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...

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+studying+osteoarthritis+genetics+in+zebrafish.">New tools for studying osteoarthritis genetics in zebrafish.</a> Osteoarthritis Cartilage. 21 (2), 269-278 (2013).</li><li>Renn, J., Buttner, A., To, T. T., Chan, S. 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E., Renn, J., Bhattacharya, D., Huysseune, A., Winkler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rankl-induced+osteoclastogenesis+leads+to+loss+of+mineralization+in+a+medaka+osteoporosis+model.">Rankl-induced osteoclastogenesis leads to loss of mineralization in a medaka osteoporosis model.</a> Development. 139 (1), 141-150 (2012).</li><li>Wakamatsu, Y., Pristyazhnyuk, S., Kinoshita, M., Tanaka, M., Ozato, 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+see-through+medaka:+a+fish+model+that+is+transparent+throughout+life.">The see-through medaka: a fish model that is transparent throughout life.</a> Proc Natl Acad Sci USA. 98 (18), 10046-10050 (2001).</li><li>Witten, P. 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The authors declare that they have no competing or financial interests.

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 style="width:140px;">A4978</td>
			<td style="width:535px;"></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>
			<td>C0875</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>
			<td></td>
			<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>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;width:249px;">LSM Image Browser (4.2.0.121)</td>
			<td>Zeiss</td>
			<td></td>
			<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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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">NIS-Elements BR 3.0 software</td>
			<td>Nikon</td>
			<td></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>
		</tr>
		<tr height="20">
			<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

9.1K Görüntüleme.  National University of Singapore. 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 specim...