Name: generation and long-term maintenance of nerve-free hydra
Uploaded: 2017-07-07T14:00:00
Description: Watch this Scientific Journal Video about Generation and Long-term Maintenance of Nerve-free Hydra at JoVE.com

The authors thank Dr. Dick Campbell (UC Irvine) for discussions about the original protocol for generating and maintaining nerve-free animals, Ms. Rui Wang for help with adapting the syringe and needle technique, and Ms. Danielle Hagstrom and Dr. Rob Steele (UC Irvine) for comments on the manuscript. This work was supported by the RCSA and NSF grant CMMI-1463572.

1. Double Colchicine Treatment
<ol>
	<li>Make a 0.4% colchicine (weight/volume) solution in Hydra medium3,4,17. 
	Caution: Colchicine is acutely toxic, fatal if swallowed, may cause genetic defects, and may cause eye damage. Handle the powder in a fume hood and wear full Personal Protective Equipment (PPE).</li>
	<li>Incubate Hydra vulgaris (AEP strain was used here) that have been starv...

<ol>
	<li>Bode, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interstitial+cell+lineage+of+Hydra:+a+stem+cell+system+that+arose+early+in+evolution.">The interstitial cell lineage of Hydra: a stem cell system that arose early in evolution.</a> J. Cell. Sci. 109, 1155-1164 (1996).</li><li>Hager, G., David, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+of+differentiated+nerve+cells+in+hydra+is+determined+by+precursor+migration.">Pattern of differentiated nerve cells in hydra is determined by precursor migration.</a> Development. 124, 569-576 (1997).</li><li>Campbell, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elimination+of+Hydra+interstitial+and+nerve+cells+by+means+of+colchicine.">Elimination of Hydra interstitial and nerve cells by means of colchicine.</a> J. Cell. Sci. 21, 1-13 (1976).</li><li>Marcum, B. A., Campbell, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Hydra+lacking+nerve+and+interstitial+cells.">Development of Hydra lacking nerve and interstitial cells.</a> J. Cell. Sci. 29, 17-33 (1978).</li><li>Burns, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3H-colchicine+binding:+Failure+to+detect+any+binding+to+soluble+proteins+from+various+lower+organisms.">3H-colchicine binding: Failure to detect any binding to soluble proteins from various lower organisms.</a> Exp. Cell. Res. 81, 285-292 (1973).</li><li>Lee, H. -. T., Campbell, R. D. <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+Behavior+of+an+Intergeneric+Chimera+of+Hydra+(Pelmatohydra+oligactis+Interstitial+Cells+Hydra+attenuata+Epithelial+Cells).">Development and Behavior of an Intergeneric Chimera of Hydra (Pelmatohydra oligactis Interstitial Cells Hydra attenuata Epithelial Cells).</a> Biol. Bull. 157 (2), 288-296 (1979).</li><li>Novak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+Hydra+viridis+with+Nerve+Cells+and+No+Interstitial+Cells,+or+with+Neither+of+These+Cell+Types.">Preparing Hydra viridis with Nerve Cells and No Interstitial Cells, or with Neither of These Cell Types.</a> Hydra: Research Methods. , 295-297 (1983).</li><li>Wanek, N., Marcum, B. A., Lee, H. -. T., Chow, M., Campbell, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hydrostatic+pressure+on+morphogenesis+in+nerve-free+Hydra.">Effect of hydrostatic pressure on morphogenesis in nerve-free Hydra.</a> J. Exp. Zool. 211, 275-280 (1980).</li><li>Minobe, S., Koizumi, O., Sugiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+cell+differentiation+in+nerve-free+tissue+of+epithelial+Hydra+from+precursor+cells+introduced+by+grafting.">Nerve cell differentiation in nerve-free tissue of epithelial Hydra from precursor cells introduced by grafting.