Towards Long Term Cultivation of Drosophila Wing Imaginal Discs In Vitro

The wing imaginal disc of Drosophila melanogaster is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with RGB cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs.

黑腹果蝇（Drosophila melanogaster）的翅成虫盘（wing imaginal disc）是研究细胞生长、增殖与死亡调控，以及器官发生过程中模式形成与形态发生的经典实验模型系统。该体系所适用的精准遗传方法，推动了发育生物学领域具有根本重要性的概念性进展。若能够在体外（in vitro）开展翅成虫盘的长期发育研究，将进一步拓展其实验可及性与应用多功能性。例如，体外培养体系可实现最高时空分辨率的活细胞成像（live imaging）。然而正如本研究明确证实的那样，标准培养方法会在解剖后的翅成虫盘培养数小时内快速引发细胞增殖停滞。利用已确立的S期与M期细胞标记物，以及新型报告转基因RGB细胞周期追踪器（RGB cell cycle tracker）开展分析后发现，体外培养会干扰整个间期的细胞周期进程，而非仅局限于G1期。此外，对诱导型转基因的增强型绿色荧光蛋白（EGFP）表达量进行定量分析后发现，翅成虫盘培养对细胞周期以外的基础细胞功能具有快速的负面影响。翅成虫盘移植实验证实，这些不利影响并非源于解剖分离过程中对成虫盘造成的致命损伤，明确表明问题源于培养基成分不足。研究评估了多种替代培养基，包括在自然生长状态下包围成虫盘的幼虫血淋巴（hemolymph）。但研究发现，分离得到的幼虫血淋巴甚至比现有培养基更不适用于培养，这可能是由于血淋巴分离过程或成虫盘培养过程中的转化反应所致。本研究还分析了主要生长调控通路在成虫盘培养过程中的作用，以及胰岛素、与作为内分泌因子潜在来源的幼虫组织共培养对成虫盘的影响。基于本研究的分析结果，我们开发出了一种可延长培养翅成虫盘细胞增殖时长的培养方案。