Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

Summary

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner ¹. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

Abstract

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 ². The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 ^{3 4}. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes⁵: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s ^{6 7 8}.

Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc⁹ and initiates the assembly of undifferentiated cells into ommatidia^10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 ^12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes ¹⁵, including rhabdomere formation, synaptogenesis and eventually rh expression.

In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Protocol

1. Introduction

In this video, we describe methods for retinal dissections and immunohistochemistry at three defined developmental periods: the third instar larval, the midpupal and the adult stage. Although our protocol also works for other pupal stages (for details on earlier stages, see ¹⁶), we chose the midpupal stage, as it is optimal for imaging all PRs in one focal plane and their nuclei are easily identifiable, which facilitates the visualization of transcription factor expression patterns. The dissection of late pupal retinas is very similar to the dissection of adult retinas.

First, we demonstr....

Discussion

1. Troubleshooting

In our experience, dissections require practice (up to several weeks) and are facilitated by achieving a comfortable hand position ²¹ by resting the elbows and forearms on the table and with the fingers making contact with the dissection dish. That way, only the thumbs, index and middle fingers perform subtle movements.

Removing the lamina without damaging the photoreceptors is probably the most challenging step. Practice with red-eyed w.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
			Reagent
Phosphate-buffered saline (PBS1x, pH 7.4)	Sigma		Prepare 10x stock solution 20. Dilute with distilled water to obtain 1x PBS and store at room temperature. Cool on ice before dissections.
Triton-X 100	Sigma	9002-93-1	Caution: Irritant! Wear gloves. Prepare 50 ml 1xPBS with 0.3% Triton X-100 (PBST). Store at room temperature.
37% formaldehyde solution	Fisher Scientific	F75P1GAL	Caution: Toxic, probable human carcinogen! Wear gloves. Before the fixation step, freshly prepare 3.7% solution in a chemical fume hood by diluting with PBS, store on ice.
5% normal horse serum	Jackson Immuno Research	008-000-001	Prepare 5% v/v dilution in PBST. Store at four degrees.
Primary and secondary antibodies (e.g. Donkey anti-sheep Alexa Fluor 488, Donkey-anti rabbit Alexa Fluor 555, Donkey anti-mouse Alexa Fluor 647)	Invitrogen Molecular Probes	A11015 A31572 A31571	Dilute secondary antibodies 1:800 in PBST and store at four degrees.
Alexa Fluor 488 Phalloidin		A12379	Dilute 1:100 in secondary antibody solution.
Slowfade Gold Antifade reagent	Invitrogen Molecular Probes	S36936	Mounting medium. Store at -20 degrees.
Glycerol	Fisher Scientific	G31-1	For mounting. Prepare 10 ml of 50% dilution with distilled water, store at room temp.
CO2			For anesthetizing adult flies.
			Equipment
Two sharp forceps (Dumont #55)	Fine Science Tools	11255-20
Sylgard 184 Silicone Elastomer Kit	Dow Corning		Prepare Sylgard dissection dish by filling a plastic Petri dish with Sylgard mixture.
Three-well glass dissection dishes	Fisher Scientific	21-379
Two minutien dissecting pins (0.1 mm diameter) and two pinholders (12 cm)	Fine Science tools	26002-10	Insert one minutien pin in each of the two pinholders and bend one of the pins to form a hook.
Microscope cover slips (22x22-1 and 24x40-1)	Fisher Scientific	12-542A
Microscope slides (25x75x1.0 mm; precleaned)	Fisher Scientific	12-550-143
1.5 ml microcentrifuge tubes
Clear nail polish or Scotch tape
Orbital shaker	Bellco
Slide holder box	Fisher Scientific
Parafilm	Bemis	PM996
Thin paintbrush
P20 and P200 micropipettes and tips
Dissecting microscope
Small bucket with ice			For cooling the glass well plates, dissected retinas and solutions.