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

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.

Figure 1:. Experimental timeline and schematics.

(A) The entire experiment, from mating to data analysis, can be carried out in approximately two weeks. Embryos are collected in the morning on the 8^th day post copulation (E8.25) for cardiac crescent stage analysis (B). Once fixed, embryos are blocked and stained with primary and secondary antibodies before mounting. Embryos are mounted with standard microscopy slides and coverslips, using double-sided tape as a spacer (C). Embryos are placed in a drop of anti-fade in the displayed orientation (C, upper) and a coverslip is slowly lowered onto the tape (C, lower). Confocal imaging and image analysis are performed to generate high resolution z- stack images (D) and 3D surface models (E). HF, head fold; CC, cardiac crescent; A, anterior; P, posterior; AF, anti-fade. Scale bars are 100 μm.

Figure 2:. Stepwise confocal image processing and 3D surface generation.

(A) Confocal z-series loaded in the volume view. The reference channel (Nkx2–5) is selected (B) and the intensity and gamma adjusted (C) before starting the Create Surfaces algorithm. After the region of interest is selected (D), and the level of surface detail is chosen (E). The initial surface (F) is thresholded to include all true signal in the surface (G). Filtering is then performed to remove small background fragments (H) to yield a final reference surface (I). By selecting the reference surface (J), the comparison channel (YFP) can be duplicated and masked (K). The intensity and gamma for this channel are then adjusted (L) before generating a second surface through the same sequence of steps (M, N). The total volume for each surface is calculated automatically and can be compared both quantitatively and visually (O, note that surface colors do not merge when overlapped). Scale bars = 100 μm. Please click here to view a larger version of this figure.