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.
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.
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 developed embryo.2,3,4,5 Genetic lineage-tracing experiments use the same concept with well-defined reporter alleles that are used to label cell populations without experimental manipulation. Key to these approaches is the ability to determine, with high spatial resolution, the locations of the experimental and reference labels. These approaches have yielded outstanding progress in pre-implantation development and explant organogenesis studies.6,7,8,9
The developmental events that underlie heart morphogenesis have been increasingly well described in recent years.10 One of the major discoveries in this area of research is the description of a number of progenitor populations that can be distinguished by expression of unique markers.11 These populations include the First and Second Heart Fields (FHF and SHF), which are present within the cardiac crescent at the anterior side of the embryo at embryonic day (E) 8.25 of mouse development.12 These populations are frequently examined through a combination of wide-field microscopy, which provides tissue-level information, and serial sectioning with immunofluorescence assays, which offers high cellular resolution but only two-dimensional spatial information.13 Thus, while these studies have greatly advanced our understanding of heart development, the available methods have limited in depth quantitative analysis of morphogenesis during these stages, creating the need for approaches to examine the organization of these populations on a whole-organism level.
The recent advances in both confocal microscopy and 3D image analysis allow for high-resolution and high-throughput algorithmic reconstructions of cells and structures in situ with relative ease, thus paving the way for detailed studies of complex cellular structures.14 With the increase of computational power and the development of big-data managing algorithms, both necessary to handle the exponential increase of the size of imaging data-sets, analyses can now be fully automated.15 Automated analysis of imaging data-sets has the benefit of being unbiased, but it is only as reliable as the quality of the input dataset; it is imperative, then, that best-practices are used during acquisition and image pre-processing to ensure the highest quality, unbiased analysis.16 Protocols can be completely automated and shared for reproducibility, and the algorithms used by proprietary software are readily available through libraries to be used by scientists who have familiarity with modern proprietary or open-source developer tools.17
The following protocol explains the necessary steps to perform such analysis on one well defined model of organogenesis, the formation of the cardiac crescent during heart development. Specifically, this protocol describes how to (1) harvest and dissect cardiac crescent stage embryos, (2) perform whole mount immunofluorescence for reference (Nkx2-5) and experimental (Foxa2Cre:YFP18,19) markers, (3) prepare and image the embryos using confocal microscopy, and finally (4) analyze and quantify the resulting images using advanced three-dimensional approaches. While the cardiac crescent is used as an example here, with appropriate modification, this protocol may be used for analysis of multiple lineages in gastrula to early somite stage embryos.
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
2. Immunofluorescence Staining
NOTE: The incubation conditions below can be adjusted to accommodate different schedules. Use of gentle shaking or rocking for all long incubation steps is recommended.
3. Mounting Embryos for Microscopy
4. Confocal Imaging
5. Image Analysis and Quantitative 3D Modeling
NOTE: For this protocol, images were analyzed using the Imaris software package. Similar analysis may be possible using alternative packages. The description below covers the analysis pipeline for a reference channel (i.e. Nkx2-5) and one experimental channel (i.e. YFP). Additional channels can be analyzed through repetition of these steps. An example dataset has been provided that can be used to replicate the analysis below.
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 antibodies will frequently bind non-specifically. However, removal of the yolk sac will make the embryos less robust, so care should be taken during downstream handling.
The combination of high quality antibodies and proper imaging settings are crucial to achieve high signal-to-noise ratio images. Figure 2A shows an example image using antibodies against Nkx2-5 as a reference marker to label the cardiac crescent, and GFP to label a lineage traced population of interest (Foxa2Cre:YFP ventricular cardiovascular progenitor cells) (See supplemental data for raw image and data files).19 This strategy allows for the comparison of a given population with the entire cardiac crescent region. For some experiments, it may be of interest to examine the FHF and SHF subdomains of the cardiac crescent. In this case, the FHF and SHF can be labeled with Hcn4 and Islet1, respectively (see reference19 for images and quantitative analysis with these antibody combinations). With the appropriate setup and secondary antibody combinations, we have successfully imaged and analyzed three lineages concurrently (i.e. the cardiac crescent, YFP, and first or second HF).
