David M. Gardiner, PhD, Scripps Institution of Oceanography - US grants

This workshop, entitled "Workshop on Mechanisms of Developmental Disruption in Amphibians," will be held November 5-7, 1998 at the San Diego Zoo. This workshop follows two previous workshops sponsored by the National Science Foundation that addressed issues pertaining to amphibian declines and die-offs. The first, "Workshop on Amphibian Declines", held May 28 - 29, at the NSF, brought together biologists who have been working on amphibian declines around the world. One area of research identified for further consideration was the function of the amphibian immune system with regards to the hypothesis that amphibian die-offs are occurring as a result of environmental factors that are altering the balance between pathogens and hosts. To focus on these issues a second, NSF-sponsored "Workshop on Amphibian Diseases and Immunity" was held at the San Diego Zoo, July 26-28, 1998. Both workshops pointed out the necessity to address these issues with a broad scientific perspective ranging from the population level to the molecular level. In particular, it was repeatedly noted that the most vulnerable life history stages of the amphibians are during embryonic and larval development, and that an understanding of the effects of environmental agents on these stages is essential. This third workshop is organized to extend the results of the previous workshops into discussions of the developmental and molecular mechanisms underlying environmentally-induced congenital malformations in amphibians ("deformed frogs"). In addition, the proposed workshop will for the first time explicitly address the question of the relationship between amphibian declines and amphibian malformations. The goal of the third workshop is to build upon the accomplishments of the earlier workshops, and to expand the research discussed to include more mechanism-oriented approaches to the problem. The workshop will bring together participants from the previous workshops, including both workshop organizers, with researchers in the areas of limb development and regeneration, endocrinology, developmental immunology, parasitology, receptor-ligand interactions, amphibian early development and metamorphosis, and endocrine disruption. The workshop will be structured to produce a consensus statement addressing issues pertaining to amphibian malformations and the relationship between declines and malformations that will be published in a basic research journal such as Teratology or Developmental Biology.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to further understand how cells communicate during the formation of the blastema in a regenerating limb. The process of limb regeneration requires cells to interact that do not normally interact -such as when epithelial cells on the dorsal and ventral sides of a wound interact during wound closure. Upon excision and healing of parts of the blastema these cells are capable of sensing that their new neighbor is different from their normal neighbor, despite the lack of obvious differences among these cells. Not surprisingly, cell-cell interactions are essential for communication of cell identity. Yet, exactly how these cells are different, and how the signal of cell identity is transmitted among cells remains a mystery. I hypothesize that the blastema undergoes metabolic oscillations, possibly utilizing secondary messengers (such as Ca++) to transmit this signal through cell-cell junctions. Thus when two cells interact that do not normally interact, they are metabolically out of phase and recognize each other as "different". I propose to utilize a calcium biosensor (called XXL) and determine whether synchronized calcium oscillations occur periodically in the blastema. This biosensor contains a short calcium-binding peptide between CFP and YFP. Upon calcium binding the short peptide changes conformation, which brings the CFP and YFP molecules in close proximity and allows exited CFP to FRET to YFP. FLIM will be used to measure oscillations in FRET in tissue culture cells, axolotl blastema tissue explants, and eventually in vivo blastemas. It is possible that a different secondary messenger such as cAMP or IP3 mediates metabolic oscillations occurring in the blastema. In this case, similar biosensors can also be utilized for these molecules.