Schmitz Manuel, Nelemans Ben K A, Smit Theodoor H
Department of Orthopaedic Surgery, VU University Medical Center Amsterdam, MOVE Research Institute Amsterdam.
Department of Anatomy, Embryology & Physiology, Academic Medical Center Amsterdam;
J Vis Exp. 2016 Dec 28(118):54636. doi: 10.3791/54636.
Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation, neurulation, somitogenesis, heart bending, and brain formation, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture) and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5) up to at least the 28-somite stage (HH16), embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture. The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).
由于其易于获取、成本低、几何形状扁平且具有透明性，卵外培养的鸡胚已成为研究早期胚胎发育过程中形态发生事件（如原肠胚形成、神经胚形成、体节发生、心脏弯曲和脑形成）的主要脊椎动物模型。理解形态发生过程的关键是通过延时成像动态跟踪这些过程。卵外培养鸡胚的延时电影拍摄要么局限于短时间窗口，要么需要使用培养箱来控制胚胎周围的温度和湿度。在此，我们展示了一种新的技术，用于利用透射光显微镜对卵外培养的鸡胚进行高分辨率延时成像。浸没式滤纸夹层是成熟的滤纸载体技术（EC培养）的一种变体，无需气候箱即可培养鸡胚。胚胎夹在两张相同的滤纸载体之间，并完全浸没在一种简单的、温度可控的培养基中，培养基上覆盖一层轻质矿物油。从原条期（汉堡-汉密尔顿第5期，HH5）到至少28体节期（HH16），胚胎可以腹侧或背侧朝上进行培养。这使得能够获取涵盖约30小时胚胎发育的延时电影。展示了代表性的延时帧和电影。将胚胎与在标准EC培养中培养的胚胎进行形态学比较。浸没式滤纸夹层为研究早期背侧和腹侧形态发生过程提供了一个稳定的环境。它还允许进行活体荧光成像和显微操作，如显微手术、珠子植入、显微注射、基因沉默和电穿孔，并且具有与浸没物镜结合用于基于激光的成像（包括光片显微镜）的强大潜力。
