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 &#176;C) to prepare Montju&#239;c salts.</li>
		<li>To prepare a 1 L stock of 5X salt solution, combine 5 mL of 1 M CaCl2, 5 mL of 1 M MgSO4, 0.5 mL of MgCl2, 0.5 mL of KCl and 1.6 mL of 5 M NaCl solutions in 900 mL ultrapure water. To this solution, add 0.504 g of NaHCO3 and stir to mix. Adjust the pH to 7.0 with hydrochloric acid.</li>
		<li>Dilute this 5X stock solution to a 1X working concentration in ultrapure water in a sterile container such as a large capacity carboy (see the Table of Materials). Use this 1X solution for maintaining asexual planarians. 
		Note: As an alternative to Montju&#239;c salt solution, use locally-purchased spring water or ultrapure water containing a commercially available aquarium salt mix (see Table of Materials) at a concentration of0.5 g/L18.</li>
		<li>To prevent bacterial infection in static culture19, maintain planarians in water containing an antibiotic. Prepare a 50 mg/mL stock solution of gentamicin sulfate in ultrapure water and filter-sterilize. To containers where animals are maintained, add gentamicin sulfate to a final concentration of 50 &#181;g/mL (1:1000 dilution).</li>
	</ol>
	</li>
	<li>Preparation of liver paste
	<ol>
		<li>As planarians thrive on a diet of organic, grass-fed beef liver, purchase fresh liver and process within 24 h. Remove the membranous capsule encapsulating the liver by peeling it off gently. Cut the liver into ~1-cm cubes, and use a blade to scrape off and discard all hepatic veins and arteries.</li>
		<li>Macerate liver pieces using a food mill or a food processor and then pass it through a wire mesh food strainer. Combine into pastry bags, and dispense into syringes or 35-mm Petri dishes. Prior to feeding, centrifuge gently to remove air bubbles.</li>
		<li>Store aliquots of liver at -80 &#176;C for up to a year and thaw prior to using. After thawing, re-freeze any leftover liver once, or store at 4 &#176;C for up to 24 h.</li>
	</ol>
	</li>
	<li>Maintenance of animals
	<ol>
		<li>Planarians will grow and shrink to various sizes depending on the frequency of feeding. Feed planarians every other week (once every 14 days). For long-term bulk cultures, use plastic containers of various sizes (see Table of Materials).</li>
		<li>To feed animals, use a metal spatula or plastic transfer pipet to place a pea-sized drop of liver in a box. Allow animals to eat for 1-2 h. Remove remaining food before cleaning the box.</li>
		<li>Clean worms twice a week if fed (once directly after feeding and once two days later), or once a week if not fed. To clean, drain water into a plastic beaker by carefully pouring out the water while retaining planarians in the box. Wipe box surface with a paper towel, then repeat on all sides until the box is clean.</li>
		<li>Replace water (to approximately &#190; of the box volume) and add gentamicin to a final concentration of 50 &#181;g/mL. Store animals in a dark cabinet or in a 20 &#176;C incubator.</li>
	</ol>
	</li>
	<li>Selection of worms
	<ol>
		<li>Select worms that were fed 5-7 days prior. 
		Note: It is significantly more difficult to amputate pharynges from worms that have been recently fed (1-2 days prior to amputation). Fed animals are also more sensitive to sodium azide.</li>
		<li>Select worms that are roughly 6 mm in length. To estimate the size of the worm, transfer it to a Petri dish using a plastic transfer pipette. Place either a flat 6-in ruler or 5 mm x 5 mm graph paper underneath the Petri dish and measure the worm length while it crawls. 
		Note: Worms smaller than 6 mm in length are more difficult to amputate.</li>
	</ol>
	</li>
	<li>Preparation of sodium azide
	<ol>
		<li>Prepare a 100 mM solution by dissolving sodium azide powder in planaria water. 
		CAUTION: Sodium azide is toxic and should be handled carefully. Do not discard sodium azide down the drain. Following use, collect it separately for disposal as hazardous waste.</li>
