We would like to thank members of the Mart&#237;n-Blanco laboratory for helpful discussions. We also thank Nic Tapon (The Crick Institute, London, UK), the Bloomington Stock Center (University of Indiana, USA) and FlyBase (for Drosophila gene annotation). Federica Mangione was supported by a JAE-CSIC predoctoral fellowship. The Mart&#237;n-Blanco laboratory was funded from the Programa Estatal de Fomento de la Investigaci&#243;n Cient&#237;fica y T&#233;cnica de Excelencia (BFU2014-57019-P and BFU2017-82876-P) and from the Fundaci&#243;n Ram&#243;n Areces.

NOTE: This protocol is divided into five steps: (1) staging the pupae, (2) preparing the pupae for imaging, (3) live imaging of the growing abdominal epithelia, (4) generation of genetic mosaics, (5) data processing and analysis (including sections describing how to analyze cell orientation dynamics from cell junction outlines and growth dynamics from cell clones).
1. Staging of Drosophila pupae before imaging
<ol>
	<li>Culture flies of the appropriate genotype on standard medium in plastic vials at 25 &#176;C for 5 days (&#177;12 h) after egg laying (AEL). 
	NOTE: Metamorphosis starts within the confinement of the third-instar larvae into the pupal case at 120 h AEL until 0 h after puparium formation (APF). This transition is easily identifiable, because larvae stop feeding and moving and the opercular region is formed (Figure 1A) at 0-12 h APF. The puparium is initially soft and white, but progressively hardens and tans.</li>
	<li>Transfer white prepupae (0 h APF) to a fresh plastic vial using a moisten paintbrush. Animals can be kept at different temperatures depending on the designed experiment until the desired age. 
	NOTE: Pupa formation (i.e., pupation) occurs at 12 h APF, when the head and the appendages of the adult fly are totally everted (Figure 1A). By this time the pupal case is fully separated from the pupa, allowing its complete removal (Figure 1A).</li>
</ol>
2. Preparing pupae for live imaging
NOTE: After staging, the pupae are dissected and mounted as described below (see also Figure 1).
<ol>
	<li>Remove staged pupae from the wall of the vial with the help of the forceps.</li>
	<li>Glue the ventral side of each pupa on a glass slide covered with double-sided sticky tape. Gently tap on the head spiracles (i.e., opercular region) and the dorsal surface of the pupa with the tips of the forceps to assure the adhesion of the pupal case to the tape (Figure 1A,B). 
	NOTE: The dorsal surface should face up to facilitate the dissection of the case and the recovery of the pupa.</li>
	<li>Begin dissection under a stereomicroscope by gently removing the operculum from the puparium with the forceps (Figure 1C).</li>
	<li>Insert one tip of the forceps in a shallow angle between the pupal case and the pupa surface through the opercular opening. Tear the case from head to tail laterally in one or more swings, avoiding pinching the pupa (Figure 1D). Fold back the cracked pupal case to the lateral sides as you keep proceeding to the posterior end (Figure 1E). 
	NOTE: The pupal case is quite rigid and cracks easily. In case of high humidity or in particular genotypic backgrounds, the pupal case becomes softer and tearing is more difficult. In these cases, the cracking of the pupal case can be helped by pricking its free edges with both tips of the forceps.</li>
	<li>Remove the pupa from the opened-up pupal case by carefully inserting the forceps under the animal and gently pulling up (Figure 1F). The pupa will stick to the tip of the forceps (Figure 1G&#8722;H).</li>
	<li>Transfer the pupa with the help of the forceps to a glass-bottom dish and deposit it on top of a small drop of gas-permeable halocarbon oil (Figure 1I). Hold the pupa gently by the ventral side to avoid any possible tissue damage. 
	NOTE: The drop of halocarbon oil has to be small, with a diameter approximately less than half the length of the pupa. Such amount is sufficient to adhere the pupa to the glass by capillarity and to correct the optics for oil immersion objectives.</li>
	<li>Roll a piece of wet tissue paper at the edges of the dish to maintain humidity. Cover the dish to avoid dehydration of the pupae during imaging. 
	NOTE: Both female and male pupae can be employed for imaging. We recommend employing the third abdominal segments (AIII) as a reference abdominal metamere since it is almost identical in both sexes in terms of size, shape, and patterning.</li>
</ol>
3. Live imaging of growing abdominal epithelia
NOTE: An inverted laser scanning confocal microscope equipped with a 40x/1.3 NA oil immersion objective was used to image pupae at different developmental stages.
<ol>
