Name: a method of permeabilization of drosophila embryos for assays of small molecule activity
Uploaded: 2014-07-13T14:00:00
Description: Watch this Scientific Journal Video about A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity at JoVE.com

This work was supported by NIH/NIEHS R03ES021581 (awarded to M.D.R.) and by the University of Rochester Environmental Health Center (NIH/NIEHS P30 ES001247).

1. Preparation of Fly Cultures, Solutions, and Embryo Handling Devices
<ol>
	<li>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 &#176;C humidity controlled incubator. NOTE:&#160;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...

<ol>
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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 &#176;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...

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 pathways3,4. Numerous phenotypes in the Drosophila embryo have been characterized and are easily interpretable, often providing a means to identify underlying molecular genetic mechanisms responsible for an abnormal trait.
Historically, a shortcoming of the fly embryo model has been the difficulty of introducing small molecules to embryonic tissues. This obstacle has posed limitations on: 1) using known bioactive small molecules as probes to interrogate developmental mechanisms and 2) using this established model to evaluate teratogenic or pharmacologic activity of uncharacterized small molecules. As a consequence, the screening potential of the fly embryo has been underutilized in characterization of small molecule activity.
Delivery of small molecules to the fly embryo can be achieved with two methods: 1) permeabilization of the eggshell and 2) microinjection. This article presents advances to the method of permeabilization that are easy to execute in the setting of a conventional Drosophila laboratory. It should be noted that recent advances in microinjection methods with microfluidics technology is also contributing to methods of introducing compounds to the embryo5,6. Introducing molecules to the embryo is prevented by a waxy layer of the eggshell7. The Drosophila eggshell consists of five layers. From the inside out they are: the vitelline membrane, the waxy layer, the inner chorionic layer, the endochorion and the exochorion8. The three outer chorionic layers can be removed by brief emersion of the embryo in dilute bleach, a step referred to as dechorionation. The exposed waxy layer can then be compromised by exposure to organic solvents, such as heptane and octane7,9, rendering the dechorionated embryo permeable, while it remains encased in the underlying vitelline membrane. However, use of these solvents introduces complications due to their toxicity and the difficulty in regulating their strong permeabilizing action, both of which have stark negative effects on embryo viability9,10.
A method of permeabilization using a composition termed embryo permeabilization solvent (EPS) has been previously described1. This solvent consists of d-limonene and plant-derived surfactants that enable the solvent to be miscible with aqueous buffers. The low toxicity of d-limonene and the ability to dilute the solvent to desired concentrations has yielded an effective method to generate permeable embryos with high viability1. However, two endogenous factors have continued to bring limitations to the application. First, embryos demonstrate heterogeneity in permeability after EPS treatment, even when care is taken to maintain close developmental staging. Second, embryos older than approximately eight hours have proven difficult to permeabilize, consistent with a hardening of the eggshell that occurs after egg laying11.
Described here are advances in the EPS method that: 1) assist in identifying and analyzing near-identically permeabilized embryos, even after fixation and immunostaining steps have been executed and 2) enable permeabilization of embryos at late developmental time points (&#62;8 hr, stage 12 and older). Specifically, application of a far-red dye, CY5 carboxylic acid, is described that serves as a permeability indicator, which persists in the embryo during development and after formaldehyde fixation. In addition, it is shown that rearing embryos at 18 &#176;C maintains the eggshell in an EPS sensitive state, enabling permeabilization of late stage embryos (stages 12-16).
These advances overcome the previously mentioned limitations to the EPS methodology. This application will therefore provide investigators with a means to introduce small molecules of interest to the embryo at distinct developmental time points while maintaining viability.

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			<td height="42" style="height:42px;width:216px;">Fly Cage</td>
			<td style="width:201px;">Flystuff.com</td>
			<td style="width:119px;">59-101</td>
			<td style="width:204px;">http://flystuff.com/general.php</td>
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			<td height="42" style="height:42px;width:216px;">Cocamide DEA [Ninol 11-CM]&#160;</td>
			<td style="width:201px;">Stepan Chemical</td>
			<td style="width:119px;">call for special order</td>
			<td>http://www.stepan.com/</td>
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			<td height="42" style="height:42px;">Ethoxylated alcohol [Bio-soft 1-7]&#160;</td>
			<td style="width:201px;">Stepan Chemical</td>
			<td style="width:119px;">call for special order</td>
			<td>http://www.stepan.com/</td>
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			<td height="42" style="height:42px;">d-limonene (Ultra high purity grade)</td>
			<td style="width:201px;">Florida Chemical Co.&#160;</td>
			<td style="width:119px;">call for special order</td>
			<td>http://www.floridachemical.com/</td>
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			<td height="21" style="height:21px;width:216px;">Sodium hypochlorite&#160;</td>
			<td style="width:201px;">Fisher</td>
			<td>SS290-4</td>
			<td>http://www.fishersci.com/</td>
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			<td height="21" style="height:21px;">Tween-20&#160;</td>
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			<td>BP337</td>
			<td>http://www.fishersci.com/</td>
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			<td height="21" style="height:21px;width:216px;">PBS powder</td>
			<td style="width:201px;">Sigma</td>
			<td>56064C</td>
			<td>http://www.sigmaaldrich.com/</td>
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			<td height="21" style="height:21px;">Rhodamine B</td>
			<td style="width:201px;">Sigma</td>
			<td>R6626</td>
			<td>http://www.sigmaaldrich.com/</td>
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			<td height="21" style="height:21px;width:216px;">CY5 carboxylic acid</td>
			<td style="width:201px;">Lumiprobe</td>
			<td>#23090</td>
			<td>http://www.lumiprobe.com/p/cy5-carboxylic-acid</td>
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			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:201px;">Sigma</td>
			<td>472310-100</td>
			<td>http://www.sigmaaldrich.com/</td>
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			<td height="21" style="height:21px;width:216px;">Shields and Sang M3 medium</td>
			<td style="width:201px;">Sigma</td>
			<td>S8398</td>
			<td>http://www.sigmaaldrich.com/</td>
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			<td height="21" style="height:21px;width:216px;">Nitex Nylon mesh&#160;</td>
			<td style="width:201px;">Flystuff.com</td>
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			<td height="21" style="height:21px;">Dissolved oxygen (DO) membrane&#160;</td>
			<td style="width:201px;">YSI</td>
			<td>#5793</td>
			<td>http://www.ysireagents.com/search.php</td>
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			<td height="21" style="height:21px;">25mm circular no.1 cover slip&#160;</td>
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			<td height="21" style="height:21px;">Grape-agar plate mix&#160;</td>
			<td style="width:201px;">Flystuff.com</td>
			<td>47-102</td>
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			<td height="21" style="height:21px;width:216px;">Nutator</td>
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a method of permeabilization of drosophila embryos for assays of small molecule activity

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 &#176;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.

Embryo handling devices are pictured in Figure 1 to assist in visualizing the &#8220;home-made&#8221; devices for manipulation in the above Protocols. Results seen in Figure 2 illustrate the robust effect of rearing embryos at 18 &#176;C on their ability to be permeabilized by EPS at late stages of development. This condition is applied in the protocol step 2.1. Efficacy of the CY5 carboxylic acid dye to reveal the various levels of permeability typically seen in EPS treated embryos is s...

Watch this Scientific Journal Video about A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity at JoVE.com

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

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.

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

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

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