Ines and Shiloh

Abstract:

Video Chen_Chih L 4_22_2022 1_37_21 AM.mp4

Introduction

Dugesia dorotocephala, or planaria, is an aquatic flatworm known to display superb regeneration abilities. Once a head of the planaria is cut, it grows back a head. It has been found that regeneration depends on the presence of stem cells called neoblasts, which make up one fifth of their body. Once cut, cells around the cut multiply forming a blastema then differentiate to form the correct limb.

Beta-catenin is a protein with a low concentration in the head and high concentration in the tail of the planaria. It has regulates multiple processes such as cell fate speciation, differentiation, and proliferation, In this experiment, we used RNA interference to reduce beta-catenin production. Planaria ingest bacteria with the double-stranded RNA plasmid that is complementary to the beta-catenin mRNA which will then enter their cells. In their cells, the dsRNA binds to the RISC complex that unzips it and goes "searching" for its complementary mRNA. It then binds to it and cuts it which prevents the now cut Beta-catenin mRNA from getting translated into the Beta-catenin protein.

It has been discovered that, when beta-catenin production is suppressed mirroring its concentrations in a planaria's head, planarias grow a head instead of a tail when its tail is cut. This suggests that Beta-catenin has a key role in planaria regeneration, most likely because of its involvement in the many cell processes including differentiation. Consequently, it is expected that after a planaria’s head and tail are cut, its body will grow two heads.

Why study regeneration?

In regenerative medicine, scientists seek solutions to regenerating tissues and organs that aren't naturally replaced in the human body. These instances include spinal cord injury as well as loss of neurons from stroke or other neurodegenerative diseases such as Parkinson's and Alzheimer's. By studying planaria regeneration, researchers hope to apply potential mechanisms for regeneration to human health matters.

Why study planaria?

Planaria display remarkable regenerative abilities in all tissues. They also share with vertebrates all of the major developmental signaling pathways responsible for the bilateral body plan. Planaria are also small and generally easy and inexpensive to rear. There has also been an extensive body of literature on planaria.

Why RNAi?

RNA interference (RNAi) is a technique used to identify specific gene function. Double-stranded RNA is inserted into the cell to silence mRNA expression of the targeted gene. In this case, we silence the B-catenin mRNA expression by cutting it thus preventing it from getting translated into the Beta-catenin protein. This was done in order to infer the Beta-catenin protein's effect on planaria regeneration. Gurley et al. finds that reducing the concentration of Beta-catenin produces a two-headed phenotype in planaria.

Methods:

Illustration of the process to prepare the food

Making Cells Competent + Transformation

After receiving our HT115 cells, it was time to make them competent then transform them to add the B catenin plasmids to their DNA. We conducted a total of three transformations: One with B catenin plasmid 2, one with B catenin plasmid 3, and a control with an OPSIN plasmid.

Plates containing Bacteria transformed to add the B-catenin 2 (right) and the B-catenin 3 (left) plasmids to their DNA.

The transformed bacteria was then plated and incubated at 37°C. Attached to the left are pictures of the plates after one day. After a few more days of incubation, a colony from each plate ( was chosen and grown overnight in LB media.

Liver paste getting filtered

Making the Food

1mL of the culture was then grown in 2xYT media. While the bacteria was incubating, connective and vascular tissue was removed from the calf liver that was then blended and filtered using a sieve (see picture to the left). When the cultures had OD600 values between 0.4 and 1, they were induced with IPTG and left to incubate for 3h30.

Frozen tubes containing the liver paste-water solution mixed with the bacteria pellets.

The cultures were then centrifuged, then the pellet was diluted in water to remove residual salts (which can be unappetizing for planarians). The tubes were centrifuged again and the water was disposed of. A mix of the liver paste with 10 percent of water were added and mixed with the pellet. The tubes were then placed in the freezer for later use.

Petri dishes with spring water and planaria

Feeding Time

5 planaria were put in each petri dish (control with liver paste only, OPSIN, B-catenin 2, B-catenin 3) for feeding.

Petri dishes covered by tin foil to create a dark environment.

Before feeding, the planaria are left in the dark (under aluminium foil) for an hour.

Planaria feeding on the liver paste-water-bacteria mix during the first feeding (14/04)

A tube of food for each petri dish was left to thaw for an hour. A micropipette was then used to transfer the food to the petri dishes. Planaria were left to eat for two hours. The feeding was repeated tso other times at two days interval of each other.

Cutting

The planaria were then cut in two places: just below the head and above the tail and left in the dark to regenerate.

Results

Beta-catenin 3

Beta-catenin 2

Opsin

Control

Control bacteria have not been inserted with plasmids. Opsin plasmids also serve as a control to ensure that differences observed from planaria growth are not a result of mishandling the transformation process. Beta-catenin 2 and 3 are both plasmids that allow the HT115 bacteria to produce beta-catenin double-stranded RNA, which cuts the mRNA and prevents the production of the beta-catenin protein in the planaria. They are expected to produce similar results to each other.

Five days after cutting off the head and tail of each planaria, we observed that the control and opsin planaria grew back a head where we cut off the head and grew back a tail where we cut off the tail. The beta-catenin 2 and beta-catenin 3 bacteria grew back two heads, both where we cut off the head as well as where we cut off the tail. Even though the presence of a head is barely discernible at the beginning stage of growth, we are able to define head growth as transparent tips and observable photoreceptors, which appear as two black dots and move the planaria according to the amount of light that it detects.

Sources:

Stowers.org. “Planarian Educational Resource: Where Cutting Class Is Required | Planarian Educational Resource,” 2022. https://cuttingclass.stowers.org/en/node/121409.

Gurley, Kyle A., Jochen C. Rink, and AlvaradoAlejandro Sánchez. “β-Catenin Defines Head versus Tail Identity during Planarian Regeneration and Homeostasis.” Science 319, no. 5861 (January 18, 2008): 323–27. https://doi.org/10.1126/science.1150029.

‌Petersen, Christian P., and Peter W. Reddien. “Smed- β Catenin-1 Is Required for Anteroposterior Blastema Polarity in Planarian Regeneration.” Science 319, no. 5861 (January 18, 2008): 327–30. https://doi.org/10.1126/science.1149943.

Alvarado, Alejandro Sánchez. "Q&A: What is regeneration, and why look to planarians for answers?" BMC Biology, no. 10 (2012). https://bmcbiol.biomedcentral.com/articles/10.1186/1741-7007-10-88

‌Stowers.org. “Planarian Educational Resource: Where Cutting Class Is Required | Planarian Educational Resource,” 2022. https://cuttingclass.stowers.org/en/node/121409.

RNAi Therapeutics | How RNA Interference Works | Alnylam® Pharmaceuticals. “RNAi Therapeutics | How RNA Interference Works | Alnylam® Pharmaceuticals,” 2018. https://www.alnylam.com/our-science/the-science-of-rnai.