We thank M.W. laboratory members for scientific discussion. This work is supported by AHA [13SDG16920099] to M.W., and by National Heart, Lung, and Blood Institute grants [R01HL121700] to M.W. Images were captured in the Imaging Core Facility at the Albany Medical College.

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Albany Medical College and performed according to the NIH Guide for the Care and Use of Laboratory Animals.
1. Solution Preparations
NOTE: The Rosa26CreERT2 15, R26R-Confetti16, &#945;MHC-Cre17,and Rosa26-mTmG (mTmG)20 mouse lines were purchased commercially. cTnT-Cre18 was a gift from Dr. Jiao at University of Alabama. Nfatc1-Cre was a gift from Dr. Bin Zhou at Albert Einstein College of Medicine19. Mice were euthanized by carbon dioxide inhalation in a closed chamber for at least 60 seconds followed by physical means of death verification by bilateral thoracotomy, decapitation or cervical dislocation.
<ol>
	<li>Prepare Tamoxifen. Dissolve 10 mg of Tamoxifen in 10 ml of sunflower seed oil. Once it is completely dissolved, aliquot and store at -20 &#176;C.</li>
	<li>Prepare phosphate buffered saline (PBS). Dissolve Na2HPO4 (1.41960 g), KH2PO4 (0.24496 g), NaCl (8.0669 g) and KCl (0.20129 g) in 1 L of distilled water and adjust the pH to 7.4.</li>
	<li>Prepare PBST:0.1% Tween-20 solution in PBS by dissolving 100 &#956;l of Tween-20 in 100 ml of PBS.</li>
	<li>Prepare blocking buffer. Make 3% bovine serum albumin (BSA) blocking solution by dissolving 3 g of BSA in 100 ml of PBST containing 0.2% Tween-20.</li>
	<li>Prepare CUBIC-1 (reagent 1). Mix 25 wt% urea, 25 wt% N, N, N&#39;, N&#39;-tetrakis (2-hydroxypropyl) ethylenediamine and 15 wt% Triton X-100 in dH2O.</li>
	<li>Prepare CUBIC-2 (reagent 2). Mix 50 wt% sucrose, 25 wt% urea, 10 wt% 2, 2&#8242;, 2&#8242;&#39;-nitrilotriethanol and 0.1% (v/v) Triton X-100 in dH2O.</li>
	<li>Prepare Scale A2: 4 M urea, 10% w/v glycerol and 0.1% w/v Triton X-100 in dH2O. Adjust the pH to 7.7.</li>
	<li>Prepare Scale B4: 8 M urea and 0.1% w/v Triton X-100 in dH2O. Adjust the pH to 8.7.</li>
	<li>Prepare Scale 70: 4 M urea, 70% w/v glycerol and 0.1% w/v Triton X-100 in dH2O.</li>
</ol>
2. Tamoxifen Induction, Embryo Isolation and Fixation
<ol>
	<li>Tamoxifen Induction and Embryo Isolation
	<ol>
		<li>Using a curved feeding tube approved protocol (ACUP 13-12007), gavage the female R26CreERT2 Confetti mice (plugged by R26CreERT2 male mice) with 10 &#181;g/g of Tamoxifen at embryonic developmental day E7.7533. At embryonic day 9.5 (E9.5) or E10.5, euthanize the female mice with CO2 and cervical dislocation.</li>
		<li>After verification of mouse death (see Section 1 Note), open the mouse abdominal cavity with scissors and dissect out the uterine horn, remove the uterine tube layers with the help of scissors and tweezers, and release the embryos from the uterine tube23,24.
		<ol>
			<li>Transfer the embryos to a petri dish containing PBS (pH 7.4). Under the dissecting microscope, remove the non-embryonic layers (chorion, yolk sac and amnion) with the help of tweezers.</li>
		</ol>
		</li>
		<li>Using tweezers and scissors, collect the female mouse and embryo tail samples for genotyping as previously reported22. Using a plastic pipette, transfer each embryo to a well of a 48 well plate containing 1 ml of 1x PBS.</li>
		<li>Under a fluorescent microscope, identify the GFP/RFP/CFP positive embryos via an inverted microscope with a camera. Peel away the pericardium using tweezers and rotate the heart/embryo with the help of curved tweezers to determine the number of fluorescent clones on both sides of the heart with the GFP/RFP/CFP filters. No cuts are needed.</li>
	</ol>
	</li>
	<li>After identifying GFP/RFP/CFP positive embryos, quickly wash the embryos twice (2 min each) with 1x PBS in the 48 well-plate on a plate shaker at room temperature (RT). This will remove excess blood.</li>
	<li>Fix the embryo/embryonic heart using 4% paraformaldehyde (PFA) at RT. The fixation time depends on the embryonic age and tissue size. For E9.5 fix the embryo for 2 hr at RT. The late embryonic stage embryos require longer fixation time, which can even be prolonged to 24 hr for postnatal heart. 
	CAUTION: Paraformaldehyde is toxic, avoid contact with skin, eyes and mucous membrane.</li>
	<li>After fixation, wash the embryo/heart in the 48 well-plate twice (10 min each) with 1x PBS on a plate shaker at RT. This removes the excess PFA from the embryo/heart. The embryos are then further processed for whole mount staining and tissue clearing.</li>
