EXCEED THE SPACE PROVIDED. Extracellular modulation of growth factor signaling represents a central mechanism for regulating thedistribution and levels of ligand activity in development. In work supported by the previous funding period ofthis competitive renewal, we have shown that the EGF-CFC gene Cripto is required for anterior-posterior (A-P)axis patterning and embryonic mesoderm formation at early stages of mouse development. Our studies haveprovided genetic evidence for a model in which Cripto acts as an essential co-receptor for signaling by Nodal, amember of the transforming growth factor-beta (TGF-P) family that has multiple roles in formation of thevertebrate body plan. In the current application, we propose to investigate the functions of Cripto in A-Paxispatterning, mesoderm formation, and axial midline development, as well as its potential role in cardiogenesis.Based on our preliminarydata, we anticipate that these functions of Cripto involve Nodal-dependent activitiesas well as Nodal-independent activities that may reflect its cooperation with other specific members of the TGF-P family. In addition, our in vivo analyses will be complemented by cell culture and biochemical investigationsof the mechanism of EGF-CFC cooperation with TGF-P signaling. We will pursue the following specific aims: I)Analysis of Cripto function in A-P axis patterning and mesoderm specification through phenotypic andchimera analysis of Cripto function at pre-gastrulation and gastrulation stages, and by microarray analysis toidentify genes downstream of Cripto function. II) Investigation of the role of Cripto in axial midlineformation by analysis of Cripto reduction-of-function mutants in the mouse and gain-of-function approachesin the chick embryo. Ill) Examination of the role of Cripto in cardiac development by tissue-specifictargeted deletion of Cripto in cardiac progenitors in vivo, and investigation of the differentiation of Cripto-deficient EScells in culture. IV) Analysis of EGF-CFC activities in a cell culture assay system to investigatethe Nodal-dependent and Nodal-independent activities of Cripto, and to identify TGF-P factors that caninteract with EGF-CFC proteins. Taken together, our continuing studies of Cripto should provide importantinsights into the molecular mechanisms of extracellular modulation of morphogenetic signals.
