Name: upright imaging of drosophila embryos
Uploaded: 2010-09-13T17:00:00
Duration: 7 min 34 s
Description: Watch this Scientific Journal Video about Upright Imaging of Drosophila Embryos at JoVE.com

The authors are especially indebted to Deborah Harris and Danielle MacKay. Support for this work was provided by the Department of Biology and the College of Arts and Sciences of CWRU, and by HHMI grant number 52005866 for support of undergraduate education in the biological sciences.

1. Imaging Methodology
<ol>
	<li>Melt glycerin jelly in a 55&deg; C degree water bath for 20-30 minutes until it becomes a homogenous liquid. Swirl, but do not shake the bottle to avoid the formation of bubbles.</li>
	<li>While the jelly melts, apply a thin layer of glycerol using a Kimwipe on a clean glass slide (optional).</li>
	<li>Pipette 1 to 1.5 mL of melted glycerin jelly using a cut P1000 pipette tip on the surface of the slide to create a thin first layer. Pipette carefully to avoid bubbles.</li>
	<li>Let the jelly solidify for 5 min on a horizontal surface, then place the slide at -20&deg;C for 5 min and proceed to embryo alignment. Alternatively, cover the slide with Saran Wrap and place it at 4&deg;C for up to 5 days.</li>
	<li>Pipette a small drop of stained embryos in 70% glycerol/PBS or other antifade glycerol-based mounting media (e.g. Slowfade or Vectashield) on the side of the jelly bedding.</li>
	<li>Begin pre-aligning the embryos by individually transferring them from the drop to the jelly surface. Transfer them using a slanted hypodermic needle, using the slanted side as a spoon.</li>
	<li>Roughly place the embryos along 2 to 3 columns. If an embryo gets stuck in the needle, stir it in the drop containing the other embryos to release it. Do not waste time carefully aligning them at this point. Leave some space between columns.</li>
	<li>Align the embryos by sliding them horizontally on the jelly medium with the aid of the hypodermic needle. While sliding, orient the heads and tails in the same direction. Make them parallel to each other along the column. Remove excess of PBS glycerol left as a trail with a piece of twisted Kimiwipe. Note: Excess glycerol will cause embryos to be loosely encased within gel, resulting in separation of the two jelly layers, making it difficult to cut and mount. Conversely, removing too much glycerol will dry and flatten embryos.</li>
	<li>Use a cut yellow tip to spread a thin layer of jelly over the embryos (50-150 &mu;L, depending on how many were aligned).</li>
	<li>Place slide in -20&deg;C freezer for 10-20 min or store it at 4&deg;C overnight. Make sure the jelly is completely rigid before cutting. Otherwise, the block of embryos will be very difficult to excise. Best results for cutting are obtained on the following day.</li>
	<li>Under a dissecting scope, use a razor blade to cut completely through the jelly on both sides of the aligned embryos. Hold the razor blade at a 90&deg; angle to make straight cuts. The cuts should be made very close to the anterior and posterior poles of the embryos to avoid excess jelly that may obstruct imaging.</li>
	<li>Add 100-200 &mu;L of cold PBT (4&deg;C, PBS+0.1% Tween 20) next to the cuts and carefully scrape off the jelly between embryos using a spatula. Note: The detergent in PBT helps the removal of the jelly from the slide without damaging the embryos.</li>
	<li>Using a needle, transfer the block of jelly with embedded embryos to a clean slide for further cutting. Cut smaller blocks with 5-7 embryos. Use PBT to easily move around the jelly block. Alternatively, place the block on a dry glass surface to let it attach to the glass, which creates more stability for precisely trimming the jelly, if necessary.</li>
	<li>Flip upright the embryos embedded in the jelly with the aid of a razor blade and a needle. For visualization under microscope, proceed with either one of the two steps below, depending on the type of microscope stand being used.</li>
	<li>Inverted microscope: place finished embryo blocks onto a long coverslip, with embryos in an upright position. Ensure that the side of the embryo with the least amount of jelly will be in closer contact with objective.</li>
	<li>Upright microscope: make two stacks with four #1 coverslips glued with superglue to be used as "supports" and place the embryo block in glycerol in between the "supports". Bridge the stacks using a long coverslip.</li>
	<li>For confocal analysis, check the working distance of the objectives to be used to find out whether you can image all the way through the embryo or only partially. For example, D. melanogaster embryos have ~ 0.55 mm in length and can be entirely imaged using a Zeiss 20x Plan-Apo or a 40x LD Neo-Fluor objective. The following website has information on available Zeiss objectives and gives you the option to search objectives according to working distances: https://www.micro-shop.zeiss.com/us/us_en/objektive.php</li>
</ol>
2. Representative Results
Because the embryos are left intact, this method can be used to take precise measurements of embryo size and distances between gene expression patterns. In Figure 1, we show a blastoderm stage embryos stained with the nuclear stain DAPI (blue), anti-Dorsal protein (red), sog mRNA (yellow) and sna mRNA (green). Morphological processes, such as invagination of the mesoderm (Figure 2A, B) and migration of mesodermal cells (Figure 3 and 4) can also be visualized using this method. Different Z-positions along the DV axis can be obtained by changing the focal plane, as shown in Figures 2-4.
<img src="/files/ftp_upload/2175/2175fig1.jpg" alt="Figure 1" /> Figure 1. Upright optical slice of an intact embryo in blastoderm stage. Double staining for mRNA and protein. Target mRNAs for in situ probes (sog and sna) and protein stained by primary antibody (anti-Dorsal) are indicated in the figure. DAPI was used to label the nuclei. Note that the expression of sog and sna are located apically in the cytoplasm, while the expression of Dorsal is nuclear. Strong signals from sog nascent transcripts that are located in nuclei can also be seen at this magnification.
<img src="/files/ftp_upload/2175/2175fig2.jpg" alt="Figure 2" /> Figure 2. Upright imaging of an intact gastrulating embryo. mRNA probes used and their respective colors are indicated in the figure. A) Note that in this stage, three genes stained with same fluor (sna, sog and dpp, in green), are expressed in spatially separated domains. The invaginating mesoderm expresses sna, the neuroblast layer expresses ind and the ectoderm expresses dpp, while sog expression is still present in ventral regions of the nervous system. B) In a deeper confocal plane of the same embryo, we note the base of the invaginating mesoderm strongly expresses sna, but its apical region expresses lower levels.
<img src="/files/ftp_upload/2175/2175fig3.jpg" alt="Figure 3" /> Figure 3. Upright imaging of an intact embryo with germ band extended. sog RNA(yellow); snail, ind and dpp mRNAs (green), Dorsal protein (red). Nuclei were stained with DAPI (blue). A) Focal plane near embryo head. B) Another focal plane in the trunk region of same embryo. Note mass of mesodermal cells before lateral migration (compare to Figure 4) lying close to the ventral midline on both sides of a germ-band extended embryo. Delaminating neuroblasts are labeled in green (ind and sna).
<img src="/files/ftp_upload/2175/2175fig4.jpg" alt="Figure 4" /> Figure 4. Upright imaging of an intact embryo during germ band extension. sog RNA(yellow); snail, ind and dpp mRNAs (green), Dorsal protein (red). A) Note the delaminating neuroblasts from the epithelium (arrow), stained both for ind and sna at this stage (green). Note also the restricted expression of sog to the ventral midline (yellow). Expression of dpp at this stage can be seen at lateral sides of the embryo (asterisk). B) Optical section in the same embryo shown in A near the end of the embryo length, which corresponds to mid-posterior region. The ventral midline cells are stained for sog.

