Dourlen Pierre, Levet Clemence, Mejat Alexandre, Gambis Alexis, Mollereau Bertrand
Laboratory of Molecular Biology of the Cell, Ecole Normale Supérieure de Lyon.
J Vis Exp. 2013 Sep 20(79):e50610. doi: 10.3791/50610.
The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.
果蝇眼睛被广泛用作研究发育和神经元退化的模型。借助强大的有丝分裂重组技术，基于克隆分析的精妙遗传筛选已鉴定出幼虫阶段眼睛发育和光感受器（PR）分化所涉及的信号通路。我们在此描述番茄/绿色荧光蛋白 - FLP/FRT方法，该方法可用于对成年活体果蝇眼睛进行快速克隆分析。利用角膜中和技术对荧光光感受器细胞进行成像，这些细胞位于通过翻转酶介导的重组产生的镶嵌克隆视网膜上。与传统的视网膜组织学切片相比，该方法具有几个主要优点：它可用于高通量筛选，并且已证明是鉴定调节PR存活和功能的因子的有效方法。它可用于在数周内对同一活体动物的PR退化进行动力学分析，以证明成年果蝇中PR存活或功能所需的特定基因。该方法对于解决发育突变体中的细胞自主性问题也很有用，例如那些平面细胞极性建立受到影响的突变体。
