Larval frogs (tadpoles) regenerate lost limbs completely at early stages of development but lose this ability as they approach metamorphosis. This decline in regenerative capacity in larvae occurs during development of several components of the adaptive immune system in these animals. Experiments designed in this study will test the hypothesis that changes in the developing frog's immune system, including the appearance of cells characteristic of an inflammatory response, modify the local limb environment to interfere with or preclude cellular events required for regeneration when the limb is lost. Previous analyses of tadpole limb regeneration by this laboratory have shown the expression of many inflammation-related genes and the appearance of various immunomodulatory proteins in the limb during the first days of regeneration. Other investigators working with skin regeneration in several mouse models have shown the regeneration seen at fetal stages of development persists in adults that are immunodeficient and have fetal-like immune systems.
This study will examine and quantify inflammation at the cellular and molecular levels during the early period of limb regeneration at larval stages when regeneration occurs successfully and at later stages when regeneration is poor. Procedures that affect inflammation positively and negatively will also be tested for their effects on regenerative capacity, using both molecular and morphological assays. The results are expected to reveal a strong correlation between loss of regenerative potential during development and strong or prolonged local inflammatory reactions to the regenerative stimulus. Such results will impact the emerging field of regenerative biology by highlighting the importance of peripheral immunity and immunomodulation in the local development of stem cells and tissue regeneration. The immune-regulating properties of various stem cells in mammalian models and their importance in tissue regeneration are increasingly recognized. Similar findings in a well-characterized amphibian model of regeneration will lead to greater understanding of the regenerative ability of these animals and the decline of this capacity in mammals. The broader impacts of the work, which are important not only for scientific advances but also for the education of graduate students in regenerative biology, include integrating considerations of the organism's immunity with cellular and molecular aspects of limb development, an integration required for a full appreciation of the factors controlling regeneration of organs in animals where the adaptive immune system is already functioning.
The ability of animals such as certain fish and amphibians to regenerate appendages lost to injury has long been studied by embryologists interested primarily in the mechanisms of limb formation. Such studies have provided little real insight into why "higher vertebrates" have lost this regenerative capacity. We have suggested that the more highly developed and effective system of adaptive immunity in reptiles and mammals interferes with the cellular changes required to regenerate a new limb, either through the inflammatory response to injury or by processes similar to those used to recognize and kill grafts of "foreign" cells and tissues. To test this general hypothesis we have investigated postamputation inflammation in larval limbs of the frog Xenopus, in which regenerative capacity declines during development. This decline is known to be correlated very closely to the period in which the system of cells that make up the adaptive immune system appears and becomes functional in this species. By examining expression of genes involved in inflammation and its resolution we found that these processes persist for longer periods and include the appearance of regulatory T cells in older limbs with less ability to regenerate normally. Similar analyses showed that this persistant inflammation had no effect on cellular reprogramming in the limb stumps, but did inhibit the normal expression of genes required for morphological processes such as new digit formation. Stimulating the inflammatory process with topical application of aqueous beryllium salts, which are not neutralized after binding exposed tissues, was found to inhibit successful regeneration in limbs at early developmental stages when complete regeneration normally occurs. These data support the hypothesis that the process of inflammation changes during larval development and negatively impacts regeneration, specifically by inhibiting molecular events required for generating new limb components. Confirming results of other investigators studying tail regeneration in a fish model, we also found that generalized suppression of inflammation in newly amputated limbs with the glucocorticoid beclomethasone reduces the number of digits formed by regeneration. These results show that cellular changes produced during inflammation are in fact required to initiate successful regeneration, while the beryllium result indicates that the timely resolution of those changes is needed for regeneration to be completed normally. To examine further how inflammation or immune mechanims interfere with limb regeneration we used more specific pharmacological inhibitors of immune-related processes. Two unrelated inhibitors of the enzyme COX-2 (which drives early events during inflammation) were found to improve digit formation significantly in limbs amputated at a larval stage when the capacity to regenerate digits is declining. Similar experiments showed that celastrol, an agent used experimentally by others to block autoimmunity, also resulted in regeneration of more digits. This result, together with similar results using celastrol in another lab investigating tail regeneration in larval Xenopus, suggests that an adaptive immune reaction against specific cells in the limb stump as it prepares for regeneration may underlie the regenerative decline in developing larval frog limbs. This project has thus provided initial data indicating that the development of the adaptive immune system and its changing involvement in the process of post-injury inflammation have direct effects on the animal's ability to complete limb regeneration successfully. These results underline the importance of including the processes of inflammation and resolution in explanations of regenerative capacity or its loss. Future work will strengthen the connection between specific mediators of inflammation and molecular events required to complete regeneration, such as the specification of progenitor cells for new digits.
