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...

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G., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FLP+recombinase+of+yeast+catalyzes+site-specific+recombination+in+the+Drosophila+genome.">The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome.</a> Cell. 59, 499-509 (1989).</li><li>Xu, T., Rubin, G. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Analysis Software</td>
			<td>-</td>
			<td>ImageJ</td>
			<td>Analyzing data</td>
		</tr>
		<tr>
			<td>Drosophila</td>
			<td>Atpa::GFP</td>
			<td>-</td>
			<td>Strains employed for data collection</td>
		</tr>
		<tr>
			<td>Drosophila</td>
			<td>hsflp1.22;FRT40A/FRT40A Ubi.RFP.nls</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 ...

Within multicellular organisms, tissue displays high degrees further in their spatial orientation. Cells align and display a range of priority over large distances over the course of morphogenesis. To understand how the planar arrangements of epithelia is reached during development, it is crucial to track cell's orientations and growth dynamics with high spacial temporal fidelity in vivo.The described protocol is designed to image and analyze cell behaviors at global and local levels in the drosophila pupal epidermis. This methodology could provide insights into different theory of research, cell biology, morphogenesis, and planar polarity and can be applied to the analysis of other tissues, structures, and models. All the steps can be performed easily after some practice.They demand precision. Visualization of this protocol helps achieving optimal results in a shorter time. The pupal dissection is complex.To begin, use a moistened paintbrush to transfer staged white pre-pupae to a fresh plastic vial, and keep them there for as long as necessary at 25 degrees Celsius. Then, remove each pupae from the wall of the vial with the help of forceps. Glue the ventral side of each pupa on a glass slide covered with double sided sticky tape.Gently tap on the head spiracles and the dorsal surface of the pupa with the tips of the forceps to assure the adhesion of the pupal case to the tape. Begin dissection under a stereo microscope by gently removing the operculum from the puparium with the forceps. Insert one tip of the forceps in a shallow 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. Fold back the cracked pupal case to the lateral sides as you keep proceeding to the posterior end. Remove the pupa from the opened up pupal case by carefully inserting the forceps under the animal and gently pulling up.The pupa sticks to the tip of the forceps. Hold the pupa gently by the ventral side, and transfer the pupa to a glass bottomed dish on top of a small drop of gas-permeable halocarbon oil. Roll a piece of wet tissue paper at the edges of the dish and cover the dish to maintain humidity.Orient the pupa over the oil drop on the glass bottomed dishes according to the domain and the process to be evaluated. For long term live imaging of the early expansion of the dorsal nests, dorsal laterally orient the pupa. To image their late expansion and tissue remodeling, dorsally orient the pupa.Transfer the glass bottomed dish containing the mounted pupae to the microscope stage and focus on the surface of the abdominal area using transmitted light. To employ mitotic recombination to induce genetic mosaics in the abdominal epithelium, prepare virgin females carrying a heat shock inducible flipase, an FRT site at a specific genomic location, and a recognizable cellular marker distal to the FRT site. Prepare mutant males carrying an FRT site at the equivalent genomic location.Cross several females and males in a three to one proportion in a plastic vial at 25 degrees Celsius for four to five days. To generate somatic clones in the histoblasts, perform heat shock treatment at the third instar larval stage of the progeny of the cross by submerging the plastic vials containing the animals in a water bath at 37 degrees Celsius for 45 minutes to an hour. In the image J software, use the maximum intensity projection function to protect the Z stack slices acquired by confocal microscopy in 2D.The nests have a characteristic shape that orients them in a planar coordinate system anterior to the left and dorsal to the top. To achieve optimal results with this analysis, the input image has to be acquired with high resolution. To obtain qualitative orientation values on local cell edges, click on the plug-in menu and choose the orientation J distribution option.Set the Gaussian window sigma to one pixel, cubic spline to gradient, minimum coherency to 20 percent, and minimum energy to one percent. Employ the color survey option of the plug-in. Set hue to orientation, saturation to coherency, and brightness to input image.This color codes cell edge orientations relative to the set planar coordinate system. Next, under the plug-in menu, use the orientation J measure option to quantify cellular orientations and directional cell to cell alignment. Generate small, adjacent non-overlapping regions of interest of uniform weight around 20 micrometers times 20 micrometers within the area occupied by the histoblasts.Pressing measure calculates the dominant local orientation and coherency from the ROIs. In this study, the methodology used is able to generate high resolution movies of the developing pupae for periods of up to 48 hours without significant photo bleaching or photo toxicity. Examples of wild type clones marked by the absence of RFP NLS and their twin spots at 26 hours after puparium formation are shown here.The clones elongated along the segmental boundaries. Twin clones arranged in parallel or in tandem. Morphology of a wild type clone at 26 hours and 47 hours after puparium formation shows complex border morphology at both stages.Box and whisker plots for geometrical parameters at 26 hours and 47 hours after puparium formation show the averaged area and perimeter increased significantly in this time window. The clone's orientation was sustained during expansion and remodeling. Shape parameters at 26 hours and 47 hours after puparium formation indicate the roughness, roundness and circularity barely changed in the wild type clones.Be sure to not damage the pupa, otherwise it will die within a few hours, affecting live imaging. This protocol does not require a sartus reagent or instrument to be employed. Avoid pinching yourself when dissecting.By applying these methodologies possible to generate high resolution movies for long term periods using different imagine techniques. Focal to photon or spinning disks. Chloral analysis can be employed to explore non-autonomous responses since random cells facilitate in the identification of cross talks or cell communication mechanisms.These methods can be easily adapted to study a full range of morphogenetic phenomena, including the coordination of epidermal, muscular, and neuro development during metamorphosis.

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JoVE Journal

Developmental Biology

8.9K visualizações.  Parc Científic de Barcelona. Dentro de organismos multicelulares, o tecido exibe altos graus em sua orientação espacial. As células se alinham e exibem uma gama de prioridades em grandes distâncias ao longo da morfogênese. Para entender como os arranjos planares da epitélio são alcançados durante o desenvolvimento, é crucial acompanhar as orientações e dinâmicas de crescimento das células com alta fidelidade temporal espacial in vivo.O protocolo descrito foi projetado para imagem e análise de comport...