We are extremely grateful to Jean-Antoine Lepesant and Nicolas Tissot who originally developed the protocol and shared some graphical elements of Figure 3 with us. We thank Fanny Roland-Gosselin who took the photos of Figure 4. We also thank other lab members for helpful discussions that contributed to the amelioration of this technique and Nathaniel Henneman for his comments that helped to improve this manuscript. We acknowledge the ImagoSeine core facility of the Institute Jacques Monod, member of France-BioImaging (ANR-10-INBS-04). Ma&#235;lys Loh is supported by a PhD fellowship from the French Ministry of Research (MESRI). Antoine Guichet and Fred Bernard were supported by the ARC (Grant PJA20181208148), the Association des Entreprises contre le Cancer (Grant Gefluc 2020 #221366) and by an Emergence grant from IdEx Universit&#233; de Paris (ANR-18-IDEX-0001).

1. Imaging medium preparation
<ol>
	<li>Prepare fresh media on the day of use. Pipette 200 &#181;L of Schneider medium (containing L-Glutamine and 0.40 g/L of NaHCO3 complemented with 10% heat-inactivated fetal calf serum, 100 U/mL of penicillin, and 100 mg/mL of streptomycin).</li>
	<li>Supplement with 30 &#181;L of insulin 10 mg/mL.</li>
	<li>Add 4 &#181;L of heat-inactivated fetal calf serum.</li>
</ol>
2. Observation-chamber preparation
<ol>
	<li>With a pipette tip, apply a small amount of silicone grease all around the hole on the underside of the punctured slide (Figure 4D).</li>
	<li>Position a 24 x 50 mm coverslip of 0.13-0.16 mm thickness.</li>
	<li>With the wide end of a pipette tip, apply pressure on the coverslip to flatten the silicone in order to seal the coverslip and create a silicone ring interior to the slide (Figure 4F,G).</li>
</ol>
3. Ovary dissection
<ol>
	<li>Anesthetize a female fly of the desired phenotype on a CO2 pad.</li>
	<li>Transfer the female in 150 &#181;L of the imaging medium in a dissecting well (Figure 4H).</li>
	<li>Open one female by grabbing its thorax with forceps and pinching the dorsal abdomen cuticle with a second pair of forceps.</li>
	<li>Isolate and detach the pair of ovaries, which should be readily visible upon cuticle opening.</li>
	<li>Carefully remove the uterus, oviduct, and muscle sheath (Figure 4I).</li>
	<li>Place a drop of 10-15 &#181;L of the imaging medium and transfer one ovary in the imaging chamber (Figure 4J,K).</li>
</ol>
4. Egg chamber isolation
<ol>
	<li>To separate the ovarioles, hold the posterior end of the ovary (toward the older stages) with the needle. Tease apart the ovarioles by carefully pulling on the germarium with another needle.</li>
	<li>Remove the remaining muscle sheath on the egg chambers; one needle holding the sheath and the other pulling on the ovariole through the larger chambers (stage 9 or older).</li>
	<li>Allow the unsheathed ovariole to sink and contact the coverslip.</li>
	<li>Remove late stages and the rest of the ovaries from the micro-chamber with the help of forceps. Carefully distance the ovarioles from the others with needles to facilitate the acquisition (Figure 4L).</li>
</ol>
5. Observation chamber closing
<ol>
	<li>Cut a small square (10 x 10 mm) of permeable membrane (Figure 4M,N).</li>
	<li>Carefully apply the membrane on top of the imaging medium to expel any air bubbles (Figure 4O).</li>
	<li>Hermetically seal the chamber with a thin layer of halocarbon oil around the well on the contour of the membrane (Figure 4P,Q).</li>
</ol>
6. Imaging
<ol>
	<li>Place the imaging set-up on the slide holder of the inverted microscope using a 40x objective (HCX PL Apo, 1.25NA, oil immersion).</li>
	<li>Locate and save positions of different stage 6 oocytes in which the nucleus is ready to migrate.</li>
	<li>Set-up the 488 and 561 nm lasers. With a measured output laser power of 150 mW, use 30% of the laser power and 300 ms and 500 ms exposure, respectively.</li>
	<li>Set-up the experiment. Take a time-lapse of 12 h with the interval of 15 min-41 sections with an interval of 1 &#181;m centered on the nucleus. 
	&#8203;NOTE: According to the exposure time described above, this setting allows the acquisition of one position in around 45 s. Since there is a delay due to position changing, it is recommended to set a maximum of 12 positions in these conditions.</li>
</ol>
7. Image analysis
<ol>
	<li>Process the movies on the software Fiji, using the plug-in Orthogonal view and manually track the nuclei.</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62688/62688fig04.jpg" /> 