<ol>
	<li>Ay, A., Fakhouri, W. D., Chiu, C., Arnosti, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+and+analysis+for+quantifying+gene+expression+from+early+Drosophila+embryos.">Image processing and analysis for quantifying gene expression from early Drosophila embryos.</a> Tissue Engineering. 14, 1517-1526 (2008).</li><li>Gr&#946;hans, J., Wieschaus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+link+between+morphogenesis+and+cell+division+during+formation+of+the+ventral+furrow+in+Drosophila.">A genetic link between morphogenesis and cell division during formation of the ventral furrow in Drosophila.</a> Cell. 101, 523-531 (2000).</li><li>Kosman, D., Mizutani, C. M., Lemons, D., Cox, W. G., McGinnis, W., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+of+RNA+expression+in+Drosophila+embryos.">Multiplex detection of RNA expression in Drosophila embryos.</a> Science. 305, 846-846 (2004).</li><li>Witzberger, M. M., Fitzpatrick, J. A. J., Crowley, J. C., Minden, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=End-on+imaging:+a+new+perspective+on+dorsoventral+development+in+Drosophila+embryos.">End-on imaging: a new perspective on dorsoventral development in Drosophila embryos.</a> Developmental Dynamics. 237, 3252-3259 (2008).</li><li>Zander, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+mounting+delicate+bryophytes+in+glycerol.">On mounting delicate bryophytes in glycerol.</a> The Bryologist. 100, 380-382 (1997).</li><li>Liberman, L. M., Reeves, G. T., Stathopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+imaging+of+the+Dorsal+nuclear+gradient+reveals+limitations+to+threshold-dependent+patterning+in+Drosophila.">Quantitative imaging of the Dorsal nuclear gradient reveals limitations to threshold-dependent patterning in Drosophila.</a> Proc Natl Acad Sci U S A. 106 (52), 22317-2222 (2009).</li></ol>

No conflicts of interest declared.

