Juveniles of many organisms have a remarkable ability to regenerate their body parts until the adult-specific stem cells begin to develop. The goal of this research is to investigate the mechanism of leg regeneration in insects and how the adult-specific stem cells begin to develop. Using the flour beetle as a model system, how these adult-specific stem cells develop in juveniles will be investigated by removing the expression of various proteins using a molecular technique called RNA interference. Furthermore, genes expressed during leg regeneration will be examined and characterized to identify factors involved in this process. The evolutionary origin of adult-specific stem cells has important implications in understanding a major contentious issue, the origin of insect metamorphosis. Identification of regeneration mechanisms conserved across the animal kingdom will provide insights into the fundamental and essential mechanisms underlying limb regeneration. The study will be conducted at Wellesley College, an undergraduate institution for women from a variety of socioeconomic backgrounds, and will actively involve undergraduate students from various backgrounds to provide them with an interdisciplinary research experience and training in modern molecular technology. Experience in multidisciplinary approaches to biology is critical for future scientists, and this study will provide students with such an opportunity. The proposed research will also be brought into the classroom to expose more students to research. The students will be encouraged to both publish their findings and present their work at scientific meetings.
Intellectual merit: Many organisms have a remarkable ability to regenerate their body parts until adult-specific stem cells differentiate. Mammals, including humans, lack the ability to regenerate limbs, but a major goal of regenerative biology is to identify the molecular basis of regeneration so that one day we may be able to develop therapies for amputees. The molecular underpinnings of limb regeneration have not been well understood due to the lack of suitable mammalian models for studying regeneration. We have therefore developed the flour beetle, Tribolium castaneum, as a model for studying 1) limb regeneration and 2) the development of adult-specific cells. We have sought to identify proteins involved in larval limb regeneration and have identified a number of key genes that regulate distinct phases of regeneration: wound healing, blastema formation and limb re-patterning. Each of these stages were found to be uniquely regulated by different proteins. Many of the proteins identified are also found in vertebrates where they play similar roles. Specifically, proteins involved in wound healing and blastema formation appear to play similar roles between invertebrates and vertebrates, thus illustrating the importance of these factors in regeneration across all animals. Our research has also identified additional candidates that regulate either blastema formation or the transition from the blastema stage to the re-patterning stage, and their targets are currently being analyzed. These findings provide insights into the essential components of regeneration and lay the foundation for future studies in limb regeneration. We have also determined the roles of three highly conserved signaling pathways during stem cell differentiation. All of these pathways were found to play key roles in the differentiation of adult-specific appendages. Specifically, one signaling pathway was found to regulate adult-specific cell proliferation: silencing this pathway led to loss of proliferation during metamorphosis, and up-regulation of the pathway during the larval stage led to the overgrowth of appendages and ectopic outgrowths that resemble tumors. Thus, this pathway appears to play a key role in the proliferation of stem cells. We are currently investigating how this signaling pathway interacts with hormonal fluctuations associated with tissue maturation and the age-dependent loss of regenerative abilities. Our findings have potential implications for understanding tumor formation and stem cell maintenance. In addition, they contribute to our understanding of the origin of insect metamorphosis which is a long standing question in insect evolution. Broader impacts: Funding for this project has provided research training for 26 female undergraduate students in my research lab. Many of these students have presented their work at national conferences and have also co-authored manuscripts in peer-reviewed journals. In addition, the funding has allowed us to host two or three female high school students each summer from the local public high school (Framingham High School). Through this Framingham-Wellesley Summer Outreach program, underrepresented students gained exposure to scientific research, and Wellesley students, in turn, gained valuable mentoring experience. Finally, the funding has allowed the PI to being the research into the classroom where students gained experience in independent project design and implementation of a semester-long molecular project involving limb regeneration or metamorphosis. Feedback on this activity has been positive, and the PI will continue to provide similar experiences in the coming years.
