TITLE: A simple axial grafting method for Hydra
AUTHORS: Gmakowski, Callen Hyland

[DESCRIPTION]
This protocol is a low cost and easy to implement method for axial grafting of Hydra. In 1742, Trembley first observed that tissues from two separate Hydra polyps can join when the cut edges are placed in contact (Lenhoff and Lenhoff, 1984). Tissue grafting in Hydra has remained a valuable tool for exploring body axis patterning and cell migration in this anatomically simple system. Ethyl Browne observed that grafting of a single tentacle onto the body column induced the development of a secondary body axis, the first demonstration of a developmental organizer in animals (Browne, 1909). Shimizu (2012) has reviewed the contribution of lateral grafting experiments to understanding body axis patterning in Hydra.

Various techniques have been developed for immobilizing tissues in close contact with each other during grafting. MacWilliams (1983) describes a method where Hydra and Hydra tissue rings are threaded onto fine glass needles held in place by wax. Other published methods describe threading Hydra tissues onto fishing line (Sugiyama and Fujisawa, 1978) and holding them in place with either pieces of Parafilm (Shimizu and Sawada, 1987) or pieces of polyethylene tubing (Forman and Javois, 1999).

Here we describe a simple method for axial grafting which allows rapid and reliable creation of multiple grafts. This method uses fishing line "skewers" attached to a microscope slide to align tissue rings cut from the body column. The slide holder petri dish allows the slide to be removed for cleaning or replaced. Attaching the skewers to a microscope slide was inspired by the lateral grafting method described by Wang and Collins (2020).

Thank you to Rob Steele for assistance with this protocol.

[BEFORE_START]
Collect all materials and review protocol. Grafting slides and slide holder should be made the day before starting the protocol.

[GUIDELINES]
For general guidelines for maintaining, working with and disposing of Hydra, please see Low Cost Methods for Hydra Care.
Hydra should be starved at least 48 hours before starting this experiment.

[STEPS]
SECTION: Prepare grafting slides
1. Working under the dissecting microscope, cut the fishing line into 10 mm segments using the razor blade or scalpel. Cut the fishing line at an angle to create a sharp point (Figure 1). We usually make slides with 10-12 fishing line "skewers" each.

 
SECTION: Prepare grafting slides
2. Prepare the two-part epoxy according to the manufacturer's instructions. Do the next two steps quickly before the epoxy dries.
SECTION: Prepare grafting slides
3. Use a micropipette tip or wooden toothpick to smear a stripe of epoxy on the edge of a microscope slide (Figure 2).
 
 
SECTION: Prepare grafting slides
4. Use forceps to place the fishing line skewers in the wet epoxy so that the sharp points are oriented perpendicular to the edge of the slide and parallel to each other (Figure 3). The pieces of fishing line can move around while in the epoxy is still wet and might need to be adjusted.

 
SECTION: Prepare grafting slides
5. Allow the epoxy to cure overnight.
SECTION: Hydra axial grafts
12. Fill your other petri dish with Hydra medium. Working under the dissecting microscope, use the scalpel to cut the Hydra horizontally through the body column to make the pieces that will be grafted together. The figures demonstrate a simple graft with a head, foot, and body column from three different polyps.
SECTION: Hydra axial grafts
13. Use the Pasteur pipette to transfer the tissue pieces to the petri dish with the grafting slide. Use forceps to slide each tissue piece onto the fishing line skewer.

 

SECTION: Hydra axial grafts
11. Place the grafting slide in your 100 mm petri dish. You can use either the custom slide holder or a piece of double-sided tape to hold the slide to the bottom of the dish. Fill the petri dish with Hydra medium so that the grafting slide is submerged.
SECTION: Hydra axial grafts
14. Use forceps to slide a small piece of filter paper or Kimwipe onto the end of the fishing line (Figure 6). This will keep the tissue pieces from coming off the end of the fishing line. Use the filter paper to gently press the tissue pieces together.

 

 
SECTION: Hydra axial grafts
16. Use forceps to remove the filter paper or Kimwipe, then gently slide the grafted Hydra off the end of the fishing line. Use the Pasteur pipette to transfer the grafts to another petri dish or plate with Hydra medium. 

Figure 7 shows the result of grafting together a head and foot piece of a transgenic Hydra expressing DsRed in the ectoderm with a body column piece from a non-fluorescent AEP-HS Hydra three hours after the tissue pieces were placed on the fishing line.

 
SECTION: Hydra axial grafts
15. Allow at least 2 hours for the tissue pieces to form the graft.
SECTION: Prepare slide holder
6. Use a razor blade to cut off lids of 0.65 mL microcentrifuge tubes. The slide holder in the figure is made with three microcentrifuge tube lids, but you could also use four to make the slide holder more secure.
SECTION: Prepare slide holder
7. Use a razor blade to cut off half of the inside part of the lid (Figure 4).

 
SECTION: Prepare slide holder
8. Prepare the two-part epoxy according to the manufacturer's instructions. Do the next step quickly before the epoxy dries.
SECTION: Prepare slide holder
9. Use a pipette tip or toothpick to smear epoxy on the flat side of the microcentrifuge lids and position the tube lids in the 100 mm petri dish to secure your microscope slide (Figure 5). Use the same type of microscope slide to position the tube caps as you used to make the grafting slides. We have found that slide brands differ slightly in their dimensions.

 
SECTION: Prepare slide holder
10. Allow the epoxy to cure overnight.