Dissection and Mounting of Drosophila Pupal Eye Discs

Summary

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Abstract

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia¹. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar². Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells³. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase^4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Introduction

The fields of developmental and cell biology have been greatly impacted by the model organism: Drosophila melanogaster. Within this model, studies of the eye disc have contributed a great deal of knowledge concerning signaling, cell biology and other areas. The late third larval instar eye disc has been studied extensively and is a powerful model to utilize, as it gives a snapshot of a series of developmental periods, each with its own unique signaling molecules and processes, as the morphogenetic furrow progresses across the eye disc⁸. However, there is a need to further expand our understanding of developmental processes into the pupal phase of d....

Protocol

This procedure is a 2 day procedure.

Day 1 (2 hr + dissecting time)

1. Pupal Eye Disc Dissection

1. Select a pupa for dissection.
   NOTE: The age of the pupa to be dissected will be determined by the experimental needs. However, if examining cell morphology, this is frequently done at 42 hr after puparium formation (APF) at 25 °C, which is the age of the pupae shown in the video. Collect white pupae (considered 0 hr APF) with a wetted paintbrush and arrange them in chronological order (based on collection time) in a new fly food vial maintained at 25 °C. Dissect the aged pupae in this same chron....

Results

As an example of the use of this protocol, results illustrating midpupal (42 hrs APF at 25 °C) eye discs immunostained with different antibodies are presented in Figure 2. By using an antibody directed against phosphotyrosine residues, the membrane of the cells can be observed (Figure 2A). This can be used to identify the regular arrangement of ommatidial cells in the pupal eye following the final patterning processes that occur prior to the midpupal stage. Another representative image depicts the nuclei.......

Discussion

While it appears that the process is simple and easy to perform, in reality, this technique requires a great deal of practice to master. Routinely, we start students off by learning to dissect and mount third instar eye discs¹², which are much easier to work with. This practice helps to develop an appropriate dissection position of the arms, hands and fingers¹³ so that manipulation of the forceps under the dissecting microscope is stable, easy and experienced. In essence, the practice period shou.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphate-buffered saline (PBS, pH 7.4)			80 g NaCl, 2g  KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4, Bring volume to 1 μl, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1x PBS with autoclaved ddH2O before using.
Triton X-100	Promega	H5142	Caution: Irritant! Wear gloves.
0.3% PBT			1.5 ml of Triton X-100, 500 ml 1X PBS.
37% formaldehyde solution	Fisher Scientific	F75P1GAL	Caution: Toxic, probable human carcinogen! Wear gloves.
Fix Solution			(≈4% Formaldehyde in PBS) 50 μl of 37% Formaldehyde, 450 μl 1x PBS, make fresh before use
Normal goat serum	Rockland antibodies & assays	B304	Aliquot in 1 ml volumes and store at -80 °C
Block Solution			10% NGS in PBT.  This can be made and stored at 4 °C for a few days prior to use.
DAPI stock solution	Life Technologies	D3571	For coutnerstaining nuclei. Prepare a 1 mg/ml solution with ddH2O.
VectaShield Mounting Medium	Vector Labs	H-1000	Mounting medium
Glycerol	Sigma	G5516	For mounting. Prepare 70% dilution with ddH2O.
Equipment
Nutating mixer	VWR	82007-202	Used to rock tissue in 3 well glass dish
SylGard 182 Silicone Elastomer Kit	Krayden	NC9897184	Used to make silicone dissection dish
Silicone dissecting dish			Mix Sylgard elastomer kit (above) according to directions gently (to avoid bubbles). Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37 °C incubator.
3 well glass dish	Corning	7220-85	The 3 well variety of these are no longer available, this is the 9 well product.
72 well microwell minitray	Nunc	438733
Sharp forceps (Dumont #55)	Fine Science Tools	11255-20
Vannas-type Micro Scissors, Straight, 5mm blade	Ted Pella	1346
100 mm Borosilicate glass capillaries	World Precision Instruments	1B100-4	Pull with needle puller to make fine point tip that allows a small stream of PBS to flow.
Disposable Transfer Pipets, Fine Tip	Samco Scientific	231
Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches
PVC tubing (1/8 x 3/16 x 1/32)	Nalgene	8000-0010	Use these with pulled needle to assemble the blower tube as shown in Figure 2.
Tygon Silicone tubing (3/32 x 5/32 x 1/32)	Saint Gobain Performance Plastics	ABW00004
Tygon Silicone tubing (1/32 x 3/32 x 1/32)	Saint Gobain Performance Plastics	ABW00001