The authors thank David Larson for developing the original protocol for imaging the live Drosophila pupal eye. We thank our colleagues Richard Carthew, Shoichiro Tsukita and Matthew Freeman, who developed the transgenic flies that we combined to generate our live-imaging fly lines. Brittany Baldwin-Hunter gave helpful comments on the manuscript. This work was supported by start-up funds awarded to Ruth Johnson by Wesleyan University.

Figure 3 shows a summary of the experimental procedure.
1. Tissue Preparation
<ol>
	<li>To determine the consequences of modified expression of a gene of interest, set up the following cross in duplicate and maintain at 25 &#176;C: UAS-transgene (males)X GMR-GAL4; UAS-&#945;-cat:GFP, ubi-DE-Cad:GFP ; + / SM5-TM6b (virgin females) NOTE: To obtain control tissue cross UAS-lacZ males to GMR-GAL4; UAS-&#945;-cat:GFP, ubi-DE-Cad:GFP fe...

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The authors declare that they have no competing financial interests.

The description of wild-type development (above) forms the basis for comparisons of patterning events in RNAi or overexpression genotypes. Comparisons of live tissue development are invaluable when determining precisely which cell behaviors are regulated by a protein of interest. Further, and not described here, live cell imaging enables one to make qualitative and/or quantitative descriptions of the role of a gene of interest in the events that pattern the eye. By 30-32 hr APF most pigment cells have acquired stable pos...

The compound Drosophila eye is characterized by the stereotyped arrangement of its ~750 ommatidia separated by a honeycomb-lattice of accessory pigment cells 4,5. These pigment cells are patterned by a coordinated combination of events: local cell movements, cell growth, changes in cell shape, and apoptosis. Live visualization of this epithelium allows one to study the molecular mechanisms underpinning these events in a physiologically relevant and unperturbed three-dimensional context.
In contrast to previous protocols 6,7, the technique outlined here incorporates an efficient method for stabilizing extraneous tissue movement that cannot be uncoupled from the imaging process. This method enhances studies of cell behavior in the developing Drosophila pupal eye epithelium &#8211; a tissue that grows, rotates, and shifts over the course of imaging. In addition the motion-stabilization technique described here will be useful for studying cells in other contexts where extraneous movement occurs.
To visualize cell boundaries in the Drosophila retinal field, transgenic fly lines were generated that express ubi-DE-Cadherin:GFP as well as UAS-&#945;-catenin:GFP under control of the eye-specific driver GMR-GAL4 1-3. The use of two GFP-tagged membrane markers allows for the visualization of cell boundaries at lower intensity light. This minimizes tissue damage and photobleaching on repeated exposure to high-energy wavelengths, enabling increased frame rate and movie duration. To enhance efficacy of RNAi transgenes, UAS-Dcr-2 was also incorporated into a second fly line 8.
In a third update from previous protocols, a simpler imaging rig that is easily assembled in most laboratories is described. This apparatus obviates the requirement to have a specialized imaging rig generated by a university&#8217;s &#8216;machine shop&#8217; or similar service. This imaging rig is similar to that used to image other pupal tissues 9,10.
Presented here is a simple live-cell imaging protocol that can be used to directly assess the morphogenetic events that contribute to eye patterning from ~17 to 42 hr after puparium formation (hr APF). Specifically, this protocol enables one to determine the consequences of modifying gene expression during pupal development.

<table border="0" cellpadding="0" cellspacing="0" width="672">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Name of Material/ Equipment</td>
			<td style="width:124px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">GMR-GAL4; UAS-a-cat:GFP, ubi-DE-Cadherin:GFP ; + / SM5-TM6b</td>
			<td>Available on request from authors</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">UAS-dcr-2; UAS-a-cat:GFP, ubi-DE-Cadherin:GFP; + / SM5-TM6b&#160;</td>
			<td>Available on request from authors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Scotch double stick tape</td>
			<td style="width:124px;">3M</td>
			<td style="width:119px;">665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Black Sylgard dissection dish</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>Fill glass petri dish to rim with Sylgard (colored black with finely-ground charcoal powder) or Sylgard 170; leave at room temperature for 24-48 h to polymerize</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Sylgard</td>
			<td style="width:124px;">Dow Corning</td>
			<td style="width:119px;">SYLG184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Sylgard (Black)</td>
			<td style="width:124px;">Dow Corning</td>
			<td style="width:119px;">SYLG170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Glass petri dish</td>
			<td style="width:124px;">Corning</td>
			<td style="width:119px;">7220-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">25 x 75 x 1 mm glass microscope slide</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">12-550-413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">22 x 40 mm glass coverslip</td>
			<td style="width:124px;">VWR</td>
			<td>48393-172</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Forceps&#160;</td>
			<td style="width:124px;">Fine Science Tools</td>
			<td style="width:119px;">91150-20</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Whatman 3mm Chromatography Paper</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">05-713-336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Vaseline</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">19-086-291</td>
			<td>Or purchase at local pharmacy.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">30ml syringe</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">S7510-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Adobe Photoshop CS5</td>
			<td style="width:124px;">Adobe</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Leica TCS SP5 DM microscope</td>
			<td style="width:124px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">LAS AF Version 2.6.0.7266 microscope software</td>
			<td style="width:124px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

live-imaging of the drosophila pupal eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the pupal eye is a successful strategy to observe cell behaviors that contribute to patterning of the neuroepithelium. The role of specific proteins can be readily assessed by expressing transgenes that modify protein levels during eye development. To do this GMR-GAL4 is used to drive transgene expression behind the morphogenetic furrow. This offers the advantage of not perturbing earlier events that establish the eye field during the first two larval instars. In addition many RNAi transgenes req...

Watch this Scientific Journal Video about Live-imaging of the Drosophila Pupal Eye at JoVE.com

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

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JoVE Journal

Developmental Biology

9.2K visualizações.  Wesleyan University. This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.