</ol>
3. Whole Mount Staining
NOTE: After PFA fixation the embryo/heart is subjected to whole mount staining as follows.
<ol>
	<li>Permeabilize the embryo/heart in a 48 well-plate using 1 ml of 0.1% PBST for 2 hr on plate shaker at RT.</li>
	<li>Following permeabilization, immerse the embryo/heart in 1 ml of blocking buffer, for 2 hr or overnight on a plate shaker at 4 &#186;C.</li>
	<li>Immerse the embryo/heart to the endothelial cell-specific antibody PECAM (CD31), diluted (1:100) in 0.3 ml blocking buffer for 48 hr on plate shaker at 4 &#186;C.</li>
	<li>Wash the embryo/heart using PBST 5 times, 30 min each at RT on plate shaker. This removes the excess non-specific bound primary antibody from the tissue.</li>
	<li>Immerse the embryo/heart in Alexa Fluor 647 goat anti-rat secondary antibody, diluted (1:1,000) in 1 ml blocking buffer for 48 hr on plate shaker at 4 &#186;C.</li>
	<li>Repeat step 3.4 to wash the secondary antibody.</li>
	<li>Post-fix the embryo/heart with 4% PFA for 1 hr at RT on plate shaker and wash twice with PBS 5 min each.</li>
	<li>Stain the embryo/heart with the nuclear stain 4&#8242;,6-diamidino-2-phenylindole (DAPI) (diluted in PBST at a concentration of 0.01 mg/mL) for 10 min on plate shaker at RT. Follow with two PBS washes of 5 min each.</li>
</ol>
4. Embryo/heart Clearing with Scale or CUBIC Method
NOTE: After whole mount staining, the embryo/heart is subjected to either the Scale or CUBIC tissue clearing method. The choice of method depends on the tissue size and embryonic age. For E11.5 or earlier age embryos, use Scale tissue clearing method, while for older embryos or postnatal hearts the CUBIC tissue clearing method is preferred.
<ol>
	<li>Scale Tissue Clearing Method
	<ol>
		<li>Incubate the embryo/heart with 1 ml of Scale A2 solution in a scintillation vial (wrapped in aluminum foil) with occasional gentle shaking (by hand) for 48 hr at 4 &#176;C.</li>
		<li>After Scale A2 incubation, incubate the embryo/heart with 1 mL of Scale B4 solution at 4 &#176;C in the same vial with occasional gentle shaking for another 48 hr.</li>
		<li>Incubate the embryo/heart for another 48 hr with 1 ml Scale 70 at 4 &#176;C with occasional gentle shaking. Mount embryo using 90% glycerol (see section 5.1).</li>
	</ol>
	</li>
	<li>CUBIC Tissue Clearing Method
	<ol>
		<li>For whole embryo/heart CUBIC clearing, immerse each embryo/heart in 1 ml of CUBIC-1 with occasional gentle shaking for 48 hr at 37 &#186;C7. After 48 hr, replace the solution with the same volume of fresh CUBIC reagent 1, and incubate the sample for an additional 48 hr at 37 &#176;C with occasional shaking with hand.</li>
		<li>Wash the CUBIC-1 treated heart/embryo with 1x PBS several times at room temperature with gentle shaking with hand. This removes the excess CUBIC-1 solution.</li>
		<li>After washing out CUBIC-1 with PBS, immerse the embryos/hearts in CUBIC-2 solution (1 g per embryo/heart) for 48 hr at 37 &#186;C with occasional gentle shaking with hand. This follows embryo/heart mounting using glycerol (see section 5.2).</li>
	</ol>
	</li>
</ol>
5. Sample Mounting and Imaging
NOTE: The cleared embryos or organs in Scale 70 or CUBIC-2 solution can be imaged directly with Scale 70 and CUBIC-2 solution as the imaging medium, respectively. However, considering the vulnerability of the embryonic samples to clearing reagents, if the samples are not be imaged immediately, but stored long-term, store the samples in 90% glycerol following the protocol below.
<ol>
	<li>Directly immerse the Scale cleared sample in 1 ml of 90% glycerol and store at 4 &#176;C until imaging.</li>
	<li>Wash the CUBIC treated sample with 1x PBS twice, 5 min each and sequentially incubate the sample with 1 ml of 30%, 50%, 70% and 90% glycerol solution each for 30 min.</li>
	<li>For imaging, transfer the sample to glass bottom Petri dish with a minimal amount of 90% glycerol and image clones with a confocal microscope equipped with two photon capabilities.</li>
	<li>Image the heart or embryo in the glycerol with a 10X/0.3 NA, a 25X/0.8 NA immersion, or a 40X/1.2 NA water corrective objective lens. Image DAPI using 2-photon excitation from a coherent titanium:sapphire chameleon laser. The choice of microscope and objective lens will depend on the purpose of the experiments, e.g. for super-resolution, a light sheet fluorescent microscope could be utilized.</li>
</ol>

