Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution
Summary
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.
Abstract
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.
Introduction
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 'Scale' (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 'CUBIC' (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, αMHC-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.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 2,2′,2′’-nitrilotriethanol | Sigma Aldrich | 90279 | |
| 4% Paraformaldehyde in PBS | Affymetrix | 19943 | |
| BSA | Fischer Scientific | BP16000 | |
| N,N,N’,N’-tetrakis(2-hydroxypropyl) ethylenediamine | Sigma Aldrich | 122262 | |
| Phosphate Buffer Saline | Sigma Aldrich | P5368-10PAK | |
| Triton X-100 | Sigma Aldrich | T8787 | |
| Urea | Sigma Aldrich | U-1250 | |
| Sucrose | Sigma Aldrich | 84097 | |
| Glycerol | Sigma Aldrich | G8773 | |
| Tamoxifen | Sigma Aldrich | T5648 | |
| Sunflower seed oil | Sigma Aldrich | S5007 | |
| Tween 20 | Sigma Aldrich | P1379 | |
| PECAM (CD31) | BD Pharmingen | 550274 | |
| Alexa Fluor 647 | Invitrogen | A-21247 | |
| DAPI nuclear stain | Sigma Aldrich | D9542 | |
| 37oC Incubator | Thermoscientific Fischer | Heratherm, Compact Microbiological Incubators | |
| 48 well plates | Cell Treat | 229148 | |
| Analytical Balance | Metler Toledo | PB153-S/FACT | |
| Confocal microscope | Zeiss | Zeiss 510 confocal microscope | |
| Disecting Microscope | Unitron | Z850 | |
| Fluorescent microscope | Zeiss | Observer. Z1 | |
| Germinator 500 Glass Bead Sterilizer | CellPoint Scientific | GER-5287-120V | |
| Light Source | SCHOTT | ACE I | |
| Pair of Scissors | Fine Science Tools | 14084-08 | |
| Petri dish 60x15mm | TPP Techno Plastic Products AG | 93060 | |
| Rocker II Platform Rocker | Boekel Scientific | 260350 | |
| Scintillating tubes | Fischer Scientific | 03-337-26 | |
| Transfer pipette | Samco Scientific | 202 | |
| Tweezers | Fine Science Tools | 11251-20 |
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