This study was supported by grants PID2019-104776RB-I00 and CB16/11/00399 (CIBER CV) from MCIN/AEI/10.13039/501100011033 to J. L. P. J.G.-B. was funded by Programa de Atracci&#243;n de Talento from Comunidad de Madrid (2020-5&#170;/BMD-19729). T.G.-C. was funded by Ayudas para la Formaci&#243;n de Profesorado Universitario (FPU18/01054). We thank the CNIC Unit of Microscopy and Dynamic Imaging, CNIC, ICTS-ReDib, co-financed by MCIN/AEI /10.13039/501100011033 and FEDER &#34;A way to make Europe&#34; (#ICTS-2018-04-CNIC-16). We also thank A. Galicia and L. M&#233;ndez for mouse husbandry. The cost of this publication was supported in part by funds from the European Regional Development Fund. The CNIC is supported by the ISCIII, the MCIN, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S) financed by MCIN/AEI /10.13039/501100011033.

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)
<ol>
	<li>Set up crosses in the afternoon between female mTmG and male VE-Cadherin-Cre-ERT/+.</li>
	<li>The next morning, check for a vaginal plug. If there is a vaginal plug, count it as half a day (E0.5). Depending on the experiment of interest, count 8 days or 9 days.</li>
	<li>24 h before dissection, gavage the pregnant female orally with 50 &#181;L of 5 mg/mL 4-OH-Tamoxifen diluted in corn oil. 
	&#8203;NOTE: This dose will induce a limited amount of CRE-mediated recombination revealed by the presence of GFP-positive clones. The appropriate dose will have to be determined with each new batch of 4-OH-Tamoxifen.</li>
	<li>Sacrifice the pregnant female at E8.5 or E9.5 by cervical dislocation.</li>
	<li>Open the abdomen of the pregnant mice by using scissors and remove the uterus containing the embryos by using scissors and fine forceps.</li>
	<li>Place the uterus in a Petri dish containing ice-cold 1x phosphate-buffered saline (PBS) with 0.1% Tween-20 (PBT).</li>
	<li>Remove the individual deciduae from the uterus under a dissecting microscope and using fine forceps.</li>
	<li>Using fine tweezers, make an incision in the white part of the decidua, which is where the embryo is, remove it gently, avoiding pulling, and transfer the embryos to a new Petri dish with fresh PBT using a P1000 with the tip cut off, leaving a diameter large enough for an embryo to pass through without breaking.</li>
	<li>For genotyping, take a little piece of the yolk sac (0.5 mm2) or a piece of the tail (from the posterior end, counting 5 to 10 somites toward the head) and digest it in 100 &#181;L of appropriate buffer.</li>
	<li>Under the fume hood, transfer the embryos to ice-cold 4% paraformaldehyde (4% PFA) in sterile-filtered PBS in a microcentrifuge (2 mL) tube at 4 &#176;C. Fix the embryos from 2 h to overnight (O/N) at 4 &#176;C on a nutator.</li>
	<li>Under the fume hood, remove the 4% PFA fixative from the microcentrifuge tubes without touching the embryos. Discard the PFA in an appropriate container.</li>
	<li>Wash the embryos 5x in cold PBT for 10 min each at room temperature (RT).</li>
	<li>Using fine forceps, remove the heart and transfer it to a new 1.5 mL microcentrifuge tube&#160;with fresh PBT.</li>
</ol>
2. Whole mount immunofluorescence of the mouse embryonic heart
<ol>
	<li>Replace PBT with PBSTr (0.4% Triton X-100 in PBS) and wash the samples 3x, 5 min each, at RT on an orbital shaker.</li>
	<li>Replace PBSTr with blocking solution (PBSTr with 10% heat-inactivated bovine serum). Incubate from 2 h to O/N at 4 &#176;C on an orbital shaker.</li>
	<li>Replace blocking solution with blocking solution containing primary antibody at the desired concentration (for anti-GFP or anti-VE-Cadherin antibody,use 1:1000 dilution). Incubate O/N on an orbital shaker at 4 &#176;C.</li>
	<li>After O/N incubation at 4 &#176;C, incubate for 1.5 h at RT on an orbital shaker. This step is very important to increase the signal-to-noise ratio.</li>
	<li>Remove and keep at 4 &#176;C the primary antibody solution for up to three future experiments (add sodium azide to a final concentration of 0.02% for best conservation).</li>
	<li>Wash the embryos 3x with PBSTr for 3 min each.</li>
	<li>Wash the embryos with PBSTr 3x for 30 min each on an orbital shaker at 4 &#176;C.</li>
	<li>After the last wash, add 1 mL of blocking solution containing 1:1000 fluorescence-conjugated secondary antibody against the primary antibody host species. Add also 1:3000 DAPI to the solution and incubate the embryos O/N on an orbital shaker at 4 &#176;C.</li>
	<li>After O/N incubation at 4 &#176;C, incubate for 1.5 h at RT on an orbital shaker. This step is very important to increase the signal-to-noise ratio.</li>
	<li>Wash the embryos 3x with PBSTr for 3 min each.</li>
	<li>Wash the embryos with PBSTr 3x-5x for 30 min each on an orbital shaker at RT.</li>
</ol>
3. Mounting the mouse embryonic AVC or OFTs
<ol>
	<li>Put the hearts on a Petri dish (35 mm) containing PBS.</li>
	<li>Using a tungsten wire and fine forceps, isolate the AVC or the OFT, and cut them longitudinally26.</li>
	<li>Prepare coverslips (22 mm x 22 mm), slides (60 mm x 24 mm), forceps, a P200 pipette with the appropriate tips, paper towel, and any aqueous glycerol-based mounting media for fluorescence, without DAPI.</li>
	<li>Using a similar approach as in Xiao, C. et al.25, stick two strips of tape on a slide, separated by 0.3-0.6 cm in order to create a 3D room space that will allow the samples to conserve it original shape without squashing them.</li>
	<li>Carefully, and using the minimum volume of buffer, drop the samples onto the slide between the tape stripes using a cut P200 tip pipette. Use a dissecting microscope to increase the accuracy.</li>
	<li>Using fine forceps, place the samples with the endocardium facing up (myocardium down on the slide).</li>
	<li>Remove excess PBS with a paper towel (avoid touching the sample), and then dry the samples for 1-2 min at RT to make the samples stick to the slide.</li>
	<li>Add 40 &#181;L of aqueous-based mounting media on the tissue.</li>
	<li>Place the coverslip over the two pieces of tape and lower it slowly onto the tissue with a needle or forceps. Avoid creating air bubbles.</li>
	<li>Seal the coverslip using one drop of nail polish on each vertex of the coverslip.</li>
	<li>Once the nail polish is dry, clean the excess mounting media from the sides of the coverslip by using an absorbent paper towel. Avoid moving the coverslip.</li>
	<li>Add nail polish along the coverslip edges to fully seal it, preventing mounting media evaporation.</li>
	<li>Using an upright or an inverted confocal microscope, take images at 10x for general view and at 63x with 3x zoom for detailed images.</li>
</ol>