<ol>
	<li>Vincent, S. D., Buckingham, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+heart:+the+origin+and+regulation+of+cardiac+progenitor+cells.">How to make a heart: the origin and regulation of cardiac progenitor cells.</a> Curr Top Dev Biol. 90, 1-41 (2010).</li><li>Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+discoveries+in+cardiac+biology.">A decade of discoveries in cardiac biology.</a> Nat Med. 10, 467-474 (2004).</li><li>Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+in+the+evolution+and+development+of+the+heart.">Gene regulatory networks in the evolution and development of the heart.</a> Science. 313, 1922-1927 (2006).</li><li>Bruneau, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+genetics+of+congenital+heart+disease.">The developmental genetics of congenital heart disease.</a> Nature. 451, 943-948 (2008).</li><li>Mohun, T. J., Weninger, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embedding+embryos+for+high-resolution+episcopic+microscopy+(HREM).">Embedding embryos for high-resolution episcopic microscopy (HREM).</a> Cold Spring Harb Protoc. , 678-680 (2012).</li><li>Soufan, A. T., 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=Three-dimensional+reconstruction+of+gene+expression+patterns+during+cardiac+development.">Three-dimensional reconstruction of gene expression patterns during cardiac development.</a> Physiol Genomics. 13, 187-195 (2003).</li><li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+Tissue+Clearing.">Clarifying Tissue Clearing.</a> Cell. 162, 246-257 (2015).</li><li>Susaki, E. 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=Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis.">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157, 726-739 (2014).</li><li>Becker, K., Jahrling, N., Saghafi, S., Weiler, R., Dodt, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+clearing+and+dehydration+of+GFP+expressing+mouse+brains.">Chemical clearing and dehydration of GFP expressing mouse brains.</a> PLoS One. 7, e33916 (2012).</li><li>Erturk, 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=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nat Protoc. 7, 1983-1995 (2012).</li><li>Ke, M. T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeDB:+a+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction.">SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nat Neurosci. 16, 1154-1161 (2013).</li><li>Chung, 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=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497, 332-337 (2013).</li><li>Yang, 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=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158, 945-958 (2014).</li><li>Hama, H., 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=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat Neurosci. 14, 1481-1488 (2011).</li><li>Badea, T. C., Wang, Y., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+noninvasive+genetic/pharmacologic+strategy+for+visualizing+cell+morphology+and+clonal+relationships+in+the+mouse.">A noninvasive genetic/pharmacologic strategy for visualizing cell morphology and clonal relationships in the mouse.</a> J Neurosci. 23, 2314-2322 (2003).</li><li>Livet, 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=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450, 56-62 (2007).</li><li>Agah, R., 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=Gene+recombination+in+postmitotic+cells.+Targeted+expression+of+Cre+recombinase+provokes+cardiac-restricted,+site-specific+rearrangement+in+adult+ventricular+muscle+in+vivo.">Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo.</a> J Clin Invest. 100, 169-179 (1997).</li><li>Jiao, 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=An+essential+role+of+Bmp4+in+the+atrioventricular+septation+of+the+mouse+heart.">An essential role of Bmp4 in the atrioventricular septation of the mouse heart.</a> Genes Dev. 17, 2362-2367 (2003).