	<li>Orient the pupa over the oil drop on the glass-bottom dish according to the domain and the process to be evaluated (e.g. dorsolaterally for long-term live imaging of the early expansion of the dorsal nests, or dorsally to image their late expansion and tissue remodeling). See Figure 1J,K and Figure 4.</li>
	<li>Transfer the glass-bottom dish containing the mounted pupae to the microscope stage and focus on the surface of the abdominal area using the transmitted light. 
	NOTE: Even if this protocol is optimized for imaging on inverted microscopes, it is also possible to perform imaging on an upright microscope. In that case, the sample is placed on the microscope stage with the glass-bottom surface facing up. The halocarbon oil holds each pupa on a meniscus.</li>
	<li>Set the acquisition parameters: 1) the number of Z-slices are usually between 20&#8722;40 to allow appropriate two-dimensional (2D) reconstruction of the abdominal epidermis; 2) step size between each slice (e.g., 1 micron); 3) time interval for recording (a 5 min interval is suitable for high fidelity analyses of cell orientation dynamics); and 4) frame resolution (e.g., 1024 x 1024).</li>
	<li>Turn on the appropriate lasers (i.e., 488 nm and 561 nm to visualize GFP and RFP fluorophores respectively) and adjust the laser power and gain/offset settings to visualize the marked cells. Use the lowest possible laser power (in the range 5%&#8722;20%) to minimize photobleaching and phototoxicity.</li>
	<li>Manually set the position and the appropriate Z-stack limits for multiple pupae using the attached motorized stage and the microscope multiposition acquisition software.</li>
</ol>
4. Generation of genetic mosaics to follow behaviors of cell clones
NOTE: We employ mitotic recombination to induce genetic mosaics in the abdominal epithelium via site-specific recombination (FLP/FRT system21,22) (Figure 2).
<ol>
	<li>Cross virgin females carrying a heat shock-inducible Flippase transgene (hs-FLP), a FLP recognition target (FRT) site at a specific genomic location (e.g., FRT site at position 40A at the L arm of chromosome 2), and a recognizable cellular marker (e.g., Ubi-RFP.nls or Ubi-GFP.nls) distal to the FRT site, to mutant males carrying an FRT site at the equivalent genomic location (Figure 2A&#8722;C). 
	NOTE: Autonomous and nonautonomous effects within or outside clones for any gene loss of function could be studied employing specific recessive alleles distal to the FRT site.</li>
	<li>Generate FLP/FRT somatic clones in the histoblasts by heat shock treatment at the third instar larval stage of the progeny of the cross. This is performed by submerging the plastic vials containing the animals in a water bath at 37 &#176;C for 45 min to 1 h at the wandering larvae (LIII) stage. 
	NOTE: The sensitive period for mitotic recombination is the G2 phase of the cell cycle. Histoblasts are arrested in G2 during the whole larval development.</li>
	<li>Score twin clones for absence (i.e., mutant cells) or enhanced (i.e., wild type twin-spot cells) levels of the fluorescent protein marker from 16 h APF onward (Figure 2D). 
	NOTE: On average, 45 min to 1 h at 37 &#176;C renders approximately only 2&#8722;3 twin clones per region of interest (e.g., the abdominal hemisegment). Pupae showing too a high clone density (e.g., more than four twin clones per hemisegment) should be discarded from further quantitative analyses.</li>
	<li>Upon clone identification, image living pupae at the desired stage and for the desired length of time as described in the previous section.</li>
</ol>
5. Data processing and analyses
NOTE: Data are processed using ImageJ (imagej.nih.gov/ij/).
<ol>
	<li>Distinguish cell orientation dynamics from cell junction outlines.
	<ol>
		<li>Project the Z-stack slices acquired by confocal microscopy in 2D using the Maximum Intensity Projection (MIP) function of ImageJ. 
		NOTE: The number of slices per stack should be kept to a minimum to avoid the out of focus noise generated by macrophages patrolling under the epidermis.</li>
		<li>Set a planar coordinate system identifying reliable tissue landmarks (e.g., A/P compartment boundaries) for analysis of each dataset (Figure 3A). 
		NOTE: Employing the same planar references for each data set will allow multiple measurements to be compared.</li>
		<li>Use the OrientationJ plugin (bigwww.epfl.ch/demo/orientation/) of ImageJ23,24 on local cell edges to obtain qualitative and quantitative orientation values. The plugin renders color-coded overlays on input images based on local orientations and provides numerical values when used in quantitative mode (Figure 3B&#8722;B&#39;&#39; and Figure C&#8722;C&#39;&#39;&#39;&#39;). 
		NOTE: The OrientationJ plugin is based on structure tensors, 2 x 2 matrix of eigenvalues derived from gradient and directional derivatives (See references23,24 for a detailed description).</li>
