Hirsinger Estelle, Steventon Ben
Department of Developmental and Stem Cell Biology, Institut Pasteur; IBPS- Laboratoire de Biologie du Developpement (LBD), CNRS, UPMC, UMR 7622, INSERM ERL U1156.
Department of Developmental and Stem Cell Biology, Institut Pasteur; Department of Genetics, University of Cambridge;
J Vis Exp. 2017 Jan 26(119):55210. doi: 10.3791/55210.
Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.
斑马鱼胚胎因其易于获取和光学透明性，为研究复杂的形态发生过程提供了理想的实验系统。特别是，胚胎后部身体延伸是胚胎发育中的一个重要过程，通过这个过程，多种组织变形共同作用以引导身体轴大部分的形成。为了通过长期延时成像观察这个过程，有必要采用一种固定技术，该技术能在将样本转移到显微镜并进行采集的过程中提供足够的支撑，以保持样本处于正确的方向。此外，固定还必须为身体后部区域的生长提供足够的活动自由度，同时不影响其正常发育。最后，固定方法必须具有一定程度的通用性，以允许在不同的成像设置上进行成像。在此，我们展示一种用于成像斑马鱼（Danio rerio）身体后部延伸发育的固定技术。该技术包括固定胚胎，使头部和卵黄囊区域几乎完全包含在琼脂糖中，同时留出身体后部区域以便正常延伸和发育。我们将展示如何将其适用于正置、倒置和垂直光片显微镜设置。虽然本方案重点在于固定胚胎以对身体后部进行成像，但它可以很容易地适用于斑马鱼发育多个方面的实时成像。
