The main objective of the proposed project is to determine the role of the energy balance hormone leptin on early developmental processes, including limb bud growth and differentiation, in the South African clawed frog (Xenopus laevis). Because leptin (product of the obese [ob] gene) has been primarily studied as an adipostat that regulates food intake and metabolism in juvenile and adult mammals, the actions of leptin in early fetal development have been largely unexplored. Recently, however, leptin has been shown to play an important role in implantation of zygotes and to act as a growth factor during early brain development. In addition, after cloning both leptin and leptin receptor in Xenopus I found a novel role for leptin in promoting limb growth and differentiation in amphibian tadpoles. Using X. laevis, the classical model of vertebrate embryogenesis and morphogenesis, the proposed research integrates physiological, morphological and molecular approaches to test the central hypothesis that leptin acts as a growth factor in embryogenesis and early development in vertebrates. To map the mRNA expression patterns of leptin and leptin receptor during early developmental stages in both high and low food resource environments, I will use in situ hybridization techniques of whole mount embryos and specific tadpole tissues. In addition, I will use a polyclonal antibody generated against Xenopus leptin to conduct whole mount in situ immunohistochemical analyses of leptin protein expression in embryos and early-staged tadpoles. To determine whether leptin promotes development in embryonic and early tadpole development, I will conduct a combination of morpholino knockdown and leptin overexpression experiments and monitor both growth and development of specific tissues. In addition, I will treat tadpole limbs (in vivo and in vitro) with recombinant Xenopus leptin and conduct a series of immunohistological and morphological analyses to determine whether leptin is involved in cell proliferation, apoptosis, and/or differentiation of skeletal tissue in the hindlimb. Given the evolutionarily conserved genetic control of developmental processes in tadpole and fetal development, the results from these experiments will lay the foundation for future studies aimed at understanding the role of leptin in early developmental processes in all vertebrates including in humans. Project Narrative Very little is understood about how nutritive condition affects growth and development during the early fetal/larval stage in vertebrates. The recent findings that leptin and leptin receptor, which are known to adjust growth and reproduction in mammals during later life stages according to nutritional condition, are expressed in a variety of tissues during this stage suggests that leptin signaling may be an important in modulating growth and development during fetal development according to nutritional resources. Evidence that leptin treatment promotes limb development in larval amphibians further pinpoints a role of leptin in regulating cartilage/bone formation in fetuses. This project will use the classic model of vertebrate early development, Xenopus laevis, to investigate the roles of leptin in the regulation of embryonic and early tadpole growth and morphogenesis, and to determine whether leptin signaling links internal and external nutritional cues to developmental timing. This research will greatly increase our understanding of the function of leptin in early life stages, and provide essential insight into how environmental cues affect growth and development during the earliest stages of life.
