Name: surgical ablation assay for studying eye regeneration in planarians
Uploaded: 2017-04-14T15:00:00
Description: Watch this Scientific Journal Video about Surgical Ablation Assay for Studying Eye Regeneration in Planarians at JoVE.com

The authors would like to thank Michelle Deochand for perfecting this eye ablation technique, Taylor Birkholz for assistance with the functional assay, Michael Levin for the anti-arrestin antibody, and Junji Morokuma for information on the Peltier plates. This work was supported by an SFSA grant from Western Michigan University to WSB.

1. Animal Culture and Handling
NOTE: This protocol uses Schmidtea mediterranea, a diploid planarian species with a sequenced genome16,17 that is commonly used for regeneration research. However, the assay is equally successful with other species, such as Girardia tigrina and Girardia dorotocephala (which are commercially available).
<ol>
	<li>Maintain worms in &#34;worm water&#34; made from 0.5 g/L ...

<ol>
	<li>Gentile, L., Cebria, F., Bartscherer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+planarian+flatworm:+an+in+vivo+model+for+stem+cell+biology+and+nervous+system+regeneration.">The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration.</a> Dis Model Mech. 4 (1), 12-19 (2011).</li><li>Elliott, S. A., Sanchez 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=The+history+and+enduring+contributions+of+planarians+to+the+study+of+animal+regeneration.">The history and enduring contributions of planarians to the study of animal regeneration.</a> Wiley Interdiscip Rev Dev Biol. 2 (3), 301-326 (2013).</li><li>Emili Sal&#243;, R. B., Tsonis, 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=Chapter+3.">Chapter 3.</a> Animal Models in Eye Research. , 15-26 (2008).</li><li>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=dlx+and+sp6-9+Control+optic+cup+regeneration+in+a+prototypic+eye.">dlx and sp6-9 Control optic cup regeneration in a prototypic eye.</a> PLoS Genet. 7 (8), e1002226 (2011).</li><li>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=Transcriptome+analysis+of+the+planarian+eye+identifies+ovo+as+a+specific+regulator+of+eye+regeneration.">Transcriptome analysis of the planarian eye identifies ovo as a specific regulator of eye regeneration.</a> Cell Rep. 2 (2), 294-307 (2012).</li><li>Inoue, T., 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=Morphological+and+functional+recovery+of+the+planarian+photosensing+system+during+head+regeneration.">Morphological and functional recovery of the planarian photosensing system during head regeneration.</a> Zoolog Sci. 21 (3), 275-283 (2004).</li><li>Pineda, D., 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=The+genetic+network+of+prototypic+planarian+eye+regeneration+is+Pax6+independent.">The genetic network of prototypic planarian eye regeneration is Pax6 independent.</a> Development. 129 (6), 1423-1434 (2002).</li><li>Paskin, T. R., Jellies, J., Bacher, J., Beane, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planarian+Phototactic+Assay+Reveals+Differential+Behavioral+Responses+Based+on+Wavelength.">Planarian Phototactic Assay Reveals Differential Behavioral Responses Based on Wavelength.</a> PLoS One. 9 (12), e114708 (2014).</li><li>Raffa, R. B., Martley, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+increase+in+planarian+locomotor+activity+and+block+by+UV+light.">Amphetamine-induced increase in planarian locomotor activity and block by UV light.</a> Brain Res. 1031 (1), 138-140 (2005).</li><li>Sandmann, T., Vogg, M. C., Owlarn, S., Boutros, M., Bartscherer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+head-regeneration+transcriptome+of+the+planarian+Schmidtea+mediterranea.">The head-regeneration transcriptome of the planarian Schmidtea mediterranea.</a> Genome Biol. 12 (8), R76 (2011).