Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Summary

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

Abstract

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between cell types or even within a given type? What kinds of spatial rules do they follow? The answers to such fundamental questions in vivo have been hampered so far by a lack of tools for high resolution single cell labeling. Here, a detailed protocol to target single cells with a MultiColor FlpOut (MCFO) technique is provided. This method relies on three differently tagged reporters (HA, FLAG and V5) under UAS control that are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of a heat shock-induced Flp recombinase, which randomly removes the FRT-stop-FRT cassettes in individual cells: expression occurs only in cells that also express a GAL4 driver. This leads to an array of differently colored cells of a given cell type that allows the visualization of individual cell morphologies at high resolution. As an example, the MCFO technique can be combined with specific glial GAL4 drivers to visualize the morphologies of the different glial subtypes in the adult Drosophila brain.

Introduction

Glia, the non-neuronal cell population of the nervous system (NS), were long believed to provide a static framework for neurons and therefore were not studied in detail. However, in humans, glia constitute the vast majority of cells in the NS (~90%) and fall into several different categories, including astrocytes, oligodendrocytes, microglia and Schwann cells. In Drosophila, glia constitute about 10% of the cells in the NS. Intriguingly, their morphologies and functions are remarkably similar to those found in vertebrates^1,2. Their morphologies include blood-brain barrier (BBB) forming epithelia, ensh....

Protocol

1. Preparing Flies for MultiColor FlpOut (MCFO) Experiments

NOTE: The MCFO technique refers to a modified version of the so called Flp-mediated stop cassette excision (FlpOut). Transgenic MCFO flies carry a heat shock promoter (hsp)-Flp recombinase and different reporters under UAS control. Each reporter consists of a common backbone of a myristoylated (myr) super folder green fluorescent protein (sfGFP), in which 10 copies of an epitope tag (e.g., HA, FLAG or V5) have been inserted. The resulting non-fluorescent proteins are named "spaghetti monster GFPs" (smGFPs)¹⁴ and can be detected using specific ....

Results

This section illustrates examples of results that can be obtained by using the MCFO technique in the adult Drosophila brain. Figure 3 shows a schematic of the method. Three differently membrane-tagged reporters (myr-smGFP-HA, myr-smGFP-FLAG and myr-smGFP-V5) under UAS control are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of Flp recombinase which rand.......

Discussion

This protocol describes an easy and efficient method to study the morphology of different cell types within a tissue of interest at high resolution. With the MCFO technique, multiple reporters with different epitope tags are used in combination for multicolor stochastic labeling (Figure 2). Similar to other methods such as Brainbow/Flybow^15,16,17, MCFO increases the label diversity through marker co.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Water bath	Grant	GD100
PCR tubes	Sarstedt	72.737.002
Forceps	Dumont	11251-20
Dissecting dish 30 mm x 12 mmm	Electron Microscopy Sciences	70543-30	Glass dissection dish
Pyrex 3 Depression Glass Spot Plate	Corning	7223-34	Glass dissection plates
Sylgard Black	SYLGARD, Sigma-Aldrich	805998	home made with charcoal
ExpressFive S2 cell culture medium	Invitrogen	10486-025
20% PFA	Electron Microscopy Sciences	15713
Triton X-100	Roth	3051.3
Normal goat serum	Jackson Laboratories	005-000-121
Normal donkey serum	Jackson Laboratories	017-000-121
Bovine Serum Albumin	Sigma	A9647
Rabbit HA-tag	Cell Signaling	C29F4	Primary antibody, dilution 1:500
Rat FLAG-tag	Novus Biologicals	NBP1-06712	Primary antibody, dilution 1:100
Mouse V5-tag:DyLight 549	AdSerotec	0411	Conjugated antibody, dilution 1:200
anti-rabbit AlexaFluor 488	Invitrogen	A11034	Secondary antibody, dilution 1:250
anti-rat DyLight 647	Jackson Laboratories	712-605-153	Secondary antibody, dilution 1:100
Vecta Shield	Vector Laboratories	H-1000
SlowFate Gold	Invitrogen	S36937
Secure Seal Spacer	Grace Biolabs		Contact company for ordering
Microscope cover glass 22 X 60 mm	Marienfeld	101152
Microscope cover glass 22 x 22 mm	Roth	H874
Stereo Microscope, Leica MZ6	Leica
Confocal laser scanning microscope LSM710	Zeiss
Immersol	Zeiss	518 F	Immersion oil for fluorescence-microscopy, halogen free
Immersol	Zeiss	W 2010	Immersion fluid for water-immersion objectives, halogen free
R56F03-GAL4 (EG)	Bloomington Stock Center	39157	GAL4 driver
R86E01-GAL4 (ALG)	Bloomington Stock Center	45914	GAL4 driver
hspFlpPestOpt; UAS-FRT-stop-FRT-myr-smGFP-HA, UAS-FRT-stop-FRT-myr-smGFP-FLAG, UAS-FRT-stop-FRT-myr-smGFP-V5	Bloomington Stock Center	64085	UAS reporter (https://bdscweb.webtest.iu.edu/stock/misc/mcfo.php)
Fiji (Image J)			Image analysis software
Multi Time Macro	Zeiss		Software for automated scanning