In Vitro Culturing and Live Imaging of Drosophila Egg Chambers: A History and Adaptable Method

Abstract

Summary/Abstract The development of the Drosophila egg chamber encompasses a myriad of diverse germline and somatic events, and as such, the egg chamber has become a widely used and influential developmental model. Advantages of this system include physical accessibility, genetic tractability, and amenability to microscopy and live culturing, the last of which is the focus of this chapter. To provide adequate context, we summarize the structure of the Drosophila ovary and egg chamber, the morphogenetic events of oogenesis, the history of egg-chamber live culturing, and many of the important discoveries that this culturing has afforded. Subsequently, we discuss various culturing methods that have facilitated analyses of different stages of egg-chamber development and different types of cells within the egg chamber, and we present an optimized protocol for live culturing Drosophila egg chambers. We designed this protocol for culturing late-stage Drosophila egg chambers and live imaging epithelial tube morphogenesis, but with appropriate modifications it can be used to culture egg chambers of any stage. The protocol employs a liquid-permeable, weighted, “blanket” to gently hold egg chambers against the coverslip in a glass-bottomed culture dish so the egg chambers can be imaged on an inverted microscope. This setup provides a more buffered, stable culturing environment than previously published methods by using a larger volume of culture media, but the setup is also compatible with small volumes. This chapter should aid researchers in their efforts to culture and live image Drosophila egg chambers, further augmenting the impressive power of this model system.

Lysosomal Machinery Drives Extracellular Acidification to Direct Non-apoptotic Cell Death

“…Live imaging was performed as described (Peters and Berg, 2016). Briefly, stage 13 egg chambers were individually dissected from flies in room temperature Schneider’s medium.…”

Proteomics Analysis Identifies Orthologs of Human Chitinase-Like Proteins as Inducers of Tube Morphogenesis Defects inDrosophila melanogaster

“…No. 81156) using a Kimwipe "blanket" to hold them in place, as described (Peters and Berg 2016b). Images were acquired on a Leica SP8X scanning confocal microscope and processed using Leica software (LAS Version X), FIJI (ImageJ Version 1.51j), and Photoshop (Version 12.1).…”

Epithelial Patterning, Morphogenesis, and Evolution: Drosophila Eggshell as a Model

“…Each eggshell is produced by an individual egg chamber (Figure 1B,C), which consists of 15 germline-derived nurse cells, a single oocyte, and a surrounding follicular epithelium. The egg chamber is a self-contained unit that can easily be cultured in vitro (Cliffe et al, 2007; Dorman et al, 2004; Manning and Starz-Gaiano, 2015; Peters and Berg, 2016a; Prasad et al, 2007). The development of an egg chamber proceeds through 14 stages (Spradling, 1993); during the stages in which the eggshell is formed (stages 10–14), there is neither cell division nor significant cell death among the follicle cells.…”