First I came up with two questions that seemed interesting, then after thinking some more I realized that I have always been interested about Planarians, and it is a related topic to developmental genetics. Then reading into planarian regeneration in depth, I managed to come up with a testable hypothesis which tests the possibility for an inhibitory long distance signalling mechanism during regeneration.

I sent you my final project in the email. I hope that it got sent correctly because my internet got disconnected !!

Also thanks for the awesome course 🙂

An inhibitory signalling mechanism is involved in regeneration of planarian eye spots

Question: How is loss of eye spots signalled to the neoblasts to produce optic cup progenitors?

Introduction:

Planarians have been used as a model organism in studies about regeneration for many years due to their astonishing regenerative abilities (Newmark 2001). These flatworms are able to regenerate their full body from a small piece that was cut off from the original planarian (Newmark 2001). This process requires the separated body portion to somehow determine the polarity of the original body and reconstruct itself with the body organs in the right locations (Newmark 2001). Many studies have been done to determine this process in great details and different models have been proposed . The most agreed upon model involves the use of concentration gradients to set up the body axis and determine wound polarity (Newmark 2001). This model also leads to the suggestion of a morphogenic gradient in the embryos during development (Newmark 2001). The mechanisms suggested for this regenerative ability involves the use of neoblasts, cells that possess abilities similar to stem cell in mammals and other organisms (Newmark 2001). During wound regeneration, a blastema is formed with accumulation of undifferentiated cells (neoblasts) (Newmark 2001). One of the unanswered questions

regarding regeneration is how wounding induces regeneration in neoblasts. To answer this question, we will focus on a much specified tissue type, the photoreceptors in the planarian head region. Planarian eyes can regenerate from tissues that are positioned far away from the head region (Newmark 2001). These photoreceptors are formed of a pigment cup (pigment cells), photoreceptor neurons and rhabdomeres (Newmark 2001).

In a study done by Lapan et.al (2011), two genes were identified that were invovled in the formation of optic cups and formation of optic cups occurred after the migration of progressively differentiated progenitors from the neoblasts. This study also suggested that there is a long distance signalling mechanism involved in the induction of progenitors. Another study done by Rand et al. (1926), also provides evidence for a long distance signalling mechanism. In this study they grafted a head excised just behind the eyes of one animal onto the side of the anterior portion of the body of another, then they removed the original head, the regeneration of a new head seemed to become inhibited and the grafted head swung into the body axis. A study was done by Kobayashi et al. (2008) on an eye defective mutant, mensashi, to regenerate eye pigment cells after irradiation (destroys neoblasts) and transplantation of neoblast-rich fraction onto the mutant. In this study, they were able to restore the eye pigment cells in the mensashi and this may suggest that there was a lack of an inhibitory signalling mechanism towards the neoblasts in the mensashi mutants due to having no eye spots (Kobayashi 2008). Finally, a study done by Wenemoser et al. (2010) suggests that injury leads to two miotic peaks in the body. The first mitotic peak is a body-wide response to any injury and the second mitotic peak is a localized neoblast response when the injury causes loss of tissue

(Wenemoser 2010). This second response causes the recruitment of neoblasts to the wound site (Wenemoser 2010). Therefore it is possible that the key determinant to the second mitotic peak is the absence of tissue (Wenemoser 2010). The neoblast accumulation and migration around the wound site suggests that regeneration initiation may involve a signal from injuries (Wenemoser 2010). Based on this evidence, I am proposing the following hypothesis: A long distance signalling mechanism involving changes in concentration gradient of a morphogen(s), which is produced specifically by the eye spots, is responsible in inhibiting the production of optic cup progenitors by the neoblasts closest to the eye spots. My prediction for this hypothesis is that the presence of the eye spots is inhibiting the regeneration of new eye spots by the neoblasts through a long distance signalling mechanism. This question fits into the broader picture of developing a model for planarian regeneration. This model is important because it gives us insight into the process of regeneration and stem cells as there seems to be a parallel between planarian stem cells and the stem cells in mammals (Newmark 2001). This question can give us the opportunity to address problems in development and stem cell differentiation. Another potential impact of this question is gaining a deeper understanding of injury and wound signalling that could also apply to humans. Finally, the findings for this question can be used in stem cell research and health care

