Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution
| Autor: | Margarida Barroso, Yangyang Lu, Joseph E. Mazurkiewicz, David Shieh, Wasay Mohiuddin Shaikh Qureshi, Mingfu Wu, Lianjie Miao, Jingjing Li, Saiyang Hu |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Pathology medicine.medical_specialty General Chemical Engineering Cellular differentiation Cell Gene Expression Biology General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Imaging Three-Dimensional medicine Cell Lineage General Immunology and Microbiology Embryonic heart General Neuroscience Resolution (electron density) Brain Cell Differentiation Heart Embryo Embryo Mammalian Embryonic stem cell Cell biology 030104 developmental biology medicine.anatomical_structure Clone (B-cell biology) Developmental biology Developmental Biology |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/54303 |
| Popis: | Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects. |
| Databáze: | OpenAIRE |
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