described below.</li>
	<li>Cut the embryo head using sharp surgical scissors and a forcep and transfer to a 1.5 mL tube with 100 &#181;L of buffer A (25 mM NaOH and 0.2 mM EDTA) for genotyping to correlate with the results of epifluorescent imaging.</li>
	<li>Open the chest of the embryo using fine forceps, remove the heart away from the lungs and vasculature using sharp surgical scissors and forceps, and transfer the dissected embryonic heart into a well of a 12 well plate with PBS using a transfer pipette. All dissection procedures are completed under a dissecting microscope with a fiber optic microscope illuminator. 
	NOTE: It was technically difficult to dissect out mouse embryonic hearts at E8.0 or E8.5, because of their extremely small size and fragile structure. Early embryonic hearts (i.e. E8.0 and E8.5) can be examined within the whole mount embryo without dissection.</li>
</ol>
2. Whole-mount epifluorescence imaging
<ol>
	<li>Place the 12 well plate with mouse embryonic hearts under an epifluorescent dissecting microscope.</li>
	<li>Under an epifluorescent dissecting microscope using fine forceps, position the embryonic heart such that developing ventricles are located close to the examiner.</li>
	<li>Adjust focusing of the image using a 0.63x objective (zoom range between 3.15x and 18.9x) in bright field mode.</li>
	<li>Take bright field exposures and capture multiple images. The images were usually obtained by one second exposure. However, exposure times may vary depending on illumination and camera specificationss and need to be optimized for each setup.</li>
	<li>Turn off a fiber optic microscope illuminator, and set the filter for red fluorescence (Ex545 nm/Em 605 nm) to visualize tdTomato expression.</li>
	<li>Re-adjust focusing of the image if necessary.</li>
	<li>Adjust brightness and contrast, take red fluorescent exposures, and capture multiple images. 
	NOTE: The following image setting was usually used: 1 s exposure time, 2x gain, 1.0 saturation, and 1.0 gamma correction. The optimal setting needs to be optimized for each experiment. Once the optimal setting is determined, the same setting needs to be used for an entire experiment.</li>
</ol>
3. Genotyping
<ol>
	<li>Boil the samples from step 1.12 for 1 h at 100 &#176;C.</li>
	<li>Centrifuge for 2 min at 11,360 x g, transfer 20 &#181;L of supernatant into a new 1.5 mL tube with 20 &#181;L of buffer B (40 mM Tris HCl, pH 5.5) and mix them.</li>
	<li>Take 4.5 &#181;L of the mixed supernatant from step 3.2 as a DNA template, combine it with 0.5 &#181;L of each of the specific forward and reverse primers (10 &#181;M), 10 &#181;L of pre-mixed polymerase and reaction buffer (2x) (see Table of Materials), and then add water to a total volume of 20 &#181;L. Primer sequences are as below. 
	F1: 5&#8217;-TACCCACGGAGAAGAGAAGGACT-3&#8217; 
	R1: 5&#8217;-TGGACTTCTTGGAACTGACTCTGT-3&#8217; 
	F2: 5&#8217;-ACGGCACGCTGATCTACAAGGT-3&#8217; 
	R2: 5&#8217;-TTTGCGCACAGCCCTGGGAT-3&#8217;</li>
	<li>Run a polymerase chain reaction (PCR) with the following PCR program (Table 1 and Table 2).</li>
	<li>Run PCR samples and DNA ladder on a 1% agarose gel at 140 V in 1x TAE (Tris-acetate-EDTA) buffer (40 mM Tris-acetate and 1 mM EDTA) for 25 min. Use a 100 bp DNA ladder to estimate the size of the PCR bands.</li>
	<li>Place the DNA gel on a UV transilluminator to identify the DNA bands and turn on the UV light.</li>
</ol>

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The authors have nothing to disclose.

