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-Cre^ERT2; 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 heart^1,2. 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 structure^5,6; however, these methods require expensive, specialized instruments, extensive labor, and lack detailed structural organization at single cell resolution in the final volumetric reconstructed image^7,8.

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 issue^8-10. The solvent based hydrophilic methods, such as SeeDB (fructose/thioglycerol) and 3DISCO (dichloromethane/dibenzylether), preserve fluorescent signals, but do not render the whole organ transparent^7,8,11. In comparison, the CLARITY tissue-clearing method renders the organ transparent, but it requires a specialized electrophoresis device to remove lipids^8,12, as does PACT (passive clarity technique), which also requires hydrogel embedding^7,13. 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 Rosa26Cre^{ERT2 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 previously^21,22 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

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 Rosa26Cre^{ERT2 15}, R26R-Confetti¹⁶, αMHC-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.

1. Prepare Tamoxifen. Dissolve 10 mg of Tamoxifen in 10 ml of sunflower seed oil. Once it is completely dissolved, aliquot and store at -20 °C.
2. 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.
3. Prepare PBST:0.1% Tween-20 solution in PBS by dissolving 100 μl of Tween-20 in 100 ml of PBS.
4. 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.
5. Prepare CUBIC-1 (reagent 1). Mix 25 wt% urea, 25 wt% N, N, N', N'-tetrakis (2-hydroxypropyl) ethylenediamine and 15 wt% Triton X-100 in dH₂O.
6. Prepare CUBIC-2 (reagent 2). Mix 50 wt% sucrose, 25 wt% urea, 10 wt% 2, 2′, 2′'-nitrilotriethanol and 0.1% (v/v) Triton X-100 in dH₂O.
7. 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.
8. Prepare Scale B4: 8 M urea and 0.1% w/v Triton X-100 in dH₂O. Adjust the pH to 8.7.
9. Prepare Scale 70: 4 M urea, 70% w/v glycerol and 0.1% w/v Triton X-100 in dH₂O.

2. Tamoxifen Induction, Embryo Isolation and Fixation

1. Tamoxifen Induction and Embryo Isolation
   1. Using a curved feeding tube approved protocol (ACUP 13-12007), gavage the female R26Cre^ERT2 Confetti mice (plugged by R26Cre^ERT2 male mice) with 10 µg/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.
   2. 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 tube^23,24.
      1. 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.
   3. 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.
   4. 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.
2. 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.
3. 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.
4. 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.

3. Whole Mount Staining

NOTE: After PFA fixation the embryo/heart is subjected to whole mount staining as follows.

1. Permeabilize the embryo/heart in a 48 well-plate using 1 ml of 0.1% PBST for 2 hr on plate shaker at RT.
2. Following permeabilization, immerse the embryo/heart in 1 ml of blocking buffer, for 2 hr or overnight on a plate shaker at 4 ºC.
3. 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 ºC.
4. 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.
5. 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 ºC.
6. Repeat step 3.4 to wash the secondary antibody.
7. Post-fix the embryo/heart with 4% PFA for 1 hr at RT on plate shaker and wash twice with PBS 5 min each.
8. Stain the embryo/heart with the nuclear stain 4′,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.

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.

1. Scale Tissue Clearing Method
   1. 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 °C.
   2. After Scale A2 incubation, incubate the embryo/heart with 1 mL of Scale B4 solution at 4 °C in the same vial with occasional gentle shaking for another 48 hr.
   3. Incubate the embryo/heart for another 48 hr with 1 ml Scale 70 at 4 °C with occasional gentle shaking. Mount embryo using 90% glycerol (see section 5.1).
2. CUBIC Tissue Clearing Method
   1. 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 ºC⁷. 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 °C with occasional shaking with hand.
   2. 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.
   3. 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 ºC with occasional gentle shaking with hand. This follows embryo/heart mounting using glycerol (see section 5.2).

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.

1. Directly immerse the Scale cleared sample in 1 ml of 90% glycerol and store at 4 °C until imaging.
2. 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.
3. 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.
4. 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.

Results

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...

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 60 mm x15 mm	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