Name: imaging and analysis of tissue orientation and growth dynamics in the developing drosophila epithelia during pupal stages
Uploaded: 2020-06-02T13:00:00
Description: 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

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...

<ol>
	<li>Gillespie, P. G., Muller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+by+hair+cells:+models,+molecules,+and+mechanisms.">Mechanotransduction by hair cells: models, molecules, and mechanisms.</a> Cell. 139, 33-44 (2009).</li><li>Deans, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+balance+of+form+and+function:+planar+polarity+and+development+of+the+vestibular+maculae.">A balance of form and function: planar polarity and development of the vestibular maculae.</a> Seminars in Cellular and Developmental Biology. 24, 490-498 (2013).</li><li>Stell, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+and+morphologic+relations+of+rods+and+cones+in+the+retina+of+the+spiny+dogfish,+Squalus.">The structure and morphologic relations of rods and cones in the retina of the spiny dogfish, Squalus.</a> Comparative Biochemistry and Physiology - Part A: Comparative Physiology. 42, 141-151 (1972).</li><li>Ninov, N., Chiarelli, D. A., Martin-Blanco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+and+intrinsic+mechanisms+directing+epithelial+cell+sheet+replacement+during+Drosophila+metamorphosis.">Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis.</a> Development. 134, 367-379 (2007).</li><li>Bosveld, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+control+of+morphogenesis+by+Fat/Dachsous/Four-jointed+planar+cell+polarity+pathway.">Mechanical control of morphogenesis by Fat/Dachsous/Four-jointed planar cell polarity pathway.</a> Science. 336, 724-727 (2012).</li><li>Puah, W. C., Wasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+muscles+in+Drosophila+metamorphosis:+Towards+high-throughput+gene+identification+and+function+analysis.">Live imaging of muscles in Drosophila metamorphosis: Towards high-throughput gene identification and function analysis.</a> Methods. 96, 103-117 (2016).</li><li>Teng, X., Qin, L., Le Borgne, R., Toyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+adhesion+and+modulation+of+mechanical+tensile+forces+during+apoptosis+in+Drosophila+epithelium.">Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium.</a> Development. 144, 95-105 (2017).</li><li>Weavers, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+Analysis+of+the+Dynamic+Inflammatory+Response+to+Tissue+Damage+Reveals+Spatiotemporal+Properties+of+the+Wound+Attractant+Gradient.">Systems Analysis of the Dynamic Inflammatory Response to Tissue Damage Reveals Spatiotemporal Properties of the Wound Attractant Gradient.</a> Current Biology. 26, 1975-1989 (2016).</li><li>Mangione, F., Martin-Blanco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Dachsous/Fat/Four-Jointed+Pathway+Directs+the+Uniform+Axial+Orientation+of+Epithelial+Cells+in+the+Drosophila+Abdomen.">The Dachsous/Fat/Four-Jointed Pathway Directs the Uniform Axial Orientation of Epithelial Cells in the Drosophila Abdomen.</a> Cell Reports. 25, 2836-2850 (2018).</li><li>Casal, J., Struhl, G., Lawrence, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+compartments+and+planar+polarity+in+Drosophila.">Developmental compartments and planar polarity in Drosophila.</a> Current Biology. 12, 1189-1198 (2002).</li><li>Wootton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+flies+fly.">How flies fly.</a> Nature. 400, 112-113 (1999).</li><li>Zallen, J. A., Wieschaus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+gene+expression+directs+bipolar+planar+polarity+in+Drosophila.">Patterned gene expression directs bipolar planar polarity in Drosophila.</a> Developmental Cell. 6, 343-355 (2004).</li><li>Gibson, M. C., Patel, A. B., Nagpal, R., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+geometric+order+in+proliferating+metazoan+epithelia.">The emergence of geometric order in proliferating metazoan epithelia.</a> Nature. 442, 1038-1041 (2006).</li><li>Robertson, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metamorphosis+of+Drosophila+melanogaster,+including+an+accurately+timed+account+of+the+principal+morphological+changes.">The metamorphosis of Drosophila melanogaster, including an accurately timed account of the principal morphological changes.</a> Journal of Morphology. 59, 351-399 (1936).</li><li>Mandaravally Madhavan, M., Schneiderman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+analysis+of+the+dynamics+of+growth+of+imaginal+discs+and+histoblast+nests+during+the+larval+development+of+Drosophila+melanogaster.">Histological analysis of the dynamics of growth of imaginal discs and histoblast nests during the larval development of Drosophila melanogaster.</a> Wilhelm Roux's archives of Developmental Biology. 183, 269-305 (1977).</li><li>Kornberg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+in+the+abdomen+of+Drosophila+and+the+role+of+the+engrailed+locus.">Compartments in the abdomen of Drosophila and the role of the engrailed locus.