Chow Renee Wei-Yan, Lamperti Paola, Steed Emily, Boselli Francesco, Vermot Julien
Institut de Génétique et de Biologie Moléculaire et Cellulaire; UMR7104, Centre National de la Recherche Scientifique; U964, Institut National de la Santé et de la Recherche Médicale; Université de Strasbourg.
Institut de Génétique et de Biologie Moléculaire et Cellulaire; UMR7104, Centre National de la Recherche Scientifique; U964, Institut National de la Santé et de la Recherche Médicale; Université de Strasbourg;
J Vis Exp. 2018 Feb 20(132):57290. doi: 10.3791/57290.
During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.
在胚胎发育过程中，细胞的行为会发生动态变化，而解读这些变化背后的细胞逻辑是发育生物学领域的一个基本目标。光转换蛋白的发现和发展通过提供一种光学标记细胞和组织的方法，极大地帮助了我们对这些动态变化的理解。然而，尽管光转换、延时显微镜以及随后的图像分析已被证明在揭示大脑或眼睛等器官中的细胞动态方面非常成功，但由于心脏在心动周期中的快速运动带来的挑战，这种方法一般不用于发育中的心脏。本方案由两部分组成。第一部分描述了一种在斑马鱼房室管（AVC）和房室心脏瓣膜发育过程中对心内膜细胞（EdC）进行光转换并随后追踪的方法。该方法包括用药物暂时使心脏停止跳动，以便进行精确的光转换。去除药物后心脏恢复跳动，胚胎发育继续正常进行，直到在稍后的发育时间点再次使心脏停止跳动，以便对光转换后的EdC进行高分辨率成像。方案的第二部分描述了一种图像分析方法，通过将三维结构中的荧光信号映射到二维图上，来量化幼体胚胎AVC中光转换或未光转换区域的长度。该方案的两个部分结合起来，使人们能够研究构成斑马鱼AVC和房室心脏瓣膜的细胞的起源和行为，并有可能应用于研究突变体、形态突变体或用破坏AVC和/或瓣膜发育的试剂处理过的胚胎。