</li><li>Vasquez-Doorman, C., Petersen, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NuRD+complex+component+p66+suppresses+photoreceptor+neuron+regeneration+in+planarians.">The NuRD complex component p66 suppresses photoreceptor neuron regeneration in planarians.</a> Regeneration (Oxf). 3 (3), 168-178 (2016).</li><li>Deochand, M. E., Birkholz, T. R., Beane, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+regulation+of+planarian+eye+regeneration.">Temporal regulation of planarian eye regeneration.</a> Regeneration. 3 (4), 209-221 (2016).</li><li>Sakai, F., Agata, K., Orii, H., Watanabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+regeneration+ability+of+spontaneous+supernumerary+eyes+in+planarians+-eye+regeneration+field+and+pathway+selection+by+optic+nerves.">Organization and regeneration ability of spontaneous supernumerary eyes in planarians -eye regeneration field and pathway selection by optic nerves.</a> Zoolog Sci. 17 (3), 375-381 (2000).</li><li>Asano, Y., Nakamura, S., Ishida, S., Azuma, K., Shinozawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin-like+proteins+in+planarian+eye+and+auricle:+detection+and+functional+analysis.">Rhodopsin-like proteins in planarian eye and auricle: detection and functional analysis.</a> J Exp Biol. 201 (Pt 9), 1263-1271 (1998).</li><li>Cross, S. D., 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=Control+of+Maintenance+and+Regeneration+of+Planarian+Eyes+by+ovo.">Control of Maintenance and Regeneration of Planarian Eyes by ovo.</a> Invest Ophthalmol Vis Sci. 56 (12), 7604-7610 (2015).</li><li>Robb, S. M., Gotting, K., Ross, E., Sanchez 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=SmedGD+2.0:+The+Schmidtea+mediterranea+genome+database.">SmedGD 2.0: The Schmidtea mediterranea genome database.</a> Genesis. 53 (8), 535-546 (2015).</li><li>Robb, S. M., Ross, E., Sanchez 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=SmedGD:+the+Schmidtea+mediterranea+genome+database.">SmedGD: the Schmidtea mediterranea genome database.</a> Nucleic Acids Res. 36, D599-D606 (2008).</li><li>Beane, W. S., Tseng, A. S., Morokuma, J., Lemire, J. M., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+planar+cell+polarity+extends+neural+growth+during+regeneration,+homeostasis,+and+development.">Inhibition of planar cell polarity extends neural growth during regeneration, homeostasis, and development.</a> Stem Cells Dev. 21 (12), 2085-2094 (2012).</li><li>Forsthoefel, D. J., Waters, F. A., 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=Generation+of+cell+type-specific+monoclonal+antibodies+for+the+planarian+and+optimization+of+sample+processing+for+immunolabeling.">Generation of cell type-specific monoclonal antibodies for the planarian and optimization of sample processing for immunolabeling.</a> BMC Dev Biol. 14, 45 (2014).</li><li>Ross, K. G., 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=Novel+monoclonal+antibodies+to+study+tissue+regeneration+in+planarians.">Novel monoclonal antibodies to study tissue regeneration in planarians.</a> BMC Dev Biol. 15, 2 (2015).</li><li>Cardona, A., Fernandez, J., Solana, J., Romero, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+situ+hybridization+protocol+for+planarian+embryos:+monitoring+myosin+heavy+chain+gene+expression.">An in situ hybridization protocol for planarian embryos: monitoring myosin heavy chain gene expression.</a> Dev Genes Evol. 215 (9), 482-488 (2005).</li><li>King, R. S., 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=In+situ+hybridization+protocol+for+enhanced+detection+of+gene+expression+in+the+planarian+Schmidtea+mediterranea.">In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea.</a> BMC Dev Biol. 13, 8 (2013).</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 (2), 443-450 (2009).</li></ol>

