Name: inducing complete polyp regeneration from the aboral physa of the starlet sea anemone nematostella vectensis
Uploaded: 2017-01-14T13:00:00
Description: Watch this Scientific Journal Video about Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis at JoVE.com

This work was supported by a New York Stem Cell Science (NYSTEM C028107) Grant to GHT.

1. Conditioning of Animals for Temperature, Nutrition and Light/Dark Cycle
<ol>
	<li>Obtain Nematostella vectensis adults from one of the numerous Nematostella labs worldwide, or a non-profit supplier (Table 1)</li>
	<li>Maintain Nematostella at constant temperature (typically between 18 and 21&#160;&#176;C) in the dark, in &#34;1/3x&#34; Artificial Sea Water (ASW) at a salinity of 12 parts per thousand (ppt). Maintain cultures in simple soda-lime glass culture dishes, typica...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+understanding+tissue+regenerative+capacity+and+mechanisms+in+animals.">Advances in understanding tissue regenerative capacity and mechanisms in animals.</a> Nat Rev Genet. 11 (10), 710-722 (2010).</li><li>Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydra,+a+fruitful+model+system+for+270+years.">Hydra, a fruitful model system for 270 years.</a> Int J Dev Biol. 56 (6-8), 411-423 (2012).</li><li>Gold, D. A., Jacobs, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+dynamics+in+Cnidaria:+are+there+unifying+principles?.">Stem cell dynamics in Cnidaria: are there unifying principles?.</a> Dev Genes Evol. 233 (1-2), 53-66 (2013).</li><li>Holstein, T. W., Hobmayer, E., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cnidarians:+an+evolutionarily+conserved+model+system+for+regeneration?.">Cnidarians: an evolutionarily conserved model system for regeneration?.</a> Dev Dyn. 226 (2), 257-267 (2003).</li><li>Amiel, A. R., 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=Characterization+of+Morphological+and+Cellular+Events+Underlying+Oral+Regeneration+in+the+Sea+Anemone,+Nematostella+vectensis.">Characterization of Morphological and Cellular Events Underlying Oral Regeneration in the Sea Anemone, Nematostella vectensis.</a> Int J Mol Sci. 16 (12), 28449-28471 (2015).</li><li>Warren, C. R., 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=Evolution+of+the+perlecan/HSPG2+gene+and+its+activation+in+regenerating+Nematostella+vectensis.">Evolution of the perlecan/HSPG2 gene and its activation in regenerating Nematostella vectensis.</a> PLoS One. 10 (4), e0124578 (2015).</li><li>Gong, Q., 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=Integrins+of+the+starlet+sea+anemone+Nematostella+vectensis.">Integrins of the starlet sea anemone Nematostella vectensis.</a> Biol Bull. 227 (3), 211-220 (2014).</li><li>Bossert, P. E., Dunn, M. P., Thomsen, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+staging+system+for+the+regeneration+of+a+polyp+from+the+aboral+physa+of+the+anthozoan+Cnidarian+Nematostella+vectensis.">A staging system for the regeneration of a polyp from the aboral physa of the anthozoan Cnidarian Nematostella vectensis.</a> Dev Dyn. 242 (11), 1320-1331 (2013).</li><li>Stefanik, D. J., Friedman, L. E., Finnerty, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting,+rearing,+spawning+and+inducing+regeneration+of+the+starlet+sea+anemone,+Nematostella+vectensis.">Collecting, rearing, spawning and inducing regeneration of the starlet sea anemone, Nematostella vectensis.</a> Nat Protoc. 8 (5), 916-923 (2013).</li><li>Tucker, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thrombospondin+in+the+anthozoan+Nematostella+vectensis+is+associated+with+the+nervous+system+and+upregulated+during+regeneration.">A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration.</a> Biol Open. 2 (2), 217-226 (2013).</li><li>Passamaneck, Y. J., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+proliferation+is+necessary+for+the+regeneration+of+oral+structures+in+the+anthozoan+cnidarian+Nematostella+vectensis.">Cell proliferation is necessary for the regeneration of oral structures in the anthozoan cnidarian Nematostella vectensis.</a> BMC Dev Biol. 12, (2012).</li><li>Trevino, M., Stefanik, D. J., Rodriguez, R., Harmon, S., Burton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+canonical+Wnt+signaling+by+alsterpaullone+is+sufficient+for+oral+tissue+fate+during+regeneration+and+embryogenesis+in+Nematostella+vectensis.">Induction of canonical Wnt signaling by alsterpaullone is sufficient for oral tissue fate during regeneration and embryogenesis in Nematostella vectensis.</a> Dev Dyn. 240 (12), 2673-2679 (2011).</li><li>Renfer, E., Amon-Hassenzahl, A., Steinmetz, P. R., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+muscle-specific+transgenic+reporter+line+of+the+sea+anemone,+Nematostella+vectensis.">A muscle-specific transgenic reporter line of the sea anemone, Nematostella vectensis.</a> Proc Natl Acad Sci U S A. 107 (1), 104-108 (2010).</li><li>Burton, P. M., Finnerty, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+and+novel+gene+expression+between+regeneration+and+asexual+fission+in+Nematostella+vectensis.">Conserved and novel gene expression between regeneration and asexual fission in Nematostella vectensis.</a> Dev Genes Evol. 219 (2), 79-87 (2009).