Figure 4:&#160;Step by step micro-chamber mounting pictures. (A,B,C) Preparation of the needed tools: dissecting well plate, forceps, needles, imaging media, silicon grease, permeable membrane, and the aluminum slide. (D) Application of the silicon grease at the back of the aluminum slide with a pipette tip. (E) A glass coverslip is glued on the silicon grease to create the bottom of the chamber. (F,G) Pressure application on the coverslip with the wider extremity of a pipette tip to create a joint inside the chamber. (H,I)&#160;Dissection of the fly ovaries in the imaging media. (J) Pipetting of a drop of imaging media in the micro-chamber. (K,L) Separation of the ovarioles in the micro-chamber using needles. (M,N,O) Permeable membrane cut into a 10 x 10 mm square and placing over the drop of the medium in the micro-chamber. (P,Q) Sealing of the micro-chamber with halocarbon oil. The samples are ready to be imaged. <a href="https://www.jove.com/files/ftp_upload/62688/62688fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A., Guichet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleus+positioning+within+Drosophila+egg+chamber.">Nucleus positioning within Drosophila egg chamber.</a> Seminars in Cell and Developmental Biology. 82, 25-33 (2017).</li><li>Koch, E. A., Spitzer, 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=Multiple+effects+of+colchicine+on+oogenesis+in+Drosophila:+Induced+sterility+and+switch+of+potential+oocyte+to+nurse-cell+developmental+pathway.">Multiple effects of colchicine on oogenesis in Drosophila: Induced sterility and switch of potential oocyte to nurse-cell developmental pathway.</a> Cell and Tissue Research. 228 (1), 21-32 (1983).</li><li>Januschke, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+centrosome-nucleus+complex+and+microtubule+organization+in+the+Drosophila+oocyte.">The centrosome-nucleus complex and microtubule organization in the Drosophila oocyte.</a> Development. 133, 129-139 (2006).</li><li>Tissot, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+molecular+cues+ensure+a+robust+microtubule-dependent+nuclear+positioning+in+the+Drosophila+oocyte.">Distinct molecular cues ensure a robust microtubule-dependent nuclear positioning in the Drosophila oocyte.</a> Nature Communications. 8, 15168 (2017).</li><li>Cetera, M., Horne-Badovinac, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Round+and+round+gets+you+somewhere:+collective+cell+migration+and+planar+polarity+in+elongating+Drosophila+egg+chambers.">Round and round gets you somewhere: collective cell migration and planar polarity in elongating Drosophila egg chambers.</a> Current Opinion in Genetics &amp; Development. 32, 10-15 (2015).</li><li>Hudson, A. M., Petrella, L. N., Tanaka, A. J., Cooley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mononuclear+muscle+cells+in+Drosophila+ovaries+revealed+by+GFP+protein+traps.">Mononuclear muscle cells in Drosophila ovaries revealed by GFP protein traps.</a> Developmental Biology. 314, 329-340 (2008).</li><li>Gervais, L., Claret, S., Januschke, J., Roth, S., Guichet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PIP5K-dependent+production+of+PIP2+sustains+microtubule+organization+to+establish+polarized+transport+in+the+Drosophila+oocyte.">PIP5K-dependent production of PIP2 sustains microtubule organization to establish polarized transport in the Drosophila oocyte.</a> Development. 135 (23), 3829-3838 (2008).</li><li>Vill&#225;nyi, Z., Debec, A., Timinszky, G., Tiri&#225;n, L., Szabad, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+persistence+of+importin-+b+explains+extended+survival+of+cells+and+zygotes+that+lack+the+encoding+gene+'+n+Villa.">Long persistence of importin- b explains extended survival of cells and zygotes that lack the encoding gene ' n Villa.</a> Mechanisms of Development. 3-4 (125), 196-206 (2008).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Prasad, M., Jang, A. C. C., Starz-Gaiano, M., Melani, M., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+culturing+drosophila+melanogaster+stage+9+egg+chambers+for+live+imaging.">A protocol for culturing drosophila melanogaster stage 9 egg chambers for live imaging.</a> Nature Protocols. 2 (10), 2467-2473 (2007).</li><li>Weil, T. T., Parton, R. M., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+individual+Drosophila+egg+chambers+for+live+imaging.">Preparing individual Drosophila egg chambers for live imaging.</a> Journal of Visualized Experiments: JoVE. (60), (2012).</li><li>Chanet, S., Huynh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+sorting+requires+contractile+cortical+waves+in+germline+cells.">Collective cell sorting requires contractile cortical waves in germline cells.</a> Current Biology. 30 (21), 4213-4226 (2020).</li><li>Zhao, T., Graham, O. 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The authors declare no competing interests.