</a> Developmental Biology. 86, 363-372 (1981).</li><li>Garcia-Bellido, A., Merriam, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+parameters+of+tergite+development+in+Drosophila.">Clonal parameters of tergite development in Drosophila.</a> Developmental Biology. 26, 264-276 (1971).</li><li>Roseland, C. R., Schneiderman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+metamorphosis+of+the+abdominal+histoblasts+of+Drosophila+melanogaster.">Regulation and metamorphosis of the abdominal histoblasts of Drosophila melanogaster.</a> Wilhelm Roux's archives of Developmental Biology. 186, 235-265 (1979).</li><li>Madhavan, M. M., Madhavan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+the+epidermis+of+adult+abdomen+of+Drosophila.">Morphogenesis of the epidermis of adult abdomen of Drosophila.</a> Journal of Embryology and Experimental Morphology. 60, 1-31 (1980).</li><li>Bischoff, M., Cseresnyes, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+rearrangements,+cell+divisions+and+cell+death+in+a+migrating+epithelial+sheet+in+the+abdomen+of+Drosophila.">Cell rearrangements, cell divisions and cell death in a migrating epithelial sheet in the abdomen of Drosophila.</a> Development. 136, 2403-2411 (2009).</li><li>Golic, K. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117, 1223-1237 (1993).</li><li>Fonck, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+aging+on+elastin+functionality+in+human+cerebral+arteries.">Effect of aging on elastin functionality in human cerebral arteries.</a> Stroke. 40, 2552-2556 (2009).</li><li>Rezakhaniha, R., Fonck, E., Genoud, C., Stergiopulos, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+elastin+anisotropy+in+structural+strain+energy+functions+of+arterial+tissue.">Role of elastin anisotropy in structural strain energy functions of arterial tissue.</a> Biomechanics and Modeling in Mechanobiology. 10, 599-611 (2011).</li><li>Hammer, &. #. 2. 1. 6. ;., Harper, D. A., Ryan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAST:+paleontological+statistics+software+package+for+education+and+data+analysis.">PAST: paleontological statistics software package for education and data analysis.</a> Palaeontologia electronica. 4, 1-9 (2001).</li><li>Gray, R. S., Roszko, I., Solnica-Krezel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+cell+polarity:+coordinating+morphogenetic+cell+behaviors+with+embryonic+polarity.">Planar cell polarity: coordinating morphogenetic cell behaviors with embryonic polarity.</a> Developmental Cell. 21, 120-133 (2011).</li><li>Vogg, M. C., Wenger, Y., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Somatic+Adult+Tissues+Develop+Organizer+Activity.">How Somatic Adult Tissues Develop Organizer Activity.</a> Current Topics in Developmental Biology. 116, 391-414 (2016).</li><li>Collinet, C., Rauzi, M., Lenne, P. F., Lecuit, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+and+tissue-scale+forces+drive+oriented+junction+growth+during+tissue+extension.">Local and tissue-scale forces drive oriented junction growth during tissue extension.</a> Nature Cell Biology. 17, 1247-1258 (2015).</li><li>Martin-Blanco, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=puckered+encodes+a+phosphatase+that+mediates+a+feedback+loop+regulating+JNK+activity+during+dorsal+closure+in+Drosophila.">puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila.</a> Genes and Development. 12, 557-570 (1998).</li><li>Dye, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+dynamics+underlying+oriented+growth+of+the+Drosophila+wing+imaginal+disc.">Cell dynamics underlying oriented growth of the Drosophila wing imaginal disc.</a> Development. 144, 4406-4421 (2017).</li><li>Williams-Masson, E. M., Malik, A. N., Hardin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+actin-mediated+two-step+mechanism+is+required+for+ventral+enclosure+of+the+C.+elegans+hypodermis.">An actin-mediated two-step mechanism is required for ventral enclosure of the C. elegans hypodermis.</a> Development. 124, 2889-2901 (1997).</li><li>Ferguson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palate+development.">Palate development.</a> Development. 103, 41-60 (1988).</li></ol>

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.

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

Research

JoVE Journal

Developmental Biology

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 ...

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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

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

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

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

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

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.

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.

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

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...