Vertebrate developmental biology heavily relies on the use of lower vertebrate models, such as Xenopus laevis, however, its use has been limited by lack of molecular tools that are necessary for following protein expression, localization and function. This proposal will evaluate the utilit of llama-derived nanobodies (single domain antibodies) for characterization and functional exploration of the Xenopus embryo proteome. In contrast to conventional monoclonal antibodies, which are expensive to generate and maintain, nanobodies are exceptionally stable and can be easily expressed in E. coli without loss of their binding activity. In our preliminary experiments, phage display libraries have been constructed from immune llama lymphocytes and shown to be suitable sources for nanobodies against frog antigens. Moreover, nanobodies specific for Xenopus antigens have been isolated from these libraries and a mass spectrometry-based approach to identify the corresponding antigens has been optimized. Based on these preliminary studies, we plan to develop a new class of antibody tools for the analysis of the Xenopus proteome. To achieve this goal, the constructed cDNA libraries will be used in several expression cloning approaches to establish a bank of Xenopus-specific nanobodies and to evaluate the utility of nanobodies in functional embryological studies. By applying the nanobody technology to embryological studies, these experiments will have a significant impact on the future systems-level characterization and functional analysis of Xenopus embryonic development and will constitute a major help to the Xenopus community. The generated nanobodies will be used for the analysis of protein-protein interactions and gene regulatory networks in diverse developmental processes. The advanced knowledge of these processes is critical for the future understanding of mechanisms underlying human birth defects and the molecular causes of cancer.
Studies proposed in this application will establish a bank of single chain antibodies specific for Xenopus embryonic antigens. These antibodies (nanobodies) will be useful for structural and functional analysis of developmental processes in Xenopus embryos. The knowledge of the molecular mechanisms of early development is relevant to cancer and is critical for the understanding of human birth defects.