Experimental design:

To test the possibility of an inhibitory signal and a concentration gradient produced by the eye spots, we will have 4 treatments. First treatment is to surgically remove the eyespots from a planarian, then transplant them into different locations on the planarian

body and measure the rate of regeneration of new eye spots at each. The second treatment would be to surgically remove the eyespots and then transplant them into their original location; this is a control treatment to observe the effects of moving the eye spots into a different location. The third treatment, also another control, is to surgically remove the eye spots and then measure the rate of regeneration of new eye spots. The fourth treatment would be performed on a ‘menashi’ mutant (unable to form functional black eye spots but forms white spots that contain most of the eye cells), in which the white eye spots are surgically removed and transplanted into different locations on the body and the regeneration time for new eye spots is measured. In the case that the transplantation experiments fail and the eye spots get reabsorbed, a second type of experiment could be performed with mutant planarians with more than 2 eyes (Kyobayashi 2007). In this experiment, the ectopic eye spots could be removed and the regeneration rate of new eye spots could be measured. The tissue transplantation protocol can be found in the study published by Guedelhoefer et al. (2012).

Discussion of possible results:

If eye spots (or the tissue around it that is carried with the eye spots during transplantation) produce a specific inhibitory signal to show their presence and this signal is responsible for inhibiting the neoblasts to produce optic cup progenitors, then the planarians and also the menashi mutants with transplanted eye spot should have a slower eye regeneration rate than the control planarian with completely removed eyes. Furthermore, if this signal is in the form of a concentration gradient, then the planarians that have their eye spots transplanted into a closer to the original eye position should have

a slower regeneration rate. If a slower regeneration rate is observed in these two cases, then this will suggest that the eye spots (or the tissues around it) are producing an inhibitory signal towards the neoblasts to stop the production of eye progenitors before an injury.

Resources:

Guedelhoefer IV. O., Alvarado. A. Planarian Immobilization, Partial Irradiation, and Tissue Transplantation. J Vis Exp. 2012; 66: 405.

Kyobayashi. C., Saito. Y., Ogawa. K., Agata. K.Wnt signaling is required for antero- posterior patterning of the planarian brain. Elsevier. 2007; 306:2.

Kobayashi. K., Hashiguchi. T., Ichikawa. T., Ishino. Y., Hoshi. M., Matsumoto., M. Neoblast-enriched fraction rescues eye formation in eye-defective planarian ‘menashi’ Dugesia ryukyuensis. Development, Growth & Differentiation. 2008; 50 (8).

Newmark, P., Alvarado. A. Regeneration in Planaria. Nature Publishing Group. 2001.

Rand. H., Browne. A. Inhibition of regeneration in planarians by grafting: technique of grafting. PNAS. 1926; 12:9

Wenemoser. D., Reddien. P. Planarian regeneration involves distinct stem cell responses to wounds and tissue absence. Elsevier. 2010; 344:2.

TEMPLATE FOR PROJECT OUTLINE

(please type!)

 Student’s name: Kattie Sepehri

Topic chosen: Planarian regeneration

SPECIFIC QUESTION: How is loss of eye spots signalled to the neoblasts to produce optic cup progenitors?

HYPOTHESIS:A long distance signalling mechanism involving changes in concentration gradient of a morphogen(s), that is produced specifically by the eye spots, is responsible in inhibiting the production of optic cup progenitors by the neoblasts closest to the eye spots.