</li><li>DuBuc, T. Q., Traylor-Knowles, N., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiating+a+regenerative+response;+cellular+and+molecular+features+of+wound+healing+in+the+cnidarian+Nematostella+vectensis.">Initiating a regenerative response; cellular and molecular features of wound healing in the cnidarian Nematostella vectensis.</a> BMC Biol. 12, (2014).</li><li>Hand, C., Uhlinger, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asexual+reproduction+by+transverse+fission+and+some+anomalies+in+the+sea+anemone+Nematostella+vectensis.">Asexual reproduction by transverse fission and some anomalies in the sea anemone Nematostella vectensis.</a> Invert Biol. 114, 9-18 (1995).</li><li>Fritzenwanker, J. H., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+gametogenesis+in+the+basal+cnidarian+Nematostella+vectensis(Anthozoa).">Induction of gametogenesis in the basal cnidarian Nematostella vectensis(Anthozoa).</a> Dev Genes Evol. 212 (2), 99-103 (2002).</li><li>Magie, C., Bossert, P., Aramli, L., Thomsen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Science's+super+star:+The+starlet+sea+anemone+is+an+ideal+tool+for+student+inquiry.">Science's super star: The starlet sea anemone is an ideal tool for student inquiry.</a> The Science Teacher. 83 (3), 33-40 (2016).</li><li>Genikhovich, G., Technau, U. <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+of+starlet+sea+anemone+(Nematostella+vectensis)+embryos,+larvae,+and+polyps.">In situ hybridization of starlet sea anemone (Nematostella vectensis) embryos, larvae, and polyps.</a> Cold Spring Harb Protoc. (9), (2009).</li><li>Magie, C. R., Pang, K., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+inventory+and+expression+of+Sox+and+Fox+genes+in+the+cnidarian+Nematostella+vectensis.">Genomic inventory and expression of Sox and Fox genes in the cnidarian Nematostella vectensis.</a> Dev Genes Evol. 215 (12), 618-630 (2005).</li><li>Chera, S., Kaloulis, K., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cAMP+response+element+binding+protein+(CREB)+as+an+integrative+HUB+selector+in+metazoans:+clues+from+the+hydra+model+system.">The cAMP response element binding protein (CREB) as an integrative HUB selector in metazoans: clues from the hydra model system.</a> Biosystems. 87 (2-3), 191-203 (2007).</li><li>Reitzel, A. M., Burton, P. M., Krone, C., Finnerty, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+developmental+trajectories+in+the+starlet+sea+anemone+Nematostella+vectensis:+embryogenesis,+regeneration,+and+two+forms+of+asexual+fission.">Comparison of developmental trajectories in the starlet sea anemone Nematostella vectensis: embryogenesis, regeneration, and two forms of asexual fission.</a> Invertebr Biol. 126, 99-112 (2007).</li><li>Ikmi, A., McKinney, S. A., Delventhal, K. M., Gibson, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALEN+and+CRISPR/Cas9-mediated+genome+editing+in+the+early-branching+metazoan+Nematostella+vectensis.">TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis.</a> Nat Commun. 5, 5486 (2014).</li><li>Jahnel, S. M., Walzl, M., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+epithelial+organisation+of+muscle+cells+in+the+sea+anemone+Nematostella+vectensis.">Development and epithelial organisation of muscle cells in the sea anemone Nematostella vectensis.</a> Front Zool. 11, 44 (2014).</li><li>Kelava, I., Rentzsch, F., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+eumetazoan+nervous+systems:+insights+from+cnidarians.">Evolution of eumetazoan nervous systems: insights from cnidarians.</a> Philos Trans R Soc Lond B Biol Sci. 370 (1684), (2015).</li><li>Nakanishi, N., Renfer, E., Technau, U., Rentzsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nervous+systems+of+the+sea+anemone+Nematostella+vectensis+are+generated+by+ectoderm+and+endoderm+and+shaped+by+distinct+mechanisms.">Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms.</a> Development. 139 (2), 347-357 (2012).</li><li>Richards, G. S., Rentzsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+analysis+of+a+SoxB+gene+reveals+neural+progenitor+cells+in+the+cnidarian+Nematostella+vectensis.">Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis.</a> Development. 141 (24), 4681-4689 (2014).</li><li>DuBuc, T. Q., 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=In+vivo+imaging+of+Nematostella+vectensis+embryogenesis+and+late+development+using+fluorescent+probes.">In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes.</a> BMC Cell Biol. 15, (2014).</li><li>Kaur, J., Debnath, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+at+the+crossroads+of+catabolism+and+anabolism.">Autophagy at the crossroads of catabolism and anabolism.</a> Nat Rev Mol Cell Biol. 16 (8), 461-472 (2015).</li><li>Carroll, B., Korolchuk, V. 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Use of Nematostella as a model of wound healing and regeneration is becoming increasingly popular. Thus, it is important to be able to visualize the morphological patterns of a particular protocol before effective cellular and molecular analyses can be assigned and compared. Nematostella have a high degree of regenerative &#34;flexibility&#34;, being able to reform almost any missing structure amputated at any location, at post-planula stages of life. Thus, various investigators have examined regenerati...

The observation of regeneration in a single hydra was the seminal event in the advent of biology as an experimental science1,2. Regeneration remains a phenomenon of extraordinarily broad appeal to biologist and lay person alike. The potential for developmental biologists, clinicians, biomedical scientists and tissue engineers to understand and overcome the limits on human regeneration makes regeneration biology more than intrinsically interesting.
Now, with the use of emerging technologies such as genome sequencing and gain and loss of function tools, the field is poised to tease apart regenerative mechanisms and to ultimately understand how various species can regenerate while others cannot. The degree of commonality in molecular, cellular and morphological responses remains to be elucidated, but so far it appears that the basic responses among animals that can regenerate is more similar than would have been imagined only a decade ago3.
Cnidarians in particular are facile at regenerating almost all of their body parts amid a broad spectrum of morphological diversity. From the solitary fresh water polyp, Hydra along with the tiny marine polyps that build immense coral reefs, to the complex colonial siphonophores, such as the Portuguese Man-O-War, regeneration is often a mode of reproduction, in addition to a mechanism for repairing or reforming damaged or lost body parts resulting from injury and predation. Whether the diverse species of Cnidaria use similar or different mechanisms for regeneration is a fundamentally interesting question4-6.
We, and others have been developing the anthozoan, Nematostella vectensis as a model for regeneration7-17. We recently developed a staging system for describing regeneration of an entire body from a morphologically uniform piece of tissue bisected from the aboral end of the polyp10. While Nematostella polyps can regenerate when bisected at any level, we chose to cut adults at an aboral position in the most morphologically simple region, the physa, in part because this is close to the normal plane of natural asexual fission18, and also because it permits observation and molecular analyses of how an entire body is reassembled from the simplest morphological components.
The Nematostella Regeneration Staging System (NRSS) provides a relatively simple set of morphological benchmarks that could be used to score the progress of any aspect of regeneration by an amputated physa, under normal culture conditions or experimentally perturbed situations such as small molecule treatments, genetic manipulation, or environmental alteration. As anticipated, the NRSS is becoming adopted as a morphological scaffold on which the cellular and molecular events of regeneration can be referenced10.
