Name: imaging of cell shape alteration and cell movement in drosophila gastrulation using de-cadherin reporter transgenic flies
Uploaded: 2016-12-29T14:00:00
Description: Watch this Scientific Journal Video about Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies at JoVE.com

This study was supported by the Astellas Foundation for Research on Metabolic Disorders (HT), Takeda Science Foundation (HT), and MEXT-Supported Program for the Strategic Research Foundation at Private Universities (HT).

NOTE: The transgenic flies used in this study include the following: DE-cad::GFP 10.
1. Preparation of Apple Plate
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	<li>Prepare a mixture of 12.5 g agar, 125 mL 100% commercially available apple juice, 12.5 g glucose, and 375 mL H2O. Heat the mixture in a microwave and pour it into a 3 cm cell culture dish. Store the mixture at 4 &#176;C for future use.</li>
	<li>After preparing the apple plate, add a thin layer of kneaded yeast paste on top of it to allow the flies to lay egg...

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	<li>Haruta, T., Warrior, R., Yonemura, S., Oda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proximal+half+of+the+Drosophila+E-cadherin+extracellular+region+is+dispensable+for+many+cadherin-dependent+events+but+required+for+ventral+furrow+formation.">The proximal half of the Drosophila E-cadherin extracellular region is dispensable for many cadherin-dependent events but required for ventral furrow formation.</a> Genes Cells. 15 (3), 193-208 (2010).</li><li>Oda, H., Takeichi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution:+structural+and+functional+diversity+of+cadherin+at+the+adherens+junction.">Evolution: structural and functional diversity of cadherin at the adherens junction.</a> J. Cell Biol. 193 (7), 1137-1146 (2011).</li><li>Fernandez-Sanchez, M. E., Serman, F., Ahmadi, P., Farge, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+induction+in+embryonic+development+and+tumor+growth+integrative+cues+through+molecular+to+multicellular+interplay+and+evolutionary+perspectives.">Mechanical induction in embryonic development and tumor growth integrative cues through molecular to multicellular interplay and evolutionary perspectives.</a> Methods Cell Biol. 98, 295-321 (2010).</li><li>Alderton, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+Epithelial+to+mesenchymal+and+back+again.">Metastasis: Epithelial to mesenchymal and back again.</a> Nat. Rev. Cancer. 13 (1), 3 (2013).</li><li>Fabregat, I., Malfettone, A., Soukupova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Insights+into+the+Crossroads+between+EMT+and+Stemness+in+the+Context+of+Cancer.">New Insights into the Crossroads between EMT and Stemness in the Context of Cancer.</a> J. Clin. Med. 5 (3), (2016).</li><li>Serrano-Gomez, S. J., Maziveyi, M., Alahari, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+epithelial-mesenchymal+transition+through+epigenetic+and+post-translational+modifications.">Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications.</a> Mol. Cancer. 15, (2016).</li><li>Yu, A. Q., Ding, Y., Li, C. L., Yang, Y., Yan, S. R., Li, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALEN-induced+disruption+of+Nanog+expression+results+in+reduced+proliferation,+invasiveness+and+migration,+increased+chemosensitivity+and+reversal+of+EMT+in+HepG2+cells.">TALEN-induced disruption of Nanog expression results in reduced proliferation, invasiveness and migration, increased chemosensitivity and reversal of EMT in HepG2 cells.</a> Oncol. Rep. 35 (3), 1657-1663 (2016).</li><li>Zheng, X., 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=Epithelial-to-mesenchymal+transition+is+dispensable+for+metastasis+but+induces+chemoresistance+in+pancreatic+cancer.">Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer.</a> Nature. 527 (7579), 525-530 (2015).</li><li>Kalluri, R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basics+of+epithelial-mesenchymal+transition.">The basics of epithelial-mesenchymal transition.</a> J. Clin. Invest. 119 (6), 1420-1428 (2009).</li><li>Huang, J., Zhou, W., Dong, W., Watson, A. M., Hong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed,+efficient,+and+versatile+modifications+of+the+Drosophila+genome+by+genomic+engineering.">Directed, efficient, and versatile modifications of the Drosophila genome by genomic engineering.</a> Proc. Natl. Acad. Sci. U.S.A. 106 (20), 8284-8289 (2009).</li></ol>

Although we have previously reported a similar procedure to capture live images of the gastrulation process in Drosophilla1, the method we describe here is detailed and easy to trace endogenous cadherin expression and thus is quite useful for genetic screening of key factors involved in gastrulation. To maximize success with this imaging procedure, it is essential to use an indented slide. Mechanical pressure sometimes causes embryonic death. Therefore, it is also important to handle the embryos as ge...