		<li>Use the OrientationJ Distribution option to color-code cell edge orientations relative to the set planar coordinate system (i.e., cell edge orientation maps) (Figure 3B). The Distribution option is found under the plugin menu in ImageJ. The settings to employ are: Gaussian window sigma = 1 pixel; Cubic Spline = Gradient; Minimum Coherency = 20%; Minimum Energy = 1%. Cell edge orientations are displayed as a color-coded image employing the Color Survey option of the plugin (Hue = orientation; Saturation = coherency; and Brightness = input image). 
		NOTE: Areas that contain background fluorescence do not provide any directional information and must be manually excluded from the analysis. High threshold settings reduce the pixels considered in the processed images.</li>
		<li>Use the OrientationJ Measure option to quantify cellular orientations and directional cell-cell alignment (i.e., coherency) (Figure 3C). The Measure option is found under the plugin menu in ImageJ. Generate small adjacent non-overlapping regions of interest (ROIs) of uniform weight (64 x 64 pixels, about 20 &#181;m x 20 &#181;m) within the area occupied by the histoblasts (Figure 3C).</li>
		<li>Calculate the dominant local orientation (i.e., the averaged orientation between neighboring cells-averaged cell edge orientation map) and coherency from the ROIs. The software computes the predominant orientation and local coherency within each ROI (Figure 3C). 
		NOTE: The largest and smallest eigenvalues of the structure tensor estimated by OrientationJ are employed to calculate coherency as the ratio between their difference and their sum. Coherency is bounded between values of 0&#8722;1. A value of 1 indicates full alignment uniformity, while a value of 0 indicates isotropic areas with no alignment.</li>
		<li>Statistically analyze the calculated orientation and coherency values from multiple images using free software packages such as PAST25. Axial data such as orientation values (in degrees) are appropriately described by directional statistics.</li>
		<li>Through the software, calculate the mean direction and circular variance for each set of orientation distributions. The statistical significance of the difference between the distribution of the orientations between different genotypes or conditions is determined using the nonparametric Mardia-Watson-Wheeler test (W-test) for equal distributions.</li>
		<li>Calculate the statistical significance of the difference in coherency applying the non-parametric Kolmogorov-Smirnov test (K-S test). Display data graphically as desired (polar plots, bar charts, box plots, etc.).</li>
	</ol>
	</li>
	<li>Growth dynamics from cell clones 
	NOTE: The following steps allow retrieval of geometrical and shape parameters for cells from 2D MIP images containing the clone(s) of interest. For comparisons between multiple clones, images must be acquired with the same settings.
	<ol>
		<li>Segment clone areas by drawing their contours with the ImageJ Freehand Selection tool.</li>
		<li>Calculate geometrical and shape parameters by using the Set Measurements tool of ImageJ under the Analyze menu. Activate the Area, Perimeter, Fit Ellipse, and Shape Descriptor options. 
		NOTE: This will allow retrieval of diverse geometrical parameters including the area (sum of the pixels within the clone), the perimeter (sum of the pixel of the clone border), the aspect ratio (AR, the ratio between the major and minor axes of the best-fit Legendre ellipse inscribed into the clone border), and the angle of orientation (i.e, the angle of the major axis of the clone relative to the anteroposterior boundary).</li>
		<li>Calculating non-dimensional ratios from these measurements retrieves shape parameters. These include roundness (4 x [area]/&#960; x [major axis]2), roughness (solidity - area/convex area), and circularity (4&#960; x [area]/[perimeter]2). 
		NOTE: Each of the shape parameters represents the degree of deviation of the clones from ideal shapes such as a circle or a bounded convex hull, and they are all bounded between values of 0 and 1. Values equal to 1 indicate maximum symmetry (i.e., minimal complexity).</li>
		<li>Statistically analyze geometrical and shape parameters between different genotypes or conditions using Microsoft Excel and/or PAST. Statistical significance of the difference is determined using an unpaired two-tailed Student&#39;s t-test for equal mean or the non-parametric Kolmogorov-Smirnov K-S-test for equal distributions between conditions. Data can be graphically displayed as desired (e.g., bar charts, box plots, etc.). See Figure 5.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interests.

