Name: chemical amputation and regeneration of the pharynx in the planarian schmidtea mediterranea
Uploaded: 2018-03-26T13:30:00
Description: Watch this Scientific Journal Video about Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea at JoVE.com

We would like to thank Alejandro S&#225;nchez Alvarado, who supported the initial optimization and development of this technique. Work in Carolyn Adler's laboratory is supported by Cornell University start-up funds.

1. Preparation
<ol>
	<li>Preparation of planaria water17
	<ol>
		<li>Maintain planarians in a 1X Montju&#239;c salt solution. To prepare planarian water, make individual stock solutions of 1 M CaCl2, 1 M MgSO4, 1 M MgCl2, 1 M KCl and 5 M NaCl in ultrapure water. Filter-sterilize with a 0.2 &#181;m bottle-top filterfor long-term storage. 
		Note: Use only ultrapure deionized water (with a resistivity of 18.2 M&#8486; at 25 &#...

<ol>
	<li>Tanaka, E. M., Reddien, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+basis+for+animal+regeneration.">The cellular basis for animal regeneration.</a> Dev. Cell. 21, 172-185 (2011).</li><li>Tanaka, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Molecular+and+Cellular+Choreography+of+Appendage+Regeneration.">The Molecular and Cellular Choreography of Appendage Regeneration.</a> Cell. 165, 1598-1608 (2016).</li><li>Curado, S., Stainier, D. Y. R., Anderson, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitroreductase-mediated+cell/tissue+ablation+in+zebrafish:+a+spatially+and+temporally+controlled+ablation+method+with+applications+in+developmental+and+regeneration+studies.">Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies.</a> Nat. Protoc. 3, 948-954 (2008).</li><li>White, D. T., Mumm, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nitroreductase+system+of+inducible+targeted+ablation+facilitates+cell-specific+regenerative+studies+in+zebrafish.">The nitroreductase system of inducible targeted ablation facilitates cell-specific regenerative studies in zebrafish.</a> Methods. 62, 232-240 (2013).</li><li>Smith-Bolton, R. K., Worley, M. I., Kanda, H., Hariharan, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+growth+in+Drosophila+imaginal+discs+is+regulated+by+Wingless+and+Myc.">Regenerative growth in Drosophila imaginal discs is regulated by Wingless and Myc.</a> Dev. Cell. 16, 797-809 (2009).</li><li>Smith-Bolton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Imaginal+Discs+as+a+Model+of+Epithelial+Wound+Repair+and+Regeneration.">Drosophila Imaginal Discs as a Model of Epithelial Wound Repair and Regeneration.</a> Adv. Wound Care. 5, 251-261 (2016).</li><li>Newmark, P. A., S&#225;nchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+your+father's+planarian:+a+classic+model+enters+the+era+of+functional+genomics.">Not your father's planarian: a classic model enters the era of functional genomics.</a> Nat. Rev. Genet. 3, 210-219 (2002).</li><li>Reddien, P. W., S&#225;nchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+planarian+regeneration.">Fundamentals of planarian regeneration.</a> Annu. Rev. Cell Dev. Biol. 20, 725-757 (2004).</li><li>Wagner, D. E., Wang, I. E., Reddien, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+neoblasts+are+pluripotent+adult+stem+cells+that+underlie+planarian+regeneration.">Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regeneration.</a> Science. 332, 811-816 (2011).</li><li>Scimone, M. L., Kravarik, K. M., Lapan, S. W., Reddien, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoblast+specialization+in+regeneration+of+the+planarian+Schmidtea+mediterranea.">Neoblast specialization in regeneration of the planarian Schmidtea mediterranea.</a> Stem Cell Reports. 3, 339-352 (2014).</li><li>van Wolfswinkel, J. C., Wagner, D. E., Reddien, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+reveals+functionally+distinct+classes+within+the+planarian+stem+cell+compartment.">Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment.</a> Cell Stem Cell. 15, 326-339 (2014).</li><li>Adler, C. E., Seidel, C. W., McKinney, S. A., S&#225;nchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+amputation+of+the+pharynx+identifies+a+FoxA-dependent+regeneration+program+in+planaria.">Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria.</a> Elife. 3, e02238 (2014).</li><li>Adler, C. E., S&#225;nchez Alvarado, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PHRED-1+is+a+divergent+neurexin-1+homolog+that+organizes+muscle+fibers+and+patterns+organs+during+regeneration.">PHRED-1 is a divergent neurexin-1 homolog that organizes muscle fibers and patterns organs during regeneration.</a> Dev. Biol. 427, 165-175 (2017).</li><li>Wong, M. C., Martynovsky, M., Schwarzbauer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell+migration+using+Caenorhabditis+elegans+as+a+model+system.">Analysis of cell migration using Caenorhabditis elegans as a model system.</a> Methods Mol. Biol. 769, 233-247 (2011).</li><li>Margie, O., Palmer, C., Chin-Sang, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C+elegans+chemotaxis+assay.">C elegans chemotaxis assay.</a> J. Vis. Exp. , e50069 (2013).</li><li>Fang-Yen, C., Gabel, C. V., Samuel, A. D. T., Bargmann, C. I., Avery, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microsurgery+in+Caenorhabditis+elegans.">Laser microsurgery in Caenorhabditis elegans.</a> Methods Cell Biol. 107, 177 (2012).</li><li>Tasaki, J., Uchiyama-Tasaki, C., Rouhana, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Stem+Cell+Motility+In+Vivo+Based+on+Immunodetection+of+Planarian+Neoblasts+and+Tracing+of+BrdU-Labeled+Cells+After+Partial+Irradiation.">Analysis of Stem Cell Motility In Vivo Based on Immunodetection of Planarian Neoblasts and Tracing of BrdU-Labeled Cells After Partial Irradiation.</a> Methods Mol. Biol. 1365, 323-338 (2016).</li><li>Roberts-Galbraith, R. H., Brubacher, J. L., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+genomics+screen+in+planarians+reveals+regulators+of+whole-brain+regeneration.">A functional genomics screen in planarians reveals regulators of whole-brain regeneration.</a> Elife. 5, (2016).</li><li>Arnold, C. P., 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=Pathogenic+shifts+in+endogenous+microbiota+impede+tissue+regeneration+via+distinct+activation+of+TAK1/MKK/p38.">Pathogenic shifts in endogenous microbiota impede tissue regeneration via distinct activation of TAK1/MKK/p38.</a> Elife. 5, (2016).</li><li>Pearson, B. J., 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=Formaldehyde-based+whole-mount+in+situ+hybridization+method+for+planarians.">Formaldehyde-based whole-mount in situ hybridization method for planarians.</a> Dev. Dyn. 238, 443-450 (2009).</li><li>LoCascio, S. A., Lapan, S. W., Reddien, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+Absence+Does+Not+Regulate+Planarian+Stem+Cells+during+Eye+Regeneration.">Eye Absence Does Not Regulate Planarian Stem Cells during Eye Regeneration.</a> Dev. Cell. 40, 381-391 (2017).</li><li>Nishimura, K., 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=Regeneration+of+dopaminergic+neurons+after+6-hydroxydopamine-induced+lesion+in+planarian+brain.">Regeneration of dopaminergic neurons after 6-hydroxydopamine-induced lesion in planarian brain.</a> J. Neurochem. 119, 1217-1231 (2011).</li><li>Wilson, D. F., Chance, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Azide+inhibition+of+mitochondrial+electron+transport.+I.+The+aerobic+steady+state+of+succinate+oxidation.">Azide inhibition of mitochondrial electron transport. I. The aerobic steady state of succinate oxidation.</a> Biochim. Biophys. Acta. 131, 421-430 (1967).</li><li>Duncan, H. 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This protocol describes a method of selective ablation of the pharynx using sodium azide. Other targeted ablation studies in planarians have used modified surgery to remove photoreceptors21 or pharmacological treatment to ablate dopaminergic neurons22. One significant advantage of chemical amputation over existing methods is that it does not require surgery. The rigid structure of the pharynx compared to the rest of the planarian body facilitates its complete removal from t...

