The therapeutic promise of stem cell biology lies in its potential for cell replacement therapies in diseases where an essential cell type of the patient malfunctions or degenerates. This is particularly evident in diseases of the nervous system where cells largely lose their ability to proliferate and thus regenerate after embryonic differentiation. Devastating neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are characterized by a progressive paralysis caused by motor neuron death and currently have no cure. Strategies for replacing specific neuronal cell types with cells derived from human embryonic stem (hES) cells will require understanding the genetic programs that control hES cell differentiation. These rapidly dividing pluripotent cells undergo a major transition in gene expression to become neuronal progenitor cells (NPC), while maintaining their proliferative ability. Another drastic change in gene expression program occurs as NP cells differentiate into neurons, where cell division has stopped. A great deal of work has described the DNA level changes that control gene expression in ES cells and during their transition to NPC and neurons. However, the production of a protein product from a gene is controlled at each step in the gene expression pathway where the DNA gene is first transcribed into RNA and the RNA then translated into protein. An important RNA level regulatory step in this pathway is the processing of the primary RNA transcript from the gene into an mRNA that can be translated into protein. One part of this processing is the pre-mRNA splicing reaction, where alternative splicing patterns in the pre-mRNA determine the structure of the final protein product of most human genes. Little is known about how this step in the gene expression pathway is regulated in ES cells or during their differentiation. Yet ALS and SMA can both be caused by the loss of components of the splicing machinery and a great deal of work is examining how splicing might be disrupted in mature neurons of ALS and SMA patients. In this study, we are examining how two important splicing regulators, the polypyrimidine tract binding protein (PTB) and its neuronal homolog nPTB, affect splicing in normal ES and NP cells. We are characterizing the programs of regulation controlled by these proteins. In the past funding period we adapted and applied two new genomewide methods of RNA analysis. The first uses new technologies for high density RNA sequencing (RNAseq) to examine the whole transcriptome of each cell under study. From this data, we can extract information on all the splicing changes occurring during a developmental transition. The second method called CLIP examines the sites of RNA binding by PTB and nPTB in the RNA of each cell type. These methods are now ready to apply to ESC, NPC and motor neurons that we have derived in culture. From this work, we will advance our understanding of how ES cells differentiate into neurons and how pre-mRNA splicing controls cell function in normal development and in disease.