	</ol>
	</li>
</ol>
2. Pharynx Amputation
<ol>
	<li>Place worms in a 35-mm diameter Petri dish. Carefully remove all planarian water from the dish by using a plastic transfer pipet. 
	Note: A maximum of 20 6-mm worms can be comfortably accommodated in a dish of this size.</li>
	<li>Place the dish under the microscope and adjust magnification so that multiple worms can be viewed simultaneously (e.g.&#160;10X magnification). Replace planarian water with 100 mM sodium azide solution. 
	Note: For a 35-mm Petri dish, approximately 5 mL of sodium azide is sufficient. The solution should completely submerge planarians. Adjust solution volume for larger dishes as necessary.</li>
	<li>Monitor the animals under the microscope&#160;and refrain from moving the dish for the first 3-4 min.</li>
	<li>Observe the extrusion of the pharynx through the microscope; the pharynx will protrude from the pharyngeal pouch and extend fully (approximately 1 mm in length) as shown in Figure 1B. 
	Note: It is important to wait until the pharynx has finished extending completely before proceeding further. Once the pharynx emerges from the animal, full extension takes roughly 1 min.</li>
	<li>Use a plastic transfer pipet to squirt animals around the dish. Suck the animals into the pipet and forcibly release them into the dish a couple of times. If the pharynx is fully extended, this vigorous pipetting will cause it to detach.</li>
	<li>Alternatively, grasp the pharynx either vertically along its length, or horizontally along its circumference using a pair of fine forceps (see Table of Materials) as shown in Figure 1C. With the pharynx pinched in the forceps, lift the animal upwards towards the meniscus. As the animal is raised, surface tension will cause the pharynx to detach from the animal. 
	Note: This method can be used to remove the pharynges of smaller animals (1-3 mm in length) where forceful swirling is not adequate. Avoid poking or otherwise injuring the body of the animal.</li>
	<li>Using a transfer pipet, move amputated animals to a new dish containing fresh planaria water.</li>
	<li>Once transferred, rinse amputated animals thoroughly in planaria water, completely exchanging the water three times. Transfer to a new dish containing planaria water with 50&#181;g/mL of gentamicin. 
	Note: Complete buffer exchange during these three washes is essential to ensure removal of sodium azide.</li>
	<li>The next day, replace water in the dish with fresh planaria water containing 50 &#181;g/mL of gentamicin. Repeat every other day thereafter.</li>
</ol>
3. Assessment of Pharynx Removal After Amputation
<ol>
	<li>Examine animals under a microscope (with 10-20X magnification) to determine whether a dark spot has appeared after successful removal of the pharynx, as shown in Figure 2A.</li>
	<li>Alternatively, fix and bleach animals according to the protocol described by Pearson et al.20. Soak animals overnight in 4&#39;,6-diamidino-2-phenylindole (DAPI) or fluorescently-conjugated streptavidin (1 mg/mL diluted 1:500 in 1X Phosphate Buffered Saline containing 0.3% Triton-X). Mount animals on slides in 80% glycerol/20% 1X Phosphate Buffered Saline and image under a fluorescent stereomicroscope (Figure 2B). 
	Note: All images in this manuscript were captured on a fluorescent stereomicroscope with a 1.0X objective.</li>
</ol>
4. Assessment of Pharynx Regeneration by Measuring Feeding Behavior
<ol>
	<li>Preparation of liver
	<ol>
		<li>Transfer required quantity of liver paste into a microcentrifuge or conical tube. Spin briefly to remove air bubbles. Estimate the volume of liver, and then add planaria water to 1/5 of its volume, and 2% of the total volume in red food coloring. For example, to 1000 &#181;L of liver paste, add 200 &#181;L of planaria water and 24 &#181;L of food coloring.</li>