Other protocols describe how to prepare and culture Drosophila egg chambers ex vivo for live-imaging assay12,13. The novelty of this protocol is the use of an imaging chamber constructed using a hollowed aluminum slide, a coverslip, and an O2/CO2 permeable membrane. The main advantage of this set-up is to limit the movement in Z without exerting pressure on the sample. Thus, the oocyte can still move freely, and this is why first, t...

For several years, the Drosophila oocyte has emerged as a model system to study nuclear migration. The Drosophila oocyte develops in a multicellular structure called the egg chamber. Egg chambers encompass 16 germ cells (15 nurse cells and the oocyte) surrounded by an epithelial layer of follicular somatic cells. Egg chamber development has been subdivided into 14 stages (Figure 1A), during which the oocyte will grow and accumulate reserves necessary for the early development of the embryo. During the development, upon microtubule reorganization and asymmetric transport of maternal determinants, the oocyte polarizes along the antero-dorsal and dorso-ventral axes. These axes determine the subsequent polarity axes of the embryo and the adult arising from the fertilization of this oocyte1. During oogenesis, the nucleus adopts an asymmetric position in the oocyte. In stage 6, the nucleus is centered in the cell. Upon a yet to be identified signal emitted by the posterior follicular cells which is received by the oocyte, the nucleus migrates toward the intersection between the anterior and lateral plasma membranes in stage 7 (Figure 1B)2,3. This asymmetric position is required to induce the determination of the dorso-ventral axis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62688/62688fig1v2.jpg" /> 
Figure 1: Drosophila melanogaster egg chambers. (A) Fixed ovariole from transgenic flies expressing Fs(2)Ket-GFP that labels the nuclear envelopes and ubi-PH-RFP that labels the plasma membranes. The ovariole is composed of developing egg chambers at different stages. Maturation increases along the antero-posterior axis with the germarium at the anterior tip (left) where the germ stem cell resides and the older stage at the posterior tip (right). (B) Z-projection of living egg chamber by spinning disk confocal microscopy at stage 6 of oogenesis (left), in which the nucleus is centered in the oocyte. The nucleus will migrate to adopt an asymmetrical position at stage 7 (right) in contact with the anterior plasma membrane (between the oocyte and the nurse cell) and the lateral plasma membrane (between the oocyte and the follicular cells). This position will induce the determination of the dorsal side and, thus, the dorso-ventral axis of the egg chamber. <a href="https://www.jove.com/files/ftp_upload/62688/62688fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For many decades, this nuclear migration has been studied on fixed tissues by immunostaining. This approach has notably made it possible to demonstrate that this process depends on a dense network of microtubules4,5. More recently, we developed a protocol offering conditions compatible with live imaging of the oocyte during several hours making it possible to study this process dynamically6.
Hence, for the first time, we have been able to describe that the nucleus has preferential and characteristic trajectories during its migration, one along the anterior plasma membrane (APM) and another along the lateral plasma membrane (LPM) of the oocyte (Figure 2). These latest results underline the importance of live-imaging protocols when studying dynamic processes such as nuclear migration.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62688/62688fig02.jpg" /> 
