A Rapid, Simple Workflow for Quantification of External Adult Drosophila Structures

Summary

Here, we present a rapid, low-cost, workflow for high-resolution imaging of adult Drosophila eyes to quantify patterning and growth defects. We describe our protocol for sample preparation by point-mounting, high-resolution image acquisition, and image analysis.

Abstract

The Drosophila compound eye is a precisely patterned tissue that has revealed molecular mechanisms and biological processes that drive morphogenesis. It is a simple structure of repeating unit eyes, termed ommatidia, that is used to characterize genetic interactions and gene functions. Mutations that affect eye architecture can be easily detected and analyzed; hence, this system is frequently used in under-resourced institutions. Further phenotypic analysis often includes a Scanning Electron Microscope (SEM) to generate high-magnification images suitable for quantitative analysis. However, SEMs are expensive and require costly reagents; sample preparation spans days; and, often, they need full-time staff for sample preparation and instrument maintenance. This limits their utility at under-resourced institutions or during budgetary austerity. In entomology, the use of high-resolution digital imaging technology is a common practice for the identification and characterization of species. This paper describes a method that combines strategies and allows for high-resolution digital imaging of adult Drosophila structures and quantitative analysis using the open software ImageJ. The workflow is a rapid and student-friendly alternative that remedies the limitations of underfunded and under-resourced research facilities with a cost-effective and rapid approach to quantitative phenotypic analysis.

Introduction

Drosophila melanogaster is a powerful genetic model organism that has been used for decades to elucidate molecular signaling pathways and cellular behaviors. Many of the evolutionarily conserved signaling pathways that are essential for multicellular development were first identified and their mechanism of action defined in Drosophila. About 65-75% of all human disease-associated genes have orthologs in Drosophila^1,2. The adult Drosophila eye is an important model that has allowed for unbiased genetic screens that facilitated the discovery of important conserved genes implic....

Protocol

1. Adult Drosophila collection and fixation

1. Set up Drosophila crosses or select strains and place them in vials containing fly food. Incubate the vials at the desired temperature (usually 25 °C) until the flies have developed and adults eclose (~10-14 days at 25 °C).
2. Anesthetize the flies with CO₂ and place them on a CO₂ pad.
3. Sort the flies with a feather and select the individuals with the desired phenotype (e.g., str.......

Discussion

Here we describe a method for sample preparation, high-resolution imaging, and analysis of adult Drosophila structures. The Drosophila eye is a genetically tractable model system that has yielded critical insights into molecular mechanisms underlying diseases including cancer¹⁹, neurodegeneration²⁰ and metabolic diseases²¹. In particular, cancer patient "avatars" are generated where transgenic Drosophila carrying on.......

Materials

Name	Company	Catalog Number	Comments
1 mL serological pipette	ThermoFisher Scientific	170353N
1.7 mL microcentrifuge tubes	Genesee Scientific	24-282LR
20x Apo Microscope Objective	Mitutoyo Corp.	378-804-3
Archival 65 lb cardstock	Neenah, Inc.	91901
Canon EF 70-200 mm USM II telephoto lens	Canon	3044C002
Canon EOS 6D Mark II DSLR Camera Body	Canon	1897C002
Diffuser Dome	Macroscopic Solutions	PA-DIF-GIM-SM
Diffuser for Mitutoyo M Plan APO Objectives	Macroscopic Solutions	mitutoyo-diffusers
Drosophila vials and plugs	Genesee Scientific	32-117BF
Dumont #5 fine-tip forceps	Fisher Scientific	NC9889584
Goose feathers	Amazon	B01CMMJI6U
Heavy-Duty Anodized Aluminum Tripod	Really Right Stuff, LLC	TFA-32G
Kimwipes	Fisher Scientific	06-666A	lint-free lab tissue
Levenhuk M1000 Plus Digital Camera	Levenhuk	70358
No. 3 mounting pin	Indigo Instruments	33414-3
Nutri-Fly Bloomington Drosophila media	Genesee Scientific	66-113	fly food
Point-Punch	M.C. Mieth Manufacturing, Inc.	448Detail
Screwknob Clamp	Really Right Stuff, LLC	SK-Clamp	For attaching the macro rail to the tripod
Stackshot Controller and Macro Rail	Cognisys Inc.	ST3X_100_BUNDLE
Step-down Ring Adapter	RAF Camera	763461174207	Lens adapter to connect the microscope objective to the camera lens
Titebond Glue	Franklin International	5013
Yongnuo YN-24-EX Macro Twin Lite Flash	Shenzhen Yongnuo Photographic Equipment Co.	YN-24EX
Software
Canon EOS Utility (v. 3.16.1).	Canon		acquisition software
FIJI	National Institutes of Health		Fiji is released as open source under the GNU General Public License.  FIJI Version 2.14.0/1.54f
GraphPad Prism	GraphPad Software, Boston, Massachusetts USA		Prism Version 10.3.1
Zerene Stacker (v.1.04)	Zerene Systems, LLC		Focus Stacking Software