Taking cross section images of Drosophila embryos can be challenging, since it requires either a careful hand dissection of slices 2 or the use of embedded media for microtome processing, which is usually detrimental for fluorescent stainings. Alternatively, Z-stacks made in a Confocal microscope can be used to make 3D reconstruction of cross-section images. However, it is time consuming to make several thin Confocal slices for an accurate 3D reconstruction and photobleaching becomes problematical. Another concern when imaging embryos that are mounted longitudinally is the light scattering that occurs when reaching deeper sections of the embryo, which also complicates taking precise measurements of fluorescent signals for the purpose of quantification of protein or mRNA expression levels1. Even with a two-photon confocal microscope, light scattering is still a concern due to the opacity of yolk material in the middle of the embryo, which makes selection of mounting media for optimal transparency of sample to be limited.
To circumvent these problems, a new technique called "End-on" imaging of positioning embryos vertically was recently developed to image both live and fixed samples 4. In this paper, we developed a new protocol for vertically mounted embryos that allows a simple preparation and visualization of fixed embryos or tissues of any size and shape that are stained with multiple in situ probes 3. Our method consists of using a mounting media with gelatinous consistency that is used to encase aligned embryos within it. Cut blocks of this mounting media containing embedded embryos can be positioned upright before imaging in any type of Confocal microscope. 
By embedding the embryos into the jelly and positioning them vertically, we are able to take horizontal cross-sections using Confocal microscopy, in which cells along the dorsal-ventral axis of the embryo are presented simultaneously. This is a significant improvement for quantification purposes since problems of light scattering and photobleaching of fluorescent dyes that occurs in conventional Z-stack reconstruction of longitudinally mounted embryos are now eliminated. Thus, quantification of gene expression or protein levels along the dorsal-ventral axis is accurate, since cells located at opposite dorsal-ventral positions are imaged at the same time and at the same confocal Z-position. In addition, the described method allows us to obtain a complete 2-D image across the dorsal-ventral axis from a 3-D embryo without using complex computational unrolling methods to visualize cells at different positions along the DV axis 6.
Some of the critical steps include removing excess glycerol after aligning embryos, to avoid split between the two layers of jelly. However, if the sample is too dehydrated, the resulting image will be misshapen and have incorrect morphology. In addition, if the jelly medium around the embryo is cut at an angle rather than straight, the resulting image may appear less round and more ovular in shape, due to an incorrect positioning of the embryos at an angle other than 90 degrees. Finally, if the jelly is not cut closely enough to the embryo on the anterior/posterior ends, it is not possible to obtain a proper image because the objective's working distance would be mainly used to focus into the jelly and not the embryo.
Another important step that may be necessary when imaging late gastrulating embryo is to carefully sort the stages of interest under the scope before aligning them. Usually, embryos are staged according to morphological characteristics that can be most easily recognized when in a longitudinal position.
Among alternative modifications that can be made with this method is to include an anti-fade reagent (e.g. p-Phenylenediamine or PPD) into the second layer of jelly to help protect against photobleaching of fluorescent dyes.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Glycerin Jelly mounting media</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences Company</td>
<td>17998-10</td>
<td>Contains low concentration of phenol as preservative and should be used in hood or in well ventilated area. Recipe for making your own media is described in Zander, 1997.</td>
</tr>
<tr>
<td>Zeiss LSM 700 Confocal</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>The following objectives were used: Plan-Apo 20x 0.8 M27 (WD 0.55mm); EC Plan-Neofluar 40x 1.3 oil (WD 0.21mm); LD Plan-Neofluar 0.6 Korr 40X (WD 2.9mm).</td>
</tr>
<tr>
<td>Phosphate buffered saline with 0.1% Tween 20 (PBT)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glass microscope slides</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-544-7</td>
<td>&nbsp;</td>
<tr>
<td>Glass cover slips</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72204-02</td>
<td>Size 1 &frac12; (specific for the objectives used in this work).</td>
</tr>
<tr>
<td>Glass cover slips (for making supports)</td>
<td>&nbsp;</td>
<td>Fisherfinest</td>
<td>12-544-10</td>
<td>Use four coverslips glued with superglue. </td>
</tr>
<tr>
<td>Dissection microscope</td>
<td>&nbsp;</td>
<td>M5 Wild Heerbrugg Wild Makroskop</td>
<td>M420</td>
<td>&nbsp;</td>
<tr>
<td>Razor blade</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Steel back single edge industrial blades</td>
</tr>
<tr>
<td>Spatula</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>21-401-10</td>
<td>&nbsp;</td>
<tr>
<td>Hypodermic needles</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>1482610</td>
<td>26 Gauge</td>
</tr>
<tr>
<td>Pipettes</td>
<td>&nbsp;</td>
<td>Labnet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

upright imaging of drosophila embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Watch this Scientific Journal Video about Upright Imaging of Drosophila Embryos at JoVE.com

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Research

JoVE Journal

Biology

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...