Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Summary

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

Abstract

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes’ large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Introduction

Cell division is often studied using cell lines grown in culture¹. While we have gained a wealth of invaluable insight and understanding of fundamental mechanisms from these studies², cells grown in culture cannot fully recapitulate the physiology of cell division as it occurs in intact, living tissue. For example, in intact tissues and organs, cells must divide at the right place and at the right time so that progeny cells are properly situated within the tissue, so that they can undergo appropriate differentiation or functional programs, and so that cell proliferation is properly coordinated with tissue growth or homeo....

Protocol

1. Prepare Animals, Tools, and Media for Dissection

1. Cross male and female flies to obtain progeny of the desired genotype. Useful transgenic markers for identification and staging of meiotic spermatocytes include GFP-tubulin to label the meiotic spindle and RFP-histone 2A to label chromsomes⁸. Use five to ten females per cross and maintain crosses on standard fly food in vials in a 25 °C incubator until third instar larvae begin crawling up the sides of the vial (4–5 days). .......

Discussion

We have described a protocol for the preparation of larval or early pupal Drosophila testes, optimized for long-term, live imaging of spermatocyte cell division. This is a powerful method for analysis of cell division in the physiological context of intact tissue. The power of this method is further expanded when combined with Drosophila genetic tools, such as specific gene mutations, tissue-specific RNAi-mediated suppression, and fluorescently labeled protein and organelle markers. In addition, the sma.......

Materials

Name	Company	Catalog Number	Comments
5" Dissecting Probe	Fisher	08-965-A
5.75" Glass Pasteur Pipet	Fisher	13-678-20A
Bovine Serum Albumin (BSA)	Fisher	BP9703-100
Dumont #5 Forceps	Fine Science Tools	11252-20	Straight forcepts with fine tips
Frosted Microscope Slides	Fisher	12-544-2	Slides for mounting fixed tissue, with frosted writing surface
Halocarbon oil 700	Sigma	H8898-100 mL
Lumox Dish 50	Sarstedt	SAR946077410	Gas-permeable tissue culture dish
Microscope Cover Glass	Fisher	12-541-B	22x22 mm, #1.5 glass coverslip
Minutien Pins	Fine Science Tools	26002-15	Insect pins used to make scalpel tool
Nickel Plated Pin Holder	Fine Science Tools	26018-17
Paraformaldehyde 32% Solution, EM Grade	Electron Microsocopy Sciences	15714
Plan Beveled Edge Microscope Slides	Fisher	12-549-5	Slides used for dissections
PYREX Spot Plates	Fisher	13-748B	9-well glass dissecting dish
Schneider's Drosophila medium	Fisher	21720-024
Syringe Filter, 0.22 µm	EMD Millipore	SLGS033SB
Triton X-100	Fisher	BP151-500
Vectashield	Vector Labs	H-1000	Microscopy mounting medium