Imaging brain development and organogenesis in zebrafish using immobilized embryonic explants
Imaging brain development and organogenesis in zebrafish using immobilized embryonic explants
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DOI：
10.1002/dvdy.10395
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发表时间：
2003-11-01
影响因子：
2.5
通讯作者：
Cooper, MS
中科院分区：
文献类型：
--
作者：
Langenberg, T;Brand, M;Cooper, MS
由于其光学清晰度和快速的发育速度，斑马鱼胚胎是研究器官发生的细胞力学的理想模型系统。不幸的是，斑马鱼胚胎的长时间延时记录经常被胚胎轴的延伸和拉直以及与发育中的肌肉组织相关的运动伪影所破坏。此外，胚胎巨大的卵黄细胞经常阻止光学进入感兴趣的组织。为了避免这些成像问题，我们已经开发了一个程序，放气和机械删除蛋黄细胞。首先将一种“麻痹”剂AMP-PNP（一种膜不可渗透的不可水解的ATP类似物）注射到胚胎的收缩性卵黄细胞中。然后用锋利的钨针取出卵黄细胞。然后，使用薄的血浆凝块将去蛋黄的胚胎或从中分离的器官雏形固定在显微镜盖玻片上。这种血浆凝块固定允许外植体的新型安装，使得腹侧、侧部甚至横截面视野可以使用高数值孔径物镜。我们发现，分离的头部雏形进行正常的形态发生和基因表达至少一天后，被植入器官型培养。这些程序可用于研究“去蛋黄”胚胎中器官发生的细胞力学，以及从绿色荧光蛋白转基因动物中获得的组织中的细胞力学。(C)2003 Wiley-Liss，Inc.
Owing to its optical clarity and rapid rate of development, the zebrafish embryo is an ideal model system for studying the cellular mechanics of organogenesis. Unfortunately, extended time-lapse recordings of zebrafish embryos are often disrupted by the extension and straightening of the embryonic axis, as well as movement artifacts associated with developing musculature. In addition, the embryo's massive yolk cell often prevents optical access to tissues of interest. To circumvent these imaging problems, we have developed a procedure to deflate and mechanically remove the yolk cell. A "paralyzing" agent, AMP-PNP (a membrane-impermeant nonhydrolyzable analog of ATP), is first injected into the embryo's contractile yolk cell. The yolk cell is then removed using sharpened tungsten needles. Deyolked embryos, or organ rudiments explanted from them, are then immobilized on a microscope coverslip using a thin plasma clot. This plasma clot immobilization allows novel mountings of the explants so that ventral, lateral, and even cross-sectional fields of views are possible using high numerical aperture objectives. We show that isolated head rudiments undergo normal morphogenesis and gene expression for at least I day after being explanted into organotypic culture. These procedures can be used to study the cellular mechanics of organogenesis in "deyolked" embryos, as well as in tissues explanted from green fluorescent protein transgenic animals. (C) 2003 Wiley-Liss, Inc.