Regeneration is a phenomenon that occurs throughout the animal kingdom, with regenerative capacities ranging from full body regeneration in certain invertebrates to more restricted abilities in vertebrates1. Replacement of functional tissues is a complex process and often entails the simultaneous restoration of multiple cell types. For example, to regenerate the salamander limb, osteoblasts, chondrocytes, neurons, muscles, and epithelial cells need to be replaced2. These newly generated cell types also need to be organized properly to facilitate new limb function. Understanding these complex processes requires techniques that focus on regeneration of specific cell types and their integration into organs.
One of the strategies employed to simplify the study of regenerative responses is the targeted ablation of either certain cell types or larger collections of tissues. For example, in zebrafish, expression of nitroreductase in specific cell types leads to their destruction after application of metronidazole3,4. In Drosophila larvae, expressing pro-apoptotic genes under tissue-specific promoters can selectively ablate specific regions of the imaginal disc5,6. Both of these strategies cause rapid but controlled damage, and have been used to dissect the molecular and cellular mechanisms responsible for regeneration.
In this manuscript, we describe a method to selectively ablate an entire organ called the pharynx in the planarian Schmidtea mediterranea. Planarians are a classical model of regeneration, known for their prolific regenerative ability, where even minute fragments can regrow whole animals 7,8. They have a large, heterogeneous population of stem cells consisting of both pluripotent cells and lineage-restricted progenitors9,10,11. These cells proliferate and differentiate to replace all missing tissues, including the pharynx, nervous, digestive and excretory systems, and muscle and epithelial cells9,10,12. While we know that these stem cells initiate regeneration, we do not fully comprehend the molecular mechanisms that drive them to replace all these different cell types. Defined wounding methods that elicit precise stem cell responses can help delineate this complex process.
The pharynx is a large, cylindrical tube required for feeding, and contains neurons, muscle, epithelial and secretory cells13,29. Normally hidden in a pouch on the ventral side of the animal, it extends through the animal&#39;s single body opening upon sensing the presence of food. To selectively amputate the pharynx, we soak planarians in a chemical called sodium azide, a commonly used anesthetic in C. elegans14,15,16. Its use in planarians was first reported by Adler et al., in 201412. Within minutes of exposure to sodium azide, planarians extrude their pharynges, and with gentle agitation, the pharynx detaches from the animal. We refer to this complete and selective loss of the pharynx as &#34;chemical amputation&#34;. One week after amputation, a fully functional pharynx is restored12. Because the pharynx is required for feeding, functional regeneration can be measured by monitoring feeding behavior. Below, we describe the protocol for chemical amputation, and for assessing the regeneration of the pharynx and restoration of feeding behavior.