Gastrulation is the first set of morphologically dynamic events that occur during embryonic development of multicellular animals such as Drosophila1,2. Interestingly, emerging evidence suggests that this process is regulated through the interplay between mechanical and molecular mechanisms3. Moreover, the epithelial to mesenchymal transition (EMT), which is a crucial process in gastrulation, is also implicated in human disease processes such as cancer metastasis4-8. As such, many genes that control apical constriction are also known to be key factors in the EMT observed in cancer metastasis9. Thus, apical constriction at the time of gastrulation is an excellent model to investigate the aforementioned regulatory mechanisms and to enhance our understanding of cancer metastasis. The advantage of this technique is that we can observe cell movement at the time of gastrulation in real-time and therefore, we will be able to screen genes involved in gastrulation as well as cancer metastasis.
Although relatively unknown, cell-to-cell adhesion is thought to play a central role in apical constriction1. Drosophila genetics is well suited for single cell level investigations exploring regulatory molecular mechanisms. This model will enable us to uncover the importance of apical constriction during gastrulation. Moreover, this method can be used to screen genes involved in cancer metastasis. Capturing live images of Drosophila gastrulation has further enabled us to understand in greater detail the molecular mechanisms governing tissue rearrangement. Herein, we provide a comprehensive description of a simple method to achieve this.

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			<td height="54" style="height:54px;width:268px;">Halocarbon oil 700</td>
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			<td height="54" style="height:54px;width:268px;">Vacuum grease Silicone</td>
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			<td height="38" style="height:38px;width:268px;">Glass coverslip&#160;</td>
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			<td style="width:129px;">Thickness No1</td>
			<td style="width:133px;">24-36mm</td>
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			<td height="38" style="height:38px;width:268px;">Embryo stariner</td>
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			<td height="38" style="height:38px;width:268px;">Plastic Drosophilla Stock Bottles</td>
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			<td height="21" style="height:21px;width:268px;">DE-Cadherin knock-in flies</td>
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			<td>REF (10)</td>
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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.

Here, we show the gastrulation events of the Drosophila embryo and a general overview of the embryo preparation procedure (Figure 1). Cell membranes are labeled using DE-cadherin::GFP and live imaging of cell movements is performed at the time of gastrulation in Drosophila (Figure 2). Since DE-cadherin GFP flies allow us to visualize cell adherence junctions, we are able to trace apical cell shape and movements using this system. More importantly...

Watch this Scientific Journal Video about Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies at JoVE.com

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.

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. ...

The overall goal of this procedure, is to better understand the cellular shape and movement, during Drosophila gastrulation. Here we provide a robustic measuring method of Drosophila gastrulation, to assay the initiation of morphogenetic cellular movement, during this stage of embryonic development. The main advantage of this technique is that, cell rearrangement at the time of gastrulation, can be identified along with apical constriction, during gastrulation.To begin the experiment, prepare a mixture of 375 milliliters of water. 125 milliliters 100%commercially available apple juice. 12.5 grams of agar.12.5 grams of glucose. Next, heat the mixture in a microwave, until the agar gets melted down. And pour it into a three centimeter cell culture dish.Store the mixture at 4 degrees celsius, for future use. After preparing the apple plate, add a thin layer of kneaded yeast paste on top of it, to allow the flies to lay eggs. Place 25 male and 25 female flies in a bottle on an apple plate covered in kneaded yeast.Wrap the plastic Drosophilas stock bottles with aluminum foil to prompt the flies to lay more eggs. Keep the bottle upside down in an incubator overnight. The next morning, replace the apple juice plate with a fresh one.Then wrap the bottle with aluminum foil and allow the flies to lay eggs for three to four hours. After the flies have laid eggs, loosen the eggs from the plate with a cotton bud. Collect the embryos in a strainer, and wash them with phosphate buffered saline, or PBS.Wash the embryos three more times with PBS, in 12-well culture dishes. Dechorionate the embryos with 50%bleach, in 12-well culture dishes. Following the dechorionation process, wash the embryos three more times with PBS.Transfer the embryos to a three centimeter cell culture dish containing PBS. And select stage five embryos under the microscope, based on the level of transparency at their borders. Using a 200 microliter pipette tip, collect the staged embryos.Next, place two selected embryos on a glass cover slip, and remove excess PBS with tissue paper. With a finely twisted tissue paper, orient the embryos dorsal side up to the cover slip. Then attach the embryos to the glass cover slip with silicone grease using a fine needle.Add a small drop of hollow carbon oil 700 on the embryos, and place the coverslip containing the embryos upside down on the indented slide. Leaving some space at the bottom of the slide. Then stick the cover slip to the slide.Finally, examine the embryos using a confocal imaging system, an argon 488 laser, and an oil-immersion objective. Time-lapse images were captured from live imaging, using DE-cadherin GFP of cell movement over 500 seconds, to analyze the apical areas of individual cells, of Drosophila during gastrulation. At 500 seconds, apical constriction, and invagination are clearly visible.By using our method. We can further understand, the molecular mechanisms, of Drosophila gastrulation, as well as, epithelial to mesenchymal transition, involved in cancer metastasis.

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Research

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Developmental Biology

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

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 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 ...

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a G&#945;12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

Here we describe a contractility assay using Drosophila S2R+ cells. The application of an exogenous ligand, folded gastrulation (Fog), leads to the activation of the Fog signaling pathway and cellular contractility. This assay can be used to investigate the regulation of cellular contractility proteins in the Fog signaling pathway.

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Here, we describe a protocol to visualize and analyze the pharyngeal arch arteries 3, 4, and 6 of mouse embryos using whole-mount immunofluorescence, tissue clearing, confocal microscopy, and 3D reconstruction.

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...