Generation and Long-term Maintenance of Nerve-free Hydra

Summary

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.

Abstract

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 Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra.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.

Introduction

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 colchicine^3,4, 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 "epithelial Hydra"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 appearance^3,4 (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 morphology^3,4,15. 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 hypostome^13,16. 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.

Protocol

1. Double Colchicine Treatment

1. Make a 0.4% colchicine (weight/volume) solution in Hydra medium^3,4,17.
   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).
2. Incubate Hydra vulgaris (AEP strain was used here) that have been starved for 24 h in 0.4% colchicine at a ratio of 5 Hydra per mL of colchicine solution in a Petri dish for 8 h at room temperature in the dark.
   Note: 5 Hydra/mL colchicine is a guideline for determining how much solution to use to avoid overcrowding the dish with Hydra. Use the same volume of solution for subsequent cleanings and solution changes.
3. Remove the colchicine solution and replace with clean Hydra medium without colchicine. Wash the Hydra 5 times by serial transfer with a glass Pasteur pipette into clean Hydra medium before transferring to 50 µg/mL rifampicin in Hydra medium. Keep the Hydra in an 18 °C incubator.
   Note: Sufficient 1,000X rifampicin stock (50 mg/mL) in dimethyl sulfoxide (DMSO) for 1-2 weeks of treatment can be stored at 4 °C. The exact amount can be determined by the total volume of solution used each day. For example, if there are 2 dishes, each containing 10 mL of solution that needs to be changed twice daily, a total of 40 mL of solution will be used in one day, corresponding to 40 µL of 1,000X stock per day or 560 µL over a two week period. The stock is then diluted 1:1000 in Hydra medium upon use.
   Caution: DMSO is a combustible liquid and readily penetrates skin, allowing other dissolved chemicals into the body. Handle the solvent in a fume hood and wear full PPE. Of note, DMSO freezes at 4 °C
4. Change the rifampicin-containing Hydra medium twice daily until the Hydra stop expelling cells into the medium, which is generally by 1 week post-treatment. At this point, the medium can be changed once daily. During the days following the treatment, the Hydra will completely lose their tentacles. Avoid having the Hydra in contact with one another, as they may fuse together and result in oddly shaped Hydra (Figure 2A).
   1. To prevent contact, avoid swirling the dish in a circular motion. Instead, gently agitate the dish in vertical and horizontal directions until the Hydra are spread apart. Inspect the dish upon placing the Hydra into the 18 °C incubator and adjust as necessary.
      Note: For the days that the medium is changed twice, change the medium once in the morning and again in the afternoon/evening.
5. Once the tentacles begin to form, begin force-feeding and burping the Hydra 3 to 4 times a week (see Sections 2 and 3). About 8-9 days following colchicine treatment, the Hydra will begin to grow their tentacles back.
   Note: Tentacle regrowth varies among individuals. Some may take longer than 8 - 9 days before showing signs of tentacle growth. Those that do not regrow their tentacles as well as oddly shaped (Figure 2B) animals or animals too small to be fed should be removed. These Hydra will likely not survive the second treatment. The Hydra may also bud. However, some of the buds may still have interstitial cells and be able to eat on their own.
   1. Remove any buds that can eat without assistance and detach from the parent animal. Identify these animals by adding live Artemia to the feeding dish and observing whether or not the animals catch and eat the Artemia on their own. 1 or 2 Hydra may also be able to eat on their own. These should also be discarded.
      Note: Nerve-free Hydra have a characteristic balloon-like morphology, and tentacles that are shorter and thinner than normal (due to loss of nematocytes) and can thus easily be distinguished from normal looking animals (Figure 1A, 1B).
6. Three weeks following the first colchicine treatment, repeat the colchicine treatment (steps 1.1 - 1.5). A second colchicine treatment is necessary to eliminate the remaining interstitial cells and nerve cells³.