This eye ablation technique improves on the current methods (such as hole punch) by excluding brain tissues and restricting excision mainly to the eye tissues. With practice, this technique can be performed by most individuals, from technicians experienced in microsurgeries to inexperienced but conscientious undergraduate students. It is recommended that this technique be practiced many times prior to using ablations in experiments, including (when possible) confirmation of complete removal of all eye tissues by immunohi...

Planarians are a powerful model organism for studying adult stem cell-mediated regeneration. These non-parasitic freshwater flatworms possess the ability to regenerate any and all missing tissues, including their central nervous system and brain1. Studied as far back as the 1700s2, technological advances in the planarian field during the past 10-15 years (such as a sequenced genome, in situ hybridization, immunohistochemistry, RNA interference (RNAi), and transcriptomics) have updated this historical model organism. Specifically, planarians have recently gained popularity as an emerging model for eye research3.
Planarians have prototypic eyes with only two tissue types, the photoreceptor neurons and pigment cells; this has enabled the characterization of its eye stem-cell population and demonstrated that many of the same genes regulating vertebrate eye development are conserved in planarians4,5. The optic cups are located dorsally and comprised of the white, unpigmented dendrites of the photoreceptor neurons and the semi-lunar black pigment cells, and the eyes innervate the brain via an optic chiasm. In addition to being a model for elucidating regenerative processes6, the planarian eye is well suited to studying the evolution of visual mechanisms7, behavioral responses to light (planarians display negative phototaxis)8, and the neurological basis of behavior9.
Eye regeneration in planarians has been largely studied in two main contexts: as part of head regeneration following decapitation4,10, and following excision of just the eye tissues11,12. Most planarian studies on eye regeneration have used the decapitation method, as it is simple and straightforward. The most common planarian eye excision method to date has been via hole punch with a fine glass capillary tube13,14, although some studies have also performed amputations just behind the eyes (partial decapitation)15. However, all of these methods involve the loss of many tissues other than just the eye (such as brain, intestines, and nephridia), potentially complicating interpretation of results. The eye ablation protocol presented here restricts excision to the eye tissues (specifically excluding brain), resulting in data that are more specific to the eye. Additionally, unlike decapitated worms that take 7-14 days to begin feeding, eye ablated worms will feed within 24 h of ablation12, allowing RNAi experiments (where RNAi is delivered via food) to be performed concurrently.
Although eye ablation is technically harder to successfully perform than decapitation, current studies involving eye excision have not included detailed instructions on their procedures. The goal of this video article is to enable researchers to consistently remove the planarian optic cup without disturbing the underlying brain tissues and removing as few other tissues as possible. This method can be used for both single and double eye ablation and is applicable to a wide range of investigations. Like most regeneration assays, eye ablation is well suited for combination with both pharmacological and genetic (RNAi) screens, as well as behavioral studies. Here we describe methods for the handling of worms, maintaining a planarian colony, and the eye ablation technique itself.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Instant Ocean sea salts</td>
			<td>Spectrum Brands&#160;</td>
			<td>SS15-10</td>
			<td>&#34;10 Gallon&#34; box&#160; (net weight 3 lbs)</td>
		</tr>
		<tr>
			<td>Kimwipes EX-L lint-free tissue wipe</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td>4.5 x 8.5 in&#160;</td>
		</tr>
		<tr>
			<td>Whatman #2 filter paper</td>
