We thank Bryne Ulmschneider for contributions to the protocol and Diane Barber for suggestions on the manuscript. This work was funded by a National Institute of Health grant GM116384 to T.G. Nystul and D.L. Barber.

NOTE: To measure pHi in the FSC lineage, we calculate the ratio of fluorescence intensities of pHluorin to mCherry in FSCs, pFCs, and follicle cells in physiological conditions, and convert the ratios into pHi values with standard calibration curves for each cell type7. First, live imaging experiments are performed to measure fluorescence intensities of pHluorin and mCherry in germaria dissected into a buffer containing NaHCO3, which mimics physiological conditions1,7. Next, standard curves are generated by measuring pHluorin and mCherry fluorescence intensities in a Na+-free, K+ buffer containing the ionophore nigericin adjusted to two different pH values, 6.5 and 7.5. In the presence of nigericin, pHi equilibrates with the pH of the buffer across the plasma membrane, causing pHi to match the extracellular pH. Lastly, the standard curves are used to convert pHluorin to mCherry ratios to estimated pHi values.
1. Pre-Trial: Preparation Before Measuring pHi In Vivo
NOTE: To measure pHi in vivo, the mCherry::pHluorin transgene must be expressed in the cell type of interest. Below are some common ways to generate transgenic pHluorin flies in the FSC lineage. Generating mCherry::pHluorin clones is particularly useful for identifying FSCs, which are located on the anterior edge of an FSC clone. Tissue specific mCherry::pHluorin expression is useful for measuring pHi across the entire tissue and is also more convenient when combining with expression of an RNAi or transgene.
<ol>
	<li>Generation of transgenic pHluorin flies
	<ol>
		<li>mCherry::pHluorin-labeled FSC clones
		<ol>
			<li>To make mCherry::pHluorin FSC clones, cross UAS-mCherry::pHluorin1 to an Act-Gal4 flipout stock or other stock with a Flp/FRT inducible Gal4.</li>
			<li>To make heatshock inducible clones, heatshock adult F1s for 2 days post eclosion in empty vials for 1 h in a 37 &#176;C water bath, four times approximately every 12 h.</li>
			<li>To ensure normal growth and maximum rates of oogenesis during this period, maintain flies by providing fresh wet yeast (approximately equal parts dry baker&#39;s yeast and water) daily for at least 6 days after clone induction.</li>
		</ol>
		</li>
		<li>Tissue specific mCherry::pHluorin expression</li>
		<li>Cross UAS-mCherry::pHluorin1 to a follicle cell specific driver, y1w*;P{GawB}10930/CyO (Bloomington ID: 7023).</li>
		<li>3 days after flies start eclosing, collect and place them in vials containing wet yeast, for at least 24 h prior to dissection.</li>
	</ol>
	</li>
	<li>Making solutions 
	NOTE: It is important to prepare the following buffers on the day of the experiment because the pH and solute concentrations in the buffer can change over time.
	<ol>
		<li>Prepare bicarbonate, nigericin, and dissection buffers. Add the components in the order listed to avoid formation of precipitates (Refer to Table 1, Table 2, and Table 3).</li>
	</ol>
	</li>
</ol>
2. Trial: Measuring pHi in the FSC Lineage
NOTE: The dissection, mounting, and live imaging steps for the bicarbonate buffer and the two nigericin buffer conditions may need to be performed twice: once to determine the microscope settings and a second time to collect experimental data. See the section 2.2 below for more information.
<ol>
	<li>Dissection and mounting 
	NOTE: For materials required to perform this step please refer to Figure 1 and Table of Materials. For 3D printed mounting chambers, 3D printer files are provided as Supplemental File 1 and Supplemental File 2.
	<ol>
		<li>Dissection:
		<ol>
			<li>Prepare mounting chambers by applying a thin coating of vacuum grease to the flatter side of the 3D printed mounting chamber. Place that side on a 22 x 40 mM coverslip to seal the coverslip to the mounting chamber.</li>
		</ol>
		</li>
		<li>Dissect ovaries from female adult flies in 500 &#181;L dissection buffer (bicarbonate or nigericin buffer, Table 3)
		<ol>
			<li>Anesthetize 3 - 4 flies using CO2 gas and transfer flies onto a fly pad perfused with CO2 gas.</li>
			<li>Pick up an anesthetized fly with forceps and place into the dissection media.</li>
			<li>Pinch the thorax of the fly with one forcep, while tugging on the tip of its abdomen until its ovaries are exposed.</li>
			<li>After detaching the ovaries from the other organs, tease apart the ovarioles using 221/2 syringe needles. Carefully separate the muscle sheath from the ovaries and separate individual ovarioles from the cluster of ovarioles. An effective technique to isolate the muscle sheath is to use one syringe needle to hold the cluster of ovarioles in place at its anterior end, and stroke downwards along the length of single ovarioles.</li>
			<li>Incubate the dissected ovaries in dissection media for exactly 10 min before proceeding to the mounting step so that there is enough time for the pHi of cells to fully equilibrate with the pH of the nigericin dissection media. 
