A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Summary

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

Abstract

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS¹) and by aging embryos at reduced temperature (18 °C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

Introduction

The Drosophila embryo continues to be a premier model for investigation of fundamental mechanisms of development². This powerful model is supported by a wide array of molecular genetic tools that permit manipulations of essentially any gene at any time point and within any developing organ. The small size, rapid development, and extensive characterization of morphogenesis of the Drosophila embryo make it a model of choice for genetic screens, many of which have uncovered fundamental developmental pathways^3,4. Numerous phenotypes in the Drosophila embryo have been characterized and are easily interpretable, often providi....

Protocol

1. Preparation of Fly Cultures, Solutions, and Embryo Handling Devices

1. Prepare a cage culture of Drosophila. Place 500+ mating flies of the desired strain in a population cage fitted with a 10 cm grape-agar plate and a spot of yeast paste. Maintain culture in a 25 °C humidity controlled incubator. NOTE: Cage cultures require a day or two of conditioning to obtain consistent embryo laying patterns. Grape plates with yeast paste are changed once in the morning and once in the evening.......

Discussion

The above method outlines a means to obtaining viable Drosophila embryos that are accessible to small molecule treatments across a wide developmental range. This method introduces the novel and simple finding that aging embryos at 18 °C enables permeabilization of late stage embryos with the same efficacy as previously seen only in early stage embryos. In addition, use of the far-red dye CY5 carboxylic acid as a permeability indicator has proven effective in post-fix applications and does not interfere with.......

Materials

Name	Company	Catalog Number	Comments
Fly Cage	Flystuff.com	59-101	http://flystuff.com/general.php
Cocamide DEA [Ninol 11-CM]	Stepan Chemical	call for special order	http://www.stepan.com/
Ethoxylated alcohol [Bio-soft 1-7]	Stepan Chemical	call for special order	http://www.stepan.com/
d-limonene (Ultra high purity grade)	Florida Chemical Co.	call for special order	http://www.floridachemical.com/
Sodium hypochlorite	Fisher	SS290-4	http://www.fishersci.com/
Tween-20	Fisher	BP337	http://www.fishersci.com/
PBS powder	Sigma	56064C	http://www.sigmaaldrich.com/
Rhodamine B	Sigma	R6626	http://www.sigmaaldrich.com/
CY5 carboxylic acid	Lumiprobe	#23090	http://www.lumiprobe.com/p/cy5-carboxylic-acid
DMSO	Sigma	472310-100	http://www.sigmaaldrich.com/
Shields and Sang M3 medium	Sigma	S8398	http://www.sigmaaldrich.com/
Nitex Nylon mesh	Flystuff.com	57-102	http://flystuff.com/misc.php
Dissolved oxygen (DO) membrane	YSI	#5793	http://www.ysireagents.com/search.php
25mm circular no.1 cover slip	VWR	48380-080	https://us.vwr.com/
Grape-agar plate mix	Flystuff.com	47-102	http://flystuff.com/media.php
Nutator	VWR	82007-202	https://us.vwr.com/