Work in the lab is supported by NSF (MCB0919933) and NIH (GM088790).

1. 	Trunk fragment regeneration assay
<ol>
<li>Stop feeding a cohort of worms for at least 5 days before the regenerative assay to ensure animals are free of waste and ingested food (&tilde;30 intact worms, each worm &tilde;8-10 mm long post-starvation).</li>
<li>On the day of the assay, rinse a pre-frozen leveled ice dish with water and cover the flat iced surface with plastic wrap. Using forceps, place one filter paper on the plastic wrap.</li>
<li>Moisten the filter paper with a few drops of spring water. Transfer planarians onto filter (&lt;20 worms per filter) using a transfer pipette. Worms can be repositioned on filter, if necessary, by repipetting with more spring water. Remove excess fluid.</li>
<li>Using a scalpel, amputate the head by a single cut made approximately halfway between the anterior apex of the animal and the anterior end of the pharynx (Figure 1A).</li> 
<li>Using a scalpel, amputate the tail region by a single cut made approximately halfway between the tail end of the animal and posterior end of the pharynx (Figure 1A). Remove excess mucus on the scalpel using a paper towel moistened with 70% ethanol after cutting. Wipe excess ethanol from scalpel.</li>
<li>Transfer filter via forceps into a Petri dish (100 x 25mm depth) containing spring water. Wait &tilde;3 minutes (for wound closure, observable by a pinching and darkening of the wound).</li>
<li>Rinse trunk fragments from filter paper into Petri dish containing desired medium for regenerative assay and transfer to thermostatted incubator (24 &deg;C).</li>
<li>Score regenerative phenotype after &tilde;1 week, when regenerated structures are identifiable (Figure 1A).</li>
</ol>
2.	Pharmacological manipulation of regeneration: bipolarity induced by praziquantel
<ol>
<li>Prepare praziquantel (PZQ) stock by solubilizing drug in DMSO (62.4mg PZQ in 1mL for 200mM stock). Use on same day.</li>
<li>Make 50mL of drug-containing solution (90 &mu;M PZQ) in buffered Montjuch salts. Vortex for &tilde;1 minute to ensure dispersion.</li>
<li>Perform trunk fragment regenerative assay as detailed above [protocol #1], exposing a cohort of trunk fragments to PZQ at the final step [1.7].</li>
<li>After 24-48 hours, exchange PZQ-containing media for spring water, washing worms several (&gt;3) times.</li>
<li>Return worms to incubator. Score bipolarity (two-heads, Figure 1B) one week after cutting.</li> 
</ol>
3. 	Genetic manipulation of regeneration: in vivo RNAi feeding protocol
<ol>
<li>Prior to assay, prepare chicken liver homogenate. Discard fatty, yellow colored sections from foodstock, and pur&eacute;e remaining liver. Filter pur&eacute;e through mesh strainer and aliquot suspension for storage (1.5mL tubes, -20 &deg;C).</li>
<li>Prior to assay, prepare bovine red blood cells (RBCs) by aliquoting supply into 1mL samples, stored at -20&deg;C.</li>
<li>Select worms for RNAi. Three cohorts are used: gene of interest, positive control (gene with known RNAi phenotype; Figure 1C, D &amp; E) and negative control (gene with no RNAi phenotype, also useful for assessment of knockdown levels compared to experimental cohort). For each cohort, use &tilde;250 worms (&tilde;8-10 mm long after starving for at least 5 days). Store in spring water in plastic tub. </li>
<li>For each RNAi cohort, thaw stock of E. coli expressing dsRNA targeting gene of interest [8], along with a tube of chicken liver homogenate [3.1] and a tube of RBCs [3.2].</li>
<li>Pellet bacteria (13,000g, 1 minute). Remove supernatant and resuspend pellet in 2xYT media (&tilde;700&mu;l). Recentrifuge, discard supernatant, place pellet on ice.</li>
<li>Create a large bore P200 pipette tip by cutting off the end of the tip. Thoroughly resuspend the bacterial pellet in a mixture of chicken liver homogenate (150 &mu;L) and RBCs (50 &mu;L) to create RNAi feeding mix. Remove air bubbles by centrifugation.</li>
<li>For feeding, remove the majority of the water from the plastic tub containing worms (leave &tilde;1 inch depth). Swirl tub to concentrate worms at the center of this container, and pipette RNAi feeding mix in a circle corralling worms. Use a transfer pipette to carefully coax escapees back onto the RNAi feeding mix without perturbing other diners!</li>