			NOTE: Ensure that the muscle sheath is removed from the ovarioles. The muscle sheath may attenuate the fluorescence signal, which makes it difficult to identify individual cells using Concanavilin-A, and may cause ovarioles to move spontaneously during live imaging.</li>
		</ol>
		</li>
		<li>Mounting
		<ol>
			<li>Place a small drop of dissection media onto the glass coverslip inside the mounting chamber.</li>
			<li>Transfer separated ovarioles using forceps.</li>
			<li>Separate later stage egg chambers from germaria associated with earlier stage egg chambers, and try to ensure that the dissected germaria are placed at the center of the dissection media drop.</li>
			<li>Add two small drops of nail polish to opposite sides of a round 12-mm coverslip and let it air dry for about 10 s.</li>
			<li>Place the round coverslip on the drop of dissection media containing separated germaria such that that side with nail polish faces down and contacts the dissection media. Press down on the parts of the edge with nail polish to flatten and secure the germaria in position. We recommend using older nail polish because it smears less over the surface of the coverslip as it is being secured in place.</li>
			<li>Fill the mounting chamber with additional dissection media. Condition specific buffer that does not contain Concanavalin-A dye can be used to fill the chamber. 
			NOTE: The total time spent dissecting and mounting should not exceed 15 min. This is important to minimize the effects of tissue damage and cell death.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Live imaging: 
	NOTE: In this step, first collect images from the bicarbonate condition and the two nigericin conditions to determine the proper microscope settings; adjust the microscope settings as necessary, and then collect images for the experimental data. Use a confocal microscope capable of imaging in 3 channels: GFP (475 excitation/509 emission), mCherry (575 excitation/610 emission), and far-red (633 excitation/647 emission). 
	NOTE: Set Microscope Settings: The pixel intensities of the images collected will be quantified to determine the estimates of pHi, so it is important that the intensity values of the signal in each image set is neither too low nor saturated. The range of intensities that can be captured in an image is defined by the image bit depth. In many cases, 8-bit images are generated by default, but we recommend acquiring the data as 16-bit images, which have a broader dynamic range. It is important to ensure that the crosstalk between fluorescent channels is minimized, so the settings should be adjusted so that the emission spectrum collected from each fluorophore does not overlap. To test these settings, take a sample image using the excitation spectrum for GFP and collect in the emission spectrum for mCherry and vice versa. If the settings have been adjusted properly, there should be no signal in either case. Set the microscope settings (i.e., voltage settings on a laser scanning confocal, laser power and pinhole size) so that the pixel intensities of the signal in all three image sets are within the dynamic range of the image file. For all our experiments, we used a white light laser scanning confocal microscope with a 40x objective to acquire 16-bit images in 1,024 &#215; 1,024 format, while we optimized voltage gain for every experiment as needed. For example, in one experiment, we set the voltage gain for GFP, mCherry, and far-red as 37.8%, 72.9%, and 260%, respectively.
	<ol>
		<li>Image ovarioles in nigericin dissection buffer at pH 6.5, and adjust settings such that pixel intensities of the pHluorin and mCherry images are low, but not below the limits of detection of the camera.</li>
		<li>Image another set of ovarioles in nigericin dissection buffer at pH 7.5 and adjust the settings such that the pixel intensities of the pHluorin and mCherry images are high, but not saturated.</li>
		<li>Image ovaries in bicarbonate dissection buffer and ensure that the pixel intensities of the pHluorin and mCherry images are not saturated with the chosen settings. If the pixels are saturated, adjust the settings as necessary. 
		NOTE: Microscope setting parameters may differ based on the exact microscope setup being used, and must be optimized for each experiment. After microscope settings have been set, do not change them for the remainder of the experiment. Settings should be consistent across control and experimental conditions. In our initial pHi experiments, we generated 2 and 3 point nigericin calibration curves and found no significant difference between calculated pHi using either method. However, in general, the addition of more points in the calibration curve would be expected to improve accuracy. Therefore, we recommend generating curves with different numbers of points to determine how many are needed for a given set of experimental conditions.</li>
	</ol>
	</li>
	<li>Data collection:
	<ol>
		<li>Perform dissection, mounting, and imaging of the samples in the bicarbonate and nigericin buffer conditions. After dissection and mounting, the maximum time spent imaging one set of samples should not exceed 45 min to ensure that the fluorescence intensity measurements are made in live, healthy tissue.</li>
