Table 2. Activity suggestions.
A. Section: “Maintaining, Feeding, and Amplifying Planarians”
Goals: Model maintenance; simple experiment set up; data collection; acquisition of knowledge about planarian ability to deal with the presence or absence of foodWhat happens to worms starved for one or two months when compared to well-fed worms?
Do the worms maintain proper body proportions after prolonged feeding and/or starvation?
Can you see the gut branches if you add food coloring to the food?
How many days after amputation do worms start eating again? (Note: Do not put too much food in the dish while the worms are regenerating as they may become sick. A sick worm develops dorsal lesions (Fig. 3C) and is no longer able to regenerate.)
B. Section: “Amputation Strategies and Regeneration Time Courses”
Goals: Model maintenance; experiment set-up; data collection; acquisition of knowledge about planarian ability to regenerate after different amputation strategies and other conditionsCan you visualize all the regeneration steps described in the “Results: Regeneration” section?
What is the smallest piece of tissue that can regenerate a complete animal?
Can you produce any regeneration abnormalities from certain types of cuts (such as double-headed or double-tailed worms)?
What is the behavior of the regenerating fragments over time (i.e., speed of swimming, swimming direction, light avoidance)?
How might the environment (i.e., light exposure or temperature changes) affect the regenerative responses of these animals?
C. Section: “WISH: A Method to Visualize Gene Expression”
Goals: Acquisition of basic molecular biology knowledge and abilities; acquisition of knowledge of transcription/translation processes and correlation between gene expression and cell featuresHow does the distribution of neoblasts correlate with the ability of the different regions of the body to regenerate a new animal?
Can a ribo probe specific to one species also work with other species?
Are you interested in a specific gene? Can you find the homologous sequence in a planarian transcriptome database and design specific primers? (Note: Consult the tools and tutorials available on the website http://cuttingclass.stowers.org.)
How can you clone a gene of interest and design a ribo probe using the suggested sequence or sequences?
D. Section: “RNAi: Disruption of Gene Function”
Goals: Acquisition of basic molecular biology knowledge and abilities; acquisition of knowledge of both the importance of mRNAs for normal cell functioning and the potential problems caused by missing, truncated or mutated mRNAs/genes.What is the minimum number of feedings needed to obtain the ß-catenin(RNAi) phenotypes?
Are ß-catenin and odf2 required for homeostasis, regeneration, or both? (Note: The RNAi-fed worms can be split into two groups: the first for monitoring homeostasis phenotypes in intact worms, and the second for following the regeneration phenotypes after amputation.) Does the phenotype emerge at the same time in both groups, and what might this mean?
If ß-catenin(RNAi) animals are kept alive, how many heads can they develop?
If odf2(RNAi) animals are kept alive without additional dsRNA feeding, do they ever start to swim straight again?
Which other genes would you like to try to knock down? What phenotype do you expect? Can you find homologous sequences in planarians and design specific primers using the available transcriptomes? (Note: Consult the tools and tutorials available at http://cuttingclass.stowers.org.)
Can you clone your own gene and transform the bacteria or synthesize and purify the dsRNA using the suggested sequences or sequences you may be interested in? (Note: Look at the tools and tutorials available at http://cuttingclass.stowers.org.)
What happens if you try to feed Girardia sp. or D. dorotocephala using the “food with dsRNA” protocol?
What happens if you try to feed P. morgani or P. gracilis using the “food with bacteria” protocol?
Which is the protocol that induces the phenotype fastest, “food with bacteria” or “food with dsRNA”? (Note: Feed one group of worms, Girardia sp. or D. dorotocephala, with “food with bacteria” and one group of worms, Girardia sp. or D. dorotocephala, with “food with dsRNA,” and monitor the emergence of the phenotype.)
B and C (above): Visualization of Gene Expression (WISH) after Amputation
Goals: Multiphase experiment set-up; acquisition of ability to correlate different regeneration stages with planarian internal anatomy and gene expression.What is the internal organization of the worms during regeneration? How are the organs remodeled in the regenerating fragments?
What is the internal organization of abnormal animals produced by amputation?
C and D (above): Visualization of Gene Expression (WISH) after Disruption of Gene Function (RNAi)
Goals: Multiphase experiment set-up; acquisition of ability to deeply investigate an RNAi phenotype, looking at planarian internal anatomy and gene expression.After ß-catenin(RNAi), what is the distribution of axial patterning markers?
After ß-catenin(RNAi), does the planarian with two heads also have two brains?
After odf2(RNAi), are there any neural defects?
After odf2(RNAi), are there any muscular defects?