<table border="0" cellpadding="0" cellspacing="0" width="1042">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Calcium Chloride Dihydrate (CaCl2)</td>
			<td style="width:204px;">Fisher Chemical</td>
			<td style="width:155px;">C79-3</td>
			<td style="width:236px;">Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate Anhydrous (MgSO4)</td>
			<td>Fisher Chemical</td>
			<td>M65-3</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride Hexahydrate (MgCl2)</td>
			<td>Acros/VWR</td>
			<td>41341-5000</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride (KCl)</td>
			<td>Acros Organics/VWR</td>
			<td>196770010</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride (NaCl)</td>
			<td>Acros Organics/VWR</td>
			<td>207790050</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bicarbonate (NaHCO3)</td>
			<td>Acros Organics/VWR</td>
			<td>123360010</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalgene autoclavable polypropylene copolymer lowboy with spigot</td>
			<td>ThermoFisher Scientific/VWR</td>
			<td>2324-0015</td>
			<td>Storing planaria water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instant Ocean Sea Salt</td>
			<td>Spectrum Brands</td>
			<td>Amazon</td>
			<td>Planaria water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin Sulfate</td>
			<td>Gemini Bio-products</td>
			<td>400-100P</td>
			<td>Planaria water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>71970</td>
			<td>Mincing liver&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable pastry bags-16&#8221;, 12 pack</td>
			<td>Wilton</td>
			<td>Amazon</td>
			<td>Liver aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mL syringes</td>
			<td>BD/VWR</td>
			<td>309647</td>
			<td>Liver aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes-35mm/60mm</td>
			<td>Greiner Bio-One/VWR</td>
			<td>82050-536/82050-544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic containers (various sizes)</td>
			<td>Ziploc</td>
			<td>Amazon</td>
			<td>Housing planarians in static culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipette</td>
			<td>Globe Scientific</td>
			<td>138030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps - Dumont Tweezer, Style 5</td>
			<td>Electron Microscopy Sciences&#160;</td>
			<td>72700-D (0203-5-PO)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI&#160;</td>
			<td>ThermoFisher</td>
			<td>62247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin, Alexa Fluor 488 conjugate</td>
			<td>ThermoFisher</td>
			<td>S11223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

chemical amputation and regeneration of the pharynx in the planarian schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to any type of injury. Common injury models in these animals remove large amounts of tissue, which damages multiple organs. To overcome this broad tissue damage, we describe here a method to selectively remove a single organ, the pharynx, in the planarian Schmidtea mediterranea. We achieve this by soaking animals in a solution containing the cytochrome oxidase inhibitor sodium azide. Brief exposure to sodium azide causes extrusion of the pharynx from the animal, which we call &#34;chemical amputation.&#34;&#160;Chemical amputation removes the entire pharynx, and generates a small wound where the pharynx attaches to the intestine. After extensive rinsing, all amputated animals regenerate a fully functional pharynx in approximately one week. Stem cells in the rest of the body drive regeneration of the new pharynx. Here, we provide a detailed protocol for chemical amputation, and describe both histological and behavioral methods to assess successful amputation and regeneration.

Exposure to sodium azide disrupts the normal motility of planarians, causing animals to stretch and writhe. These movements force the pharynx to emerge from the ventral side of the animal, and after approximately 6 min in sodium azide solution, the white tip of the pharynx can be seen (Figure 1B-left panel). A few minutes later, animals actively contract and fully extend the pharynx by forcefully pushing it out of the body. (<strong c...

Watch this Scientific Journal Video about Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea at JoVE.com

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to ...

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Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. ...

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

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 ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Methods for the Study of Regeneration in Stentor

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...