Imaging Through the Pupal Case of Drosophila melanogaster

Summary

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Abstract

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 µm of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Introduction

The vinegar fly, Drosophila melanogaster, is a well-established model for studying many aspects of biology. Drosophila research has a rich history of genetic experimentation that allows sophisticated forms of gene manipulation including expression, knockdown and mutation. With the advent of fluorescent protein labels, this repertoire has expanded to include studies of cells and proteins in living animals. The fly embryo is an excellent system for such studies as it is small and optically clear allowing deep, high-resolution imaging in vivo^1-3. Other stages of fly development have proven to be less tractable, requiring anaesthetiza....

Protocol

1. Fly Work

1. Maintain flies on standard cornmeal-agar-molasses-yeast medium at room temperature¹⁰.
2. For crosses, isolate virgins within 6 hr of eclosion. After crossing to males of the desired genotype, change flies to new vials every 3-4 days.

Note: For these experiments, Gal4 line A9 was used to drive expression of transgenes in the wing. Fly stocks can be obtained from the stock center in Bloomington. Stocks used in these experiments include A9-Gal4 (Bl#8761), His2Av-GFP (Bl#5941), Sco/CyO HsCre (Bl#1092), UAS-ChRFP-Tub (Bl#25773), lollibow¹¹.

2. Selection and Mou....

Results

Cells in pseudostratified epithelia, such as the developing Drosophila eye, or the ventricular layer of the developing vertebrate central nervous system, undergo nuclear movements, termed interkinetic nuclear migration, in time with the cell cycle. DNA replication occurs when nuclei are at or near the basal surface and cells enter mitosis when the nuclei reach the apical surface^15,16. The pupal wing cells form a rapidly dividing monolayer epithelium during the first several hours after head eversion. .......

Discussion

To visualize, measure, and quantify features of dividing cells, required development of a simple preparation for observing mitosis in the living pupal wing of Drosophila by means of confocal analysis of His2AvGFP expressing cells. This method was used to document that the cell cycle in the pupal wing bears strong similarities to cell cycles in pseudostratified epithelia in that nuclei move to the apical surface of the epithelium where they enter mitosis. Following telophase, nuclei drop back into the epithe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fly stuff fly pad	Genesee Scientific	59-114	for fly anesthetization
CO2 gas	Airgas South	CD50	For fly anesthetization
Regulator	Airgas South		CO2 regulator
Fly vials	Genesee Scientific	32-113RLBF	Fly culture
Drosophila lines:A9-Gal4 (Bl#8761), His2Av-GFP (Bl#5941), Sco/CyO HsCre (Bl#1092), UAS-ChRFP-Tub (Bl#25773)	Bloomington Stock Center
Glass bottom dishes #1 1/2	WillCo Wells BV		For microscopy
Thiodiethylene Glycol	Fluka	88559	mountant
Modeling clay	art supply store		Support to position pupae against
Paintbrushes	art supply store		To manipulate flies
Fine Forceps, Inox #5	Fine Science Tools	11252-20	Dumont #5
computer	any		8 Gb RAM for image/movie analysis
Fiji software		Free ware http://fiji.sc/Fiji	Image analysis software
Confocal microscope			Any fast scanning confocal should be sufficient
20X dry, and 40X or 63X oil immersion lenses	any		For imaging tissue, cellular, and subcellular features
Immersion oil (nonfluorescent)
Stereomicroscope	any		For fly manipulation