		<li>For each condition, acquire images for at least 5 germaria.</li>
	</ol>
	</li>
</ol>
3. Post-trial: Image Analysis
<ol>
	<li>Measure fluorescence intensities in FSCs, pFCs, and follicle cells
	<ol>
		<li>Background subtraction:
		<ol>
			<li>Open unprocessed images in FIJI with each channel in a separate window (Figure 4C).</li>
			<li>Use the rectangle tool to draw a region of interest (ROI) in the pHluorin channel window in a part of the image without signal.</li>
			<li>Optional step: Set the lower limit of the threshold so that pixels with intensity values below background are excluded and set the upper limit of the threshold to maximum. When the threshold is set in this way, the areas of the image without signal are mostly blue and the signal is clearly visible (Figure 4A).</li>
			<li>Measure the mean fluorescence intensity in the ROI. If the threshold was set in step 3, make sure to check the &#34;Limit to threshold&#34; box in the Set Measurements dialog box.</li>
			<li>Subtract the measured background intensity from each channel and slice of the image using the Subtract function, found in the Process &#8594; Math menu.</li>
			<li>Repeat steps 3.1.1.2-6 for the mCherry channel window except that, in step 3.1.1.2, instead of drawing a new rectangle with the rectangle tool, use the Restore selection function, found in the Edit&#8594; Selection menu, to add a rectangle with the same dimensions and position on the image.</li>
		</ol>
		</li>
		<li>Identify FSCs, pFCs, and follicle cells:
		<ol>
			<li>Identify the FSCs, pFCs, and follicle cells by morphology and location using the Concanavalin-A staining to find cell boundaries (Figure 2).</li>
			<li>FSCs are thin triangular cells located on the edge of the germarium at the Region 2a/2b border. If mCherry::pHluorin is expressed in an FSC clone, FSC can also be identified as the anterior most cell of the clone.</li>
			<li>pFCs are irregular shaped cells in Region 2b, immediately adjacent to and downstream of FSCs.</li>
			<li>Follicle cells are square or columnar cells that surround the germ cell cyst in Region 3.</li>
		</ol>
		</li>
		<li>Obtaining fluorescence intensity ratios
		<ol>
			<li>Choose one or more slices in which the cell of interest has highest fluorescence intensity in the mCherry channel and make sure that the Concanavalin-A staining, seen in the far-red channel, is in focus within the selected slice.</li>
			<li>Draw ROI around each FSC and measure the mean fluorescence intensity of pHluorin and mCherry in all slices chosen for measurements.</li>
			<li>Divide the mean fluorescence intensity of the pHluorin channel by the mean fluorescence intensity of the mCherry channel to calculate a ratio of pHluorin to mCherry fluorescence.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Derive pHi values and generate pseudocolored images
	<ol>
		<li>Derive pHi values
		<ol>
			<li>In all cases, the linear regression curve should be generated using ovaries from flies of the same genotype for both experimental and control conditions. Generate a linear regression curve for each cell type using the data from two nigericin buffer conditions (Figure 3). In Excel, this can be done by plotting the data on a graph and adding a linear trendline. Alternately, see:</li>
			<li>Use the slope and the y-intercept of the linear regression curve calculated from the nigericin conditions and the equation of a line (y = mx + b) to convert the pHluorin to mCherry ratio values calculated from the samples in the bicarbonate condition to pH values. In this equation, substitute the slope form (the y-intercept) for b, put the ratio value in for x, and solve for y.</li>
		</ol>
		</li>
		<li>Generate a pseudocolored image reflecting the ratio values in FIJI.
		<ol>
			<li>Follow the procedure above for background subtraction (step 3.1.1 and Figure 4).</li>
			<li>Divide the pHluorin channel from the mCherry channel using the Image Calculator function, found in the Process menu. Be sure the &#34;Create new window&#34; and &#34;32-bit (float) result&#34; checkboxes are both checked. Change the lookup table (LUT), found under the Image menu, to Thermal or LUT of choice. See Figure 4 for other suitable options.</li>
			<li>In the adjust brightness and contrast dialog box (Image &#8594; Adjust &#8594; Brightness/Contrast), click the &#34;Set&#34; button. Set the Minimum displayed value to 0 and the Maximum displayed value to a ratio that best captures the data, usually 1.0-2.0 (Figure 5).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Here, we describe a method for measuring the pHi of cells in the FSC lineage within wildtype tissue. This protocol has been developed and refined over the past five years, since we first began to study pHi in Drosophila ovarian tissue. During that time, the protocol has been used successfully by multiple investigators in our lab and on at least four different spinning disc and laser scanning microscopes. The reproducibility of our original observation, that pHi increases as cells in the FSC lineage differentiate...

