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.<sup c...

<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 ce...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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) w...

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 ...

This protocol allows the analysis of primitive endocardium in a planar orientation. We can analyze the distribution of endocardial cells, the anisotropy of other injunctions, and describe the shape of a single endocardial cell during valve development. Compared to the classical tissue sections, the en face imaging of the inner valve regenerative region allows the analysis of planar cell polarity and dynamics of endocardial cells during valve development in intact embryos.This method can be used to deepen the knowledge of planar cell polarity during cardio development. Begin by placing the uterus excised from euthanized pregnant mice in a Petri dish containing ice-cold PBT. Remove the individual deciduye from the uterus under a dissecting microscope using fine forceps.Using fine tweezers, make an incision in the white part of the decidua, which is where the embryo is. Remove it gently, avoid pulling, and transfer the embryos to a new Petri dish with fresh PBT using a P-1000 with the tip cut off, leaving a diameter large enough for an embryo to pass through without breaking. For genotyping, take a little piece of the yolk sac approximately 0.5 square millimeters or a piece of the tail from the posterior end, counting five to 10 somites toward the head and digest it in 100 milliliters of the appropriate buffer.Under the fume hood, transfer the embryos to ice cold 4%PFA in a two-milliliter microcentrifuge tube at four degrees Celsius. Fix the embryos from two hours to overnight at four degree Celsius on a nutator. Under the fume hood, remove the 4%PFA fixative from the microcentrifuge tubes without touching the embryos.Discard the PFA in an appropriate container. Wash the embryos five times in cold PBT for 10 minutes at room temperature. Using fine forceps, remove the heart and transfer it to a new 1.5-milliliter microcentrifuge tube with fresh PBT.Replace PBT with PBS Triton and wash the samples thrice, five minutes each, at room temperature on an orbital shaker. Replace PBS Triton with a blocking solution containing PBS Triton with 10%heat-inactivated bovine serum. Incubate from two hours to overnight at four degree Celsius on an orbital shaker.Replace the blocking solution with blocking solution containing primary antibody at the desired concentration. Incubate overnight on an orbital shaker at four degrees Celsius. After overnight incubation at four degrees Celsius, incubate the samples for 1.5 hours at room temperature on an orbital shaker.This step is crucial to increase the signal-to-noise ratio. Remove and keep the primary antibody solution at four degrees Celsius, up to three future experiments. Add sodium azide to a final concentration of 0.02%for best conservation.Then, wash the embryos thrice with PBS Triton for three minutes each. Wash the embryos with PBS Triton thrice for 30 minutes and keep them on an orbital shaker at four degrees Celsius. After the last wash, add one milliliter of blocking solution containing fluorescence-conjugated secondary antibody against the primary antibody host species.Then add DAPI to the solution and incubate the embryos overnight on an orbital shaker at four degrees Celsius. After the overnight incubation at four degrees Celsius, incubate the embryos for 1.5 hours at room temperature on an orbital shaker. This step is essential to increase the signal-to-noise ratio.Repeat the PBS Triton wash steps as mentioned in the text manuscript. Put the hearts on a 35-millimeter Petri dish containing PBS. Using a tungsten wire and fine forceps, isolate the atrioventricular canal or the outflow tract and cut them longitudinally.Prepare cover slips, slides, forceps, a P-200 pipette with the appropriate tips, a paper towel, and any aqueous glycerol-based mounting media for fluorescence without DAPI. Stick two strips of tape on a slide separated by 0.3 to 0.6 centimeters to create a 3D room space that will allow the samples to conserve their original shape without squashing them. Then, using approximately 20 microliters of buffer, carefully drop the samples onto the slide between the tape strips using a cut P-200 pipette tip.Use a dissecting microscope to increase the accuracy. Using fine forceps, place the samples with the endocardium facing up and the myocardium down on the slide. Remove excess PBS with a paper towel and avoid touching the sample.Then, dry the samples for one to two minutes at room temperature to make the samples stick to the slide. Add 40 microliters of aqueous-based mounting media to the tissue. Place the cover slip over the two pieces of tape and lower it slowly onto the tissue with a needle or forceps.Avoid creating air bubbles. Seal the cover slip using one drop of nail polish on each vertex of the cover slip. Once the nail polish is dry, clean the excess mounting media from the sides of the cover slip by using an absorbent paper towel.Avoid moving the cover slip. Add nail polish along the cover slip edges to fully seal it, preventing mounting media evaporation. Using an upright or an inverted confocal microscope, take images at 10X magnification for general view and at 63X with 3X zoom for detailed images.The cell shape of the endocardium was analyzed during the formation of the valves at a cellular resolution. Single cells or clones of a few cells expressed GFP in the endocardium. The GFP expression and combination with VE-cadherin subcellular localization was an effective approach to study the relationship between AJ dynamics and filopodia formation and pre-EMT cells, since these cells developed membrane protrusions as AJs dissolve.Differences in the intensity of VE-cadherin staining in the endocardium indicated the presence of anisotropic contractility in the embryonic endocardium of the atrioventricular canal at pre-valvular stages. The whole heart was stained with VE-cadherin antibody at E8.5 and E9.5. At E8.5, the endocardium of the atrioventricular canal tended to have a stable epithelial organization, and vertices formed by more than four cells were not detected.At E9.5, vertices of up to six cells were observed forming rosette-like structures, similar to the cellular organization previously described in epithelial undergoing active cell rearrangements, suggesting that the atrioventricular canal endocardium was undergoing dynamic cellular rearrangement during valve development. A learning curve is necessary to become an expert in this technique. Moreover, it is highly recommended to use Sovereign forceps and tungsten wire.Today, visualization of pre-valvular endocardium was limited to cross-sections. Our approach offers the possibility to study embryonic valvular endocardium behavior from a planar point of view.

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

Research

JoVE Journal

Developmental Biology

1.6K visualizações.  Centro Nacional de Investigaciones Cardiovasculares (CNIC). Este protocolo permite a análise do endocárdio primitivo em orientação plana. Podemos analisar a distribuição das células endocárdicas, a anisotropia de outras injunções e descrever a forma de uma única célula endocárdica durante o desenvolvimento valvar. Em comparação com as seções de tecido clássicas, a imagem en face da região regenerativa da válvula interna permite a análise da polaridade e dinâmica das células planares das células endocárdicas durante o desenvolv...