			<td>Sigma&#160;</td>
			<td>WHA1002125</td>
			<td>Circles, 125 mm diameter, white</td>
		</tr>
		<tr>
			<td>Easy Touch Insulin syringe (with needle)</td>
			<td>Pet Health Market</td>
			<td>17175-04</td>
			<td>&#160;U-100 1 cc syringe, 31-gauge 5/16 in needle</td>
		</tr>
		<tr>
			<td>100 mm Petri dish</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td>100 x 15 mm</td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>VWR</td>
			<td>25384-092</td>
			<td>60 x 15 mm</td>
		</tr>
		<tr>
			<td>Dumont #5&#160; forceps&#160;</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>Inox, straight tip , 11 cm</td>
		</tr>
		<tr>
			<td>Transfer pipettes&#160;</td>
			<td>Samco Scientific&#160;</td>
			<td>225</td>
			<td>Graduated, large bulb, 7.5 mL, non sterile</td>
		</tr>
		<tr>
			<td>Parafilm M paraffin film</td>
			<td>Brand&#160;</td>
			<td>701606</td>
			<td>&#160;4 in x 125 ft roll</td>
		</tr>
		<tr>
			<td>12-well untreated tissue culture plate</td>
			<td>VWR</td>
			<td>15705-059</td>
			<td>Untreated, flat bottom, sterile, Falcon brand</td>
		</tr>
		<tr>
			<td>Plastic food containers (for colony)&#160;</td>
			<td>Ziploc</td>
			<td>Large rectangle</td>
			<td>2.25 qt (2.12 L), 10&#34; x 6 -3/4 &#34; x 3 -3/16&#34;&#160;</td>
		</tr>
		<tr>
			<td>Planaria (Girardia tigrina)</td>
			<td>Carolina Biological</td>
			<td>132954</td>
			<td>Sold as &#34;Brown&#34; Planaria; most often they are G. tigrina (aka Dugesia tigrina), but sometimes are G. dorotocephala (aka Dugesia dorotocephala); either will work.</td>
		</tr>
		<tr>
			<td>Planaria (Schmidtea mediterranea)</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>S. mediterranea are not commercially available. At this time animals are only obtainable from laboratories that use them and have extra animals.</td>
		</tr>
		<tr>
			<td>Brown paper towels&#160;</td>
			<td>Grainger</td>
			<td>2U229</td>
			<td>9-3/16 x 9-3/8&#34; 1-Ply Multifold Paper Towel, UNBLEACHED</td>
		</tr>
		<tr>
			<td>Wash bottle (for worm water), optional</td>
			<td>VWR</td>
			<td>16650-275</td>
			<td>Wash Bottles, Low-Density Polyethylene, Wide Mouth, 500 mL</td>
		</tr>
		<tr>
			<td>Anti-synapsin antibody, optional</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>3C11</td>
			<td>Supernatant</td>
		</tr>
		<tr>
			<td>Anti-arrestin antibody, optional</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Not commercially available. Kind gift from Michael Levin, Tufts University</td>
		</tr>
		<tr>
			<td>Nalgene Lowboy carboy with spigot (for storing worm water), optional</td>
			<td>Nalge Nunc International Corporation</td>
			<td>2324-0015</td>
			<td>15 L,&#160; polypropylene, low profile makes it easier to fill plastic colony containers&#160;</td>
		</tr>
		<tr>
			<td>Custom Peltier plate, optional</td>
			<td>Williams Machine, Foxboro, MA&#160;</td>
			<td>n/a</td>
			<td>Design specifics courtesy of Junji Morokuma, Tufts University:&#160; Peltier plate is constructed of a standard thermoelectric heat pump (for example, All Electronics Corp Catalog # PJT-1, 30 mm2).&#160; The square heat pump is covered with a thin mirrored surface, then placed inside a 30 mm2 square hole in a circular plexiglass form (~50 mm in diameter). This form is of similar thickness to the heat pump, and fits flush into a well tooled in the center of a round heat sink (~115 mm in diameter). The form/heat pump is&#160; &#34;anchored&#34; to the sink with silicone base heat sink compound. The leads are threaded through holes drilled through both the form and the the heat sink. The bottom half of the heat sink is tooled into a &#34;foot&#34; that fits into the opening of your microscope&#39;s base plate.&#160;</td>
		</tr>
		<tr>
			<td>DC power source (for Peltier plate), optional</td>
			<td>B &#38; K Precision</td>
			<td>1665</td>
			<td>Regulated Low Voltage DC Power Supply, 1-18 volts (DC), 1-10 amps.</td>
		</tr>
		<tr>
			<td>Other common supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scope with gooseneck lighting</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chopstick rests, optional</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