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The authors have nothing to disclose.

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 cells6,8,24,32. This approach allowed the analysis of the expression of signaling pathways and other molecules involved in valve development, but it was not possible to analyze spatial aspects of the pre-valvular endocardium.
AVC or OFT explants were mainly used to study transforming EC on a 3D collagen gel. Explants are useful for EMT quantification, the analysis of transformation capacity, or testing drugs after few days in culture6,7,11,33. These ex vivo experiments also allow the expansion of the endocardium on top of the collagen gel as a monolayer, but the environmental conditions in which ex vivo cardiac explants are grown differ from in utero conditions. For example, unidirectional blood flow is essential for proper valve development34,35 and is only achieved in in utero, not in ex vivo, explants. 
This novel approach allows us to observe the intact valvular endocardium en face during the onset of valve development without any environmental manipulation. It allows analysis of the cellular distribution of the valvular endocardium, as well as single-cell shape analysis before and during EMT in in utero conditions. We can also observe and analyze the intensity and organization of the cell-cell contacts and the subcellular distribution of relevant molecules during EMT. This technique also offers the possibility of analyzing subcellular structures of the endocardium, the activation of signaling pathways by using reporter genes, the effect that gene inactivation may have on cellular behavior, and how it may affect wild-type neighboring cells.
To obtain high-quality samples, it is important to be very careful when handling them, since impurities (e.g., microfibers) that stick to the tissue can result in artifacts during the capture of images. In addition, as they are very small samples, the risk of breaking is high, so one must wash the tissue (step 1. and step 2.) in slow shakers. During the change of media with pipettes, we recommend cutting off the tip to prevent the tissue from collapsing as it passes through the tip in the event that it is accidentally absorbed. To become an expert in this technique, a learning curve is necessary, which will be longer or shorter depending on the researcher's previous experience in micromanipulation.
Choosing the right fixative and time of fixation can be important in the case of the use of transgenes that express fluorescent proteins. The anatomical structure is not affected by different fixatives such as ethanol 70%, methanol, PFA 4%, or formalin 10%.
For counterstaining of nuclei, we recommend the incubation of samples with DAPI because penetration is much more efficient than using mounting media with DAPI.