</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=Endocardial+cells+form+the+coronary+arteries+by+angiogenesis+through+myocardial-endocardial+VEGF+signaling.">Endocardial cells form the coronary arteries by angiogenesis through myocardial-endocardial VEGF signaling.</a> Cell. 151, 1083-1096 (2012).</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, 593-605 (2007).</li><li>Wu, 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=Epicardial+spindle+orientation+controls+cell+entry+into+the+myocardium.">Epicardial spindle orientation controls cell entry into the myocardium.</a> Dev Cell. 19, 114-125 (2010).</li><li>Zhao, C., 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=Numb+family+proteins+are+essential+for+cardiac+morphogenesis+and+progenitor+differentiation.">Numb family proteins are essential for cardiac morphogenesis and progenitor differentiation.</a> Development. 141, 281-295 (2014).</li><li>Kaufman, M. H., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The atlas of mouse development. , (1992).</li><li>Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo : a laboratory manual. , (2003).</li><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> Nat Rev Genet. 6, 826-835 (2005).</li><li>Kelly, R. G., Buckingham, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anterior+heart-forming+field:+voyage+to+the+arterial+pole+of+the+heart.">The anterior heart-forming field: voyage to the arterial pole of the heart.</a> Trends Genet. 18, 210-216 (2002).</li><li>Verzi, M. P., McCulley, D. J., De Val, S., Dodou, E., Black, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+right+ventricle,+outflow+tract,+and+ventricular+septum+comprise+a+restricted+expression+domain+within+the+secondary/anterior+heart+field.">The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field.</a> Dev Biol. 287, 134-145 (2005).</li><li>Ward, C., Stadt, H., Hutson, M., Kirby, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+of+the+secondary+heart+field+leads+to+tetralogy+of+Fallot+and+pulmonary+atresia.">Ablation of the secondary heart field leads to tetralogy of Fallot and pulmonary atresia.</a> Dev Biol. 284, 72-83 (2005).</li><li>Echelard, Y., Vassileva, G., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cis-acting+regulatory+sequences+governing+Wnt-1+expression+in+the+developing+mouse+CNS.">Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS.</a> Development. 120, 2213-2224 (1994).</li><li>Jiang, X., Rowitch, D. H., Soriano, P., McMahon, A. P., Sucov, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+the+mammalian+cardiac+neural.">Fate of the mammalian cardiac neural.</a> Developement. 127, 1607-1616 (2000).</li><li>Viragh, S., Challice, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+the+epicardium+and+the+embryonic+myocardial+circulation+in+the+mouse.">The origin of the epicardium and the embryonic myocardial circulation in the mouse.</a> Anat Rec. 201, 157-168 (1981).</li><li>Wu, M., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numb+family+proteins:+novel+players+in+cardiac+morphogenesis+and+cardiac+progenitor+cell+differentiation.">Numb family proteins: novel players in cardiac morphogenesis and cardiac progenitor cell differentiation.</a> Biomolecular concepts. 6, 137-148 (2015).</li><li>Li, 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=Single-Cell+Lineage+Tracing+Reveals+that+Oriented+Cell+Division+Contributes+to+Trabecular+Morphogenesis+and+Regional+Specification.">Single-Cell Lineage Tracing Reveals that Oriented Cell Division Contributes to Trabecular Morphogenesis and Regional Specification.</a> Cell reports. 15, 158-170 (2016).</li><li>Alkaabi, K. M., Yafea, A., Ashraf, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pH+on+thermal-+and+chemical-induced+denaturation+of+GFP.">Effect of pH on thermal- and chemical-induced denaturation of GFP.</a> Appl Biochem Biotechnol. 126, 149-156 (2005).</li><li>Captur, G., 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=Morphogenesis+of+myocardial+trabeculae+in+the+mouse+embryo.">Morphogenesis of myocardial trabeculae in the mouse embryo.</a> J Anat. , (2016).</li></ol>