Figure 2: Schematic representation of the different migration paths of the nucleus. At stage 6 of the oogenesis, the oocyte is a large cell with a central nucleus. At this stage, the antero-posterior polarity axis is set with a posterior/lateral plasma membrane of the oocyte in contact with the follicular cells and the anterior plasma membrane (in yellow) is in contact with the nurse cells2. We have previously reported that the nucleus could migrate either along the anterior plasma membrane (APM), along the lateral plasma membrane (LPM), or through the cytoplasm (STAD, straight to the antero-dorsal cortex)6. <a href="https://www.jove.com/files/ftp_upload/62688/62688fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The oocyte nucleus migration is a phenomenon of about 3 h6, and so far, the event triggering the start of the actual migration is unknown. The start of the migration can also be delayed by protein mutants used to study this mechanism. These unknown variables motivated us to acquire images over long time periods (10-12 h). It is, therefore, important to ensure that the oocytes remain alive. As the egg chamber develops, it elongates along the antero-posterior axis from a spherical to an elliptical shape. This elongation is driven by the rotation of follicular cells, which occurs from stage 1 to stage 8, perpendicular to the antero-posterior axis7. In addition, a tubular sheath of muscle with pulsatile property surrounds the egg chambers. Its physiological function is to push the developing follicles toward the oviduct continuously8. In order to limit the movements that induce oscillations of the egg chambers after their dissection, we designed an observation micro-chamber measuring 150 &#181;m in height (Figure 3A). This height is marginally higher than the size of a follicle at stages 10 and 11. It considerably limits the vertical movements of the sample while preserving the rotation of the egg chamber, thereby resulting in limited defects in follicle development. We then perform live imaging for 12 h on dissected egg chambers by multi-position time-lapse acquisitions using a spinning-disk confocal microscope. Here we describe our protocol for studying the oocyte nuclear migration between stages 6 and 7.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62688/62688fig3v3.jpg" /> 
Figure 3: Schematic representation of the observation chamber. (A) (Top view) Precise dimensions of the aluminum slide with the heights (A&#39;) and circumferences (A&#39;&#39;) of the well drilled in the middle of the slide. (B) (Bottom view) A coverslip blocking the well is sealed to the slide with silicon grease. (C) (Top view) Dissected ovarioles develop in an imaging medium that is covered by a gas permeable membrane. Halocarbon oil is used to stabilize the membrane. <a href="https://www.jove.com/files/ftp_upload/62688/62688fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In order to follow the nuclear migration and precisely assess trajectories in the oocyte, markers for both the nuclear envelope and plasma membrane are needed. With this aim, two transgenes that have a high signal/noise ratio and do not fade over the course of live imaging have been selected. To label the plasma membrane, the use of a P[ubi-PH-RFP] that encodes the Pleckstrin Homology (PH) domain of the Human Phospholipase C &#8706;1 (PLC&#8706;1) fused to RFP is recommended. This PH domain binds to the phosphoinositide PI(4,5)P2 distributed along the plasma membrane of the oocyte9. For the nuclear envelope, the P[PPT-un1]Fs(2)Ket-GFP protein-trap strain where GFP is inserted within the gene encoding the Drosophila &#223;-importin displays a homogeneous and an intense signal10. Young flies (1-2 days old) are placed in fresh vials containing dry yeast 24-48 h prior to ovary dissection.