<li>After feeding (&tilde;1 hour), identify planarians that fed well. These worms show a deep red coloration from ingested RBCs. Discard others.</li>
<li>Carefully replace feeding solution with fresh spring water, minimizing disturbance to prevent egestion of food. To do this, gently localize the worms to one side of the tube using a transfer pipette, pour out turbid water, and carefully replace with fresh water poured down the opposite wall of the tub. Resubmerge planarians quickly as prolonged exposure to air also results in food egestion.</li>
<li>Loosely seal container lid and return to incubator (24&deg;C)</li>
<li>Repeat RNAi feeding protocol over multiple cycles (&tilde;2-3 days apart), interspersed with regenerative cycles [protocol #1] to screen for RNAi phenotypes. A standard protocol for feeding and regeneration effective for many genes is shown in Figure 2, which takes &tilde;1 month in total duration.</li> 
</ol>
Modify this scheme for different genes, as optimal protocol will depend on factors such as mRNA stability, tissue localization, protein perdurance, or development of a phenotype that precludes multiple feeding cycles after regeneration. Assess knockdown levels by simply screening for penetrance of phenotype (if apparent), or by qPCR approaches to compare targeted mRNA levels with negative control cohort.
4. Representative results:
The trunk fragment regeneration assay is robust such that all worms should regenerate with normal anterior-posterior ('head to tail') polarity. This can be scored as soon as five days after cutting, facilitated by the emergence of the anterior eyespots ( asterixed, Figure 1A). However, complete morphological restoration of lost structures occurs after one week. Pharmacological manipulation of trunk fragment regeneration by PZQ to yield two-headed worms (Figure 1B) is also robust (EC50=87 (+-) 11% bipolar fragments, 70&mu;M PZQ for 48 hours20). Bipolarity occurs over a single regenerative cycle. Lower efficacy in these assays likely relates to issues with drug solubility and/or storage, or use of a different flatworm species where PZQ is ineffective. For drugs with lower penetrance, an anteriorization index is often used to score intermediate phenotypes aside from complete bipolarity22. Phenotypes resulting from RNAi of positive control constructs are shown in Figure 1: RNAi of Dugesia japonica six-1 (Dj-six-1) results in an eyeless phenotype23 (Figure 1C), RNAi of Dugesia japonica PC2 (Dj-PC2) results in a loss of the light aversion response24 (Figure 1D) and RNAi of Dugesia japonica &beta;catenin-1 (Dj-&beta;catenin-1) results in a two-headed phenotype16-19, 21 from trunk fragments (Figure 1E). These phenotypes can be achieved using the following simple RNAi protocols: Dj-six-1 (FFxFx), Dj-PC2 (FFFx), Dj-&beta;catenin-1 (FFxFx), where F=feeding cycle and x=regenerative cycle respectively.
<img src="/files/ftp_upload/3058/3058fig1.jpg" alt="Figure 1"/> 
Figure 1: Trunk fragment regeneration assay. A Left, Image of planarian (top) and schematic (middle) to show location of excised trunk fragment. Right, timecourse of trunk fragment regeneration showing appearance of regenerating fragment at indicated times (days). B Image of two-headed planarian produced by PZQ exposure. C 'Eyeless' worm produced by Dj-six-1 RNAi. D Loss of light aversion response in immobile Dj-PC2 RNAi worms (stained red), compared to mobile control worm (stained green). E Bipolar planarian produced by Dj-&beta;catenin-1 RNAi. In B-E, the original anterior end of the RNAi worms is oriented to the left.
<img src="/files/ftp_upload/3058/3058fig2.jpg" alt="Figure 2"/> 
Figure 2: Sequence of feeding and cutting cycles for typical RNAi protocol comprising multiple feedings (F), and regenerative cycles (x) that is effective for knockdown of many genes in D. japonica. Modification of this protocol for individual genes may be needed to yield optimal results, for example using a fewer (parentheses) or a greater number of feeding and regenerative cycles.