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 rightward looping. The looped heart has different anatomical regions with specific molecular signatures such as the outflow tract (OFT), the ventricles, and the atrio-ventricular canal (AVC)3. Although initially the heart tube expands at its inflow side through the addition of cells4, at E9.5, intensive cardiac proliferation results in ballooning of the chambers and establishment of the trabecular network5. Valve formation takes place in the AVC (future mitral and tricuspid valves) and in the OFT (future aortic and pulmonary valves).
The endocardium plays crucial roles in valve development. Endocardial cells undergo epithelial-mesenchymal transition (EMT) in the AVC and OFT to form the endocardial cushions, a structure that appears at the onset of valve development. Different signaling pathways activate this process; at E9.5 in mice, NOTCH activated in the endocardium in response to myocardial-derived BMP2 promotes invasive EMT of endocardial cells in the AVC and OFT regions through activation of TGF&#946;2 and SNAIL (SNAI1), which directly represses the expression of vascular endothelial cadherin (VE-cadherin), a transmembrane component of adherens junctions (AJs)6,7,8. In the OFT, activation of the endocardium to initiate EMT is mediated by FGF8 and BMP4, whose expression is activated by NOTCH9,10,11,12.
Progression of EMT involves cellular dynamics as cells change shape, break and remake junctions with their neighbors, delaminate, and begin to migrate13. These changes include AJ remodel and gradual disassembling14,15, planar cell polarity (PCP) signaling, the loss of apico-basal polarity (ABP), apical constriction, and cytoskeletal organization16,17. ABP refers to the distribution of proteins along the anterior-posterior axis of a cell. In the developing heart, ABP regulation in cardiomyocytes is required for ventricular development18. PCP refers to a polarized distribution of proteins within cells across the plane of a tissue and regulates cellular distribution; epithelia with a stable geometry are made up of hexagon-shaped cells, where only three cells converge at the vertices19,20,21,22. Different cellular processes, such as cell division, neighbor exchange, or delamination occurring during epithelial morphogenesis, produce an increase in the number of cells that converge on a vertex and the number of neighboring cells that a given cell has22. These cellular behaviors related to PCP can be regulated by different signaling pathways, actin dynamics, or intracellular trafficking23.
The data generated studying valve development in mice have been obtained from transversal, coronal, or sagittal sections of E8.5 and E9.5 embryonic hearts, where the endocardium is shown as a line of cells instead of as a field of cells-the endocardium covers the entire inner surface of the heart tube24. Embryonic sections do not allow the analysis of PCP in the endocardium of mouse embryos. Our novel experimental method allows the analysis of endocardial cell distribution, AJ anisotropy, and single-cell shape analysis, as shown in the representative results. This type of data is required for PCP analysis, together with the description of other molecules related to PCP, not shown in this report. Whole-mount immunofluorescence, specific sample preparation and the use of genetically modified mice enable planar polarity analysis in the endocardium at the onset of valve development in mice.

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

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.