Finally our method of cutting produces a gaping hole several orders of magnitude greater than the pin point puncture used in a recent study17, yet both wounds heal in around 6 hours. Documenting the visually arresting and distinct phases of wound closure should suggest experimental approaches to explain the apparent independence of the size of a wound and the time it takes to close. Thus, a deeper visual understanding of the aboral amputation process, provided by this protocol, will aid further investigations into this model regeneration system and broaden the application of this staging system using Nematostella vectensis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Nematostella vectensis, adults</td>
			<td>Marine Biological Lab (MBL)</td>
			<td></td>
			<td>non-profit supplier</td>
		</tr>
		<tr>
			<td>Glass Culture Dish, 250 ml</td>
			<td>Carolina Biological Supply</td>
			<td>741004</td>
			<td>250 ml</td>
		</tr>
		<tr>
			<td>Glass Culture Dish, 1,500 ml</td>
			<td>Carolina Biological Supply</td>
			<td>741006</td>
			<td>1,500 ml</td>
		</tr>
		<tr>
			<td>Polyethylene transfer pipette, 5ml&#160;</td>
			<td>USA Scientific&#160;</td>
			<td>1022-2500</td>
			<td>narrow bore, graduated</td>
		</tr>
		<tr>
			<td>Polyethylene transfer pipet, tapered</td>
			<td>Samco</td>
			<td>202-205</td>
			<td>cut off 1 inch of tip to make wide bore</td>
		</tr>
		<tr>
			<td>Disposable Scalpel</td>
			<td>Feather Safety Razor Co. Ltd</td>
			<td>no. 10</td>
			<td>blade should be curved</td>
		</tr>
		<tr>
			<td>#5 Dumont Fine point tweezers</td>
			<td>Roboz</td>
			<td>RS5045</td>
			<td>alternative suppliers available</td>
		</tr>
		<tr>
			<td>Pyrex petri dish, 100 mm diameter</td>
			<td>Corning&#160;</td>
			<td>3160</td>
			<td>can substitute other glass petri plates</td>
		</tr>
		<tr>
			<td>Sterile 6 well plate</td>
			<td>Corning Falcon&#160;</td>
			<td>353046</td>
			<td>or similar from other manufacturer</td>
		</tr>
		<tr>
			<td>Sterile 12 well plate</td>
			<td>Nunc&#160;</td>
			<td>150628</td>
			<td>or similar from other manufacturer</td>
		</tr>
		<tr>
			<td>Sterile 24 well plate</td>
			<td>Cellstar, Greiner bio-one</td>
			<td>662-160</td>
			<td>or similar from other manufacturer</td>
		</tr>
		<tr>
			<td>Brine shrimp hathery kit</td>
			<td>San Francisco Bay; drsfostersmith.com</td>
			<td>CD-154005</td>
			<td>option for growing brine shrimp</td>
		</tr>
		<tr>
			<td>pyrex baking dish</td>
			<td>common in grocery stores</td>
			<td></td>
			<td>option for growing brine shrimp</td>
		</tr>
		<tr>
			<td>artificial seawater mix 50 gal or more&#160;</td>
			<td>Instant Ocean; drsfoster-smith.com</td>
			<td>CD-116528</td>
			<td>others brands may suffice</td>
		</tr>
		<tr>
			<td>Plastic tub for stock ASW preparation</td>
			<td>various</td>
			<td></td>
			<td>common 25 gallon plastic trash can OK</td>
		</tr>
		<tr>
			<td>Polypropylene Carboy</td>
			<td>Carolina Biological Supply</td>
			<td>716391</td>
			<td>For working stock of ASW @ 12 ppt</td>
		</tr>
		<tr>
			<td>Beaker, Graduated, 4,000ml</td>
			<td>PhytoTechnology Laboratories</td>
			<td>B199</td>
			<td>For dilution of 36 ppt ASW to 12 ppt</td>
		</tr>
		<tr>
			<td>Stereomicroscope and light source</td>
			<td>various&#160;</td>
			<td></td>
			<td>with continuous 1 - 40x magnification&#160;</td>
		</tr>
	</tbody>
</table>

