Name: drug treatment and in vivo imaging of osteoblast-osteoclast interactions in a medaka fish osteoporosis model
Uploaded: 2017-01-01T14:00:00
Description: Watch this Scientific Journal Video about Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model at JoVE.com

This project was funded by grants from the Singapore Ministry of Education (MOE, grant number 2013-T2-2-126) and the National Institute of Health, USA (NIH, grant number 1R21AT008452-01A1). T.Y. received a graduate scholarship from the NUS Department of Biological Sciences. We thank the confocal unit of the NUS Centre for Bioimaging Sciences (CBIS) for their constant support.

All experiments were performed in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols of the National University of Singapore (R14-293).
1. Fish Husbandry and the Collection of Embryos
<ol>
	<li>Raise WT, ctsk:nlGFP24, RANKL:HSE:CFP24, and osx:mCherry21 single- or compound-transgenic medaka fish at 26 &#176;C under a controlled light cycle (14 h light, 10 h dark) to induce spawning.</li>
	<li>Daily...

<ol>
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Critical Steps within the Protocol
It is essential that the conditions for heat&#160;shock treatment are consistent and stable when comparing different samples. Stable temperature conditions guarantee similar levels of RANKL induction in the transgenic larvae and, consequently, comparable osteoclast formation, which can be confirmed by screening for ctsk:nlGFP expression. Ultimately, this leads to a similar degree of induced ectopic bone resorption and osteoporosis-like lesions, as valid...

The vertebrate skeleton provides structural support and protection for organs, allows mobility, and serves as a source of calcium. Throughout life, the extracellular bone matrix is continuously turned over to maintain bone stability and rigidity. This process requires the tightly coordinated activity and interplay of bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts are derived from multipotent mesenchymal progenitors and produce collagen to form the osteoid, the proteinaceous part of the bone matrix10. Osteoblasts interact with osteoclasts to achieve a balanced activity of both cell types, which is required to control bone homeostasis7. Because of these intricate regulatory interactions, responses to drug treatment and bone homeostasis cannot be fully examined using in vitro studies. Hence, there is a strong demand for animal models. Compared to the cell&#160;culture settings, in vivo models can provide valuable insight into the multicellular networks within the bone environment.
Numerous mouse models exist for a variety of human bone disorders including osteoporosis16. However, the size and accessibility of mouse embryos represent significant limitations for live imaging of skeletal processes. Small teleost fish, on the other hand, serve as an attractive alternative for in vivo imaging. Zebrafish (Danio rerio) and medaka (Oryzias latipes) have become popular animal models for skeletal research over the last two decades17, 19, 22, 24. Bone in teleost fish and in mammals is very similar, both on a structural and on a physiological level, and many of the key regulatory genes and signaling pathways are conserved3. As in mammals, teleost fish carefully regulate the activity of osteoblasts and osteoclasts to balance bone formation and resorption26. Most importantly, the optical clarity of fish larvae allows the use of fluorescent reporters to label bone cells and the calcified skeletal matrix8, 9, 12, 21, 23, which&#160;facilitates the observation of cellular processes in the living animal. In addition, a series of genetic tools has been generated to facilitate biomedically relevant research in fish. For medaka in particular, methods for targeted gene mutation by CrispR/Cas92, cell-lineage tracing6, and site-specific transgenesis14 have been recently established and are now widely in use15.
Small teleost larvae have been successfully used for chemical screens, which led to the discovery of several pharmacologically relevant drugs1, 18.
Fish larvae are tolerant to low concentrations of DMSO and are able to absorb compounds from their aquatic environment, either through the skin or through the gastrointestinal tract1, 5. Our lab previously reported transgenic medaka lines that express fluorescent reporters in bone cells under the control of various osteoblast- and osteoclast-specific promoters. These include premature osteoblasts (collagen 10a1, col10a1; osterix, osx)20, 21, mature osteoblasts (osteocalcin, osc)27, and osteoclasts (cathepsin K, ctsk)24. We also generated a transgenic line that expresses the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter24.
Induction of RANKL in this system results in the ectopic formation of active osteoclasts. This leads to increased bone resorption and a severe osteoporosis-like phenotype, with drastically reduced mineralization in the vertebral bodies. We recently showed that osteoclast activity in this model can be blocked by the bisphosphonates etidronate and alendronate, two drugs commonly used in human osteoporosis therapy, thus validating medaka as a suitable model system for osteoporosis27.
Due to their large brood size, rapid development, and small size of embryos, transgenic medaka larvae are uniquely suited for the large-scale screening of osteoporosis drugs and for the in vivo analysis of bone cell behavior. Studies in medaka thus can efficiently complement experiments in cell cultures and in mice that are aimed at discovering new therapeutic targets and novel therapies for human bone disorders.
In the present study, we describe a protocol to treat medaka bone-reporter larvae with the common osteoporosis drug, alendronate. We also describe in detail how treated larvae are mounted and prepared for the live imaging of bone matrix and bone cells. These protocols can be easily adapted to other small chemical compounds that either work as bone anabolic or antiresorptive drugs.

