Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Summary

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

Abstract

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Introduction

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior studies, and behavioral evolution^1,2,3,4. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons^1,5,6. The overall goal of this manuscript is to demonstrate how to stage and dissect de....

Protocol

The following protocol is consistent with the ethics guidelines established by the Duke University Research Ethics Committee.

1. Tissue Preparation and Developmental Staging of Drosophila melanogaster Pupa

1. First, identify how many flies will be necessary for the dissection. For RT-PCR and RNAseq, collect 100 - 200 antennal discs and antennae from individuals which are necessary at prepupal, 8 h APF, 40 h APF, and adult stages. For immunohistochemistry, dissect 10 - 20 individual.
2. Clean all materials, including dissection pads and forceps, using wipes with 70% EtOH. If the dissected material is to be used for R....

Results

The dissected developing olfactory tissue can be used for RNA extraction followed by RT-PCR to assess gene expression or can be taken through immunohistochemistry protocols to determine its expression pattern, and the sub-cellular localization of genes of interest. In this section, we present representative results from either protocol. Figure 1 is the representative quantitative RT-PCR showing the transcription of cell surface receptor and olfactory receptor.......

Discussion

This protocol is a useful resource for labs interested in identifying the genetic and transcriptional programs operating in developing Drosophila olfactory tissue, as well as a comparative analysis of these programs across Drosophila species. It is especially useful if systems-level transcriptional profiles from different developmental stages are needed as a primer for future studies. The protocol described here demonstrates how to stage and isolate developing olfactory tissue from Drosophila t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10X Phosphate-buffered Saline	Gibco	70011-044	Dilute to 1X in RNase free water
CO2 tank	Air Gas	CD R200
Two sharp forceps (Dumont #55)	Fine Science Tools	11255-20
Trizol	Invitrogen	15596-026	RNA isolation solutions
Dissecting Microscope
Small bucket with ice
P20 and P200 micropipettes and tips
1.7 mL Microfuge tubes			Purchase nuclease free for best results
0.2 mL PCR tubes
Paraformaldehyde powder	Polysciences	380
10% Triton X-100	Teknova	T1105	Dilute to 0.2% v/v in 1X PBS
2" petri dishes
55mm filter paper	Whatman	1001-055	Wet with water and place in a petri dish to keep pupae moist
25 C incubator
deionized H2O			Purchase nuclease free or treat with DEPC
Sylgard 184 Silicone Elastomer Kit	Dow Corning		to be used for making dissection pads from Terizaki plate covers.
MicroWell Mini Trays with lids	Nunc	438733	Lids can be used to make silicone dissection pads
Rabbit anti-GFP antibody	MBL international corpor	PM005	primary antibody
Mouse anti RAT CD2	AbD seroTec	MCA154RHDL	primary antibody
ADL195 (anti lamin, mouse)	Dev studies hydoma ban	ADL195-s	primary antibody
Alexa Fluor® 488 goat anti-rabbit IgG (H+L)	invitrogen	A11008	Secondary antibody
Cy3 Goat Anti-Mouse IgG	Jackson ImmunoResearch	115-166-003	Secondary antibody
Triton X-100, Protein Grade Detergent, 10% Solution, Sterile-Filtered	CALBIOCHEM	648463	detergent
Lab Rotator	Thermo scientific		Rotator
Nuclease Free Water	Growcells.com	NUPW-0125	Water
Light-Duty Tissue Wipers	VWR	82003-822	For cleaning dissection supplies
Superscript II	invitrogen	18064014	Reverse Transcriptase
QIAshredder (50)	RNA extraction	QIAGEN	79654
Oligo(dT)12-18 Primer	RT	life technologies	18418012
RNeasy MinElute Cleanup Kit (50)	RNA extraction	QIAGEN	74204
FASTSTART UNIV SG MASTER (5 ML)(500 RXN)	qPCR	Roche	04913850001
FASTSTART ESSENTIAL GREEN DNA MASTER	qPCR	Roche	06402712001
LIGHTCYCLER 480 - MULTIWELL PLATE 96	qPCR	Roche	04729692001
NEBNext High-Fidelity 2X PCR Master Mix	qPCR	NEB	M0541S