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.

<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

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. 
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

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 ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

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

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living 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 the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

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

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 ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...