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.