</a> Dev. Biol. 172 (1), 170-181 (1995).</li><li>Miljkovic-Licina, M., Chera, S., Ghila, L., 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=Head+regeneration+in+wild-type+hydra+requires+de+novo+neurogenesis.">Head regeneration in wild-type hydra requires de novo neurogenesis.</a> Development. 134 (6), 1191-1201 (2007).</li><li>Wenger, Y., Buzgariu, W., 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=Loss+of+neurogenesis+in+Hydra+leads+to+compensatory+regulation+of+neurogenic+and+neurotransmission+genes+in+epithelial+cells.">Loss of neurogenesis in Hydra leads to compensatory regulation of neurogenic and neurotransmission genes in epithelial cells.</a> Phil. Trans. R. Soc. B. 371 (20150040), (2015).</li><li>Campbell, R. D., Josephson, R. K., Schwab, W. E., Rushforth, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitability+of+nerve-free+Hydra.">Excitability of nerve-free Hydra.</a> Nature. 262, 388-390 (1976).</li><li>Takaku, Y., Hwang, J. S., 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=Innexin+gap+junctions+in+nerve+cells+coordinate+spontaneous+contractile+behavior+in+Hydra+polyps.">Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps.</a> Sci. Rep. 4 (3573), (2014).</li><li>Marcum, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+Epithelial+Hydra.">Culturing Epithelial Hydra.</a> Hydra: Research Methods. , 287-290 (1983).</li><li>David, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+method+for+maceration+of+Hydra+tissue.">A quantitative method for maceration of Hydra tissue.</a> Wilhelm Roux Arch. Entwickl. Mech. Org. 171, 259-268 (1973).</li><li>Gr&#246;ger, H., Schmid, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+development+in+Cnidaria:+A+connection+to+Bilateria.">Larval development in Cnidaria: A connection to Bilateria.</a> Genesis. 29 (3), 110-114 (2001).</li><li>Lenhoff, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water,+Culture+Solutions,+and+Buffers.">Water, Culture Solutions, and Buffers.</a> Hydra: Research Methods. , 29-34 (1983).</li><li>Shenk, M. A., Bode, H. R., Steele, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Cnox-2,+a+HOM/HOX+homeobox+gene+in+hydra,+is+correlated+with+axial+pattern+formation.">Expression of Cnox-2, a HOM/HOX homeobox gene in hydra, is correlated with axial pattern formation.</a> Development. 117, 657-667 (1993).</li><li>B&#246;ttger, A., 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=Horizontal+Gene+Transfer+Contributed+to+the+Evolution+of+Extracellular+Surface+Structures:+The+Freshwater+Polyp+Hydra+Is+Covered+by+a+Complex+Fibrous+Cuticle+Containing+Glycosaminoglycans+and+Proteins+of+the+PPOD+and+SWT+(Sweet+Tooth)+Families.">Horizontal Gene Transfer Contributed to the Evolution of Extracellular Surface Structures: The Freshwater Polyp Hydra Is Covered by a Complex Fibrous Cuticle Containing Glycosaminoglycans and Proteins of the PPOD and SWT (Sweet Tooth) Families.</a> PLoS One. 7 (12), (2012).</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></ol>

Hydra interstitial cells can be eliminated through a double colchicine treatment3,4. In the days following the first treatment, it is critical to prevent contact between individual Hydra to avoid fusion of Hydra pieces into deformed Hydra. Also, animals that can eat unassisted following the first treatment must be removed as the second colchicine treatment may not be sufficient to eliminate the remaining interstitial cells from...

