Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction
Summary
Here, we describe a protocol to visualize and analyze the pharyngeal arch arteries 3, 4, and 6 of mouse embryos using whole-mount immunofluorescence, tissue clearing, confocal microscopy, and 3D reconstruction.
Abstract
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 disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.
Introduction
During mouse embryogenesis, pharyngeal arch arteries (PAAs) arise as symmetrical, bi-lateral pairs of arteries that connect the heart with the dorsal aortae1. As the embryo develops, the first and second pairs of PAAs regress, while the 3rd, 4th, and 6th PAAs undergo a series of asymmetrical remodeling events to form the aortic arch arteries2.
The PAAs 3, 4 and 6 develop via vasculogenesis, which is the de novo formation of blood vessels3. Defects in the formation or remodeling of these arch arteries give rise to various congenital heart defects, such as those seen in patients with DiGeorge Syndrome4,5. Therefore, understanding mechanisms that regulate the development of PAAs can lead to a better understanding of congenital heart disease (CHD) etiology.
Current approaches for visualizing and analyzing PAA development include immunofluorescence of tissue sections, vascular casts, India ink injection, high resolution episcopic microscopy, and/or whole-mount immunohistochemistry1,4,5,6,7. Herein, we describe a protocol combining whole-mount immunofluorescence, confocal microscopy and 3D image rendering in order to gather, analyze, and quantify volumetric data, vascular connectivity and cell identity. Further, we detail a method to compartmentalize and quantify the numbers of ECs in each pharyngeal arch as a means to study formation of the pharyngeal arch vascular plexus and its remodeling into the PAAs. While this protocol is designed for analyzing PAA development, it can be used to analyze other developing vascular networks.
Protocol
Representative Results
Discussion
The ability to visualize the endothelium in mouse embryos in 3D has provided new insights into their development3. Here we present a protocol that allows for high-resolution 3D imaging of embryos, visualization of vascular connectivity, and quantitative analyses of PAA formation. This protocol can be employed to see how genetic alterations or environmental insults impact PAA development. The procedure reported here uses antibodies against VEGFR2 and ERG to visualize PAA formation and quantify EC n…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
We thank Brianna Alexander, Caolan O'Donnell and Michael Warkala for careful reading and editing of this manuscript. This work was supported by the funding from the National Heart, Lung and Blood Institute of the NIH R01 HL103920, R01 HL134935, R21 OD025323-01 to SA; AJR is supported by NHLBI HL103920-08S1 and the National Institute of Arthritis and Musculoskeletal and Skin Diseases Training grant T32052283-11.
Materials
| 10x PBS | MP Biomedicals | PBS10X02 | |
| 20x water immersion objective | Nikon | MRD77200 | |
| Agarose | Bio-Rad Laboratories | 1613101 | |
| Alexa Fluor 488 anti-goat | Invitrogen | A-11055 | |
| Alexa Fluor 555 anti-mouse | Invitrogen | A-31570 | |
| Analysis Software | Imaris 9.2.0 | ||
| Benzyl Alcohol | Sigma-Aldrich | 305197 | |
| Benzyl Benzoate | Sigma-Aldrich | 8.18701.0100 | |
| Cover Slips | VWR | 16004-312 | |
| DAPI (5 mg/mL stock) | Fisher Scientific | D3571 | |
| Eppendorf Tubes (2.0 mL) | Fisher Scientific | 05-408-138 | |
| Ethanol | VWR | 89370-084 | |
| Falcon tubes (50 mL) | Corning | 352098 | |
| Fast wells | Grace Bio Labs | 664113 | |
| Forceps | Roboz | RS-5015 | |
| Goat anti-VEGFR2 | R&D Systems, Inc. | AF644 | |
| Methanol | VWR | BDH1135-4LP | |
| Microscope | Nikon | A1HD25 | |
| Mouse anti-ERG | Abcam | ab214341 | |
| Normal Donkey Serum | Sigma-Aldrich | D9663 | |
| Paraformaldehyde | Electron Microscopy Sciences | 15710 | |
| Pasteur pipets | Fisher Scientific | 13-678-20D | |
| Petri dishes (35 mm) | Genesee Scientific | 32-103 | |
| Petri dishes (60 mm) | Genesee Scientific | 32-105 | |
| Plastic Molds | VWR | 18000-128 | |
| Scapels | Exelint International Co. | 29552 | |
| Triton-X-100 | Fisher Scientific | BP 151-500 |
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