Long-range order is an essential characteristic of most functional physiological units. During morphogenesis, order is achieved through the integration of complex instructions implemented with high temporal and spatial precision. Multiple and multilevel constrains are integrated into stereotyped tissue arrangements.
Polarity and directionality are critical to ordered spatial arrangement during development. Polarity implies symmetry breaking during development. The achievement of asymmetry is n...

To achieve their roles, epithelial tissues fully rely on the spatial organization of their cellular components. In most epithelia, cells are not only packed against each other to create a precise cobblestone layer but they orient themselves relative to the body axes.
The functional importance of precise tissue organization is obvious in sensory epithelia, such as the vertebrate inner ear and retina. In the first case, hair and supporting cells align in a specific axial direction to efficiently sense mechanical inputs such as sound and motion1,2. Similarly, photoreceptor cell spatial organization is essential for achieving optimal optical properties by the retina3. Spatial control of cell position and orientation is thus of particular relevance for proper physiological function.
Drosophila is a holometabolous insect that undergoes a complete transformation of its larval body structures through metamorphosis, giving rise to its adult tissues. The Drosophila pupa is an excellent model for the noninvasive live imaging of a variety of dynamic events, including developmental cell migration4, cell division and growth dynamics5, muscle contraction6, cell death7, wound repair8, and cell orientation9. In the adult Drosophila, the external epithelium shows a high degree of order. This is easily observed on the arrangements of trichomes (i.e., cell protrusions originating from single epithelial cells) and sensory bristles all over the fly&#39;s body surface10. Indeed, trichomes are aligned in parallel rows guiding airflow11. The morphogenesis of the adult epithelia and the ordered arrangement of the individual cells starts during embryogenesis and culminates during pupal stages. While in embryos cell divisions, intercalations, and shape changes all decrease tissue order12,13, this is reverted at later stages of development, especially at pupal stages, when the fly approaches maturity9.
The immobile Drosophila pupa provides an ideal system to evaluate cell shape and orientation changes. The pupal abdominal epidermis presents special advantages. While the precursors of the adult head, thorax, genitalia, and appendages grow and get patterned from larval stages, the histoblasts, which are integrated into the larval epidermis, start growing and differentiating only at pupariation14. This feature allows the tracking of all spatiotemporal events involved in the establishment of tissue order in its entirety9.
Histoblasts are specified during embryonic development at contralateral positions in each presumptive abdominal segment. The dorsal abdominal epidermis of the adult derives from dorsolaterally located histoblast nests present at the anterior and posterior compartments15,16. As histoblasts expand, replacing the larval epithelial cells (LECs), the contralateral nests fuse at the dorsal midline forming a confluent sheet17,18,19,20.
This work describes 1) a methodology for dissection, mounting, and long-term live imaging of the Drosophila pupae, and 2) analytical methods to study the dynamics of cellular orientation and growth at high spatiotemporal resolution. A detailed protocol is provided here, covering all the steps required from the initial pupae preparation (i.e., staging and imaging) to the extraction and quantification of directionality and orientation features. We also describe how to infer local tissue properties from the analysis of cell clones. All the steps described are minimally invasive and allow long-term live analyses. The methods described here can be easily adapted and applied to other developmental stages, tissues, or model organisms.