The authors declare no competing financial interests.

The embryo isolation is a very critical step. E9.5 embryos are very fragile and small in size, so extra care should be taken not to damage the embryo/heart structures during isolation. The non-embryonic extra layers enveloping the embryo/heart should be removed carefully especially when imaging the whole embryo. This allows antibody and clearing mixture penetration deep inside the embryonic tissues, and also helps in removing the background signal when imaging. Multiple antibodies including antibodies against PECAM, acetylated &#945;-tubulin and integrin &#946;1 were examined and all were successfully detected (data not shown)33. This demonstrates that the molecular integrity, cellular integrity and antigen compatibility were not disrupted during this clearing process.
Transfer the embryo/heart using a wide mouth transfer pipette (cut the tip). This avoids damage during handling. The GFP/YFP/RFP/CFP expression pattern and number of clones should be carefully recorded in the heart to determine the appropriate dosage to label one clone per heart or any other organs. Due to the low dosage of Tamoxifen (10 &#956;g/g), sometimes the heart does not contain any clones or is difficult to detect any clones. Therefore, it is recommended to use the yolk sac to determine if the embryo has any GFP/YFP/RFP/CFP clones.
The embryo or heart fixation is a critical step, as over-fixation will result in a high background while under-fixation will result in poor staining. It is recommended to fix the E8.5-E10.5 whole embryo for 2 hr at RT on a plate shaker. While for E11.5-E15.5 embryos, the recommendation is to carefully isolate the heart and fix it with 4% PFA for 2 hr at RT. After ensuring to remove the layers surrounding the heart if processing the whole embryo, the E11.5- E13.5 embryos can also be fixed for 3-4 hr at RT but that requires increased tissue clearing time. The E16.5-P1 (postnatal day 1) hearts are fixed for 3-4 hr at RT on a plate shaker. When fixing the whole embryo, the fore and hind limbs should be removed as they hinder the heart imaging.
The samples should be treated with CUBIC and Scale in scintillation vials to minimize sample drying. We have observed better clearing of samples with the CUBIC tissue clearing method at 37 &#176;C, while Scale clearing is optimally done at 4 &#176;C. After imaging, the samples can be stored short-term in CUBIC-2 or Scale 70. The samples should always be covered with aluminum foil to prevent fluorescence bleaching.
Embryonic tissues and organs have less lipid and extracellular matrix deposition, which results in less clearing time. On the other hand, they are more vulnerable to the clearing reagents. The clearing reagents might affect the integrity of the tissue structure and antigen, resulting in the quenching of CFP, RFP and antibody conjugated fluorophore. Therefore, longer fixation times might prevent the quenching. The quenching of the fluorescent proteins could also be due to their sensitivity to chemicals and pH as previously reported34. Other chemical recipes, pH, fixatives, and incubation length should be explored to minimize the quenching of the fluorescent proteins.
The method of whole organ transparentizing or tissue clearing has revolutionized volumetric 3D imaging. The previous imaging method for cardiac morphogenesis, which involved the manual 2D or 3D reconstruction of serial sections, is time consuming and instrument demanding5,6. High resolution episcopic microscopy (HREM) in conjunction with 3D modeling can be applied to the study of the morphogenesis of the mouse heart and provides for excellent anatomic description of the trabecular architecture in the developing mouse embryo35. However, HREM does not allow the identification of the fluorescent protein labeled cells or antibody stained cells from the rest of cells35. Using the current protocol, with minimal effort and high cost effectiveness, we have successfully preserved the fluorescent signals in the embryonic heart using Scale or CUBIC tissue clearing methods (Figures 1-3). Combined with a low dose (10 &#956;g/g) of Tamoxifen to induce Cre recombination events and generate a few clones inside the whole embryo and in the heart, this system can be used to trace and study single cardiac progenitor cell differentiation and cellular behaviors in vivo (Figure 2A)33. In addition, combined with Cre mediated gene deletion or overexpression and Cre mediated single cell labeling, this imaging system can be applied to study genetic loss of function or gain of function on progenitor cell differentiation and cellular behavior during cardiac morphogenesis, and thus provides a robust tool to study the etiology of congenital heart defects.
The two-tissue clearing systems Sca/e and CUBIC used in this study showed different clearing efficiencies, which further differentiates the systems based on embryonic age, tissue thickness and duration of tissue clearing time. For early stage embryos E9.5-E10.5, the Sca/e system not only rendered the tissue clear but also protected the embryo morphology and preserved the fluorescent signals, while the CUBIC treatment resulted in embryo dissolution. This might be the result of additional aminoalcohols and a higher concentration of Triton X-100 in the CUBIC solutions8. For older hearts, the Sca/e system failed to clear the tissue completely even with a longer incubation time. The CUBIC clearing solution rendered the older tissue clear (Figure 3). However, increased incubation time resulted in weaker signals and only protected endogenous GFP, while RFP, CFP and antibody conjugated signals were lost. For this reason, a more optimized recipe of the clearing reagents and better incubation time would be required to volumetrically image hearts or organs with a greater spectrum of fluorescent labels.
This study demonstrated that one can label, trace, and image single cardiac clones in combination with a whole mount staining of endocardial/endothelial cell marker PECAM of the developing heart. The whole mount staining of PECAM and DAPI in this system not only helps to identify the cell types and cell morphology of the labeled cells inside a heart, but also allows the study of the clonal pattern, migration and behavior during cardiac morphogenesis. In Figure 2A we observed a clone with 8 cells in OFT. Using the number of cell present in this clone with reference to the labeling time, the cell proliferation rate can be easily calculated. In another study using this same system we identified the oriented cell division pattern and migration pattern of single cardiomyocyte during trabeculation process33. The application of this protocol to study the effects of genetic loss of function or gain of function spatiotemporally on the labeled cardiac progenitor cell differentiation, clonal pattern and cellular behavior is feasible, and will help in studies of the etiology of congenital heart defects at genetic and molecular level in a 3D context. In addition, this protocol can be applied to study of morphogenesis in other organ systems.

