Name: en face endocardial cushion preparation for planar morphogenesis analysis in mouse embryos
Uploaded: 2022-07-27T14:30:00
Description: Watch this Scientific Journal Video about En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos at JoVE.com

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>
	<li>Buckingham, M., Meilhac, S., Zaffran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+mammalian+heart+from+two+sources+of+myocardial+cells.">Building the mammalian heart from two sources of myocardial cells.</a> Nature Reviews Genetics. 6 (11), 826-835 (2005).</li><li>Kelly, R. G., Buckingham, M. E., Moorman, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+fields+and+cardiac+morphogenesis.">Heart fields and cardiac morphogenesis.</a> Cold Spring Harbour Perspectives in Medicine. 4 (10), 015750 (2014).</li><li>Ivanovitch, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=outflow+tract+heart+progenitors+arise+from+spatially+and+molecularly+distinct+regions+of+the+primitive+streak.">outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak.</a> PLoS Biology. 19 (5), 3001200 (2021).</li><li>Rochais, F., Mesbah, K., Kelly, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+controlling+second+heart+field+development.">Signaling pathways controlling second heart field development.</a> Circulation Research. 104 (8), 933-942 (2009).</li><li>Moorman, A. F., Christoffels, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+chamber+formation:+Development,+genes,+and+evolution.">Cardiac chamber formation: Development, genes, and evolution.</a> Physiological Reviews. 83 (4), 1223-1267 (2003).</li><li>Timmerman, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+promotes+epithelial-mesenchymal+transition+during+cardiac+development+and+oncogenic+transformation.">Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation.</a> Genes &amp; Development. 18 (1), 99-115 (2004).</li><li>Luna-Zurita, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+a+Notch-dependent+mesenchymal+gene+program+and+Bmp2-driven+cell+invasiveness+regulates+murine+cardiac+valve+formation.">Integration of a Notch-dependent mesenchymal gene program and Bmp2-driven cell invasiveness regulates murine cardiac valve formation.</a> Journal of Clinical Investigation. 120 (10), 3493-3507 (2010).</li><li>Papoutsi, T., Luna-Zurita, L., Prados, B., Zaffran, S., de la Pompa, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmp2+and+Notch+cooperate+to+pattern+the+embryonic+endocardium.">Bmp2 and Notch cooperate to pattern the embryonic endocardium.</a> Development. 145 (13), (2018).</li><li>MacGrogan, D., Luna-Zurita, L., de la Pompa, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+in+cardiac+valve+development+and+disease.">Notch signaling in cardiac valve development and disease.</a> Birth Defects Research Part A: Clinical and Molecular Teratology. 91 (6), 449-459 (2011).</li><li>de la Pompa, J. L., Epstein, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinating+tissue+interactions:+Notch+signaling+in+cardiac+development+and+disease.">Coordinating tissue interactions: Notch signaling in cardiac development and disease.</a> Developmental Cell. 22 (2), 244-254 (2012).</li><li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+a+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue.</a> Developmental Cell. 95 (1), 108-114 (1983).</li><li>Wu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nfatc1+coordinates+valve+endocardial+cell+lineage+development+required+for+heart+valve+formation.">Nfatc1 coordinates valve endocardial cell lineage development required for heart valve formation.</a> Circulation Research. 109 (2), 183-192 (2011).</li><li>Amack, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+dynamics+of+EMT:+Lessons+from+live+in+vivo+imaging+of+embryonic+development.">Cellular dynamics of EMT: Lessons from live in vivo imaging of embryonic development.</a> Cell Communication and Signaling. 19 (1), 79 (2021).</li><li>Cano, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+snail+controls+epithelial-mesenchymal+transitions+by+repressing+E-cadherin+expression.">The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression.</a> Nature Cell Biology. 2 (2), 76-83 (2000).</li><li>Batlle, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+snail+is+a+repressor+of+E-cadherin+gene+expression+in+epithelial+tumour+cells.">The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells.</a> Nature Cell Biology. 2 (2), 84-89 (2000).</li><li>Weng, M., Wieschaus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin-dependent+remodeling+of+adherens+junctions+protects+junctions+from+Snail-dependent+disassembly.">Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly.</a> Journal of Cell Biology. 212 (2), 219-229 (2016).</li><li>Weng, M., Wieschaus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+protein+Par3/Bazooka+follows+myosin-dependent+junction+repositioning.">Polarity protein Par3/Bazooka follows myosin-dependent junction repositioning.</a> Developmental Biology. 422 (2), 125-134 (2017).</li><li>Jimenez-Amilburu, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualization+of+cardiomyocyte+apicobasal+polarity+reveals+epithelial+to+mesenchymal-like+transition+during+cardiac+trabeculation.">In vivo visualization of cardiomyocyte apicobasal polarity reveals epithelial to mesenchymal-like transition during cardiac trabeculation.</a> Cell Reports. 17 (10), 2687-2699 (2016).</li><li>Davey, C. F., Moens, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+cell+polarity+in+moving+cells:+Think+globally,+act+locally.">Planar cell polarity in moving cells: Think globally, act locally.