Studies of spontaneous regeneration in a novel amniote model

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Although most vertebrates are capable of replacing certain individual tissues, complete organ and appendage regeneration is rare. Among amniotes (reptiles + mammals), the most striking example is observed following tail loss in lizards. Unlike mammals, many lizard species are able to spontaneously regenerate the tail complete with a spinal cord, blood vessels, skeletal support and muscle. Using the lizard tail regeneration model, the LONG TERM GOAL of my research program is to identify and understand the biological mechanisms that permit and promote scar-free wound healing and tissue regeneration. Over the past 5 years, my NSERC funded research program has established that wound healing and regeneration in the lizard follows a highly conserved sequence of cellular and tissue-level events similar to those of other regenerating species. In addition, my lab has identified many of the key proteins involved in these events. I now propose to employ the lizard tail regeneration model to explore one of the most fundamental questions in regenerative biology: why some tissues, organs and species are capable of regenerating, whereas others cannot. I HYPOTHESIZE that regeneration requires a coordinated interaction between resident populations of stem/progenitor cells and a permissive wound environment. My SHORT TERM OBJECTIVES are to: (1) identify and track resident cells present in original tissues that contribute to the stem/progenitor pool following injury; and (2) determine the molecular factors and tissue-level characteristics of the wound healing environment that enable and promote regeneration. My proposed research program will employ a hierarchical and integrated approach, including: cell and tissue cultures; pharmacological, molecular and experimental manipulations of tissue within the regenerating tail; and the use of electroporation for labeling cells both in vitro and in vivo. For objective (1), my primary foci will be the spinal cord and axial skeleton. In lizards the spinal cord is continuous throughout the length of the tail. Loss of the tail results in the regeneration of a new spinal cord. Similarly, the axial skeleton is also regenerated. However, whereas the original skeleton is composed of bony vertebrae, the regenerated organ is an unsegmented cartilaginous cone that never ossifies. Presently, the original source and identity of cells contributing to the formation of these structures remains unknown. Experiments will include tracing studies (e.g., with fluorescent protein electroporation) to establish the source of cells from the original tissues into the regenerating tail. Other experiments will use explant cultures or, in the case of the spinal cord, generating neurospheres to screen for markers of stemness and neural/chondrogenic lineage. For objective (2), I will experimentally modulate the wound healing environment. Experiments will include altering the endogenous inflammatory response and modulating the transforming growth factor beta (TGFß)/activin signaling pathway. Conceptually, this ORIGINAL RESEARCH PROGRAM represents an INNOVATIVE approach to regenerative biology. Comparative studies involving regeneration-competent species provide unique opportunities to probe endogenous and naturally evolved mechanisms of tissue restoration. Lizards are one of the only amniotes capable of spontaneous multi-tissue regeneration. Lizards have evolved the ability to rapidly self-detach the tail as an anti-predation strategy. Structural adaptations of the tail minimize damage to adjacent tissues, and hence initiating regeneration is arguably less invasive than for other species. The entire tail can be repeatedly lost and regenerated (with functional recovery) without the need for clinical intervention.