HYPOTHESIS: Loss of function of the transcriptional regulator of c-ski results in perturbation of the cranial neural crest and associated cranial neural tube defects.
SPECIFIC AIMS : 1- Identify the impact of c-ski loss of function of patterns of cranial neural crest formation, proliferation, apoptosis and migration among embryos of differing c-ski genotypes. 2- Establish a comprehensive """"""""gene expression profile"""""""" or """"""""molecular fingerprint"""""""" of isolated cranial neural crest cells during normal neural tube morphogenesis in vivo and during the genetic of neural tube defects (NTDs) in embryos of differing c-ski genotypes. 3- Define the molecular and functional sequelae of c-ski loss of function on the transforming growth factor beta (TGFbeta) signaling pathway in the cranial neural crest. The hypothesis to be examined in the present application is that an abnormal c-ski genotype in the mouse increases the risk for cranial NTDs, the phenotype of which is associated with alterations in cranial neural crest gene expression (i.e. neural crest function). Through breeding of existing Wnt1-Cre/LoxP reporter mice to c-ski knockout mice, a novel """"""""composite"""""""" mouse model will be generated which is genetically sensitive to NTDs due to c-ski loss of function and in which the neural crest and their derivatives are indelibly marked with enhanced green fluorescent protein (EGFP). Such a mouse model will enable analysis of the effects of c-ski loss of function on cranial neural crest formation, migration, proliferation, and apoptosis (Specific Aim #1). In addition, the application of laser capture microdissection of neural crest cells and DNA microarray technologies to this animal model will facilitate generation of isolated neural crest """"""""gene expression profiles"""""""" during normal neural tube morphogenesis and during the genesis of neural tube defects in embryos of differing c-ski genotypes (Specific Aim #2). Neural cress cells synthesize TGFbetas during development. However, under normal conditions, c-Ski is known to moderate/repress signaling initiated by this family of growth factors. Thus, the molecular and functional sequelae of c-ski loss of function also will be examined with particular emphasis on effects on the transforming growth factor beta (TGFbeta) signaling pathway in the neural crest (Specific Aim #3). The effect of c-ski loss of function and persistent de-repression of TGFbeta signaling on cranial neural crest gene expression in vivo (whole embryo culture) and in vitro (primary neural crest cell culture) will be examined. The proposed studies offer a novel approach to address issues of genetic differences in susceptibility to cranial NTDs, as well as serve to initiate investigation, at a mechanistic level, of potential cellular and molecular targets of c-ski loss of function during early embryonic development. The integration of our novel """"""""composite"""""""" animal model with the new technologies of laser capture microdissection and DNA microarray provides a powerful strategy to elucidate mechanisms underlying the role of c-Ski in the cranial neural crest during neural tube morphogenesis.
Showing the most recent 10 out of 114 publications
