Clonal Analysis of the Neonatal Mouse Heart using Nearest Neighbor Modeling

Summary

Presented here is a protocol for using multicolor lineage tracing and nearest-neighbor modeling to identify clonally derived cardiomyocytes during growth and regeneration in mice. This approach is objective, works across different labeling conditions, and can be adapted to incorporate a variety of image analysis pipelines.

Abstract

By replacing lost or dysfunctional myocardium, tissue regeneration is a promising approach to treat heart failure. However, the challenge of detecting bona fide heart regeneration limits the validation of potential regenerative factors. One method to detect new cardiomyocytes is multicolor lineage tracing with clonal analysis. Clonal analysis experiments can be difficult to undertake, because labeling conditions that are too sparse lack sensitivity for rare events such as cardiomyocyte proliferation, and diffuse labeling limits the ability to resolve clones. Presented here is a protocol to undertake clonal analysis of the neonatal mouse heart by using statistical modeling of nearest neighbor distributions to resolve cardiomyocyte clones. This approach enables resolution of clones over a range of labeling conditions and provides a robust analytical approach for quantifying cardiomyocyte proliferation and regeneration. This protocol can be adapted to other tissues and can be broadly used to study tissue regeneration.

Introduction

A histologic hallmark of heart failure is the loss of cardiomyocytes (CMs), either following injury, senescence, or apoptosis¹. Replenishing lost or dysfunctional myocardium through tissue regeneration represents a potential therapeutic strategy for curing patients with heart failure. Over the past several decades, seminal advances in developmental and regenerative biology have unearthed a limited ability for the mammalian heart to replenish lost CMs^2,3,4,5. This exciting work has raised the possibility that inna....

Protocol

All procedures for handling mice, performing survival surgeries, and for harvesting hearts require approval by a local institutional animal use committee.

1. Mice for clonal analysis of CMs

1. Cross Myh6-MerCreMer mice²⁰ with Gt(ROSA)26Sor^{tm1(CAG-EGFP,-mCerulean,-mOrange,-mCherry)Ilw} mice²¹ to generate Myh6-MerCreMer; R26R-Rainbow bitransgenic mice.
   1. Maintain stocks of mice homozygous for the R26R-Rainbow allele and cross with mice heterozygous for the Myh6....

Results

Following the protocol for neonatal cryoinjury should yield P21 hearts with and without injury. Cryoinjured hearts have a well-circumscribed injury while the surface of sham hearts is smooth and homogeneous. In cryoinjured hearts, the area of injury should be consistent from heart to heart. After microscopy, images similar to Figure 1 should be obtained. Note that the image resolution allows for identification of individual CMs and imaging conditions allow for each fluorophore to be resolved.......

Discussion

Multicolor lineage tracing is a powerful approach to identify patterns of organ growth with single cell resolution. However, a major limitation to multicolor lineage tracing is the need for sparse labeling of cells, which can reduce the sensitivity for identifying rare events. For organs like the heart with notoriously low levels of parenchymal cell turnover, this can lead to underestimates of growth responses. Presented here is a step-by-step protocol for performing clonal analysis of CM expansion during growth and rege.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#1.5 glass coverslip	FisherScientific	12-544E
6-O prolene	Ethicon	8706H
Anti-fade mounting medium	FisherScientific	00-4958-02
CO2 inhalational chamber
Cold pack
Cryomolds	VWR	15160-215
Cryoprobe	World Precision Instruments	501313
Filter cubes
Gt(ROSA)26Sortm1(CAG-EGFP,-mCerulean,-mOrange,-mCherry)Ilw mice
ImageJ software			https://imagej.net
KCl 1M	FisherScientific	LC187951
Leica CM3050 cryostat
Liquid Nitrogen
Microscissors, 6mm	World Precision Instruments	14003
Myh6-CreERT2 mice	The Jackson Laboratory	005657
Needler holder	World Precision Instruments	14109
Paraformaldehyde 4%	FisherScientific	AC416785000
Phosphate buffered saline
Python			https://www.python.org/
R			https://cran.r-project.org/
Rotating Shaker
Stereoscope
Sucrose 30% (wt/vol)	FisherScientific	BP220
Surgical dissecting scissors	World Precision Instruments	14393
Syringe for tamoxifen	VWR	BD328438
Tamoxifen, 20 μg	Sigma	T5648
Tissue Freezing Media	VWR	15148-031
White Frosted/Plus slides	Globe Scientific	1358W
Zeiss Axio Imager M1 upright widefield fluorescence system
Zen 2.5 Blue software