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			<td height="20" style="height:20px;width:249px;">Alendronate&#160;</td>
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			<td height="40" style="height:40px;width:249px;">alizarin-3-methyliminodiacetic acid, Alizarin Complexone</td>
			<td>Sigma</td>
			<td>A3882</td>
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			<td height="20" style="height:20px;">Calcein</td>
			<td>Sigma</td>
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			<td height="40" style="height:40px;width:249px;">ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td>Sigma</td>
			<td>A5040</td>
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			<td height="40" style="height:40px;">ImageJ (1.4.3.67)</td>
			<td style="width:177px;">National Institute of Health (NIH)</td>
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			<td>https://imagej.nih.gov/ij/</td>
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			<td height="20" style="height:20px;width:249px;">LSM 510 Meta confocal&#160;</td>
			<td>Zeiss</td>
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			<td height="20" style="height:20px;width:249px;">LSM Image Browser (4.2.0.121)</td>
			<td>Zeiss</td>
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			<td>http://www.zeiss.com/microscopy/en_de/downloads/lsm-5-series.html</td>
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			<td height="20" style="height:20px;">Micro-loader</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td>Eppendorf ep T.I.P.S 20 &#956;l</td>
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			<td height="20" style="height:20px;width:249px;">NIS-Elements BR 3.0 software</td>
			<td>Nikon</td>
			<td></td>
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			<td height="20" style="height:20px;width:249px;">Photoshop CS6 (13.0.0.0)</td>
			<td>Adobe</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;width:249px;">SMZ1000 stereomicroscope&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
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drug treatment and in vivo imaging of osteoblast-osteoclast interactions in a medaka fish osteoporosis model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Abundant egg numbers, as well as the small size of the larvae, make medaka an excellent model for drug screening. A single six-well plate was used to culture up to 36 larvae, which was sufficient to provide statistically significant data. Another big advantage of using fish for skeletal analysis is the possibility of doing live imaging. The transparency of fish larvae allows the use of fluorescent proteins to label bone cells, as well as the use of dyes that bind to bone matrix in order to visualize mineralization. Fish ...

Watch this Scientific Journal Video about Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model at JoVE.com

