Name: imaging cleared embryonic and postnatal hearts at single-cell resolution
Uploaded: 2016-10-07T13:00:00
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 ...

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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.

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			<td height="21" style="height:21px;width:381px;">2,2&#8242;,2&#8242;&#8217;-nitrilotriethanol&#160;</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">90279</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4% Paraformaldehyde in PBS</td>
			<td style="width:192px;">Affymetrix</td>
			<td>19943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td style="width:192px;">Fischer Scientific&#160;</td>
			<td style="width:211px;">BP16000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N,N&#8217;,N&#8217;-tetrakis(2-hydroxypropyl) ethylenediamine&#160;</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">122262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">P5368-10PAK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton&#160;X-100</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">U-1250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Sucrose</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">84097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Glycerol</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">G8773</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Tamoxifen</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">T5648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Sunflower seed oil</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">S5007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">PECAM (CD31)</td>
			<td>BD Pharmingen&#160;</td>
			<td style="width:211px;">550274</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647&#160;</td>
			<td>Invitrogen</td>
			<td>A-21247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">DAPI nuclear stain</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">D9542</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">37oC Incubator</td>
			<td style="width:192px;">Thermoscientific Fischer</td>
			<td style="width:211px;">Heratherm, Compact Microbiological Incubators</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">48 well plates</td>
			<td style="width:192px;">Cell Treat</td>
			<td style="width:211px;">229148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Analytical Balance</td>
			<td style="width:192px;">Metler Toledo</td>
			<td>PB153-S/FACT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope</td>
			<td>Zeiss</td>
			<td>Zeiss 510 confocal microscope</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Disecting Microscope</td>
			<td style="width:192px;">Unitron</td>
			<td style="width:211px;">Z850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescent microscope</td>
			<td>Zeiss</td>
			<td>Observer. Z1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Germinator 500 Glass Bead Sterilizer</td>
			<td style="width:192px;">CellPoint Scientific</td>
			<td style="width:211px;">GER-5287-120V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Light Source</td>
			<td style="width:192px;">SCHOTT</td>
			<td style="width:211px;">ACE I</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pair of Scissors</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dish 60x15mm&#160;</td>
			<td align="left">TPP Techno Plastic Products AG</td>
			<td>93060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Rocker II&#160; Platform Rocker</td>
			<td>Boekel Scientific</td>
			<td style="width:211px;">260350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scintillating tubes</td>
			<td style="width:192px;">Fischer Scientific&#160;</td>
			<td>03-337-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipette</td>
			<td style="width:192px;">Samco Scientific</td>
			<td style="width:211px;">202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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...

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, ...

The overall goal of this procedure is to volumetrically image fluorescent protein labeled cells deep inside intact hearts, to study gene function at single cell-resolution during cardiac development and in postnatal hearts. This method can help to answer key questions during cardiac development, such as the study of the gene functions and helping image at single-cell resolution during cardiac development and animidal artifacts. The main advantage of this method is it is relatively simple, inexpensive, and pro-wise in that special temporal analysis of the gene and protein functions at single-cell resolution in intact embryo or heart.Although we use this system to study heart development in mice, you can also use it to study other organ systems, such as muscles, brain, and lungs and in other model organisms as well. Fluorescent protein positive embryos from Tamoxifren-treated R-26 Cre-RET2 confetti dams are used here. Begin staining by immersing each paraformaldehyde-fixed embryo in one milliliter of 0.1%PBST in the wells of a 48 well plate.Incubate for two hours on a plate shaker at room temperature to permeabilize the tissue. Then, replace the permeabilization solution with one mL of blocking buffer and incubate, with shaking, for two hours at room temperature or overnight at four degrees celsius. Next, prepare a one to 100 dilution of the endothelial-cell specific antibody, PECAM, in 0.3 milliliters of blocking buffer for each well and add this to the wells containing the embryos.Incubate for 48 hours with shaking at four degrees celsius. Following the incubation, wash the embryos five times with PBST for 30 minutes each time to remove excess primary antibody from the tissue. Next, immerse the embryos in Alexa Fluor 647 goat antivat secondary antibody solution and incubate with shaking for 48 hours at four degrees celsius, while protected from light.48 hours later, after washing out before, post-fix the embryos with 4%PFA for one hour at room temperature with shaking. Then, after washing twice with PBS for five minutes each time, incubate the embryos with DAPI nuclear stain for ten minutes. Follow with two PBS washes of five minutes each, then proceed to tissue clearing.The tissue clearing is the most critical step in this protocol in order to image the heart at a single-cell resolution. Also, the selection of the clearing method either scale or CUBIC, is also very important in order to preserve fluorescent signals in the intact embryonic heart. Transfer the washed, stained embryos into a scintillation vial wrapped in aluminum foil, that contains one milliliter of scale A2 solution.Incubate at four degrees celsius with occasional gentle shaking by hand for 48 hours. After the scale A2 incubation, replace the solution in the vial with one milliliter of scale B4 solution, and incubate at four degrees celsius with occasional gentle shaking for another 48 hours. Incubate the embryos for another 48 hours with one milliliter of scale 70 at four degrees celsius with occasional gentle shaking as before.Lastly, immerse the scale cleared sample in one milliliter of 90%glycerol and store at four degrees celsius until imaging. To image, transfer the sample to a glass-bottom petri dish with a minimal amount of 90%glycerol and image with a confocal microscope equipped with two photon capabilities. For CUBIC clearing, immerse each embryo in one milliliter of CUBIC one with occasional gentle shaking for 48 hours at 37 degrees celsius.After 48 hours, replace the solution with the same volume of fresh CUBIC reagent one and incubate the sample for an additional 48 hours, as before. Following the incubation, wash the CUBIC one treated embryos with PBS several times with gentle shaking by hand to remove the excess CUBIC one solution. Then, immerse the embryos in CUBIC two solution for 48 hours at 37 degrees celsius with occasional gentle shaking by hand.If there will be a delay before imaging, sequentially incubate the samples with one milliliter of 30%50%and 70%glycerol and PBS for 30 minutes each. After moving through the series, transfer to 90%glycerol solution for storage at four degrees celsius. When ready, image in a glass-bottom petri dish with 90%glycerol, as before.This movie shows optical sections of a scale-treated E 8.75 heart, from heart surface to lumen. Yellow marks the PECAM expression and all the cardiomyocytes appear green. The depth is 110.4 microns and there are 93 slices in total.This is the 3D surface picture of the reconstructed heart just shown. Again, yellow marks PECAM expression, and green labels all the cardiomyocytes. This movie shows sections of a single progenitor clone from a scale-treated E 9.5 heart in the outflow tract region.Yellow marks the PECAM expression and the clone appears green. The depth is 36 microns and there are 10 slices in total. This movie shows sections of a CUBIC-treated P two heart from surface to lumen.The depth is 130 microns with three microns per section. This movie shows sections of a CUBIC-treated E 17.5 heart from surface to lumen. The depth is 630 microns with six microns per section.This is a 3D surface picture of the heart just shown, reconstructed via the 3D reconstruction software. When attempting this procedure, it is important to remember that the embryo is fragile and heart pathology can be disturbed easily. Following this procedure, other protein can be realized, using staining to study the gene function at a post-ost lation and lather.

Research

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