Cardiac morphogenesis is a sequential event that requires the spatiotemporal organization of four different types of cardiac progenitor cells into distinct sectors of the heart, and also requires multiple genetic regulatory networks to orchestrate this process to form the functional heart1,2. Cardiac specification, differentiation, patterning, and chamber maturation are regulated by cardiogenic transcription factors3. Genetic mutation or posttranscriptional aberration of these factors in cardiac progenitor cells could result in either embryonic lethality or congenital heart defects (CHD)4. The study of cardiac morphogenesis requires an understanding of inherent structural details in three dimensions (3D) and single labeled cardiac progenitor cell lineage tracing during cardiac development will promote the understanding of cardiac morphogenesis. A number of high-resolution section based tomography methods have been developed in the past few decades to image organ structure5,6; however, these methods require expensive, specialized instruments, extensive labor, and lack detailed structural organization at single cell resolution in the final volumetric reconstructed image7,8.
3D volumetric imaging at the single cell level provides a means to study progenitor cell differentiation and cellular behavior in vivo7. However, tissue light scattering remains the primary obstacle to imaging cells and structures in 3D deep inside the intact heart. Lipids are a major source of light scattering, and the removal of lipids and/or adjustment of the refractive index difference between lipids and their surrounding areas are potential approaches for increasing tissue transparency8. In the past several years, a number of tissue clearing methods were developed, which reduce tissue opacity and light scattering, like BABB (benzyl alcohol and benzyl benzoate mixture) and DBE (tetrahydrofuran and dibenzylether); but in these methods, fluorescence quenching remains an issue8-10. The solvent based hydrophilic methods, such as SeeDB (fructose/thioglycerol) and 3DISCO (dichloromethane/dibenzylether), preserve fluorescent signals, but do not render the whole organ transparent7,8,11. In comparison, the CLARITY tissue-clearing method renders the organ transparent, but it requires a specialized electrophoresis device to remove lipids8,12, as does PACT (passive clarity technique), which also requires hydrogel embedding7,13. For detailed information regarding all available tissue-clearing methods, refer to Table 1 in Richardson and Lichtman, et al.7.
In 2011, Hama et al. serendipitously discovered a hydrophilic mixture &#39;Scale&#39; (urea, glycerol and Triton X-100 mixture) that renders the mouse brain and embryo transparent while completely preserving fluorescent signals from labeled clones14. This allows for the imaging of the intact brain at a depth of several millimeters and large-scale reconstruction of neuronal populations and projections at a subcellular resolution. Susaki et al. further improved Scale by adding aminoalcohols and developed the &#39;CUBIC&#39; (clear, unobstructed brain imaging cocktails and computational analysis) tissue clearing method, which increased phospholipid solubilization, reduced clearing time, and allowed for multicolor fluorescent imaging8. In the present study, taking advantage of the Scale and CUBIC tissue clearing techniques and high resolution 3D optical sectioning, individual clones inside the heart during cardiogenesis were traced using Rosa26CreERT2 15, R26R-Confetti16, &#945;MHC-Cre17, cTnT-Cre18, Nfatc1-Cre19, and Rosa26-mTmG (mTmG)20 mouse lines. The combination of the whole mount staining (WMS) method developed previously21,22 with tissue clearing methods further allowed for the staining of other proteins in labeled clones and for the study of their behavior in a 3D volumetric context. The combination of tissue clearing and WMS allows for a better understanding of the roles of different genes and proteins during cardiac development, and the etiology of congenital heart defects. This protocol can be applied to study other progenitor cell differentiation, cellular behavior, and organ morphogenesis events during development.

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			<td>Zeiss</td>
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			<td height="21" style="height:21px;width:381px;">Germinator 500 Glass Bead Sterilizer</td>
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imaging cleared embryonic and postnatal hearts at single-cell resolution