The nervous system of Hydra consists of a nerve net, with neurons associated with both epithelial tissue layers1. The nerve net is denser in the hypostome and peduncle and less dense in the body column2. The nerve cells originate from interstitial stem cells, which are multipotent stem cells that give rise to secretory cells, nematocytes, germ cells, and neurons1. It is possible to eliminate the interstitial cells of Hydra vulgaris through treatment with colchicine3,4, a plant-derived toxin that kills dividing cells. Although colchicine has been found to inhibit microtubule polymerization in other organisms, a previous study has shown that microtubules are present in Hydra throughout the entire treatment, suggesting that colchicine does not act this way in Hydra3. Another study suggests that colchicine does not bind efficiently to tubulin in some organisms, including Tetrahymena pyriformis, Zea mays, Chlamydomonas, and Schizosaccharomyces pombe, which may explain this difference5. The colchicine treatment induces phagocytosis of the interstitial cells by the endodermal epithelial cells3 and thus allows for the creation of animals that are lacking nerve cells, gland cells, and nematocytes. It is unclear why the interstitial cells are particularly susceptible to colchicine treatment. Given that both post-mitotic interstitial cells and the interstitial stem cell lineage are damaged and phagocytosed, Campbell concluded that colchicine was not directly affecting mitotic activity3. Notably, the colchicine treatment works well in Hydra vulgaris, but has been shown to not work as well in other species, such as Hydra oligactis6. A modified treatment with colchicine and hydroxyurea can be used to produce nerve-free Hydra viridis7. Nerve-free Hydra (also sometimes referred to as &#34;epithelial Hydra&#34;8) are therefore a useful tool for studying the roles of these specialized cell types from the interstitial cell lineage in tissue homeostasis and regeneration.
Hydra may be the only known example of an animal capable of living without a nervous system. Nerve-free Hydra serve as a particularly useful model for dissecting the role of the nerve net in regulating Hydra regeneration, homeostasis, and behavior. For example, the introduction of interstitial cells into nerve-free Hydra via grafting allowed for the characterization of nerve cell differentiation as highly region-specific9. Furthermore, because nerve-free Hydra can regenerate, they enable the investigation of alternative, nervous system-independent regeneration pathways. One such example is apical neurogenesis and head formation, which has been shown to depend on cnox-2 function in the nervous system in wildtype Hydra, but appears to be dispensable in nerve-free Hydra, suggesting that there may be an alternative head regeneration process10.
Nerve-free Hydra have also been used to study epithelial cell expression and regulation of neurogenic and neurotransmission genes after the loss of neurogenesis11. Nerve-free Hydra do not exhibit spontaneous contraction bursts12, indicating that these bursts are regulated by the nervous system. Nerve-free Hydra do, however, contract in response to pinching the body column with forceps, suggesting that contraction in response to mechanical stimuli is mediated by coupling through gap junctions in epithelial cells, while spontaneous contractile behavior is mediated by coupling through gap junctions in nerve cells13.
Nerve-free Hydra do not open their mouths when presented with food or reduced glutathione3, suggesting that sensory neurons are necessary to detect the presence of food and signal the mouth to open. In addition, the nerve net seems to play a role in sensing osmotic pressure, because nerve-free animals are unable to autonomously regulate their internal hydrostatic pressure through mouth opening, causing their characteristic balloon-like appearance3,4 (Figure 1B). Regulation of hydrostatic pressure in nerve-free Hydra by frequent manual deflation led to a loss of some abnormal morphology in the hypostome and body column. However, chronic deflation led to interference with growth, elongation, budding, and tissue organization8.
Although nerve-free Hydra are unable to feed and egest on their own, it is possible to maintain them indefinitely in the laboratory by manually force-feeding and burping each animal. Previous publications have described methods of force-feeding and burping nerve-free Hydra, however these protocols involved the use of micropipette tips that must be hand pulled carefully to the appropriate size as well as use of a mouthpiece connected to the pipette by tubing14. Here, a simpler, safer, and more time-efficient method of feeding and burping is described.
In addition, previous studies involved checking for the absence of nerve cells through dissociation of fixed animals into individual cells and examination of cell morphology3,4,15. Here, immunohistochemistry with a monoclonal antibody against the tyrosinated carboxyl-terminus of alpha-tubulin was used as a complimentary method to maceration to check for the depletion of neurons in the hypostome13,16. Previous studies have shown that neurons in the peduncle can also be visualized using this antibody13, however these neurons as well as those in the body column are more difficult to make out. While immunohistochemistry is sufficient to confirm the absence of nerve cells in the hypostome and does not require expertise on cell type morphology, it cannot be used to check for the absence of the interstitial stem cells and the other derivatives of these cells. Dissociation and cell morphology studies are more rigorous and can give a quantitative account of the numbers of each cell type remaining following each stage of the treatment.

