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Developmental Biology

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Published: October 7th, 2016

DOI:

10.3791/54303

1Department of Molecular and Cellular Physiology, Albany Medical College, 2Department of Neuroscience and Experimental Therapeutics, 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.

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.

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....

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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, αMHC-Cre17,and Rosa26-mTmG (mTmG)20 mouse lines were purchased commercially. cTnT-Cre18 .......

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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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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.......

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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.

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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 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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