School of BioSciences, University of Melbourne, Parkville, VIC, Australia.
Methods Mol Biol. 2021;2179:115-134. doi: 10.1007/978-1-0716-0779-4_11.
In the early stages of Drosophila melanogaster (Drosophila) metamorphosis, a partial epithelial-mesenchymal transition (pEMT) takes place in the peripodial epithelium of wing imaginal discs. Blocking this pEMT results in adults with internalized wings and missing thoracic tissue. Using peripodial GAL4 drivers, GAL80 temporal control, and UAS RNAi transgenes, one can use these phenotypes to screen for genes involved in the pEMT. Dominant modifier tests can then be employed to identify genetic enhancers and suppressors. To analyze a gene's role in the pEMT, one can then visualize peripodial cells in vivo at the time of eversion within the pupal case using live markers, and by dissecting, fixing, and immunostaining the prepupae. Alternatively, one can analyze the pEMT ex vivo by dissecting out wing discs and culturing them in the presence of ecdysone to induce eversion. This can provide a clearer view of the cellular processes involved and permit drug treatments to be easily applied.
在黑腹果蝇(Drosophila melanogaster)变态发育的早期阶段,翅 imaginal 盘的体壁上皮中会发生部分上皮-间充质转化(pEMT)。阻断这种 pEMT 会导致成虫的翅膀内陷,胸部组织缺失。使用体壁 GAL4 驱动子、GAL80 时间控制和 UAS RNAi 转基因,可以利用这些表型筛选参与 pEMT 的基因。然后可以进行显性修饰测试,以鉴定遗传增强子和抑制子。为了分析基因在 pEMT 中的作用,可以在蛹壳内翻出时使用活体标记物在体内可视化体壁细胞,并通过解剖、固定和免疫染色预蛹。或者,可以通过解剖出翅膀盘并在存在蜕皮激素的情况下培养它们来离体分析 pEMT,以诱导翻出。这可以更清楚地观察到所涉及的细胞过程,并允许轻松应用药物处理。