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 estimate the embryonic age of the mice by identifying vaginal plugs in female mice. However, the presence of a vaginal plug does not necessarily indicate pregnancy. It only indicates that sexual intercourse occurred within an approximate 8 to 24 h period. Even if vaginal plugs are found, it is often difficult to determine whether mice are indeed pregnant or not until 10-11 days post coitum by examining the abdomen of the female mice. Breeding multiple pairs of male and female mice is a safe breeding strategy to obtain the expected age of the mouse embryos. Isolating developing mouse hearts without significant damages to their structures is critical to accurately examine cardiac chamber development during mouse embryogenesis as illustrated in Figure 1. Careful dissection under a dissecting microscope and understanding developmental cardiac anatomy at each embryonic stage before dissection are necessary for successfully performing this protocol.
Since MLC-2v-tdTomato reporter expression is first observed at E8.0, it is not possible to examine the earlier embryonic heart tube or cardiac crescent stage12. The various reporter mouse lines labeling earlier cardiac markers (e.g. Mesp1Cre: Rosa26EYFP13, Isl1Cre: Rosa26EYFP13, Hcn4H2BGFP14, Hcn4Cre: Rosa mT/mG14, Nkx2-5Cre: Rosa mT/mG14, Hcn4-eGFP15, Isl1Cre: Rosa mT/mG14, and Nkx2.5Cre: Rosa26tdTomato15, TgMef2c-AHF-GFP16 mice) can be used to examine earlier stages of chamber development using the method described in this article.

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><tbody><tr><td>dissecting microscope</td><td>Leica</td><td>MZ125</td><td></td></tr><tr><td>DNA ladder (100 bp)</td><td>Promega</td><td>G2101</td><td></td></tr><tr><td>epifluorescence dissecting microscope</td><td>Leica</td><td>M165 FC</td><td></td></tr><tr><td>GoTaq Green master Mix</td><td>Promega</td><td>M712</td><td></td></tr><tr><td>PCR machine (master cycler)</td><td>Eppendorf</td><td>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 epifluorescence whole mount imaging as early as at E8.012 (Figure 2). Relatively weak expression of tdTomato in the linear heart tube at E8.0 becomes stronger at E8.5. At E10.5, MLC-2v-tdTomato reporter expression was demonstrated in the ventricular portion of the dissected looped heart from a whole mouse embryo, while it was not shown in the inflow tract, the outflow tract or the future atria. At E12.5-E13.5, whole mount epifluorescent imaging of the dissected heart of MLC-2v-tdTomato knock-in reporter mouse embryo showed that the tdTomato reporter is exclusively expressed in the ventricles of the four-chambered heart. The similar ventricular specific expression pattern of MLC-2v-tdTomato reporter was shown in the dissected mouse embryo at E16.5. Using this method, we can easily track down ventricular chamber formation during mouse heart development.
After whole mount epifluorescent imaging of mouse embryos or dissected developing hearts, we retrospectively confirmed the genotype of the embryo using the head of mouse embryos. Using two sets of primers as illustrated in Figure 3A, we performed PCR genotyping. The embryos carrying the wild type allele showed a 383 bp PCR product using the F1 and R1 primer set. The embryos carrying the tdTomato knock-in allele showed the 497 bp PCR product using the F2 and R2 primer set (Figure 3B). Heterozygous embryos were defined by demonstrating both the 383 bp and the 487 bp bands, while the wild type or homozygous genotype was determined by demonstrating a single 383 bp or 497 bp band, respectively.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59413/59413fig1.jpg" /> 
Figure 1. Outline of stepwise experimental procedure for whole mount epifluorescent imaging of the embryonic MLC-2v-tdTomato reporter mouse hearts. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59413/59413fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59413/59413fig2.jpg" /> 
Figure 2. Representative epifluorescent images of whole embryos and developing hearts dissected from MLC-2v-tdTomato reporter knock-in mice. Epifluorescent imaging of whole mount embryos and dissected hearts at different embryonic stages demonstrates specific expression of tdTomato in the ventricles of developing hearts.&#160; (A) Whole embryo at E8.0, (B) Whole embryo at E8.5, (C) Whole embryo at E9.0, (D) Whole embryo at E10.5, (E) Whole embryo at E13.5, (F) Whole embryo at E16.5, (G) Embryonic heart at E9.0, (H) Embryonic heart at E10.5, (I) Embryonic heart at E13.5, and (J) Embryonic heart at E16.5.&#160; A, atrium; V, ventricle; IFT, inflow tract; OFT, outflow tract. Scale bar (A&#8210;F) = 1 mm; Scale bar (G-J) = 500 &#181;m.&#160; This figure has been modified from reference #12 with permission. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59413/59413fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59413/59413fig3.jpg" /> 
Figure 3. Genotyping of MLC-2v-tdTomato reporter knock-in embryos. (A) Illustration of genotyping primer design. (B) Representative genotyping results using F1 and R1 primers (left) and F2 and R2 primers (right). +/+: homozygous, +/-: heterozygous, -/-: wild type. Exon: a segment of a gene that contains information required for protein synthesis; IRES: Internal ribosome entry site; FRT: flippase recombinase-recombination&#160;target. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59413/59413fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Temp&#160;&#176;C</td>
			<td>Time</td>
			<td>Note</td>
		</tr>
		<tr>
			<td>1</td>
			<td>94</td>
			<td>3 min</td>
			<td></td>
		</tr>
		<tr>
			<td>2</td>
			<td>94</td>
			<td>30 s</td>
			<td></td>
		</tr>
		<tr>
			<td>3</td>
			<td>60</td>
			<td>35 s</td>
			<td></td>
		</tr>
		<tr>
			<td>4</td>
			<td>72</td>
			<td>35 s</td>
			<td></td>
		</tr>
		<tr>
			<td>5</td>
			<td></td>
			<td></td>
			<td>repeat steps 2-4 for 38 cycles</td>
		</tr>
		<tr>
			<td>6</td>
			<td>72</td>
			<td>5 min</td>
			<td></td>
		</tr>
		<tr>
			<td>7</td>
			<td>10</td>
			<td>Hold</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: PCR program using F1 and R1 primers
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Temp&#160;&#176;C</td>
			<td>Time</td>
			<td>Note</td>
		</tr>
		<tr>
			<td>1</td>
			<td>94</td>
			<td>3 min</td>
			<td></td>
		</tr>
		<tr>
			<td>2</td>
			<td>94</td>
			<td>30 s</td>
			<td></td>
		</tr>
		<tr>
			<td>3</td>
			<td>61.7</td>
			<td>35 s</td>
			<td></td>
		</tr>
		<tr>
			<td>4</td>
			<td>72</td>
			<td>35 s</td>
			<td></td>
		</tr>
		<tr>
			<td>5</td>
			<td></td>
			<td></td>
			<td>repeat steps 2-4 for 38 cycles</td>
		</tr>
		<tr>
			<td>6</td>
			<td>72</td>
			<td>5 min</td>
			<td></td>
		</tr>
		<tr>
			<td>7</td>
			<td>10</td>
			<td>Hold</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: PCR program using F2 and R2 primers