2. Force-Feeding

1. Add Artemia cysts to a narrow and tall glass container that tapers at the bottom. Fill the container with Artemia water (6.72 M NaCl). Avoid exceeding 1 g of cysts per 1 L of water for a higher hatch yield.
   1. Cover the top of the container with parafilm and insert a 10 mL serological pipette through the parafilm into the container. Provide aeration by attaching tubing to an aquarium air pump and fitting the tubing around the pipette. Make sure that the pipette tip reaches the bottom of the container and that no cysts are settled at the bottom. The Artemia will hatch after 48 h.
      Note: There are many different ways to hatch Artemia that may work just as well.
2. Strain the Artemia from the Artemia water in an Artemia net (available from aquarium supply companies) and wash them for about 20 s in DI water before placing them in a dish with Hydra medium. The salinity of Artemia water is too high for Hydra, so the Artemia must be washed before they are used.
3. Under a dissecting microscope, euthanize the Artemia by lightly squeezing them with a pair of fine forceps. Avoid squeezing hard enough to expel the guts, as this will stick to the forceps and make feeding difficult. Lightly squeezing the Artemia before feeding it to the Hydra will help with the digestion process¹⁴. Place the freshly killed Artemia close to the Hydra in the dish to facilitate fast feeding.
   Note: All subsequent steps are done under a dissecting microscope.
4. Use one pair of forceps to hold the Hydra by the peduncle. Continue holding the peduncle during the process of feeding the Hydra. Using a second pair of forceps, pinch the body column in order to make the Hydra contract.
5. While holding the tips of the second pair of forceps together, tap at the center of the hypostome (the domed structure at the oral end of the animal). This sometimes causes the mouth to open. If the mouth does not open by tapping, puncture the mouth by inserting the forceps into the center of the hypostome and then slightly release the pressure on the tips of the forceps. This should stretch out the mouth opening made by the puncture.
   Note: The Hydra may deflate and collapse when the mouth is opened (Figure 2C). If this occurs, the Artemia may still be inserted into the Hydra. If it is too difficult to do so, move on to the next Hydra and return to the original Hydra some time later and try again. The Hydra tend not to deflate as much during subsequent mouth openings.
6. Quickly pick up Artemia with the forceps and insert them into the Hydra's gastric cavity. Insert as many Artemia as possible until the gastric cavity is full and more Artemia cannot be inserted without damaging the Hydra, accidentally pulling out any Artemia inside, or preventing the mouth from closing. On average, this is 5 - 6 Artemia, however up to 12 have been fed to the larger animals.
   1. If the mouth begins to close, stretch open again with the forceps as described above; however, caution should be taken as any Artemia already inside the animal may begin to come out when the mouth is reopened.
   2. If the Hydra is too small for a whole Artemia, cut the Artemia using a scalpel and feed the Hydra smaller pieces. If the Artemia does not stay inside the Hydra, it may be necessary to press the forceps against the Artemia to keep it inside the Hydra until its mouth closes.
7. Transfer fed Hydra with a glass Pasteur pipette, carefully to avoid accidental burping, to a dish with fresh 50 µg/mL rifampicin in Hydra medium. If an Artemia was expelled from the Hydra while transferring, reinsert it while in the new dish.
   Note: A glass pipette is used to transfer the Hydra, as it has been found that Hydra stick less to glass compared to plastic. The length of the pipette does not matter, but using 5 inch pipettes may be somewhat easier.

3. Burping

1. Burp the Hydra to remove undigested material any time between 8 and 20 h after feeding.
2. Sand down the point of a 30G hypodermic needle with P320 or similar grit sandpaper until the tip is flat.
   Note: A 27G hypodermic needle would also work, but the smaller tip of the higher gauge is preferable.
3. Attach the needle to a disposable 1 mL plastic syringe and fill the syringe with fresh 50 µg/mL rifampicin in Hydra medium.
   Note: Burping a single Hydra takes about 0.05 mL to 0.1 mL of solution. A 1 mL syringe is preferable since controlling the force and volume of the solution coming out is more difficult with a larger volume syringe.
4. Under a dissecting microscope, hold the peduncle of the Hydra with fine point forceps and tap at the center of the hypostome with the needle. If the mouth does not open, insert forceps into the hypostome and open the mouth as described above for feeding.
   Note: All subsequent steps are done under a dissecting microscope.
5. Use the syringe to flush, very gently to avoid blowing away the entire animal, the gastric cavity with the 50 µg/mL rifampicin solution until all debris has been expelled. The needle does not necessarily need to be inserted into the Hydra if the size of the Hydra does not permit. The needle may be held close to the mouth opening and pointed directly towards the mouth.
6. Using a glass Pasteur pipette, transfer burped Hydra to a dish with fresh 50 µg/mL rifampicin in Hydra medium.

4. Quality Control Via Immunohistochemistry

NOTE: The following protocol is adapted from protocols by Shenk, M. A, et al.¹⁸, and Böttger, A¹⁹. All steps are done at room temperature (RT) unless otherwise noted. A nutator can be used for the incubation steps and may improve staining quality. However, if the Hydra get tangled up with one another, all steps can also be performed without.