Recent studies revealed a role for changes in pHi during cellular differentiation and dysplasia in vivo1,2. These studies found that pHi is remarkably consistent in cells of the same type at the same stage of differentiation, but that it changes as cells transition from one stage to another. In some cases, blocking the changes in pHi partially disrupts differentiation, suggesting that the change in pHi is not just a consequence of changes in cell fate but instead helps to promote the change in cell fate, perhaps through effects on pH-sensitive regulatory proteins or chemical reactions required for differentiation. Future studies have the potential to reveal more insight into the many different roles of pHi dynamics in vivo. However, one of the challenges of studying pHi during differentiation in vivo is obtaining accurate measurements of pHi. Unlike other features of differentiation, such as changes in cellular morphology and gene expression, pHi is a labile chemical property of the cell that is not preserved in cells that have been fixed and permeabilized with standard methods. In addition, pHi may not be stable in cells that are stressed or dying as a result of experimental manipulation. Thus, it is important to keep cells alive and as healthy as possible when measuring pHi. Several vital dyes are available that work well for measuring the pHi of cells in culture3, but in many cases they are not suitable for in vivo studies because they do not penetrate the tissue deeply or evenly enough to provide accurate measurements.
To circumvent the problem of poor dye penetration, we and others have used a genetically encoded probe, mCherry::pHluorin4,5,6,7, that can be expressed specifically in the cell types of interest and imaged in live tissue. pHluorin is a variant of GFP with a higher pKa (~ 7.0 vs. ~ 4.0) that folds more readily at higher pH; so the total fluorescence intensity emitted from a population of pHluorin molecules in the cell increases with increasing pHi8. Importantly, fluorescence is linear within the normal cytosolic range of pHi values. In contrast, the fluorescence of mCherry (pKa ~ 4.5) is insensitive to pH changes within the cytosolic range. These two reporters are covalently linked together as a single chimeric protein, encoded by a single open reading frame, so they are always present in equal amounts. Therefore, the ratio of pHluorin to mCherry fluorescence intensity provides a measurement of pHi that is normalized to the probe concentration in each cell. The ratios can then be converted to estimates of pHi values using a standard curve that is generated by obtaining the pHluorin to mCherry ratios from tissues that have been equilibrated to known pH values.
Here, we describe the methods for using mCherry::pHluorin to measure the pHi of the epithelial FSC lineage in the Drosophila ovary. This well-characterized tissue has been used to model many different aspects of epithelial biology, such as stem cell self-renewal and differentiation9,10,11, collective cell migration12, and the development and maintenance of cell polarity13,14. The follicle epithelium is produced by two FSCs that reside at the anterior edge of the tissue in a structure called the germarium15,16. These cells divide regularly during adulthood to self-renew and produce progeny, called prefollicle cells (pFCs), that can either re-enter the niche and become an FSC or differentiate into one of three different follicle cell types: polar cells, stalk cells, or main body follicle cells. We showed previously that in wildtype tissue, the pHi increases steadily during the early stages of differentiation, from a pHi of approximately 6.8 in FSCs to 7.0 in pFCs, to 7.3 in follicle cells2. Blocking this increase by RNAi knockdown of a ubiquitously expressed sodium/proton exchanger, DNhe2, severely impairs pFC differentiation, whereas increasing pHi by overexpression of DNhe2 causes a mild excess differentiation phenotype. These findings demonstrate that pHi is stably maintained in the early FSC lineage and that it can be experimentally increased or decreased in vivo. The methods described here can be used to measure pHi in either wildtype tissue or various forms of mutant tissues, including RNAi knockdown or overexpression using a Gal4 of interest, and mitotic clones.

