Joseph Mathieu, Hipfner David R
Institut de recherches cliniques de Montréal (IRCM); Programmes de biologie moléculaire, Université de Montréal.
Institut de recherches cliniques de Montréal (IRCM); Programmes de biologie moléculaire, Université de Montréal; Département de Médecine, Université de Montréal;
J Vis Exp. 2025 Aug 22(222). doi: 10.3791/68411.
Embryonic tissue growth and patterning are largely controlled by signals exchanged locally between cell populations within the tissues themselves. Cytonemes are a type of signaling filopodia first identified in Drosophila that connect and mediate exchange between signal-producing and signal-receiving cells. In the developing Drosophila wing imaginal disc, cytonemes are involved in signal exchange between distinct populations of cells within the disc proper (DP) epithelium, which will form the adult wing, as well as between DP cells and cells in adjacent disc-associated tissues. Cytonemes synapse with target cells to form intimate membrane contacts. Here, we present a protocol for quantifying cytoneme-mediated contact between DP cells and cells of the adjacent peripodial membrane (PerM) epithelium, which is separated from the DP cells by the disc lumen, using a GFP reconstitution approach in live wing discs. Using the GAL4-UAS and LexA-LexAop systems, complementary fragments of split-GFP (spGFP1-10, spGFP11), each fused to the transmembrane domain of CD4, are expressed on either side of the disc lumen. Imaging of reconstituted GFP fluorescence in live wing disc preparations by confocal microscopy is then used to generate image stacks from which reconstituted GFP fluorescence can be localized and quantified. Using this system it is possible to co-express protein-coding or RNA interference transgenes in either cytoneme-producing or target cells to gauge their effect on DP-PerM cell contacts. This system, easily adaptable to other tissues, thus enables the identification of factors important for cytoneme formation or function.
胚胎组织的生长和模式形成在很大程度上受组织内细胞群体之间局部交换信号的控制。胞质桥粒是一种信号丝状伪足，最初在果蝇中被鉴定出来，它连接信号产生细胞和信号接收细胞并介导它们之间的信号交换。在发育中的果蝇翅成虫盘，胞质桥粒参与成虫盘固有（DP）上皮内不同细胞群体之间的信号交换，DP上皮将形成成虫的翅膀，同时也参与DP细胞与相邻盘相关组织中的细胞之间的信号交换。胞质桥粒与靶细胞形成突触，形成紧密的膜接触。在这里，我们展示了一种使用活翅成虫盘中的绿色荧光蛋白（GFP）重组方法来量化DP细胞与相邻围膜（PerM）上皮细胞之间胞质桥粒介导的接触的方案，PerM上皮细胞通过盘腔与DP细胞分隔开。利用GAL4-UAS和LexA-LexAop系统，将分裂型GFP（spGFP1-10、spGFP11）的互补片段分别与CD4的跨膜结构域融合，在盘腔两侧表达。然后通过共聚焦显微镜对活翅成虫盘制剂中重组的GFP荧光进行成像，以生成图像堆栈，从中可以定位和量化重组的GFP荧光。使用该系统，可以在产生胞质桥粒的细胞或靶细胞中共表达蛋白质编码或RNA干扰转基因，以评估它们对DP-PerM细胞接触的影响。该系统易于应用于其他组织，因此能够鉴定对胞质桥粒形成或功能重要的因子。