1. Prepare the blocking solution: 10% fetal bovine serum (FBS) and 1% DMSO in 1x phosphate-buffered saline (PBS). Store this solution at 4 °C until use (steps 4.6 and 4.11). The solution can be stored for 1 - 2 days.
   Note: All solutions used in this protocol must be properly collected as hazardous waste.
2. Relax Hydra in 200 µL of 2% urethane in Hydra medium for 1 min in 1.7 mL microcentrifuge tubes. Use 5 Hydra per tube for each of the 4 conditions: nerve-free, untreated, untreated with no primary antibody, and untreated with no secondary antibody.
   Note: Do not exceed 1 min. The timing here is critical as animals will be in bad shape after spending too much time in urethane.
3. Remove the urethane and fix the Hydra in 200 µL of 4% paraformaldehyde (PFA) in Hydra medium for 15 min.
4. Wash the samples 3 times with 1x PBS for 10 min each.
   Note: All washes with PBS and PBSTx are done with 500 µL of solution.
5. Permeabilize with 500 µL of 0.5% Triton X-100 in PBS for 15 min.
6. Remove the 0.5% Triton X-100 and add 500 µL of the blocking solution. Block the samples for at least 1 h.
7. Dilute the anti-tyrosine-tubulin antibody 1:200 in blocking solution.
   Note: This concentration was determined experimentally. If the signal in the controls is found to be too weak, the concentration may need to be increased. If there is nonspecific binding, the concentration may need to be decreased. Pre-incubation of the antibody may also help reduce nonspecific binding.
8. Remove the blocking solution from the samples and add 200 µL of the primary antibody to the nerve-free Hydra, untreated controls, and untreated controls with no secondary. For the untreated controls with no primary, only add 200 µL of blocking solution without the antibody.
9. Incubate the samples overnight (>12 h) at 4 °C.
   Note: Alternatively, incubate 5 - 6 h at RT.
10. Remove the primary antibody and wash the samples extensively with 0.3% Triton X-100 in 1x PBS (PBSTx).
    Note: Save the primary antibody and store at 4 °C, as it can be reused at least 2 - 3 times.
11. Dilute goat anti-mouse hP secondary antibody 1:500 in blocking solution. Add 200 µL to the nerve-free Hydra, untreated controls, and untreated controls with no primary. For the untreated controls with no secondary, only add 200 µL of blocking solution without the antibody.
    Note: Alternatively, a fluorescent secondary antibody can be used, for which steps 4.13 - 4.17 are not needed. Using a fluorescent secondary saves time and is generally adequate, but the hP secondary provides increased sensitivity. The concentration of secondary was determined experimentally. If the signals in the controls are found to be too weak, the concentration may need to be increased. If there is nonspecific binding, the concentration may need to be decreased.
12. Incubate the samples overnight at 4 °C.
13. Remove the secondary antibody and wash the samples extensively with PBSTx.
14. Prepare 1x PBT: 0.2% bovine serum albumin (weight/volume), and 0.05% Tween20 in 1x PBS. Incubate the samples in 1x PBT for 30 min.
15. Remove the 1x PBT and incubate the samples in 1:1,000 NHS-fluorescein and 1:10,000 H₂O₂ in 1x PBT for 15 min in the dark. From this step on, keep the samples in the dark as much as possible, as the fluorescent signal will decrease as they are exposed to light.
    Note: NHS-fluorescein was prepared following a detailed FISH protocol by King, R. S. and Newmark, P. A.²⁰
16. Wash the samples 3 times with PBSTx quickly, then 2 times for 30 min.
17. Leave the samples in PBSTx overnight at 4 °C to continue washing.
18. Do a few more washes with 1x PBSTx. To visualize cell nuclei, carry out the following optional steps for DNA counterstaining using 4', 6-diamindino-2-phenylindole, dihydrochloride (DAPI). Otherwise, the samples may be imaged now.
19. Dilute 5 mg/mL DAPI stock to a working concentration of 1:500 in PBSTx and add 200 µL to each tube. Incubate the samples for 30 min.
    Caution: DAPI is a known carcinogen. Wear full PPE.
20. Remove the DAPI and wash the samples 2 - 3 times with PBSTx before imaging with fluorescence microscopy.

Results

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...

Discussion

Hydra interstitial cells can be eliminated through a double colchicine treatment^3,4. 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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Colchicine	Acros Organics	227120010
1 mL Syringe	BD	301025
Brine Shrimp Eggs	Brine Shrimp Direct	N/A	Can be purchased locally
Brine Shrimp Hatchery Dish	Brine Shrimp Direct	N/A
60 mm x 15 mm Petri Dish	Celltreat	229663
30 G x 3/4" Hypodermic Needle	Covidien	1188830340	A 27G needle may also be used
2 x Fine-tip Tweezers	Dumont	0109-5-PO
Rifampicin	EMD Millipore	557303
Goat anti-mouse lgG, Pab (HRP Conjugate)	Enzo	ADI-SAB-100-J
Bovine Serum Albumin (BSA)	Fisher	BP9703-100
Scalpel	Fisher	08-920A
Fetal Bovine Serum (FBS)	Gibco	10437028
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Invitrogen	D1306
PBS Tablets	MP Bio	2810305
Monoclonal Anti-Tubulin, Tyrosine	Sigma	T9028
Dimethyl Sulfoxide (DMSO)	Sigma	D2650
Hydrogen Peroxide	Sigma	216763
Paraformaldehyde (PFA)	Sigma	P6148
Triton X - 100	Sigma	T9284
Tween 20	Sigma	P1379
Urethane	Sigma	U2500
Air Pump	Tetra	77846-00
Glass Pasteur Pipette	VWR	53283-916	Length of the pipet does not matter
15 mL Tube	VWR	89039-670
P320 Sandpaper	3M	IBGABBV00397	Can be purchased at local home improvement store