Name: quantitative whole-mount immunofluorescence analysis of cardiac progenitor populations in mouse embryos
Uploaded: 2017-10-12T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos at JoVE.com

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 &#8211; Cancer Center Support Grant.

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
<ol>
	<li>Mate a fertile female mouse with a fertile stud male.</li>
	<li>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)...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Mount+Dissection+and+Immunofluorescence+of+the+Adult+Mouse+Cochlea.">Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea.</a> J Vis Exp. , (2016).</li><li>Calderon, D., Bardot, E., Dubois, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Early+Heart+Development+to+Instruct+Stem+Cell+Differentiation+Strategies.">Probing Early Heart Development to Instruct Stem Cell Differentiation Strategies.</a> Dev. Dyn. , (2016).</li><li>Meilhac, S., Lescroart, F., Blanpain, C., Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Cell+Lineages+that+Form+the+Heart.">Cardiac Cell Lineages that Form the Heart.</a> Cold Spring Harb Perspect. Med. 4, a013888-a013888 (2014).</li><li>Kelly, R., Buckingham, M., Moorman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Fields+and+Cardiac+Morphogenesis.">Heart Fields and Cardiac Morphogenesis.</a> Cold Spring Harb Perspect. Med. 4, a015750-a015750 (2014).</li><li>Cai, C. -. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isl1+Identifies+a+Cardiac+Progenitor+Population+that+Proliferates+Prior+to+Differentiation+and+Contributes+a+Majority+of+Cells+to+the+Heart.">Isl1 Identifies a Cardiac Progenitor Population that Proliferates Prior to Differentiation and Contributes a Majority of Cells to the Heart.</a> Dev Cell. 5, 877-889 (2003).</li><li>Sbalzarini, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing:+Quantifying+is+convincing:+Computational+image+analysis+in+biology.">Seeing is believing: Quantifying is convincing: Computational image analysis in biology.</a> Adv. Anat. Embryol. Cell Biol. 219, 1-39 (2016).</li><li>Myers, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+bioimage+informatics+matters.">Why bioimage informatics matters.</a> Nat. Methods. 9, 659-660 (2012).</li><li>North, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing?+A+beginners'+guide+to+practical+pitfalls+in+image+acquisition.">Seeing is believing? A beginners' guide to practical pitfalls in image acquisition.</a> J. Cell Biol. 172, 9-18 (2006).</li><li>Eliceiri, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+imaging+software+tools.">Biological imaging software tools.</a> Nat. Methods. 9, 697-710 (2012).</li><li>Horn, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mind+bomb+1+is+required+for+pancreatic+&#946;-cell+formation.">Mind bomb 1 is required for pancreatic &#946;-cell formation.</a> Proc. Natl. Acad. Sci. U. S. A. 109, 7356-7361 (2012).</li><li>Bardot, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxa2+identifies+a+cardiac+progenitor+population+with+ventricular+differentiation+potential.">Foxa2 identifies a cardiac progenitor population with ventricular differentiation potential.</a> Nat Commun. 8, 14428 (2017).</li><li>Adams, M. W., Loftus, A. F., Dunn, S. E., Joens, M. S., Fitzpatrick, J. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sheet+fluorescence+microscopy+(LSFM).">Light sheet fluorescence microscopy (LSFM).</a> Curr. Protoc. Cytom. 2015, (2015).</li><li>Shaner, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nat. Biotechnol. 22, 1567-1572 (2004).</li><li>Johnson, S., Rabinovitch, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+imaging+of+excised+tissue+using+vital+dyes+and+confocal+microscopy.">Ex vivo imaging of excised tissue using vital dyes and confocal microscopy.</a> Curr. Protoc. Cytom. , (2012).</li><li>Burtscher, I., Lickert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxa2+regulates+polarity+and+epithelialization+in+the+endoderm+germ+layer+of+the+mouse+embryo.">Foxa2 regulates polarity and epithelialization in the endoderm germ layer of the mouse embryo.</a> Development. 136, 1029-1038 (2009).</li><li>Baron, M. 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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.
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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.