Inability to achieve strong signal will also limit the ability to generate high confidence 3D models, as it will become difficult to remove background signal from the final surfaces. In these cases, care should be taken when adjusting image gamma (during pre-processing, Figure 2B, 2C, 2N, 2O) or surface thresholding (during surface generation algorithm, Figure 2G, 2H, 2L) and similar settings should be used for all images within an experiment. Failure to do so will result in inconsistent volumetric data that would be inappropriate to use for direct comparison. Often, erroneous surfaces resulting from background signal can be removed through size filtering (Figure 2I, 2J), though doing so from high-background images will be difficult without losing true surface fragments as well.
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 8th 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. Please click here to view a larger version of this figure.
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.
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.
We believe that this approach offers a distinct advantage over existing approaches. Whole mount immunofluorescence with wide-field imaging can offer tissue-level information but lacks cellular resolution. Conversely, immunofluorescence of serial sections can give detailed cellular-level resolution, though these data lack the three-dimensional advantage of whole mount imaging. While three-dimensional confocal imaging addresses these shortcomings, few users take advantage of the quantitative potential of this data, and we hope that our protocol will allow more researchers to take full advantage of the data they collect.
Here, this approach has been exemplified using the marker Nkx2-5 to delineate the cardiac crescent and examine the presence of Foxa2Cre:YFP lineage-traced cells within that domain. However, due to the ability to image at least three lineages concurrently and the high resolution three-dimension data generated, this protocol will likely be useful to researchers in many fields. We propose that this approach can be applied to multiple embryonic stages, organ systems, and lineages through minor adjustments to the protocol based on the specific needs of the system. In our experience, this protocol is directly applicable to a range of embryonic ages (E6.5-E9.5 at minimum) with limited adaptations.19 We have not encountered issues with permeability or antibody penetration in embryos as large as E9.5 with the incubation times listed, though we expect that with thicker or denser tissues these would have to be elongated to achieve full penetration.
The analysis described here was performed using a proprietary software package, but can be adapted to be performed in alternative proprietary packages optimized for big-data handling. Alternatively, similar pipelines can easily be implemented using computational languages or open-source software packages, which will lead to the rapid development of a shareable protocols database. The growth of a global network of scientists, with access to freely available tools and protocols for image acquisition and analysis, will rapidly advance the field and increase the reproducibility of studies.17
In our experience, the major limitation to this approach was related to imaging depth, especially for later embryonic stages, and will depend heavily on the specifics of the microscopy setup being used. Similarly, how samples are mounted for imaging will depend on the specific needs of the user. We have found that placing the region of interest closest to the lens improves imaging depth and decreases background signal, and we suggest using layered double-sided tape for mounting, as it enables users to create a chamber that fits their specific needs. Use of a glycerol-based mounting media with an anti-fade reagent, and equilibrating samples in this media before mounting, is highly recommended to suppress photobleaching. Finally, the samples used in this analysis are nearly transparent; users may need to incorporate clearing steps for more advanced embryonic stages, tissue fragments, or organoids, and determining the best clearing protocol for a given application will require empirical testing.
In the case of cardiac morphogenesis, various imaging methods can be used. While confocal microscopy is used here, light-sheet microscopy is extremely well suited for these studies because of the speed, resolution, and signal-to-noise ratio of the acquired image.20 We expect the increased availability of this new technology to further advance the utility and richness of similar imaging approaches. Similarly, the continuous advancement of fluorescent reporters,21 chromophores, dyes,22 and live-embryo culture23,24 will lead to these studies moving to four dimensions, adding the element of time, and yielding yet more information about how embryonic structures form during development.
The authors have nothing to disclose.
This work was funded by NIH/NHLBI R56 HL128646 and the Mindich Child Health and Development Institute (MCHDI) at ISMMS (to N.D.). E.B. is supported by an NIH/NIDCR Traineeship T32 HD075735.Microscopy and image analysis was performed at the Microscopy Core at the Icahn School of Medicine at Mount Sinai, which is supported in part by the Tisch Cancer Institute at Mount Sinai P30 CA196521 – Cancer Center Support Grant.
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 | |
22×22 mm coverslips | VWR Scientific | 48366-227 | |
Imaris 8.4.1 | Bitplane |