EVIDENCE ON WHICH THE HYPOTHESIS IS BASED (INCLUDE REFERENCES):

• S., Reddien. P. dlxand sp6-9 Control Optic Cup Regeneration in a Prototypic Eye, PLOS. 2011;7.
• Identified two genes that make TFs, expressed specifically in the optic cup.
• RNAi of these genes prevented the formation of optic cups
• Optic cup formation occurred after migration of progressively differentiated progenitor cells from the neoblasts.
• Suggests that there might be a long distance signaling mechanism

• H., Browne. A. Inhibition of regeneration in planarians by grafting: technique of grafting. PNAS. 1926; 12:9.
• Graft a head excised just behind the eyes of one animal onto the side of the anterior portion of the body of another, remove the original head, the regeneration of a new head seems to become inhibited and the grafted head swung into the body axis.

• K., Hashiguchi. T., Ichikawa. T., Ishino. Y., Hoshi. M., Matsumoto., M. Neoblast-enriched fraction rescues eye formation in eye-defective planarian ‘menashi’ Dugesia ryukyuensis. Development, Growth & Differentiation. 2008; 50 (8).
• An eye defective mutant, mensashi, was able to regenerate eye pigment cells after irradiation and transplantation of neoblast-rich fraction.
• May suggest a lack of an inhibitory signalling mechanism that is not present in the mensashi mutants due to having no eye spots.

·         Wenemoser. D., Reddien. P. Planarian regeneration involves distinct stem cell responses to wounds and tissue absence. Elsevier. 2010; 344:2.

o   Suggests that injury leads to two miotic peaks in the body. The  first mitotic peak is a body-wide response to any injury and the second mitotic peak is a localized neoblast response when the injury causes loss of tissue. This second response causes the recruitment of neoblasts to the wound site. Therefore it is possible that the key determinant to the second mitotic peak is the absence of tissue.

o   The neoblast accumulation and migration around the wound site suggests that regeneration initiation may involve a signal from injuries.

PREDICTION(S):

• The regeneration rate of the eye spots will change/stay the same when more eye spots are removed or displaced due to a possible inhibitory signal that is produced by the eyes that stops the formation of progenitors by the neoblasts.

EXPERIMENTAL APPROACH TO TEST PREDICTION (INCLUDE ANY DETAILS THAT YOU HAVE WORKED OUT SO FAR):

First approach: To test the possibility of a signal and a concentration gradient produced by the eye spots, we will surgically remove the eyespots and at the same time we will transplant eye spots into a different location. If eye spots have a specific signal to show their presence and this signal is responsible in inducing the optic cup progenitors, then the planarian with transplanted eye spot should have a slower eye regeneration rate.

Second approach: Another method to test the presence of a signal is to remove ectopic eye spots from mutant planarians with more than 2 eyes, these mutants should have slower eye regeneration than wild type planarians as there will be a smaller change in signal.

Third approach: Transplant eye regions from a defective mutant planarian ‘menashi’ (unable to form functional black eye spots but forms white spots that contain most of the eye cells) into a wild type planarian that has its eye spots removed. If an inhibitory long distance signal is produced by these cells then the planarian should not regenerate new eye spots.

LIST OF RELEVANT PRIMARY AND REVIEW ARTICLES READ, AND SUMMARY OF RELEVANT INFORMATION FROM EACH (this is the start of an annotated bibliography):

• F. Studies on transplantation in planaria. Biological Bulletin. 1929; 57:188-197.
• Transplanting different areas of the planarian, including the areas around the eyes

• Guedelhoefer IV. O., Alvarado. A. Planarian Immobilization, Partial Irradiation, and Tissue Transplantation. J Vis Exp. 2012; 66: 405.
• Detailed protocols for tissue transplantation

• C., Saito. Y., Ogawa. K., Agata. K.Wnt signaling is required for antero-posterior patterning of the planarian brain. Elsevier. 2007; 306:2.
• The mutants created in this study have more than 2 eye spots and can be used in my experiment.

·         Cebria. F., Newmark . P. Morphogenesis defects are associated with abnormal nervous system regeneration following roboA RNAi in planarians. Development. 2007; 134.

o   Role of nervous system in regulating morphogenesis during the regeneration of the anterior part of planarian. Smed-roboA is important in guidance of visual axons, and the RNAi mutants of this gene have ectopic projections and visual defects. In the case of my question this could suggest that the long distance signal is carried by the nervous system.

