Ninov Nikolay, Martín-Blanco Enrique
Instituto de Biología Molecular de Barcelona (Consejo Superior de Investigaciones Científicas), Parc Cientific de Barcelona, Josep Samitier 1-5, Barcelona 08028, Spain.
Nat Protoc. 2007;2(12):3074-80. doi: 10.1038/nprot.2007.417.
During larval stages of Drosophila development, the abdominal epidermis is composed of histoblasts (adult precursors) and larval epidermal cells (LECs). During metamorphosis, histoblasts proliferate and colonize the territories occupied by the LECs, which die and become engulfed by macrophages. This morphogenetic process is an excellent model for in vivo analysis of epithelial migration, cell division, cell death, patterning and differentiation. Here, we describe a protocol for time-lapse recording of the developing epidermis during metamorphosis. The protocol describes the removal of the pupal case (which acts as an opaque barrier to effective imaging) and mounting and imaging of specimens of different stages so that normal developmental processes are preserved. This method enables high-resolution studies over long time periods using fluorescent markers and confocal microscopy. The protocol requires 1 h for pupal dissection and mounting and, depending on the stages and genotypes to be analyzed, several more hours for preprocessing and aging and developmental staging of flies and pupae.
在果蝇发育的幼虫阶段，腹部表皮由成组织细胞（成虫前体）和幼虫表皮细胞（LECs）组成。在变态过程中，成组织细胞增殖并占据LECs所占据的区域，LECs死亡并被巨噬细胞吞噬。这种形态发生过程是上皮迁移、细胞分裂、细胞死亡、模式形成和分化体内分析的优秀模型。在这里，我们描述了一种在变态过程中对发育中的表皮进行延时记录的方案。该方案描述了去除蛹壳（它对有效成像起到不透明屏障的作用）以及对不同阶段的标本进行固定和成像，从而保留正常发育过程。这种方法能够使用荧光标记和共聚焦显微镜在长时间内进行高分辨率研究。该方案需要1小时进行蛹解剖和固定，并且根据要分析的阶段和基因型，还需要几个小时对果蝇和蛹进行预处理、老化以及发育分期。
