Name: imaging cleared embryonic and postnatal hearts at single-cell resolution
Uploaded: 2016-10-07T13:00:00.000+00:00
Duration: 7 min 30 s
Description: Watch this Scientific Journal Video about Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution at JoVE.com

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>

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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, acet...

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.

<table><tbody><tr><td>2,2&amp;prime;,2&amp;prime;&amp;rsquo;-nitrilotriethanol&amp;nbsp;</td><td>Sigma Aldrich</td><td>90279</td><td></td></tr><tr><td>4% Paraformaldehyde in PBS</td><td>Affymetrix</td><td>19943</td><td></td></tr><tr><td>BSA</td><td>Fischer Scientific&amp;nbsp;</td><td>BP16000</td><td></td></tr><tr><td>N,N,N&amp;rsquo;,N&amp;rsquo;-tetrakis(2-hydroxypropyl) ethylenediamine&amp;nbsp;</td><td>Sigma Aldrich</td><td>122262</td><td></td></tr><tr><td>Phosphate Buffer Saline</td><td>Sigma Aldrich</td><td>P5368-10PAK</td><td></td></tr><tr><td>Triton&amp;nbsp;X-100</td><td>Sigma Aldrich</td><td>T8787</td><td></td></tr><tr><td>Urea</td><td>Sigma Aldrich</td><td>U-1250</td><td></td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>84097</td><td></td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G8773</td><td></td></tr><tr><td>Tamoxifen</td><td>Sigma Aldrich</td><td>T5648</td><td></td></tr><tr><td>Sunflower seed oil</td><td>Sigma Aldrich</td><td>S5007</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P1379</td><td></td></tr><tr><td>PECAM (CD31)</td><td>BD Pharmingen&amp;nbsp;</td><td>550274</td><td></td></tr><tr><td>Alexa Fluor 647&amp;nbsp;</td><td>Invitrogen</td><td>A-21247</td><td></td></tr><tr><td>DAPI nuclear stain</td><td>Sigma Aldrich</td><td>D9542</td><td></td></tr><tr><td>37&lt;sup&gt;o&lt;/sup&gt;C Incubator</td><td>Thermoscientific Fischer</td><td>Heratherm, Compact Microbiological Incubators</td><td></td></tr><tr><td>48 well plates</td><td>Cell Treat</td><td>229148</td><td></td></tr><tr><td>Analytical Balance</td><td>Metler Toledo</td><td>PB153-S/FACT</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>Zeiss 510 confocal microscope</td><td></td></tr><tr><td>Disecting Microscope</td><td>Unitron</td><td>Z850</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Zeiss</td><td>Observer. Z1</td><td></td></tr><tr><td>Germinator 500 Glass Bead Sterilizer</td><td>CellPoint Scientific</td><td>GER-5287-120V</td><td></td></tr><tr><td>Light Source</td><td>SCHOTT</td><td>ACE I</td><td></td></tr><tr><td>Pair of Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Petri dish 60 mm x15 mm&amp;nbsp;</td><td>TPP Techno Plastic Products AG</td><td>93060</td><td></td></tr><tr><td>Rocker II&amp;nbsp; Platform Rocker</td><td>Boekel Scientific</td><td>260350</td><td></td></tr><tr><td>Scintillating tubes</td><td>Fischer Scientific&amp;nbsp;</td><td>03-337-26</td><td></td></tr><tr><td>Transfer pipette</td><td>Samco Scientific</td><td>202</td><td></td></tr><tr><td>Tweezers</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr></tbody></table>

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 fi...

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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, ...

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Universidad de Cartagena UDEC

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

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

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

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

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

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Biology

Light-Sheet Microscopy and Tissue Clearing for Comprehensive Cardiac Imaging

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights into fundamental cardiac physiology and pathologic responses. We hereby delve into the development and implementation of the LSM technique to elucidate the micro-architecture of the heart in mouse models. The methodology integrates a customized LSM system with tissue clearing techniques, mitigating light scattering within cardiac tissues for volumetric imaging. The combination of conventional LSM with image stitching and multiview deconvolution approaches allows for the capture of the entire heart. To address the inherent trade-off between axial resolution and field of view (FOV), we further introduce an axially swept light-sheet microscopy (ASLM) method to minimize out-of-focus light and uniformly illuminate the heart across the propagation direction. In the meanwhile, tissue clearing methods such as iDISCO enhance light penetration, facilitating the visualization of deep structures and ensuring a comprehensive examination of the myocardium throughout the entire heart. The combination of the proposed LSM and tissue clearing methods presents a promising platform for researchers in resolving cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

Author Spotlight: Customized Light-Sheet Imaging for Investigating Myocardial Structures in Rodent Hearts

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights ...

Bioengineering

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

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

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

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

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid ...

Mesoscopic Optical Imaging of Whole Mouse Heart

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Summary

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Chapters in this video

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

Immunostaining of Dissected Zebrafish Embryonic Heart

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Mesoscopic Optical Imaging of Whole Mouse Heart