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) ROSAmT/mG is a two-color fluorescent Cre reporter allele (tdTomato/mT and EGFP/mG) whose fluorescence is detected at the cell membrane. Without Cre-mediated activation, the transgene expresses tdTomato (mT). Upon Cre-mediated activation, tdTomato expression is replaced by EGFP fluorescence expression (mG) in specific cells and clonal derivatives27. (2) The Cdh5CreERT2/+ mouse line expresses tamoxifen-inducible CRE-recombinase (CreERT2) under the regulation of the vascular endothelial cadherin promoter (Cdh5), which is expressed in the vascular endothelium and endocardium in the embryonic stages28. We crossed a male and a female of each genotype to obtain embryos carrying the two transgenes in order to activate the expression of GFP in individual cells of the endocardium. To achieve this, we inoculated low dosages of tamoxifen to activate CRE expression in a reduced number of cells. As a result, single cells, or clones of few cells, expressed GFP in the endocardium (Figure 1A). GFP expression (Figure 1A,B) in combination with VE-Cadherin subcellular localization (Figure 1C) is an effective approach to study the relationship between AJ dynamics and filopodia formation in pre-EMT cells since these cells develop membrane protrusions as AJs dissolve29. PCP signals can confer anisotropic contractility on the AJs, producing cellular forces that promote polarized morphogenesis in the epithelia30. We found differences in the intensity of VE-Cadherin staining in the endocardium (Figure 2), indicating that we cannot discard anisotropic contractility in the embryonic endocardium of AVC at pre-valvular stages.
The second objective was to analyze the planar topology of the endocardium during valve development (Figure 2). In order to define the number of cells converging in a single vertex, we stained the whole heart with VE-Cadherin antibody at E8.5 and E9.5 (Figure 2A,B). Other molecules localized at cell-cell contacts of the endocardial cell would also be useful for that purpose (e.g., &#946;-Catenin). At E8.5, the endocardium of the AVC tends to have a stable epithelial organization, and we could not detect vertices formed by more than four cells (Figure 2A,C). Later at E9.5, we found vertices made up of up to six cells forming rosette-like structures (Figure 2B,D), similar to the cellular organization previously described in epithelia undergoing active cell rearrangements31, suggesting that the AVC endocardium is undergoing active cellular rearrangement during valve development.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64207/64207fig01.jpg" /> 
Figure 1: Single-cell shape analysis in prevalvular endocardium. (A) AVC from E9.5 mouse embryo opened longitudinally showing scattered GFP positive cells. (B) GFP expression in single cells defines cell shape at cellular resolution. (C) Whole-mount staining of VE-Cadherin highlights AJs. Filopodia are generated in domains of the cell that do not localize VE-Cadherin (purple arrows), and vice versa, VE-Cadherin localizes in domains of the cell that are smooth and do not form filopodia (blue arrowheads). v, ventral; d, dorsal. Scale bar in A is 100 &#181;m; in B and C is 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64207/64207fig01large.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/64207/64207fig02.jpg" /> 
Figure 2: Planar topology and anisotropy of planarly localized proteins can be defined in en face images of endocardium at the AV canal. VE-Cadherin is localized at the cell-cell contacts of the endocardium.&#160;(A) E8.5 and (B)&#160;E9.5 AVC from embryos were opened longitudinally. Magnifications in (C) and (D)&#160;allowed us to observe the number of cells that converge in a vertex. In addition, we can distinguish different intensities in the VE-Cadherin staining, indicating that its location in the AVC is anisotropic. Scale bar in A and B is 100 &#181;m; in C and D is 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64207/64207fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos at JoVE.com

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.

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 ...

en-face-endocardial-cushion-preparation-for-planar-morphogenesis

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

1.7K Views.  Centro Nacional de Investigaciones Cardiovasculares (CNIC). Dit protocol maakt de analyse van primitief endocardium in een vlakke oriëntatie mogelijk. We kunnen de verdeling van endocardcellen analyseren, de anisotropie van andere injuncties, en de vorm van een enkele endocardiale cel beschrijven tijdens de ontwikkeling van de klep. Vergeleken met de klassieke weefselsecties maakt de enface-beeldvorming van het regeneratieve gebied van de binnenklep de analyse mogelijk van de polariteit van planaire cellen en de dynamiek van endocardcellen tijdens de...

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

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 ...

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 ...

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.

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 ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Biology

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 ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

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

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