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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7.5K Views.  Vanderbilt University Medical Center. 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.

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

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

Medicine

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...

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

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.

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

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.

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart ...

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe CHD died before adulthood, with the maximum mortality during the first week of life. Remarkable advances in surgical techniques, diagnostic approaches, and medical management have led to marked improvements in outcomes. To address the critical research needs of understanding congenital heart defects, murine models have provided an ideal research platform, as they have very similar heart anatomy to humans and short gestation rates. The combination of genetic engineering with high-throughput phenotyping tools has allowed for the replication and diagnosis of structural heart defects to further elucidate the molecular pathways behind CHDs. The use of noninvasive fetal echocardiography to screen the cardiac phenotypes in mouse models coupled with the high fidelity of Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) histopathology with three-dimensional (3D) reconstructions enables a detailed view into the anatomy of various congenital heart defects. This protocol outlines a complete workflow of these methods to obtain an accurate diagnosis of murine congenital heart defects. Applying this phenotyping protocol to model organisms will allow for accurate CHD diagnosis, yielding insights into the mechanisms of CHD. Identifying the underlying mechanisms of CHD provide opportunities for potential therapies and interventions.

This article details murine congenital heart disease (CHD) diagnostic methods using fetal echocardiography, necropsy, and Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) followed by three-dimensional (3D) reconstruction.

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe ...

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

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Rozdziały w tym wideo

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Murine Fetal Echocardiography

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

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

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse