Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

Summary

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

Abstract

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.

This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Introduction

The study of organogenesis has long relied on the observation of morphogenetic events in the developing embryo. These studies frequently rely on the use of fluorescent dyes or lineage tracing reporters in combination with labeling of defined reference populations.¹ By comparing relative positions of these labels, information can be gleaned on the origin, movement, or ultimate contribution of a population of interest. Transplantation and fate mapping experiments use either morphological landmarks or injection of dyes into non-motile lineages to define the starting point of the cells of interest, which are then examined for contribution to the de....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai.

1. Harvesting and Processing Cardiac Crescent Stage Embryos

1. Mate a fertile female mouse with a fertile stud male.
2. Check for the presence of a vaginal copulation plug using a blunt probe or forceps. Noon on the day of plug detection is considered embryonic day (E) 0.5 (See Figure 1A for full timeline).
   NOTE: Plugs should be checked for in the morning, as they are lost throughout the day.
3. On the morning of the 8^th day (E8....

Results

The quality of the final data and analysis depends greatly on (1) the integrity and morphology of the dissected embryos, (2) the use of high specificity antibodies, and (3) the proper setup of imaging parameters. Damaged embryos will confound the surface generation process and hinder quantitative analysis. Examples of properly dissected and staged embryos are shown in Figure 2B. The embryos can be further dissected by removing the yolk sac, to which antibodie.......

Discussion

The protocol above describes a strategy for obtaining quantitative data from high quality whole mount immunofluorescence images of post-implantation mouse embryos. When performed correctly, the 3D volumetric data generated through these steps can be used for comparative and intersectional analysis of multiple domains within the embryo. The surface signal masking approach described is of particular use when investigating novel cell populations in comparison with well-established reference structures.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Blunt probe	Roboz	RS-9580
Forceps	Roboz	RS-8100
Fine forceps	Roboz	RS-5015
Dissection scissors	Roboz	RS-5912
Saponin	Sigma Aldrich	84510
Bovine Serum Albumin	Sigma Aldrich	A8022
Triton	RPI	A4490
Goat anti-Nkx2-5	Santa Cruz Biotech	sc-8697	Used at 1:100-1:500
Chicken anti-GFP	abcam	ab13970	Used at 1:500
Rabbit anti-Islet1	abcam	ab109517	Used at 1:100
Rabbit anti-Hcn4	Millipore	AB5808	Used at 1:100
488 anti-chicken	Jackson Immunoresearch	703-546-155	Reconstituted in water and stored at -20 °C in final concentration of 50% glycerol. Used at 1:500.
555 anti-goat	Thermo Fisher Scientific	A21432	Used at 1:500
647 anti-rabbit	Jackson Immunoresearch	711-606-152	Reconstituted in water and stored at -20 °C in final concentration of 50% glycerol. Used at 1:500.
DAPI	Sigma Aldrich	D9542
n-Propyl gallate	Sigma Aldrich	2370	Stock solution is 20% w/v in DMSO. Working solution prepared by mixing 1 part 10x PBS with 9 parts 100% glycerol and slowly adding 0.1 part stock solution.
Superfrost Plus microscopy slides	VWR Scientific	48311-703
22x22 mm coverslips	VWR Scientific	48366-227
Imaris 8.4.1	Bitplane