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.

The progression of morphological events during regeneration in severed physa is shown in Figure 1A, which includes representative views of physa at each NRSS stage. The typical physa cut site is indicated on the adult (arrowheads). The photographs in Figure 1A show progressive regeneration of oral and body structures from freshly cut physa through fully formed polyp. Figure 1B, C show&#160;the arrangement of internal septa, the mesenterie...

Watch this Scientific Journal Video about Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis at JoVE.com

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.

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 ...

The overall goal of this procedure is to illustrate the predictable series of morphological changes that occurred during the regeneration of a complete polyp from a tiny section of tissue removed from the aboral tip of nematostella vectensis This method can help answer key questions in regeneration biology, such as how cells at the site of a wound reorganize and deploy to remake the missing structures. I first had the idea for this method because I wanted to see how relatively complex polyp could put itself back together starting with the least amount of morphological information. Visual demonstration of this method is critical because stage progression after the initial wounding is sometimes obscured by a mucous membrane.Generally individuals new to this method will struggle to obtain a complete separation of the tissue at the wound site, because of the soft, sticky, gelatinous nature of the polyp. Begin this procedure with conditioning of nematostella vectensis as described in the text protocol. It's especially important when using this technique that the animals selected are the same size, and have the same culture history, such as identical feeding and other condition parameters.Remove the dish of selected animals from the incubator into room light at least one hour prior to amputation. Use a wide bore plastic pipette to transfer the five animals from the pool to be amputated into the bottom of a sterile glass cutting dish of 100 millimeter diameter containing artificial seawater at a salinity of 12 parts per thousand. Place the dish onto the stage of a stereo microscope with variable magnification between 10 to 40x.Select size-matched polyps of approximately the same length and place them in a bowl separated from the colony for three days prior to amputation. Using a sterile scalpel, amputate the aboral physa from each polyp to obtain a section of the physa that is approximately as long as it is wide and containing no mesentery. To achieve this, place the scalpel blade in contact with the animal at the desired site of amputation.Do this either unassisted or by gently grasping the animal's body with a number five forceps. Cut through the tissue by leveraging the curved blade of the scalpel in a rocking motion across the body. Remove each amputated donor polyp from the dish and return it to a separate bowl labeled pooled amputees.After dating the bowl, return it to stock culture. Rinse the excised physa that remain in the cutting dish in artificial seawater at a salinity of 12 parts per 1, 000. Then, transfer each physa to a separate sterile well in a multi-well cell culture plate that has 10 milliliters of artificial seawater at a salinity of 12 parts per 1, 000 in each well.Repeat these steps to collect at least five physa in each well to be reserved for each experimental treatment. Place the plate containing the physa into a temperature-regulated incubator at a fixed temperature determined by the desired rate of regeneration. Score the physa using a stereo compound microscope with variable magnification.Score the freshly cut nematostella physa as stage zero, and continue scoring at the same time each day post-amputation, or DPA, using the NRSS. Score the physa as stage zero if a freshly cut physa appears as a cup-shaped mass resembling a flacid ballon with an open wound site that is likely visible. Score a physa as stage one if the amputation wound appears closed.Score a physa as stage two if the surface of the aural pole appears inflated, revealing eight raised arches arranged in a radially symmetric pattern and separated by grooves. At stage two, observe small hemispherical blebs at the apex of the arches that are about as tall as wide, likely transient, and initial comprised by a single ectodermal cell layer. As the physa ages, it may become encased in mucous, which should be carefully removed with forceps.Score a physa as stage three if the buds of the tentacles containing endodermal and ectodermal tissue layers are stably formed at the aural end of at least some radial arches. Score a physa as stage four if the physa contains eight distinct visible mesenteries that extend in a coelenteron from insertions in the body wall with aural aboral lengths that are more than twice their radial width measured from where they appear to connect to the pharynx at its aboral end, or enterostome. Finally, score a physa as stage five if the physa has more than four mesenteries with pleating, and the pleating is more full and sinuous than at stage four.At stage five, the animal has an almost normal adult appearance, but there are no visible gonadal cells. This group of control nematostella physa treated with 0.1 percent DMSO have regenerated completely to stage five. Physa treated with chloroquine, however, failed to completely form completed mesenteries.A closeup shows both the presence and lack of pleating in the mesenteries in a physa that was incubated in chloroquine. This graph illustrates the aggregate results of chloroquine treatment. Any treatment between 10 and 50 micromolar caused similar defects, mostly the lack of complete reformation of mesenteries in that the mesenteries lack pleating.Once mastered, this technique can be done in less than an hour for each six-well plate containing 30 cut physa. Scoring is rapid, perhaps requiring a half hour for each replicate plate. After watching this video, you should have a good understanding of how to amputate the physa to visualize the morphological changes that occur during the regeneration of a complete polyp from a tiny section of aboral tissue.The main advantage of this technique is that it provides morphological stages in which to pin the molecular events underlying the regeneration of a complete polyp from a cup of visually uniform tissue. While attempting this procedure, it's important to remember to precondition the animals with respect to nutritional history, to bisect to exclude adult mesentery, and to standardize the size of the regenerating physa to reduce variation within treatments. Following this procedure, the effects of genetic mutations or signaling pathway perturbations with small molecules, for example, could be assessed.Or morphogenetic changes could be correlated with microscopic analysis of self-proliferation, lineage tracing, or site two gene expression. This technique will pave the way for researchers in the field of regeneration biology to explore the cell biology, signaling pathways, and morphogenetic processes that underlie the remarkable regenerative capacity of this model organism.

Research

JoVE Journal

Developmental Biology

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Assaying Blood Cell Populations of the Drosophila melanogaster Larva

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Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science.  One of the most demanding technical aspects of ...

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

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

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

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Methods for the Study of Regeneration in Stentor

Methods for the Study of Regeneration in Stentor

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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 ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Genotyping of Sea Anemone during Early Development

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the ...