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

The overall goals of this procedure are to use the embryos of transgenic Medaka bone reporter lines for drug testing and for live imaging of bone-cell behavior after the induction of osteoporosis like conditions. This method can help answer key questions in skeletal research such as how do different type of bone cells interact with each other in vivo? The main advantage of this technique is that the optical clarity of Medaka larvae allows live imaging of bone-cell behavior in an intact specimen.The implications of this technique extends towards the development of novel therapeutics for common bone diseases as it allows the visualization of long term effects of drug treatment in C2.All procedures described in this video were approved by IACUC of the National University of Singapore. To harvest the embryos during the first few hours of the light cycle, first use a fine meshed net to capture female adults carrying an egg cluster. Let the fish briefly rest in the net, and then gently massage the fish's abdomen to carefully strip the fertilized egg cluster from the abdomen of the female.Transfer 20 to 30 eggs into individual plastic 60 millimeter petri dishes, and use a plastic pipette to rinse the embryos in each plate with 5 to 10 milliliters of daneous solution. Gently roll egg cluster to form a knot of attachment filaments. Using forceps, carefully remove the attachment filaments from the fertilized egg clusters to obtain individual embryos.Then, culture the embryos in a 28 degrees Celsius incubator with daily medium changes to ensure normal embryonic development. For transgenic embryo screening, at two days post fertilization or later, heat shock the larvae for 20 minutes at 39 degrees Celsius. Then, use a stereo microscope equipped with a mercury lamp for fluorescence imaging to identify ubiquitous CFP expression.On day 5 post fertilization, mCherry reporter expression can be observed in osteoblasts of the early forming cranial bones on both sides of the posterior head and at a central position in the ventral cranium. Starting from day 6 post fertilization, robust nlGFP expression can be visualized in cathepsin positive cells of the head and tail. For the induction of an osteoporosis-like phenotype, subject the embryos to a 1.5 to 2 hour heat shock treatment at 9 days post fertilization or later to induce large numbers of ectopic osteoclasts in the trunk region.For bisphosphonate treatment, first transfer 6 Medaka larvae per well into a 6-well plate. Then use a clean, plastic pipette to carefully remove the fish medium. Add approximately 0.5 milliliters of bisphosphonate to each well to dilute any traces of medium.Then replace the wash solution with 4 milliliters of fresh bisphosphonate solution, and return the larvae to the incubator with daily medium changes for 2 to 6 days. At the appropriate experimental time point, filter freshly prepared bone staining solution through a single use 0.2 micron strainer. And incubate the larvae in a 1 and 10 dilution of the stain in fresh fish medium, for the appropriate staining period in a 28 degree Celsius incubator in the dark.At the end of the incubation, use a clean plastic pipette to transfer the larvae into fresh fish medium, and replace the stain contaminated medium with another aliquot of fresh medium. When there is no visible stain left in the medium, allow the larvae to rest in the fish medium for 30 to 60 minutes before imaging. For live fluorescence imaging of the Medaka larvae, use a plastic microloader to orient the anesthetized larvae according to the region of interest.And place them under a stereo microscope with fluorescence illumination. Select the appropriate magnification and image individual sections of each larvae using a suitable image processing software to stitch together the images at the appropriate overlapping regions. After each larvae has been imaged, return the animal to its well in the 6-well plate in fresh fish medium for recovery.For live confocal imaging, add 0.5 to 1 milliliter of 30 degree Celsius 1.5%liquid low melting agarose in fish medium to a glass bottom petri dish. Before the agarose solidifies, transfer an anesthetized larvae to the agarose and use a plastic microloader to push the larvae to the bottom of the dish. And use the yoke extension to orient the larvae according to the region of interest.After the agar has solidified, image the larvae by confocal microscopy. Then, use a pair of fine syringe needles to release the larvae from the agarose. And transfer each larvae and residually attached agarose into a new petri dish containing fresh fish medium for recovery.In these images, triple transgenic larvae were used for the simultaneous detection of premature osteoblasts and differentiated osteoclasts. Overview images were taken with a stereo microscope, while confocal microscopy was used to visualize processes at the cellular level. ALC stained bone matrix along the neural arches and sentry, was used as a reference for determining the position of the fluorescently labeled bone cells.RankL induction resulted in extensive formation of ectopic osteoclasts around the vertebral bodies. These excessive osteoclasts cause an osteoporosis-like phenotype which are observed as cavities in the mineralized centra, and as absent mineralization in the neural arches. After incubation in alendronate, osteoclasts formed normally but vertebral columns are intact, with no lesions and fully mineralized arches suggesting an efficient inhibition of osteoclast activity.Further, successive staining of the bone matrix with ALC followed by calcein, reveals a de novo mineralized bone matrix, with a simultaneous visualization of the osteoclasts and osteoblasts in the same intact larvae. Once mastered, this technique can be completed in 3 days from the induction of the osteoporotic lesions, until the imaging of the drug effect if it is performed properly. While attempting this procedure, it is important to remember to handle the living larvae with extreme care and to mound the larvae properly so that the region of interest will be in one focal plane.Following this procedure, other methods like noninvasive sulfate checking of individual bone precursor sites can be performed to answer additional questions such as bone-cell origin, activation, and attraction to the lesion sites for subsequent bone repair. After the development of this technique, it pave the way for scientists in the field of bone research to explore the effects of various chemical compounds, or genetic mutations on the dynamic behavior of cells during bone homeostasis. After watching this video, you should now have a good understanding of how to use Medaka bone transgenic lines for drug screening and live imaging of bone-cell behavior.

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Research

JoVE Journal

Developmental Biology

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

Automated Quantification of Hematopoietic Cell &amp;#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

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 ...

Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.

Transcriptional  Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

Trophoblast giant cells (TGCs) play a key role in the placenta to ensure a healthy pregnancy. We present a protocol for assessing the transcriptional status of genes in TGCs by nascent fluorescent in situ hybridization on cryostat sections of post-implantation embryos or short-term cultures of embryonic day 7 ectoplacental cones.

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells ...

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 ...

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

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 ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

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 ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...