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">Colchicine</td>
			<td style="width:75px;">Acros Organics</td>
			<td style="width:119px;">227120010</td>
			<td style="width:167px;"></td>
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			<td height="60" style="height:60px;">Brine Shrimp Eggs</td>
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			<td style="width:167px;">Can be purchased locally</td>
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			<td height="60" style="height:60px;">Brine Shrimp Hatchery Dish</td>
			<td style="width:75px;">Brine Shrimp Direct</td>
			<td style="width:119px;">N/A</td>
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			<td height="20" style="height:20px;">60 mm x 15 mm Petri Dish</td>
			<td style="width:75px;">Celltreat</td>
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			<td height="40" style="height:40px;">30 G x 3/4&#34; Hypodermic Needle</td>
			<td style="width:75px;">Covidien</td>
			<td style="width:119px;">1188830340</td>
			<td style="width:167px;">A 27G needle may also be used</td>
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			<td height="20" style="height:20px;">2 x Fine-tip Tweezers</td>
			<td style="width:75px;">Dumont</td>
			<td style="width:119px;">0109-5-PO</td>
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			<td style="width:119px;">557303</td>
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			<td style="width:75px;">Fisher</td>
			<td style="width:119px;">BP9703-100</td>
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			<td height="20" style="height:20px;">Scalpel</td>
			<td style="width:75px;">Fisher</td>
			<td style="width:119px;">08-920A</td>
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			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:75px;">Gibco</td>
			<td style="width:119px;">10437028</td>
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			<td height="20" style="height:20px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:75px;">Invitrogen</td>
			<td style="width:119px;">D1306</td>
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			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">T9028</td>
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			<td height="20" style="height:20px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">D2650</td>
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			<td height="20" style="height:20px;width:303px;">Hydrogen Peroxide</td>
			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">216763</td>
			<td style="width:167px;"></td>
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			<td height="20" style="height:20px;">Paraformaldehyde (PFA)</td>
			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">P6148</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X - 100</td>
			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">T9284</td>
			<td style="width:167px;"></td>
		</tr>
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			<td height="20" style="height:20px;">Tween 20</td>
			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">P1379</td>
			<td style="width:167px;"></td>
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			<td height="20" style="height:20px;width:303px;">Urethane</td>
			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">U2500</td>
			<td style="width:167px;"></td>
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			<td height="40" style="height:40px;">Glass Pasteur Pipette</td>
			<td style="width:75px;">VWR</td>
			<td style="width:119px;">53283-916</td>
			<td style="width:167px;">Length of the pipet does not matter</td>
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			<td height="20" style="height:20px;">15 ml Tube</td>
			<td style="width:75px;">VWR</td>
			<td style="width:119px;">89039-670</td>
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			<td height="60" style="height:60px;">P320 Sandpaper</td>
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			<td style="width:119px;">IBGABBV00397</td>
			<td style="width:167px;">Can be purchased at local home improvement store</td>
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</table>

generation and long-term maintenance of nerve-free hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Immediately following the initial 8 h colchicine treatment, all Hydra survive. Some of these Hydra will have only tentacle stubs remaining (Figure 3A), while others will have completely lost their tentacles (Figure 3B). Over the following 1 or 2 days, the tentacles will continue to shrink until all Hydra have lost their tentacles. About 1 week following the treatment, t...

Watch this Scientific Journal Video about Generation and Long-term Maintenance of Nerve-free Hydra at JoVE.com

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

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