Planarian Head Regeneration Lab Report

Samantha Rogers & Sarah Grill
July 6, 2015
Stem Cell Biology and Regenerative Medicine

Analyzing the Effects of PZQ on Planarian Head Regeneration

Abstract This paper focuses on the question of whether or not the drug PZQ (Praziquantel) has an effect on the double-headed formation of planarians. As the rest of the report will describe in more detail, we used multiple concentrations of PZQ reconstituted in 95% ethanol to observe its effects on planarians cut in a shape that promoted double headed growth. We used concentrations that we believed to be above, at, and below the optimal concentration of PZQ. In addition, we did tests on planarians with concentrations of plain ethanol to rule it out as a confounding variable. Our conclusion once
the experiment finished was not ideal and did not support our hypothesis about the optimal concentrations of PZQ. This was due to the fact the concentrations appeared to have absolutely no effect on the double headed formation on our planarians since no planarians actually had two headed growth.

Introduction Planarians are a type of flatworm that have long been known for their regenerative abilities. This experiment sought to find out what the addition of PZQ would have on these abilities, specifically relating to their regeneration of two possible heads. As mentioned, planarians have been used in research for many years because of their ability to regenerate, which is due to the fact that they have an extraordinary amount of stem cells that are ready to differentiate into whatever part of their body is needed. The stem cells that they use are called neoblasts, which are pluripotent adult stem cells, meaning they are able to differentiate into any of the three germ layers. These stem cells compose around 20% of the cells in a planarians body, which is a much larger percentage of stem cells in proportion to alternate cells than in nearly any other animal. They use these stem cells to asexually reproduce, creating perfect cloned offspring by literally tearing themselves in half to make a copy of themselves.
Praziquantel, or PZQ, is a drug that we used in this experiment that is an isoquinoline drug that has been shown to block certain pathways in planarians, encouraging two headed growth when used in optimal concentrations.
This information led us to our hypothesis that if we placed planarians that were cut in a T-shape in an 80 µm PZQ concentration, they would develop two heads a greater part of the time compared to those placed in dishes containing 40 µm PZQ concentration and 120 µm PZQ concentration.
We formed our hypothesis based on the fact that it has been shown that 70-90 µm PZQ concentration is the optimal value to two-headed planarian growth. Our overall purpose of this experiment was to see if our hypothesis was correct, and that PZQ truly did increase double head formation when added in the correct concentration to planarians. Our experimental design included 3 concentrations of PZQ and 3 equal concentrations of 95% ethanol. Our independent variable in this experiment was the concentration of the solution our planarians were in, and the dependent variable was whether or not the cut planarians grew two
heads.

Planarian Head Regeneration Data

40 µm PZQ
80 µm PZQ
120 µm PZQ
40 µm ethanol
80 µm ethanol
120 µm ethanol
Double Headed
0
0
0
0
0
0
Single Headed
2
2
2
2
2
2
*This figure shows planarian head regeneration after 48 hours in labeled solutions and 48 hours in pure water.

Materials & Methods
In our experiment, a six well sterile container was used as the vessel to hold our planarians. These six wells were filled with six different solutions as follows: 80 µm PZQ reconstituted in 95% ethanol (.001 mL stock solution), same amount of plain 95% ethanol, 120 µm PZQ reconstituted in 95% ethanol (.0015 mL stock solution), same amount of plain 95% ethanol, 40 µm PZQ reconstituted in 95% ethanol (.0005 mL stock solution), and same amount of plain 95% ethanol. Enough water was added to each well to make the total amount of liquid 2 mL. The amounts of water were respectively, 1.999 mL H20, 1.9985 mL H20, 1.9995 ml H20. This ensured that every planarian had the same amount of liquid to regenerate in, removing it from our list of possible confounding variables.
PZQ was the independent variable in our experiment, which means that all other substances had to be completely planned and observed, ensuring that no other substance could be a factor on our dependent variable. This is the reason ethanol was put into three of the six wells as controls. If the control planarians were put into plain water, the results could not be conclusive as to whether the regeneration patterns of the planarians later observed were due to the PZQ or the ethanol.
Once we had our wells prepared, we added the cut planarians. The cut we decided to make was T-shaped, cutting horizontally across the planarians to remove the head, and slicing around 1/3 of the way down the body. We cut them in this fashion because it seemed to be the best way to promote two headed growth, which is what our experiment is centered around. We added two of these cut planarians to each of our six wells. After 48 hours in their original solution, we changed the planarians conditions to pure water, and recut them ⅓ way down their abdomen since they all appeared to be growing back with only one head. We did this to try and further promote two headed growth.
Results
As you can see in the chart, our results did not support our hypothesis, or any hypothesis, that expected PZQ to have an effect on the planarian regeneration.
Out of all 12 worms that we tested with PZQ and with ethanol, not one grew two heads, even after the second cutting 48 hours after their original dissection.

Discussion The results did not provide an accurate answer to our hypothesis as it shows that the PZQ had no effect on the planarians two headed growth. We know this to be inaccurate because it is known that the PZQ does have an effect on double headedness when used correctly in certain concentrations according to reputable sources.
Our findings could not agree with what other experiments have shown due to flaws in our experiment. Our T-shaped planarian cuts were not perfect so they all had to be recut later on which was not ideal. The process of transferring our planarians back and forth to their petri dish after being cut was very hard on the planarians as it required them to be pipetted up. This pipetting also affected our PZQ concentrations as it caused unequal amounts of spring water to be added to the petri dishes. In order for the experiment to provide more accurate results more planarians should be used and a more delicate and efficient transferring process would need to be
found.

Literature Cited
El-Showk, Sedeer. "Unravelling How Planaria Regenerate." Nature.com. Nature Publishing Group. Web. 4 July 2015.
http://www.nature.com/scitable/blog/accumulating-glitches/unravelling_regeneration_in_planaria