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

Imaging the cleared embryonic heart
Vertebrate heart formation is a spatiotemporally regulated morphogenic process and depends on the organization and differentiation of progenitor cells from four different sources1. Cells from the first heart field of the cardiac crescent will fold toward the ventral midline to form a linear heart tube. The cells from the second heart field, initially residing dorsomedially to the first heart field, subsequently translocate to the pharyngeal and splanchnic mesoderm, from where they migrate to the pre-existing scaffold of the linear heart tube. The cells of second heart field will contribute to the right ventricle, outflow tract (OFT) myocardium and to some endocardium25-28. Cardiac neural crest cells (CNCC), originating from postotic rhombomeres 6, 7 and 8, will migrate to the caudal pharynx and contribute significantly to the smooth muscle layer and endocardial cushions in the OFT. They also contribute to the formation of the aorticopulmonary septum29,30. The fourth population consists of epicardial cells derived from the pro-epicardial organ (PEO), and contributes to fibroblasts, smooth muscle cells and potentially other cardiac cell types31. The epicardium regulates coronary vascular development, cardiac growth and morphogenesis21.
The most important concept about cardiac morphogenesis is that during heart development, abnormal cell migration/differentiation of the four progenitor cell populations will result in malformations or congenital heart defects4,22,32, which is the number one cause of birth defects in the world. Determining the mechanisms of cardiac morphogenesis at the cellular and molecular level is an essential step toward improving diagnosis and potential treatments for CHDs. Therefore, it is critical to image the process of heart morphogenesis at the whole heart level with single cell resolution. To achieve that, we labeled the cardiomyocytes by crossing the Cre line under the control of cardiac specific troponin T (cTnT), which is specifically expressed in cardiomyocytes18, with mTmG reporter line. The embryos at E8.5 were harvested and stained for PECAM to distinguish endocardial and endothelial cells from cardiomyocytes. The embryos were then cleared by Scale and the heart was imaged. The myocardium was labeled by GFP due to cTnT-driven Cre-mediated recombination of the mTmG reporter, and the endocardium was labeled with PECAM (Figure 1A and Supplementary Movie 1). The cardiomyocytes can be observed at a single cell resolution (Figure 1A). The heart structures of the imaged portion, such as the primitive ventricle and OFT can be clearly identified after 3D reconstruction using 3D reconstruction software (Figure 1B and Supplementary Movie 2).
Imaging single clones of the cleared embryonic heart
Cardiac morphogenesis depends on cardiac lineage differentiation and individual cell organization at the whole heart level22, and thus, it is essential to image single cardiac progenitor cell differentiation to different cardiac cell types and to also image cell morphology at single cell resolution. To achieve that, Rosa26CreERT2 15 was crossed to R26R-Confetti16, and the pregnant female was gavaged with Tamoxifen at a low concentration. After 48 hr, the embryo was harvested at E9.5, and whole mount stained for PECAM, and then the whole heart was imaged. We found that there was only one cluster of cells, localized to the OFT, and this clone had 8 cells based on the reconstructed image and consecutive sections (Figures 2A, 2B and Supplementary Movie 3), indicating that these cells are derived from a single progenitor cell. The cellular morphology and cellular behavior such as cellular proliferation and migration can be inferred from the ultimate positioning of the cells (Figure 2A). We also applied the CUBIC method to clear the embryonic heart at E9.5 and E10.5, and found that even after only 24 hr of incubation with reagent 1, the embryos were subtly dissolved and the tissue structure was adversely affected (Data not shown).
Imaging of cleared postnatal and late embryonic hearts
We applied both Scale and CUBIC to clear postnatal hearts. P2 hearts with the genotype of &#945;MHC-Cre; mTmG (&#945;MHC-Cre is specifically expressed in cardiomyocytes17) were cleared with Scale for 48 hr, but did not display any more transparency than the hearts treated with PBS only (Data not shown), indicating that Scale clearing is not effective for postnatal hearts. When P2 hearts were cleared with CUBIC reagent 1 for 48 hr, and reagent 2 for 96 hr, they were much more transparent than the hearts cleared with reagent 1 for 48 hr and reagent 2 for 48 hr. The imaging depth was significantly increased with 96 hr of reagent 2 incubation (Supplementary Movies 4 and 5), indicating that longer incubation with the CUBIC reagents promotes transparency and provides for an increase in imaging depth. The shape and size of each cardiomyocyte can be identified by the membrane localized GFP (Figure 3A), which can be used to identify hypertrophy.
We also cleared Nfatc1-Cre;Confetti (Confetti female plugged with Nfatc1Cre male) E17.5 hearts (Nfatc1-Cre is expressed in cardiac endocardial cells19) with CUBIC reagent 1 for 48 hr and reagent 2 for 48 hr. The hearts are transparent, and fluorescent proteins including GFP, YFP, CFP and RFP were expected to label the endocardial cells and their derived cells; however, only GFP and YFP could be imaged through the whole heart (Figures 3B, 3C and Supplementary Movies 6 and 7), and the CFP and RFP could not be detected (Data not shown). The staining for PECAM (Alexia Fluor 647) also could not be detected (Data not shown), suggesting that the CUBIC reagent quenches the CFP, RFP and the antibody conjugated fluorophore in this protocol.