<table>
	<tbody>
		<tr>
			<td>Blunt probe</td>
			<td>Roboz</td>
			<td>RS-9580</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS-8100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Roboz</td>
			<td>RS-5015</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Roboz</td>
			<td>RS-5912</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma Aldrich</td>
			<td>84510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A8022</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>RPI</td>
			<td>A4490</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Nkx2-5</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-8697</td>
			<td>Used at 1:100-1:500</td>
		</tr>
		<tr>
			<td>Chicken anti-GFP</td>
			<td>abcam</td>
			<td>ab13970</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>Rabbit anti-Islet1</td>
			<td>abcam</td>
			<td>ab109517</td>
			<td>Used at 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti-Hcn4</td>
			<td>Millipore</td>
			<td>AB5808</td>
			<td>Used at 1:100</td>
		</tr>
		<tr>
			<td>488 anti-chicken</td>
			<td>Jackson Immunoresearch</td>
			<td>703-546-155</td>
			<td>Reconstituted in water and stored at -20 &#176;C in final concentration of 50% glycerol. Used at 1:500.</td>
		</tr>
		<tr>
			<td>555 anti-goat</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21432</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>647 anti-rabbit</td>
			<td>Jackson Immunoresearch</td>
			<td>711-606-152</td>
			<td>Reconstituted in water and stored at -20 &#176;C in final concentration of 50% glycerol. Used at 1:500.</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Propyl gallate</td>
			<td>Sigma Aldrich</td>
			<td>2370</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Superfrost Plus microscopy slides</td>
			<td>VWR Scientific</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>22x22 mm coverslips</td>
			<td>VWR Scientific</td>
			<td>48366-227</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris 8.4.1</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative whole-mount immunofluorescence analysis of cardiac progenitor populations in mouse embryos

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 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...

Watch this Scientific Journal Video about Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos at JoVE.com

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

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 ...

The overall goal of this method is to quantitatively assess cardiac progenitor cell localization and distribution during heart organogenesis. This method can help answer key answers in the heart development field by allowing us to observe the localization of cardiac progenitor populations during normal embryonic development. The main advantages of this technique are that the volumetric imagery can yield quantitative information about tissue level dynamics and that the analysis pipeline can be automated for improved reproducibility.Though this method provides information about early cardiac organogenesis, it can also be applied to the general study of early embryogenesis or to the morphogenesis of other organs of interest. On the morning of embryonic day eight, disinfect the pregnant mouse abdomen with 70%ethanol and make an abdominal incision through both the skin and body wall. Beginning above one oviduct, carefully dissect the fat along the side of the uterus down to the cervix.Make an incision through the cervix and continue to the upper end of the other oviduct cutting to release the entire uterus. Wash the excess blood from the uterus in a 10 centimeter dish containing fresh PBS and cut the mesometrium between each deciduom. Transfer the embryos to a six centimeter dish containing fresh PBS under a dissecting microscope and use number five fine forceps to remove the uterine tissue from the decidual tissue.Next, carefully slice the tip of the embryonic half of the deciduom to reveal the embryos, pinching the deciduom to carefully push and pull the first embryo out. Dissect away as much extra embryonic tissue as possible without damaging the morphology of the animal and use a transfer pipette to place the embryo into a 1.5 milliliter tube containing fresh PBS on ice. When all of the embryos have been harvested, aspirate the PBS from each tube and rinse the embryos with fresh PBS.After thoroughly aspirating the PBS from each tube, fix the embryos in 4%paraformaldehyde for one hour at room temperature, followed by three washes with fresh PBS, then store the embryos at four degrees Celsius. Begin by replacing the PBS with one milliliter of blocking buffer for a four-hour incubation at room temperature. At the end of the incubation, replace the blocking buffer with the appropriate primary antibody mixture and diluted blocking buffer for an overnight incubation at four degrees Celsius.The next morning, aspirate the excess antibody and wash the embryos three times for one hour per wash in 0.1%Triton in PBS. After the third wash, add the appropriate secondary antibody mixture to the embryos for a three-hour incubation at room temperature, followed by three washes as just demonstrated. Next, counterstain the embryos with DAPI in PBS for 10 minutes followed by two five-minute washes, then slowly suspend the embryos in anti-fade mounting medium allowing the embryos to equilibrate for at least one hour with periodic gentle flicking to help the specimens drop into the solution.Before mounting the embryos, make two parallel stacks of five to six layers of double stick tape 15 to 20 millimeters apart on one microscope slide per embryo and place a 15 microliter drop of anti-fade medium onto each slide between the tape stacks. Carefully transfer one embryo into each drop and use a dissection microscope and fine forceps to position the embryos with their anterior sides facing away from the slide and with their body axis in line with the long axis of the slides. To place the coverslips, first rest one side of the glass on one stack of double stick tape and use forceps to gently lower the coverslip until it contacts the other tape stack.It's particularly important to carefully position and mount the embryos. Embryos that aren't positioned correctly will be much more difficult to image and may require additional processing to get good quantifiable data. Use a pipette to add additional anti-fade medium between the slide and the coverslip to keep the sample from drying out during the imaging and select the highest magnification objective available that allows the whole cardiac crescent region to be captured in one field of view.Then set up the imaging parameters using standard best imaging practices and use Nyquist sampling rates to determine the XYZ voxel dimensions of each embryo. To analyze the embryo images, load the raw image data sets into the appropriate image analysis software and open the first file in the surpass view. The data will load in a 3D view in the maximum intensity projection mode.Open the display adjustment window and select the reference channel only. Adjust the threshold of the image as necessary. If the signal to noise ratio is too low for segmentation, adjust the gamma.Following best practices during image acquisition is critical for obtaining suitable data and will depend on the specific microscopy setup used. If adjustments to the gamma are needed, these images should not be used for comparing fluorescence intensities. Use the surface algorithm dialogue to create a new surface for the reference channel and check the segment only origin of interest box.Adjust the bounding box region so that it contains the region of interest as delineated by the reference stain and select the reference channel from the source channel dropdown menu. Set the smoothing to half of the Z voxel size and use the threshold sliders to adjust the lower limit to its absolute intensity to ensure that the surface does not extend beyond the channel signal. Filter the objects by voxel number or volume rejecting any background objects that the algorithm might have generated and finish the algorithm.Next, select the newly created surface and choose the mask set function from the edit window. Select the experimental channel of interest taking care that the duplicate channel before applying mask checkbox is selected and select the mask channel only in the display adjustment window. Then create a new surface as just demonstrated.To obtain the volumetric data with each surface selected, open the detail sub tab in the statistics tab and select average values from the dropdown menu. The total volume of the surface will be found in the sum column of the generated table. Here, an embryonic day 8.25 mouse embryo with GFP positive ventricular cardiovascular progenitor cells labeled with an anti-cardiac crescent antibody as just demonstrated is shown.After selecting the reference channel for the stained tissue, the intensity and gamma are adjusted before beginning the create surfaces algorithm. The region of interest is selected and the level of surface detail is chosen. The initial surface is thresholded to include all true signal in the surface.Filtering is then performed to remove small background fragments to yield a final reference surface. By selecting the reference surface, the comparison channel can be duplicated and masked. The intensity and gamma for this channel are adjusted before generating a second surface through the same sequence of steps.The total volume for each surface is then calculated automatically and can be compared both quantitatively and visually. While attempting this procedure, it's important to remember that the quality of the dissected embryos, the antibody efficiency, the mounting orientation, and the imaging setup all affect the quality of the final data. In addition to this quantitative analysis pipeline, other methods such as deconvolution can be performed to yield additional qualitative data.After its development, this technology paved the way for researchers in cardiac development to explore the dynamics of novel cardiac progenitor populations during early embryogenesis.

Research

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Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...