		<li>Using a plastic pestle, pipette tip or metal spatula, mix thoroughly until food coloring is evenly distributed in the liver paste. Spin again briefly. Liver paste can be stored at 4 &#176;C for 24 h or frozen in aliquots at -80 &#176;C for long-term storage.</li>
		<li>If testing up to 25 animals, prepare 25 &#181;L of liver paste per dish (approximately 1 &#181;L of liver paste per animal).</li>
	</ol>
	</li>
	<li>Feeding animals
	<ol>
		<li>Seven days after chemical amputation, transfer animals to a new Petri dish. If testing 10-15 animals, use a 35 mm dish; more than 15 animals should be tested in a 60-mm Petri dish. Keep animals in the dark, undisturbed, for approximately 1 h prior to feeding.</li>
		<li>Using scissors, increase the width of a P200 pipette tip by trimming roughly &#189; cm off of the narrow end. Pipet 25 &#181;L of red liver paste into the dish. 
		Note: Trimming the end makes pipetting the viscous liver paste easier. To prevent the liver from floating on the surface, touch the tip to the bottom of the dish while dispensing.</li>
		<li>Allow animals to feed for 30 min.</li>
		<li>Score the number of animals that have eaten by placing the animals on a white background or examining them under a microscope with 10-20X magnification.</li>
		<li>After feeding, remove liver from the dish and clean.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 the animal, which is much softer-bodied. Also, the pharynx detaches from the animal at a small junction (the esophagus)30 joining the pharynx and the intestine, generating a more reproducible wound than one induced by a larger surgical tool. Based on its physical properties and small anatomical linkage to the rest of the animal, pharynx removal therefore generates more homogeneous wounds than other surgical amputations. 
Length of exposure to sodium azide is also critical for the success of this technique. Although brief exposure to sodium azide does not adversely affect planarians, prolonged exposure is toxic and will eventually kill animals. Sodium azide is known to inhibit energy-dependent processes through inhibition of cytochrome oxidase23,24. In addition, it suppresses transcription and translation, likely by accumulation of key components of these reactions in stress granules25. In planarians, sodium azide exposure suppresses mitosis for 24 h, after which cells resume proliferation12. Modifications such as limiting the time of azide exposure, removing azide with thorough rinsing, and accounting for the transient mitotic suppression in experimental design can help overcome these limitations. 
The unique anatomical position of the pharynx and its characteristic radial symmetry allow for the easy distinction of newly regenerated pharyngeal tissue from pre-existing stem cells. Pharynx regeneration can be monitored at the cellular and anatomical levels using tissue stains like streptavidin and DAPI, immunohistochemistry, or by in situ hybridization26. The feeding assay described here assays functional regeneration of the pharynx. Chemotaxis towards food requires the pharynx27,28 and animals without a fully functional pharynx cannot ingest food. This simple, quantitative assessment of organ function therefore complements structural regeneration. Additionally, chemical amputation can be performed on populations of animals, and can generate numerous animals with identical injuries. The procedure is therefore convenient for large-scale studies. These advantages, coupled with the fact that regeneration of a mature pharynx requires stem cells, makes pharynx amputation an ideal model to study organ regeneration. Used in combination with broader amputations, pharynx removal can be used to test hypotheses about how wound size and anatomical position impact the stem cell response to injury, which is a key question in the regeneration field21.