For this live-imaging assay, a 1 mm thick piece of aluminum, which is nonreactive for the sample, has been cut into the dimensions of a microscopy slide. It has a 16 mm diameter hole in the center of the slide that has been counterbored to 0.85 mm. This counterbore has an additional 6 mm diameter hole with a depth of 150 &#181;m (Figure 3A). A coverslip is glued with silicone grease (inert for the sample) at the bottom of the aluminum chamber (Figure 3B). After placing the samples in the medium-filled well, a membrane permeable to O2/CO2 exchange is placed over the medium and surrounded by halocarbon oil (Figure 3C).
For the dissection, it is recommended to use stainless steel forceps with a tip dimension of 0.05 x 0.02 mm, and 0.20 mm diameter needles for the separation of the ovarioles (Figure 4B,C). The migrating nuclei are imaged on a spinning-disk confocal inverted microscope CSU-X1 equipped with a camera. Multi-position images were acquired by time-lapse every 15 min at 24 &#176;C. A 15 min interval allows performing multi-position acquisitions with limited photobleaching of the fluorescent proteins and phototoxicity for the samples. Furthermore, a shorter interval would not provide much more informative data to follow the nuclear trajectories. The movies are processed and analyzed via Fiji software11.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthetize CO2 pad</td>
			<td>Dutscher</td>
			<td>789060</td>
			<td>Anesthetize flies</td>
		</tr>
		<tr>
			<td>Coverslip (24x50 mm)</td>
			<td>Knittel Glass</td>
			<td>VD12450Y100A</td>
			<td>Observation-chamber preparation</td>
		</tr>
		<tr>
			<td>Forceps Dumont #5</td>
			<td>Carl Roth</td>
			<td>K342.1</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Stainless steel needles</td>
			<td>Entosphinx</td>
			<td>20</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Heat-inactivated fetal calf serum</td>
			<td>SIGMA-ALDRICH</td>
			<td>F7524</td>
			<td>Imaging medium</td>
		</tr>
		<tr>
			<td>Insulin solution bovine pancreas</td>
			<td>SIGMA-ALDRICH</td>
			<td>10516 - 5ml</td>
			<td>Imaging medium</td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin solution</td>
			<td>SIGMA-ALDRICH</td>
			<td>P0781</td>
			<td>Imaging medium</td>
		</tr>
		<tr>
			<td>Permeable membrane</td>
			<td>Leica</td>
			<td>11521746</td>
			<td>Observation-chamber preparation</td>
		</tr>
		<tr>
			<td>Schneider Medium</td>
			<td>Pan Biotech</td>
			<td>P04-91500</td>
			<td>Imaging medium</td>
		</tr>
		<tr>
			<td>Silicon grease</td>
			<td>BECKMAN COULTER</td>
			<td>335148</td>
			<td>Observation-chamber preparation</td>
		</tr>
		<tr>
			<td>Spinning disk confocal</td>
			<td>Zeiss</td>
			<td>CSU-X1</td>
			<td>Nuclear migration observation</td>
		</tr>
		<tr>
			<td>Voltalef oil 10S</td>
			<td>VWR</td>
			<td>24627 - 188</td>
			<td>Observation-chamber preparation</td>
		</tr>
	</tbody>
</table>

nuclear migration in the drosophila oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Before migration, the nucleus is dynamic and oscillates around a central position during a period defined as pre-migration. These small movements reflect a balance of pushing and pulling forces that maintain equilibrium in the middle of the oocyte. By quantifying the trajectories of the nuclei, we have shown that the APM and LPM trajectories had similar proportions. We define the nature of the trajectory by the first contact between the nucleus and the plasma membrane6. Thus, the nucleus reaches e...

Watch this Scientific Journal Video about Nuclear Migration in the Drosophila Oocyte at JoVE.com

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

nuclear-migration-in-the-drosophila-oocyte

Research

JoVE Journal

Developmental Biology

3.7K Views.  Université de Paris, CNRS, Institut Jacques Monod. In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

Video: Nuclear Migration in the Drosophila Oocyte

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

Methods Article

Nuclear Migration in the Drosophila Oocyte