Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Ashish Misra; Zhonghui Feng; Jiasheng Zhang; Zhi-Yin Lou; Daniel M. Greif

doi:10.3791/56039

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

7.8K views

⸱

DOI:

10.3791/56039

⸱

September 12th, 2017

Ashish Misra¹ , Zhonghui Feng¹ , Jiasheng Zhang¹ , Zhi-Yin Lou²^,³ , Daniel M. Greif¹

¹Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, ²Department of Neurology, Yale University School of Medicine, ³Department of Neurology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine

Summary

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

Abstract

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Introduction

The circulatory systems of multicellular organisms function to deliver nutrients and oxygen to cells that are not in contact with the external environment and to remove waste products and carbon dioxide from these cells. In vertebrates, the primary circulatory system consists of the heart, which pumps blood through a series of blood vessels. The walls of large blood vessels, such as arteries and veins, consist of three layers: i) the intima, or inner layer of endothelial cells; ii) the media, or middle layer of alternating circumferentially elongated smooth muscle cells SMCs and elastic lamellae; and iii) the adventitia, or outer layer of connective tissue and fibrobl....