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><tbody><tr><td>Calcium Chloride Dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Chemical</td><td>C79-3</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Magnesium Sulfate Anhydrous (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Chemical</td><td>M65-3</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Magnesium Chloride Hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Acros/VWR</td><td>41341-5000</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Potassium Chloride (KCl)</td><td>Acros Organics/VWR</td><td>196770010</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Acros Organics/VWR</td><td>207790050</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Sodium Bicarbonate (NaHCO3)</td><td>Acros Organics/VWR</td><td>123360010</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Nalgene autoclavable polypropylene copolymer lowboy with spigot</td><td>ThermoFisher Scientific/VWR</td><td>2324-0015</td><td>Storing planaria water</td></tr><tr><td>Instant Ocean Sea Salt</td><td>Spectrum Brands</td><td>Amazon</td><td>Planaria water</td></tr><tr><td>Gentamicin Sulfate</td><td>Gemini Bio-products</td><td>400-100P</td><td>Planaria water</td></tr><tr><td>Razor blades</td><td>Electron Microscopy Sciences</td><td>71970</td><td>Mincing liver&amp;nbsp;</td></tr><tr><td>Disposable pastry bags-16&amp;rdquo;, 12 pack</td><td>Wilton</td><td>Amazon</td><td>Liver aliquots</td></tr><tr><td>5 mL syringes</td><td>BD/VWR</td><td>309647</td><td>Liver aliquots</td></tr><tr><td>Petri dishes-35mm/60mm</td><td>Greiner Bio-One/VWR</td><td>82050-536/82050-544</td><td></td></tr><tr><td>Plastic containers (various sizes)</td><td>Ziploc</td><td>Amazon</td><td>Housing planarians in static culture</td></tr><tr><td>Sodium Azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td>Transfer pipette</td><td>Globe Scientific</td><td>138030</td><td></td></tr><tr><td>Forceps - Dumont Tweezer, Style 5</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>72700-D (0203-5-PO)</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>DAPI&amp;nbsp;</td><td>ThermoFisher</td><td>62247</td><td></td></tr><tr><td>Streptavidin, Alexa Fluor 488 conjugate</td><td>ThermoFisher</td><td>S11223</td><td></td></tr><tr><td>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. (Figure 1B-middle panel). Approximately 11 min after azide exposure, the animals and the pharynx relax. At this stage, the characteristic bell shape of the pharynx is clearly visible (Figure 1B-right panel). For easy amputation, it is important to wait for the pharynx to relax.
Because the pharynx is a large, unpigmented mass of tissue, removing it results in the appearance of a dark spot on the dorsal side of amputated animals (Figure 2A). This darkened region is a visual indicator of successful amputation in live animals. The spot is visible immediately after amputation and becomes more prominent the next day. As the pharynx regenerates, this region lightens. To monitor pharynx regeneration more precisely, animals can be stained with DAPI or fluorescently-conjugated streptavidin overnight (Figure 2B). To quantitatively assess the extent of pharynx regeneration, its area can be measured using ImageJ software.
The pharynx is an essential organ for chemosensation and food ingestion. To determine when these functions are restored during regeneration, we used feeding assays to monitor animal behavior. By combining food coloring with liver paste, we were able to distinguish animals that ate from those that did not (Figure 3A). We then quantified the number of red animals to assess the percentage of animals with a functional pharynx. Based on the results shown in Figure 3B, successful feeding behavior was restored 7 days after amputation, indicating that one week after pharynx removal, animals have regenerated a functional pharynx. To test whether sodium azide exposure affected feeding behavior, we soaked animals in sodium azide but washed it out immediately prior to pharynx ejection. One day later, we evaluated whether food-seeking behavior was altered by sodium azide exposure with the feeding assay. In contrast to chemically-amputated animals lacking a pharynx, all animals retaining an intact pharynx after sodium azide exposure ate (n=30 animals for each condition) the food. This result indicates that sodium azide exposure does not affect feeding behavior, as long as the pharynx remains intact.
<img alt="Planarian regeneration: diagram, pharynx emergence and amputation process with grip techniques." class="xfigimg" src="/files/ftp_upload/57168/57168fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Pharynx ejection and amputation.(A) Schematic of planarian anatomy. (B) Images of live animals showing pharynx ejection upon soaking in 100 mM sodium azide. Red arrows highlight pharynx. Scale bars = 750 µm. (C) Image of live animal (left) and schematic (right) of amputation using forceps. Panels show two different options for gripping the pharynx. Ventral side of the animal faces up. Scale bars = 500 µm. <a href="https://cloudflare.jove.com/files/ftp_upload/57168/57168fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Planarian regeneration process, stages intact, amputated, regenerated; microscopy images showing progress." class="xfigimg" src="/files/ftp_upload/57168/57168fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Assessment of pharynx amputation and regeneration. (A) Representative images of live animals at specified times before and after amputation. Middle panel shows dark spot in pharyngeal region after amputation, highlighted by dashed white box. Dorsal side faces up. Scale bars = 500 µm. (B) Representative images of animals stained with Alexa488-streptavidin at specified times before and after amputation. Middle panel shows absence of pharynx after amputation. Pharyngeal region highlighted by white box. Ventral side faces up. Scale bar = 500 µm. <a href="https://cloudflare.jove.com/files/ftp_upload/57168/57168fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Planarian feeding behavior post-pharynx amputation; schematic, experimental setup, food uptake graph." class="xfigimg" src="/files/ftp_upload/57168/57168fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Evaluation of pharynx regeneration. (A) Images and schematic of feeding assay, conducted by placing 25 µl of liver in the center of a 60 mm Petri dish. Schematic (top, left) and image (middle) show feeding behavior in animals with intact pharynges. Top right, image of animal that has eaten. Schematic (bottom left) and image (middle) of feeding behavior in animals 1 day after amputation. Bottom right, animal that has not eaten. Scale bar = 750 µm. (B) Results of feeding assay. Percentage of animals that have ingested food (quantified by red coloration) at specific times after amputation. For each time point, n=10 animals, repeated in triplicate. Error bar = SD. <a href="https://cloudflare.jove.com/files/ftp_upload/57168/57168fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Regeneration and Repair