<table><tbody><tr><td>Analysis Software</td><td>-</td><td>ImageJ</td><td>Analyzing data</td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt;</td><td>&lt;em&gt;Atpa::GFP&lt;/em&gt;</td><td>-</td><td>Strains employed for data collection</td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt;</td><td>&lt;em&gt;hsflp1.22;FRT40A/FRT40A Ubi.RFP.nls&lt;/em&gt;</td><td>-</td><td>Strains employed for data collection</td></tr><tr><td>Dumont 5 Forceps</td><td>FST</td><td>11251-20</td><td>1.5 mm diameter for dissection</td></tr><tr><td>Glass Bottom Plates</td><td>Mat Tek</td><td>P35G-0.170-14-C</td><td>Mounting pupae for data collection</td></tr><tr><td>Halocarbon Oil 27</td><td>Sigma-Aldrich</td><td>9002-83-9</td><td>mounting pupae</td></tr><tr><td>Inverted Confocal microscope</td><td>Zeiss</td><td>LSM700</td><td>Data collection</td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>DFC365FX</td><td>Visualization of the pupae during dissection</td></tr></tbody></table>

imaging and analysis of tissue orientation and growth dynamics in the developing drosophila epithelia during pupal stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

The protocol described above covers the preparation of Drosophila pupae for long-term live imaging and the procedures for the analysis of cell orientation and growth dynamics of the abdominal epidermis. By applying this methodology it is possible to generate high-resolution movies of the developing pupae for periods of up to 48 h without significant photobleaching or phototoxicity. Snapshots depicting the abdominal epidermis (e.g., histoblasts and LECs) at different time points a...

Watch this Scientific Journal Video about Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages at JoVE.com

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

imaging-analysis-tissue-orientation-growth-dynamics-developing

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.2K Views.  Parc Científic de Barcelona. This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Video: Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

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 ...

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. This morphological alteration is also recognized as epithelial to mesenchymal transition (EMT). Dysregulation of EMT is associated with fibrosis and cancer metastasis. There is emerging evidence that EMT is controlled by a number of molecular mechanisms. As such, many key genes that control apical constriction are also known to be important factors in the EMT observed in cancer metastasis. Like EMT during Drosophila gastrulation, epithelial cells can be induced to change their shape and be reprogrammed to redirect cell fate towards various other cell types. Here we provide a robust imaging method of Drosophila gastrulation to assay the initiation of morphogenetic cellular movements and cell fate identification during this stage of embryonic development. Using this method, we identify cell rearrangement at the time of gastrulation and demonstrate the importance of apical constriction during gastrulation using GFP labeled DE-cadherin.

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. ...

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Biology

Imaging Cell Shape Change in Living Drosophila Embryos

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

Imaging Cell Shape Change in Living Drosophila Embryos

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

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

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Extended Live Imaging of Drosophila Pupal Leg Morphogenesis Without Puparium

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which fate-determined cells form three-dimensional organismal shapes remains unclear. Recent advances in confocal microscopy have facilitated efforts to observe cell dynamics during development through live imaging. The Drosophila melanogaster pupa is ideal for live imaging due to its immobility, transparent pupal cuticle, and the availability of fluorescent reporter lines. A primary challenge for imaging is the puparium, the cuticle surrounding the pupa, which obstructs optical imaging. While previous methods involved either partial or complete removal of the puparium, maintaining pupal viability for extended periods after this procedure has remained challenging. Here, a simple method is presented for days-long live imaging of the Drosophila leg during the pupal stage, involving complete puparium removal. The method includes removing the puparium from a pupa adhered to double-sided tape, followed by assembling a small chamber on a glass-bottom dish to enclose the pupa and a drop of water. This setup is straightforward, reliable, and supports extended pupal survival by preventing desiccation. Long-term live imaging of the Drosophila pupa has significantly contributed to capturing how the adult leg undergoes dramatic three-dimensional structural changes over 2-3 days. These changes include the transient formation of an intriguing structure (the Parthenon-like structure) by epithelial cells, rapid tissue narrowing, joint formation, and bristle elongation. This method is applicable to the observation of various tissues and can potentially be combined with other techniques, such as optical gene induction, to advance the understanding of cell dynamics during the final shape formation of tissues in the pupal stage.

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Here, a protocol is presented for long-term live imaging of the Drosophila leg during the pupal stage, involving complete removal of the puparium. This protocol can also be applied to other tissues.

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which ...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

Immunology and Infection

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

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Live-imaging of the Drosophila Pupal Eye

Upright Imaging of Drosophila Egg Chambers

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Long-term Live Imaging of Drosophila Eye Disc

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Imaging Cell Shape Change in Living Drosophila Embryos

Drosophila Pupal Abdomen Immunohistochemistry

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Imaging Through the Pupal Case of Drosophila melanogaster