Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.
Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, Georgia, USA.
Curr Protoc. 2023 Oct;3(10):e924. doi: 10.1002/cpz1.924.
The rapid succession of events during development poses an inherent challenge to achieve precise synchronization required for rigorous, quantitative phenotypic and genotypic analyses in multicellular model organisms. Drosophila melanogaster is an indispensable model for studying the development and function of higher order organisms due to extensive genome homology, tractability, and its relatively short lifespan. Presently, nine Nobel prizes serve as a testament to the utility of this elegant model system. Ongoing advancements in genetic and molecular tools allow for the underlying mechanisms of human disease to be investigated in Drosophila. However, the absence of a method to precisely age-match tissues during larval development prevents further capitalization of this powerful model organism. Drosophila spends nearly half of its life cycle progressing through three morphologically distinct larval instar stages, during which the imaginal discs, precursors of mature adult external structures (e.g., eyes, legs, wings), grow and develop distinct cell fates. Other tissues, such as the central nervous system, undergo massive morphological changes during larval development. While these three larval stages and subsequent pupal stages have historically been identified based on the number of hours post egg-laying under standard laboratory conditions, a reproducible, efficient, and inexpensive method is required to accurately age-match larvae within the third instar. The third instar stage is of particular interest, as this developmental stage spans a 48-hr window during which larval tissues switch from proliferative to differentiation programs. Moreover, some genetic manipulations can lead to developmental delays, further compounding the need for precise age-matching between control and experimental samples. This article provides a protocol optimized for synchronous staging of Drosophila third instar larvae by colorimetric characterization and is useful for age-matching a variety of tissues for numerous downstream applications. We also provide a brief discussion of the technical challenges associated with successful application of this protocol. © 2023 Wiley Periodicals LLC. Basic Protocol: Synchronization of third instar Drosophila larvae.
发育过程中的事件快速连续发生，这对精确同步提出了固有挑战，而精确同步对于在多细胞模式生物中进行严格的、定量的表型和基因型分析是必需的。由于广泛的基因组同源性、可操作性以及相对较短的寿命，黑腹果蝇是研究高等生物发育和功能的不可或缺的模型。目前，九个诺贝尔奖证明了这个优雅的模型系统的实用性。遗传和分子工具的不断进步使得可以在果蝇中研究人类疾病的潜在机制。然而，在幼虫发育过程中精确匹配组织年龄的方法的缺失，阻碍了这个强大的模式生物的进一步利用。果蝇在其生命周期中近一半的时间都在经历三个形态上明显不同的幼虫龄期，在此期间，成虫外部结构（例如眼睛、腿、翅膀）的前体细胞—— imaginal discs 生长并发育出不同的细胞命运。其他组织，如中枢神经系统，在幼虫发育过程中经历了巨大的形态变化。虽然这三个幼虫龄期和随后的蛹期历来是根据在标准实验室条件下产卵后小时数来确定的，但需要一种可重复、高效和廉价的方法来在第三龄期内精确匹配幼虫。第三龄期特别有趣，因为这个发育阶段跨越了一个 48 小时的窗口，在此期间，幼虫组织从增殖程序切换到分化程序。此外，一些遗传操作可能导致发育延迟，进一步加剧了对照和实验样本之间精确年龄匹配的需要。本文提供了一种通过比色特征化来优化同步分级果蝇第三龄幼虫的方案，该方案可用于为众多下游应用匹配各种组织的年龄。我们还简要讨论了成功应用此方案所涉及的技术挑战。 © 2023 Wiley Periodicals LLC. 基本方案：同步分级果蝇第三龄幼虫。