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No conflicts of interest declared.

The protocols described here detail assays for studying and manipulating regeneration of the planarian Dugesia japonica. They are simple and do not require specialized equipment such that they can be easily performed in the laboratory or classroom. Assays can be performed individually, or combined (for chemical genetic screening of drug efficacies in vivo) and can be performed at the candidate gene level, or are adaptable to unbiased, higher throughput screening8. Whether for studying the intriguing biology of planarians in their own right, or for assessing in vivo function of mammalian homologues in a alternative model for studying tissue regeneration, these approaches should catalyze interest from a diverse range of researchers.

<table><tbody><tr><td>Spring water</td><td>Kandiyohi. Premium Waters Inc. Minneapolis, MN</td><td>n/a</td><td>Other forms of spring water work well also. Trial first in viability assays.</td></tr><tr><td>1 x buffered Montju&amp;#239;ch salts: NaCl (1.6mM), CaCl2 (1mM), MgSO4 (1mM), MgCl2 (0.1mM), KCl (0.1mM), NaHCO3 (1.2mM), HEPES (1.5mM). pH 7.4 at 24&amp;#x00B0;C.</td><td>Multiple Suppliers</td><td>n/a</td><td>Artificial water for drug treatments during regenerative assays to ensure pH buffering. 5/8 Holtfreter&amp;rsquo;s solution is an alternative.</td></tr><tr><td>2xYT Broth</td><td>Fisher Scientific</td><td>BP2467-500</td><td>Media = 31 g/L . Autoclaved. </td></tr><tr><td>Petri Dish (100x25mm)</td><td>Fisher Scientific</td><td>08-757-11</td><td>Housing worms during regeneration cycles</td></tr><tr><td>Square Dish (100x100x15mm)</td><td>Fisher Scientific</td><td>08-757-11A</td><td>Fill with water, freeze for ice tray used as worm cutting surface</td></tr><tr><td>Plastic tub: Ziploc Twist &amp;rsquo;n Loc (16oz).</td><td>Various</td><td>n/a</td><td>Convenient water tight containers for RNAi cohorts</td></tr><tr><td>Chicken Liver</td><td>Commercial grocery</td><td>n/a</td><td>Bias towards organic supplies, owing to avoidance of antibiotics.</td></tr><tr><td>Hand Blender</td><td>Any Supplier</td><td>n/a</td><td>Use for making chicken liver puree</td></tr><tr><td>Wire 1mm Mesh strainer</td><td>Any Supplier</td><td>n/a</td><td>Use for straining chicken liver puree</td></tr><tr><td>Bovine red blood cells</td><td>Lampire Biological Laboratories</td><td>7240807</td><td>100% Washed and pooled RBC suspension</td></tr><tr><td>Circular filter papers</td><td>Whatman, GE Healthcare</td><td>1003 055</td><td></td></tr><tr><td>Transfer Pipette</td><td>Fisher Scientific</td><td>13-711-41</td><td></td></tr><tr><td>Sterile, surgical blades</td><td>Multiple Suppliers</td><td>n/a</td><td></td></tr><tr><td>&amp;#177;Praziquantel</td><td>Sigma-Aldrich</td><td>P4668</td><td>Store powder aliquots in the dark at 4&amp;#x00B0;C. Desiccate. </td></tr><tr><td>&lt;em&gt;Dj-six-1&lt;/em&gt;</td><td></td><td>GenBank AJ557022.1</td><td>RNAi positive control for &amp;#8220;eye-less&amp;#8221; phenotype&lt;sup&gt;23&lt;/sup&gt;</td></tr><tr><td>&lt;em&gt;Dj-&amp;#946;catenin-1&lt;/em&gt;</td><td></td><td>GenBank AB181913.1</td><td>RNAi positive control for two-headed phenotype&lt;sup&gt;17&lt;/sup&gt;</td></tr><tr><td>&lt;em&gt;Dj-PC2&lt;/em&gt;</td><td></td><td></td><td>RNAi positive control for loss of light aversion phenotype&lt;sup&gt;24&lt;/sup&gt;</td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica at JoVE.com

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.

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 ...

pharmacological-functional-genetic-assays-to-manipulate-regeneration

Nanyang Technological University

Research

JoVE Journal

Biology

36.8K Views.  University of Minnesota Medical School. 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.

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

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

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 ...

Developmental Biology

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Neuroscience

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.

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.

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 ...

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

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 ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Medicine

Advanced In Vitro Model for Studying Neuronal Hypersensitivity in DRG Cultures

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.

Author Spotlight: In Vitro and In Vivo Models to Enhance Chronic Pain Treatment Efficacy

This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

Bioengineering

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

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 ...

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

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Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

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

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

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing