Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Summary

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Abstract

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Protocol

Part 1: Pre-experimental set-up. One week in advance, mate flies to generate larva that express GFP under the control of a glial-specific promoter. For our experiment we visualized GFP tagged with a nuclear localization sequence expressed in glial cells using the reversed polarity (repo) promoter (3, 4). Prepare cover slips at least one day in advance by soaking 18 mm round cover slips for 10 minutes in 1% poly-L-lysine solution and air dry overnight. On the day before the experim…

Discussion

In this protocol we describe observation of glial cell migration into the eye imaginal disc using live microscopy. In our wild type example (figure 1 A-D), we used a nuclear GFP marker to observe glial cell movement in the eye disc over the course of one hour. In a mutant for a candidate gene required for glial cell migration currently under study in our laboratory, we observed a stalling of glial cell nuclei within the optic stalk during a one-hour period (figure 1 E-H). Our strategy can be adapted to visual…

Materials

Material Name	Type	Company	Catalogue Number	Comment
Poly-L-lysine	Reagent	Sigma	P8920
Schneider’s Insect Media	Reagent	Sigma	S0146
Penicillin-Streptomycin	Reagent	Sigma	P4458
Insulin solution from bovine pancreas	Reagent	Sigma	I0516
Chamlide Magnetic chamber	Tool	Live cell Instrument	CM-R-10	35 mm dish type chamber for 18 mm coverslip
Ultra fine clipper scissors	Tool	Fine scientific tools	15200-00
Dumont #5 forceps	Tool	Fine scientific tools	11251-20
Fluorescent microscope	Microscope	Zeiss		Any fluorescent imaging system that has the necessary filters and excitation for GFP can be used.