<img alt="Figure 1" src="/files/ftp_upload/54303/54303fig1.jpg" /> 
Figure 1: Imaging the Sac/e cleared embryonic heart. (A) One optical slice of an E8.75 heart (cTnT-Cre;mTmG) is shown. V: ventricle; OFT: outflow tract. (B) Shows the reconstructed heart structure of 93 consecutive optical slices with a total depth of 110.4 &#181;m. Scale bar = 20 &#181;m in A. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54303/54303fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54303/54303fig2.jpg" /> 
Figure 2: Imaging single clone of Sca/e cleared embryonic heart. (A) One optical section of a clone from an E9.5 heart with the genotype of ROSA26-CreERT2; ROSA26-Confetti. The arrow points to a cellular protrusion of a cardiomyocyte. (B) Shows the reconstructed clone in the OFT region of the E9.5 heart with 10 optical slices and 36 &#181;m total depth. Scale bar = 20 &#181;m in A. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54303/54303fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54303/54303fig3.jpg" /> 
Figure 3: Imaging the CUBIC cleared postnatal and late embryonic hearts. (A) Shows one optical section of a P2 heart with the genotype of &#945;MHC-Cre;mTmG; all of the optical sections are shown in Supplementary Movie 5. (B) Shows one section of an E17.5 heart with the genotype of Nfatc1-Cre;ROSA26-Confetti. (C) Shows the reconstructed heart structure from 106 optical slices with a total depth of 630 &#181;m. The Scale bar = 20 &#181;m in A and is 100 &#181;m in B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54303/54303fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/54303/54303mov1.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54303/Suppl_Movie_1.mp4" target="_blank">Supplementary Movie 1: Imaging the Scale cleared embryonic heart (Right click to download). </a>Movie 1 shows optical sections of a Scale treated E8.75 heart (cTnT-Cre;mTmG) from heart surface to heart lumen. The depth is 110.4 &#181;m and there are 93 slices in total.
<img alt="Movie 2" src="/files/ftp_upload/54303/54303mov2.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54303/Suppl_Movie_2.mpg" target="_blank">Supplementary Movie 2: Imaging the Scale cleared embryonic heart (Right click to download). </a>Movie 2 shows the 3D surface picture of the reconstructed heart in Movie 1, reconstructed via the 3D reconstruction software. Yellow marks the PECAM expression and green labels all the cardiomyocytes.
<img alt="Movie 3" src="/files/ftp_upload/54303/54303mov3.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54303/Suppl_Movie_3.mp4" target="_blank">Supplementary Movie 3: Imaging a single clone of the Scale cleared embryonic heart (Right click to download). </a>Movie 3 shows sections of a clone from an E9.5 heart with the genotype of ROSA26-CreERT2.
<img alt="Movie 4" src="/files/ftp_upload/54303/54303mov4.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54303/Suppl_Movie_4.mp4" target="_blank">Supplementary Movie 4: Imaging the CUBIC cleared postnatal hearts (Right click to download). </a>Movie 4 shows sections of a CUBIC treated P2 heart (&#945;MHC-Cre;mTmG) from heart surface to heart lumen. This P2 heart was cleared with CUBIC reagent 1 for 48 hr, and CUBIC reagent 2 for 96 hr.
<img alt="Movie 5" src="/files/ftp_upload/54303/54303mov5.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54303/Suppl_Movie_5.mp4" target="_blank">Supplementary Movie 5: Imaging the CUBIC cleared postnatal hearts (Right click to download). </a>Movie 5 shows a heart that was cleared with reagent CUBIC 1 for 48 hr and reagent CUBIC 2 for 48 hr. The depth is 136 &#181;m with 6 &#181;m per section in movie 4 and the thickness is 76 &#181;m with 6 &#181;m per section in movie 5.
<img alt="Movie 6" src="/files/ftp_upload/54303/54303mov6.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54303/Suppl_Movie_6.mp4" target="_blank">Supplementary Movie 6: Imaging the CUBIC cleared late embryonic heart (Right click to download). </a>Movie 6 shows sections of a CUBIC treated E17.5 heart (Nfatc1-Cre;ROSA26-Confetti) from heart surface to heart lumen. The depth is 630 &#181;m with 6 &#181;m per section.
<img alt="Movie 7" src="/files/ftp_upload/54303/54303mov7.jpg" /> 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54303/Suppl_Movie_7.mpg" target="_blank">Supplementary Movie 7: Imaging the CUBIC cleared late embryonic heart (Right click to download). </a>Movie 7 shows the 3D surface picture of the heart in Movie 6, reconstructed via the 3D reconstruction software.

Watch this Scientific Journal Video about Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution at JoVE.com

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

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

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

imaging-cleared-embryonic-postnatal-hearts-at-single-cell

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.1K Views.  Albany Medical College. We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately. 

Video: Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

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

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

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

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

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Chapters in this video

Imaging Calcium in Drosophila at Egg Activation

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging