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....

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

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