• R., Newmark. P. The cell biology of regeneration. The Journal of Cell Biology. 2012; 196: 5.
• Discussion of regeneration in other organisms such and also planarians. Evidence suggests conservation of regenerative abilities through evolution.

·         Adell. T., Cebria. F., Salo. E. Gradients in Planarian Regeneration and Homeostasis. CSH Perspectives. 2012; 2:1.

o   Discusses the gradients that are involved in planarian regeneration and polarity, such as BMP, Wnt and FGF. These gradients could also be important in my experiment and should be taken into consideration.

• Newmark, P., Alvarado. A. Regeneration in Planaria. Nature Publishing Group. 2001.
• Contains an introduction to regeneration in planarian and some histroy of research in this field. Also a proposed mechanism in regeneration such as the role of neoblasts is discussed.

HOW DOES THE QUESTION FIT INTO THE BROADER PICTURE, AND WHAT IS ITS IMPACT?

• This question fits into the broader picture of developing a model for planarian regeneration. This model is important because it gives us insight into the process of regeneration and stem cells.
• This question can give us the opportunity to address problems in development and stem cell differentiation.
• Another potential impact of this question is gaining a deeper understanding of injury and wound signalling that could also apply to humans.
• There seems to be a parallel between planarian stem cells and the stem cells in mammals.

POTENTIAL WAYS TO MAKE YOUR QUESTION KNOWN TO THE PUBLIC AT LARGE (OR TO YOUR NON-BIOLOGIST FAMILY AND FRIENDS):

• The findings for this question can be used in stem cell research and health care. Also the concept of planarian regeneration is very easy to demonstrate without having a significant knowledge about biology.
• Demonstrations of the cool regenerative powers of planarians can easily be shown by cutting them into tinny pieces and letting them form a new organism.  This demonstration could raise the curiosity of the public about this complex process.

ANY OTHER PARTS OF THE PROJECT COMPLETED SO FAR:

ANYTHING YOU WOULD LIKE SPECIFIC FEEDBACK ON:

• The experimental procedure, since I have doubts about whether it is possible to transplant an eye spot – cannot find any research that successfully transplanted an eye spot into another part of the body, but there is some research/techniques on transplantation of the eye spots and the tissue around it. Therefore my 1st and 3rd approaches  could lead to failure if the transplanted eyes spots are simply reabsorbed….
• Ways to make my question known to the public…

Initial questions for final project … they were very broad

1) The human heart is initially similar to a fish heart in the embryo and the hands are initially formed as paddles. Is this a necessary step or is this simply a remainder of the evolutionary history ?

2) When are maternal RNA or proteins deposited into the embryo? Is it before fertilization or after? If it is before fertilization, then at what stage of the egg development are they deposited? If it is after fertilization then what are the mechanisms that signals the mother to deposit them, and how would this work in the case of external fertilization?

If I was a developmental geneticist, I would ask the following two questions:

1) The human heart is initially similar to a  fish heart in the embryo and the hands are initially formed as paddles. Is this a necessary step or is this simply a remainder of the evolutionary history ? 

This question is interesting for me because the stages of development for human embryo  has often been used as an evidence for evolution. If it is found that these traits are simply a remainder of the evolutionary history then the evidence for evolution could become much stronger. This can have great impacts on science and the community by influencing the arguments around evolution vs. revolution. On the other hand, if it is found that these traits are a necessary step in development, then it could have interesting implications for developmental genetics. Why should the hands initially form as paddles and then turn into fingers?  Is there a benefit to having this course of development?

2) When are maternal RNA or proteins deposited into the embryo? Is it before fertilization or after? If it is before fertilization, then at what stage of the egg development are they deposited? If it is after fertilization then what are the mechanisms that signals the mother to deposit them, and how would this work in the case of external fertilization?