<table><tbody><tr><td>&lt;strong&gt;Fly Stocks&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>UAS-mCherry::pHluorin[1]</td><td></td><td></td><td></td></tr><tr><td>y1 w*;P{GawB}10930/CyO</td><td>Bloomington Stock Center</td><td>7023</td><td></td></tr><tr><td>Act-Gal4 flipout stock</td><td>Bloomington Stock Center</td><td>4409</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals for Buffer preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S5886</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P-3911</td><td></td></tr><tr><td>glucose</td><td>Mallinckrodt</td><td>4912</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher Scientific</td><td>BP310</td><td></td></tr><tr><td>MgSO4</td><td>Thermo Fisher Scientific</td><td>M63</td><td></td></tr><tr><td>CaCl2</td><td>Sigma Aldrich</td><td>C-5080</td><td></td></tr><tr><td>HCO3</td><td>Sigma Aldrich</td><td>S-5761</td><td></td></tr><tr><td>MgCl2</td><td>Sigma Aldrich</td><td>M-9272</td><td></td></tr><tr><td>NMDG+</td><td>Sigma Aldrich</td><td>M-2004</td><td></td></tr><tr><td>K2HPO4</td><td>Mallinckrodt</td><td>7088</td><td>Use to Make KHPO4 pH 7.4</td></tr><tr><td>KH2PO4</td><td>Thermo Fisher Scientific</td><td>BP362</td><td>Use to Make KHPO4 pH 7.4</td></tr><tr><td>Concanavalin A, Alexa Fluor 647 Conjugate</td><td>Thermo Fisher Scientific</td><td>C21421</td><td>0.25 mg/ml dilution</td></tr><tr><td>Nigericin</td><td>Thermo Fisher Scientific</td><td>N1495</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Dissection and mounting tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2 Dumont Inox forceps (Size 5)</td><td>Thermo Fisher Scientific</td><td>NC9473431</td><td></td></tr><tr><td>2 23-gauge syringe needles</td><td>Sigma Aldrich</td><td>Z192457</td><td></td></tr><tr><td>9-well glass dissecting dish</td><td>Thermo Fisher Scientific</td><td>13-748B</td><td></td></tr><tr><td>Vacuum Grease</td><td>Dow Corning</td><td>1018817</td><td></td></tr><tr><td>22 X 40 mM glass coverslips</td><td>Thermo Fisher Scientific</td><td>12545C</td><td></td></tr><tr><td>Round Glass Coverslips, 12mm diameter, 0.13-0.16mm thickness</td><td>Ted Pella, Inc.</td><td>26023</td><td></td></tr><tr><td>3-D mounting chamber</td><td>custom manufactured</td><td></td><td>.stl and .ipt files for 3-D printer included as supplemental files</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Other equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pH meter</td><td>Thermo Fisher Scientific</td><td>13-620-183A</td><td>Model: Accumet AB15</td></tr><tr><td>Dissection microscope</td><td>Olympus Corporation</td><td>0H11436</td><td>Model: SZ2-ST</td></tr><tr><td>Confocal Microscope</td><td>Leica Biosystems</td><td></td><td>SP5 or SP8 laser-scanning confocal microscope with a 40&amp;times; objective with a numerical aperture of 1.3</td></tr></tbody></table>

methods for imaging intracellular ph of the follicle stem cell lineage in live drosophila ovarian tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Here we have described the process of measuring pHi in the follicle epithelium, which involves several steps. First, the ovaries are dissected from flies of the appropriate genotype using tools for dissecting and mounting (Figure 1). The ovarioles are then imaged using quantitative fluorescence microscopy and the images are analyzed to obtain measurements of pHi. For each image, the cell types of interest are identified as described in Section 3.1 (<strong cl...

Watch this Scientific Journal Video about Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue at JoVE.com

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

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Research

JoVE Journal

Developmental Biology

6.8K Views.  University of California, San Francisco. We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

Video: Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

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 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.

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

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Biology

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Neuroscience

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.

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.

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

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Summary

Explore More Videos

Chapters in this video

Live-imaging of the Drosophila Pupal Eye

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Live Imaging of Drosophila Larval Neuroblasts

Nuclear Migration in the Drosophila Oocyte

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

Dissection and Staining of Drosophila Larval Ovaries

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation