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

摘要

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.

摘要

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.

引言

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....

方案

All mouse protocols are approved by the Institutional Animal Care and Use Committee at Yale University.

1. In Vivo Embryonic Fate Mapping and Clonal Analysis

Note: We have used these approaches widely to evaluate the origins of cells and their clonal architecture in development and disease models^7,8,9,10.

1. Set up mating between mice with a CreER and mice with a Cre reporter.
   NOTE: A CreER is used for SMC marking; Myh11-CreERT2 or Acta2-CreERT2 mice

结果

In a representative clonal analysis of SMCs in embryos mutant for Eln (the gene encoding the extracellular matrix protein elastin), Eln^(+/-), Acta2-CreER^T2 mice were mated to Eln^(+/-) mice also carrying the multi-color ROSA26R^(Rb/Rb) reporter. As described in step 1, plugs were checked, pregnant dams were induced with a single tamoxifen injection (1.5 mg) at E12.5, and they were sacrificed at E18.5. Embryos were harves.......

讨论

In contrast to the extensive investigations of the murine aorta and its major branches in adult pathological conditions, such as models of atherosclerosis, less is known regarding the morphogenesis and the pathogenesis of the embryonic and perinatal aorta. Here, we focus on the embryonic/perinatal aorta, specifically the SMCs, and provide protocols to study the aorta through in vivo, tissue explant, and SMC isolation approaches. These complimentary approaches provide the investigator with diverse approaches to s.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
他莫昔芬	Sigma	T5648
玉米油	Sigma	C-8267	他莫昔芬
4-OH-他莫昔芬	Sigma	H7904	他莫昔芬
黄体酮	的	P8783-5G	以他莫昔芬
OCT 化合物	Sakura tissue tek	4583	用于制作冷冻块
Cryomolds	Polysciences inc	18986
DAPI	Sigma	D9542	细胞核 IHC 染色，终浓度 5 mg/mL
Cy3 直接偶联抗 SMA 抗体	Sigma	A2547	SMA IHC 染色，终稀释度 1：500
抗 CD31 抗体	BD Pharmingen	550274	GFP IHC 染色，终浓度 0.006 mg/mL
抗 GFP 抗体	Thermo Fisher Scientific	A-11121	IHC 染色 CD31，终浓度 0.0016 mg/mL
山羊抗兔二抗，Alexa 647	Life Technologies	a21244	IHC 染色，终浓度 0.004 mg/mL
山羊抗兔二抗，Alexa 488	Life Technologies	a11008	IHC 染色，终浓度 0.004 mg/mL
DMEM	Thermo Fisher Scientific	10567-014	用于细胞培养
FBS，Thermo	Fisher Scientific	10437028
抗整合素 β3 封闭抗体	BD Biosciences 553343		克隆 2C9。G2，终浓度 0.02 mg/mL
胶原酶	Worthington Biochemical Corp	44H14977A	用于消化主动脉
弹性蛋白酶	Worthington Biochemical Corp	34K15139	用于消化主动脉
抗生素-抗真菌剂 （100X）	Thermo Fisher Scientific	15240062
重组人 FGF	Promega	G5071
重组人 EGF	Promega	G5021
青霉素/链霉素 （10,000 U/mL）	Thermo Fisher Scientific	15140122
两性霉素 B	Thermo Fisher Scientific	15290026
组织培养板	康宁	CLS430165
Alzet 渗透微型泵	Durect Corporation	2001
ECLIPSE 80i 立式荧光显微镜	尼康
TCS SP5	Leica
Branson Sonifier 450	VWR
Myh11-CreERT2 小鼠	杰克逊实验室	19079
Acta2-CreERT2小鼠			从Pierre Chambon博士和Daniel Metzger的实验室获得
ROSA26R-CreERT2小鼠 杰	克逊实验室	8463
ROSA26R（mTmG/mTmG） 小鼠	杰克逊实验室
ROSA26R （EYFP/EYFP） 小鼠	杰克逊实验室	006148
ROSA26R（Confetti/Confetti） 小鼠	The Jackson Laboratory	13731
ROSA26R（Rb/Rb） 小鼠	Irv Weissman		从 Irv Weissman 博士实验室获得