For the first 1-2 h post surgery, animals may exhibit decreased movement compared to intact worms (however they will still move). If desired, worms will eat within 24 h of surgery (for instance, for RNAi feeding). When following eye regeneration in the same individuals over time, make sure to take a photograph of each worm both prior to surgery (intact) and at 1 h post ablation (hpa). By 4 days post ablation (dpa) regenerating pigment cells should be visible, and by 14 dpa the entire eye ...

Watch this Scientific Journal Video about Surgical Ablation Assay for Studying Eye Regeneration in Planarians at JoVE.com

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

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 ...

The overall goal of this procedure is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues so that the technique can be implemented by researchers and students at all levels of education. This method can help answer key questions in the regenerative field, such as determining the mechanisms that regulate endogenous eye stem cell maintenance and differentiation in vivo. The main advantage of this technique is that it allows to researchers to remove just the eye tissues with minimal disturbance of other tissues.Generally, practice is required before individuals new to this method can reliably remove the correct amount of tissue, ablating the entire eye while avoiding ablation of surrounding tissues. To begin, select worms that have not been fed for at least one week and are at least five to seven millimeters long. Transfer 15 to 30 worms to a 100 millimeter Petri dish two-thirds full of worm water and immediately replace the cover.Observe the worms for missing or unpigmented tissues, such as white heads or tails, to ensure that they are undamaged and not recently regenerating. First, wipe the workspace and dissecting microscope base with a 70%ethanol solution, and let it dry completely. Place the dish with the selected worms on the left side of the microscope.Then, on the right side of the microscope, place a pair of number five forceps, a 31 gauge 5/16ths inch insulin needle with a one milliliter syringe, a clean transfer pipette, and a labeled 12-well plate for collecting ablated worms. Place a box of lint-free tissue wipes, a plastic wash bottle filled with fresh worm water, and additional transfer pipettes so that they are easily accessible during the surgery. Position a Peltier plate at the center of the dissecting microscope base, and set the output on the DC power source so that the surface of the Peltier plate is sufficiently cool to immobilize worms.Alternatively, an ice-filled Petri dish can be used in place of a Peltier plate. To prepare the surgery surface, first cut a five centimeter by ten centimeter piece of plastic paraffin film and place it over the center of the Peltier plate. Next, fold a lint-free tissue wipe into a square of approximately two centimeters, and place it on top of the film.Then, hold the folded wipe in place, moisten it with worm water, and roll the wipe flat with a transfer pipette. Finally, cut a piece of white filter paper of 1.5 to two centimeters squared, and lay it on top of the wipe. To begin the surgery, place one worm dorsal side up onto the filter paper, using the transfer pipette.Turn the light on and focus its beam on the worm. Adjust the dissecting microscope focus and magnification so that the eyes of the worm are clearly visible. Rotate the paraffin film to adjust the position of the worm so that its head is pointed towards the researcher and angled 30 to 40 degrees to the right.If the worm is positioned ventral side facing up, and the pharyngeal opening is visible, use the dull side of the forceps to gently change the position of the worm to dorsal side up with the eyes visible. Readjust the microscope settings so that the eyes are clearly in focus. Also ensure that both the eyes and surrounding head tissues are in view.Hold a clean syringe in the right hand between thumb and index finger. Support the bottom part of the syringe on the left thumb, braced against the Peltier plate. Look through the microscope to ensure the bevel of the needle is visible.Place the left index finger on the surgery surface so that it makes a 40-degree angle with the left thumb to ensure that the surface remains stable during the procedure. Position the needle at a 90-degree angle to the eye with the bevel of the needle facing upward. With the tip of the needle, gently penetrate the thin layer of tissue overlying the optic cup of the eye visible as white, unpigmented region.Scooping from right to left, very gently remove any black pigmented tissue located within the optic cup as well as all of the white tissues. If performing double eye ablation, ablate the second eye now. After completing the surgery, backload the pipette with a small amount of worm water, and release the water onto the worm to lift it off the filter paper.Immediately draw the worm into the transfer pipette. Move the worm to a labeled 12-well plate two-thirds full of fresh worm water. Once all worms are transferred, wash them by replacing the water from the wells with fresh worm water.Incubate the worms protected from light in a 20-degree Celsius incubator and monitor the process of regeneration. To sacrifice live worms, remove worm water from the plate and replace it with 70%ethanol. Incubate the worms for three to five minutes and observe whether the worms lyse and turn gray.Presented here are photographs of flatworms subjected to double eye ablation and single eye ablation, taken before, four days after, and two weeks after the surgical procedure. The images demonstrate regeneration progress over time. Eye regeneration in ablated worms was also confirmed by immunohistochemical staining of arrestin and newly developed photoreceptor neurons.The recovery of the visual system in flatworms following double eye ablation was also studied in a functional assay based on the wildtype photophobic response to light. 24 hours after the ablation, eyeless worms do not avoid light and travel through light spots. Photophobia is restored by seven days after the procedure, indicating that the regenerated visual system is functional.Once mastered, the procedure of a double eye ablation of one worm can be done approximately three minutes. When ablating, it's important to excise all of the pigmented and unpigmented tissues of the eye, while making sure not to damage the tissue between the eyes or to tear the tissue later into the eye. Following this procedure, other methods like immunohistochemistry and RNA interference can be performed in order to examine the role of specific genes and their regenerative process.Additionally, behavioral assays can be performed to test eye function following regrowth. After watching this video, you should have a good understanding of how to excise planarian eyes without disturbing surrounding tissues. Don't forget that working with needles can be hazardous, and precautions such as wearing gloves and holding the needle pointing away from you should be taken.

surgical-ablation-assay-for-studying-eye-regeneration-in-planarians

Research

JoVE Journal

Developmental Biology

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

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 organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

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

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, and stress response. More than a decade ago, stem cells were identified in the pituitary gland. However, despite the application of transgenic in vivo approaches, their phenotype, biology, and role remain unclear. To tackle this enigma, a new and innovative organoid in vitro model is developed to deeply unravel pituitary stem cell biology. Organoids represent 3D cell structures that, under defined culture conditions, self-develop from a tissue's (epithelial) stem cells and recapitulate multiple hallmarks of those stem cells and their tissue. It is shown here that mouse pituitary-derived organoids develop from the gland's stem cells and faithfully recapitulate their in vivo phenotypic and functional characteristics. Among others, they reproduce the activation state of the stem cells as in vivo occurring in response to transgenically inflicted local damage. The organoids are long-term expandable while robustly retaining their stemness phenotype. The new research model is highly valuable to decipher the stem cells' phenotype and behavior during key conditions of pituitary remodeling, ranging from neonatal maturation to aging-associated fading, and from healthy to diseased glands. Here, a detailed protocol is presented to establish mouse pituitary-derived organoids, which provide a powerful tool to dive into the yet enigmatic world of pituitary stem cells.

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary gland is the key regulator of the body's endocrine system. This article describes the development of organoids from the mouse pituitary as a novel 3D in vitro model to study the gland's stem cell population of which the biology and function remain poorly understood.

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...