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10.8K Views.  Cornell University. De planarian Schmidtea mediterranea is een uitstekend model voor het bestuderen van stamcellen en weefselregeneratie. Deze publicatie beschrijft een methode om selectief verwijderen van één orgaan, de keelholte, door dieren naar de chemische natriumazide bloot te leggen. Dit protocol wordt ook uitgelegd welke methoden voor de controle van de keelholte regeneratie.

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

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

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

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

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

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

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and withdrawal properties of natural products is described. Experimental assays benefitting from unique aspects of planarian physiology have been applied to studies on wound healing, regeneration, and tumorigenesis. In addition, because planarians exhibit sensitivity to a variety of environmental stimuli and are capable of learning and developing conditioned responses, they can be used in behavioral studies examining learning and memory. Planarians possess a basic bilateral symmetry and a central nervous system that uses neurotransmitter systems amenable to studies examining the effects of neuromuscular biomodulators. Consequently, experimental systems monitoring planarian movement and motility have been developed to examine substance addiction and withdrawal. Because planarian motility offers the potential for a sensitive, easily standardized motility assay system to monitor the effect of stimuli, the planarian locomotor velocity (pLmV) test was adapted to monitor both stimulation and withdrawal behaviors by planarians through the determination of the number of grid lines crossed by the animals with time. Here, the technique and its application are demonstrated and explained.

Planarian motility is used to gauge the stimulant and withdrawal properties of natural products when compared to the movement of the animals in spring water alone.

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

Biochemistry

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 regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

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

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Biology

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...

Planarian Ovary Dissection for Ultrastructural Analysis and Antibody Staining

Accessibility to germ cells allows the study of germ cell development, meiosis, and recombination. The sexual biotype of the freshwater planarian, Schmidtea mediterranea, is a powerful invertebrate model to study the epigenetic specification of germ cells. Unlike the large number of testis and male germ cells, planarian oocytes are relatively difficult to locate and examine, as there are only two ovaries, each with 5-20 oocytes. Deeper localization within the planarian body and lack of protective epithelial tissues also make it challenging to dissect planarian ovaries directly. 
This protocol uses a brief fixation step to facilitate the localization and dissection of planarian ovaries for downstream analysis to overcome these difficulties. The dissected ovary is compatible for ultrastructural examination by transmission electron microscopy (TEM) and antibody immunostaining. The dissection technique outlined in this protocol also allows for gene perturbation experiments, in which the ovaries are examined under different RNA interference (RNAi) conditions. Direct access to the intact germ cells in the ovary achieved by this protocol will greatly improve the imaging depth and quality and allow cellular and subcellular interrogation of oocyte biology.

This protocol presents steps taken to dissect ovaries in the freshwater planarians, Schmidtea mediterranea. The dissected ovaries are compatible for antibody immunostaining and ultrastructural analysis with transmission electron microscopy to study the cell biology of the oocytes and somatic cells, providing an imaging depth and quality that were previously inaccessible.

Accessibility to germ cells allows the study of germ cell development, meiosis, and recombination. The sexual biotype of the freshwater planarian, ...

Whole Body Regeneration

Regeneration is the process of restoring injured or lost tissues, organs, or&#160;body parts. While simpler organisms generally show greater ability to regenerate their whole body, few complex animals show similarly exceptional regeneration. For example, planarian flatworms have a unique regenerative potential making them a popular study organism among biologists to understand the mechanisms of whole body regeneration. Other organisms, such as hydra, also show extreme regeneration potential; even when cut into several pieces, each piece can turn into an individual organism.
Planarian Stem Cells
Planarians are among the few animals that maintain pluripotent stem cells throughout life. First described in the 1800s, these pluripotent stem cells are now called neoblasts. Neoblasts are essential for the regeneration of the endoderm, mesoderm, and ectoderm, thus, facilitating whole body regeneration. These neoblasts are present throughout the planarian body, including the space surrounding the gut. As much as 30 percent of cells in planaria are neoblasts. Thus, a planarian worm can regenerate a whole body even when cut into small pieces.
Regeneration Polarity in Planarians
Regeneration requires the restoration of complex anatomical structures and specific integration of organs within the body through precise control of the organs&#8217; size, location, and identity. When a planarian flatworm is amputated transversely, two fragments are generated &#8212; a fragment containing the head that regenerates a tail; and a tail fragment that regenerates the head. The location along the anterior-posterior axis determines whether the tissue differentiates into the head or tail. The Wnt proteins are a major determinant of this axis, with high Wnt expression in the anterior cells and low Wnt expression in the posterior cells. At the wound site, posterior cells express the Wnt1 gene, which triggers tail regeneration. In contrast, anterior cells express the notum gene, which inhibits the Wnt signaling and facilitates regeneration of the head.

Regeneration is the process of restoring injured or lost tissues, organs, or&#160;body parts. While simpler organisms generally show greater ability to ...

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

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