Sam

At the start of my project, I was researching possible regeneration inhibitors in G. Dorotocephala and one of the first-mentioned inhibitors was an over-the-counter supplement that anybody can buy at a local pharmacy. Melatonin is a fairly simple compound (Img. 1) that can be either endogenous, produced inside the body, or exogenous, ingested, or injected into the body to supplement or replace the endogenous production of the same hormone. In humans, melatonin is commonly used to treat temporary insomnia (jet lag, etc.). It is produced and secreted by the pineal gland in the brain and binds to melatonin receptors.(2) This, in turn, leads to a signaling pathway in the human brain that causes you to feel tired.

In Planarians, specifically G. Dorotocephala based on results from a study done in 1991, it is suggested that the presence of endogenous melatonin in the anterior ends of planarian worms, may be partly responsible to the regulation of regeneration polarities, in a similar way to the Wnt-signaling process we studied earlier this year. (3) Understanding the process of stem-cell reproduction and regeneration, especially in planarians, could lead to a better understanding of how to apply stem cell regeneration treatments to human medical care. So, in order to gain a better understanding of the effect that melatonin has on the regeneration of planarians, I decided to recreate a similar experiment to the one cited above.

First, three concentrations were decided on for the different runs. Preliminary tests showed an almost 100% mortality rate in concentrations higher than 0.49 mmol (melatonin)/1 liter of water. So, out of caution, the final concentrations used in testing were 0.2 mmol/liter, 0.1 mmol/liter, and a 0.0mmol/liter negative control. Before the G. Dorotocephala were amputated, 6 individuals were soaked in each concentration overnight.

During amputation, three G. Dorotocephala from each trial were amputated, using a scalpel, at the anterior end (the head), and the remaining three were amputated at the posterior end. To prevent confusion during observations, the posterior and anterior amputees from each run were stored in separate solutions, both solutions had been taken from the original soaking solution. The G. Dorotocephala were left soaking in these solutions for 1.5 weeks, occasionally they were observed under a microscope.

During the growth period, differences were noticed in the generation of blastemas in the G. Dorotocephala individuals soaked in melatonin and the blastema growth on the individuals soaked in spring water. In individuals, soaked in the two melatonin solutions, the blastema generation was notably smaller (figures 1,2, and 3) in comparison to the blastema growth in the G. Dorotocephala soaked in spring water. (Fig. 3) The surviving control individuals displayed full recovery from the amputations, the blastemas had transformed into functioning posterior and anterior portions of the worm.

Interestingly, the introduction of melatonin seemed to have a greater inhibitory effect on the growth of posterior blastemas than the anterior, directly contrasting with the findings of the 1991 experiment. (3) posterior blastemas displayed little growth when compared to anterior blastemas from the same run, 0.2 mmol/liter. (fig. 1)

Another interesting thing to note, was the slightly higher mortality rate, in the 0.1 mmol/liter run and the negative control. both of these runs only resulted in four planarians, after 1.5 weeks of growth. However, this result can probably be attributed to an unsterilized environment or rough handling post-amputation.

In a similar way to the signaling polarity described in a study done on RNAi of different planarian signaling genes (4), I believe that endogenous melatonin secretion inside of G. Dorotocephala is responsible for regulating basic regeneration functions. When an overload of melatonin is detected, the exposed individual stops the regeneration of posterior blastemas.

Endnotes:

1. Unknown Author, “Melatonin-Wikipedia”, Wikipedia, Unknown Date, https://en.wikipedia.org/wiki/Melatonin#/media/File:Melatonin.svg

2. Unknown. “Melatonin.” NHS Choices, NHS, 8 Nov. 2019, www.nhs.uk/medicines/melatonin/

3. Yoshizawa, Yasuhiro, et al. “Inhibition of Planarian Regeneration by Melatonin.” Hydrobiologia, Kluwer Academic Publishers, Dec. 1991, link.springer.com/article/10.1007/BF00027578#:~:text=Melatonin%2C%20which%20is%20a%20substance,japonica%20japonica%20Ichikawa%20et%20Kawakatsu.&text=Melatonin%2C%20thus%2C%20appears%20to%20play,positional%20information%20in%20that%20process.

4. Gurley, Kyle, et al. “Beta-Catenin Defines Head Versus Tail Identity During Planarian Regeneration and Homeostasis.” Science Magazine, vol. 319, 18 Jan. 2008, pp. 323–327.

Planarian species have a fairly unique ability to regenerate almost any part of the body if it is cut off from the rest. In fact, if both parts are kept in spring water after the amputation, both will regenerate into fully functional individual worms.

This regeneration property has evolved through the stem cells of the Planaria, specifically c-Neoblasts. These cells function as pluripotent stem cells and can proliferate into any kind of tissue needed for regeneration. In humans, this regeneration is not possible, since we no longer have usable pluripotent stem cells after all of our tissue has been created once. All of our once pluripotent cells have differentiated into specialized tissue-producing stem cells. Planaria, including G. Dorotocephala, have evolved a regenerative system, using long-lasting pluripotent stem cells, that can proliferate into new tissue, to regenerate vast parts of their bodies. (1) it is logical to assume that individual G. Dorotocephala with the ability to regenerate, were selected for because they could survive longer, and could pass down their own genes in multiple different ways.

Planarian stem cells have evolved, to a point where they can recreate any of their own bodily tissue, and in many worms, intentional splitting is used as a method of asexual reproduction. If worms with the regenerative neoblasts began asexually reproducing, it is possible that this gene pool quickly overtook the other possible combinations, until the regenerative properties were an inherent part of the species.

In addition to the stem cells, G. Dorotocephala have developed complex signaling pathways, like Wnt-signaling, to account for regeneration polarity (knowing which end is anterior and which is posterior). (2) In addition to Wnt-signaling, it seems that endogenous melatonin concentration may have an effect on posterior regeneration, however, the exact pathway is unknown, and response is unknown.

If we can continue to experiment on the regenerative properties of G. Dorotocephala, then we may be able to better understand the evolution of human stem cells, and why the two species have taken such drastically different evolutionary pathways. We may also be able to discover new ways to apply our own stem cells in medical therapies, or procedures.

Endnotes:

1. Wagner, Daniel E, et al. “Clonogenic Neoblasts Are Pluripotent Adult Stem Cells That Underlie Planarian Regeneration. .” Science Magazine, vol. 332, 13 May 2011, pp. 811–816.

2. Gurley, Kyle, et al. “Beta-Catenin Defines Head Versus Tail Identity During Planarian Regeneration and Homeostasis.” Science Magazine, vol. 319, 18 Jan. 2008, pp. 323–327.