</a> Development. 144 (2), 187-200 (2017).</li><li>Grego-Bessa, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumor+suppressor+PTEN+and+the+PDK1+kinase+regulate+formation+of+the+columnar+neural+epithelium.">The tumor suppressor PTEN and the PDK1 kinase regulate formation of the columnar neural epithelium.</a> Elife. 5, 12034 (2016).</li><li>Jones, C., Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+cell+polarity+signaling+in+vertebrates.">Planar cell polarity signaling in vertebrates.</a> Bioessays. 29 (2), 120-132 (2007).</li><li>Mahaffey, J. P., Grego-Bessa, J., Liem, K. F., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofilin+and+Vangl2+cooperate+in+the+initiation+of+planar+cell+polarity+in+the+mouse+embryo.">Cofilin and Vangl2 cooperate in the initiation of planar cell polarity in the mouse embryo.</a> Development. 140 (6), 1262-1271 (2013).</li><li>Devenport, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+morphodynamics:+Translating+planar+polarity+cues+into+polarized+cell+behaviors.">Tissue morphodynamics: Translating planar polarity cues into polarized cell behaviors.</a> Seminars in Cell and Developmental Biology. 55, 99-110 (2016).</li><li>Del Monte, G., Grego-Bessa, J., Gonzalez-Rajal, A., Bolos, V., De La Pompa, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+Notch1+activity+in+development:+Evidence+for+a+feedback+regulatory+loop.">Monitoring Notch1 activity in development: Evidence for a feedback regulatory loop.</a> Developmental Dynamics. 236 (9), 2594-2614 (2007).</li><li>Xiao, C., Nitsche, F., Bazzi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+node+and+notochordal+plate+in+gastrulating+mouse+embryos+using+scanning+electron+microscopy+and+whole+mount+immunofluorescence.">Visualizing the node and notochordal plate in gastrulating mouse embryos using scanning electron microscopy and whole mount immunofluorescence.</a> Journal of Visualized Experiments. (141), e58321 (2018).</li><li>Mahler, G., Gould, R., Butcher, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+avian+embryonic+valvular+progenitor+cells.">Isolation and culture of avian embryonic valvular progenitor cells.</a> Journal of Visualized Experiments. (44), e2159 (2010).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45 (9), 593-605 (2007).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephrin-B2+controls+VEGF-induced+angiogenesis+and+lymphangiogenesis.">Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis.</a> Nature. 465 (7297), 483-486 (2010).</li><li>Yilmaz, M., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMT,+the+cytoskeleton,+and+cancer+cell+invasion.">EMT, the cytoskeleton, and cancer cell invasion.</a> Cancer and Metastasis Reviews. 28 (1-2), 15-33 (2009).</li><li>Nishimura, T., Honda, H., Takeichi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+cell+polarity+links+axes+of+spatial+dynamics+in+neural-tube+closure.">Planar cell polarity links axes of spatial dynamics in neural-tube closure.</a> Cell. 149 (5), 1084-1097 (2012).</li><li>Blankenship, J. T., Backovic, S. T., Sanny, J. S., Weitz, O., Zallen, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+rosette+formation+links+planar+cell+polarity+to+tissue+morphogenesis.">Multicellular rosette formation links planar cell polarity to tissue morphogenesis.</a> Developmental Cell. 11 (4), 459-470 (2006).</li><li>Prados, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+Bmp2+gain+causes+ectopic+EMT+and+promotes+cardiomyocyte+proliferation+and+immaturity.">Myocardial Bmp2 gain causes ectopic EMT and promotes cardiomyocyte proliferation and immaturity.</a> Cell Death and Disease. 9 (3), 399 (2018).</li><li>Camenisch, T. D., Biesterfeldt, J., Brehm-Gibson, T., Bradley, J., McDonald, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cardiac+cushion+development+by+hyaluronan.">Regulation of cardiac cushion development by hyaluronan.</a> Experimental &amp; Clinical Cardiology. 6 (1), 4-10 (2001).</li><li>Courchaine, K., Rykiel, G., Rugonyi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+blood+flow+on+cardiac+development.">Influence of blood flow on cardiac development.</a> Progress in Biophysics and Molecular Biology. 137, 95-110 (2018).</li><li>Goddard, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+forces+sculpt+developing+heart+valves+through+a+KLF2-WNT9B+paracrine+signaling+axis.">Hemodynamic forces sculpt developing heart valves through a KLF2-WNT9B paracrine signaling axis.</a> Developmental Cell. 43 (3), 274-289 (2017).</li></ol>

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.

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			<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>
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		<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>
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			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
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			<td>Tween 20</td>
			<td>A4974,0500</td>
			<td>AppliChem</td>
			<td></td>
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		<tr>
			<td>Vectashield Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000-10</td>
			<td></td>
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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 ...

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During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Wholemount In Situ Hybridization for Astyanax Embryos

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...