Generation and Long-term Maintenance of Nerve-free Hydra

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.