The long-term goal of this project is to elucidate and characterize the molecular mechanism by which the Hoxa1 transcription factor directs the differentiation of embryonic stem cells into neurons. Expression of the Hoxa1 gene in cells and tissues can be activated by retinoic acid (RA), a derivative of vitamin A. Inactivation of both alleles of the Hoxa1 gene in mice results in numerous developmental defects, including hindbrain deficiencies and abnormal skull ossification, and ultimately, in neonatal death. In humans, truncating mutations of the HOXA1 gene have been associated to autism susceptibility. This project will characterize the function of Hoxa1 during the RA-induced differentiation of mouse embryonic stem (ES) cells grown in suspension as embryoid bodies (EBs). We hypothesize that RA can induce the differentiation of mouse ES cells into all three germ layers and that the role of Hoxa1 is to promote neuronal cell differentiation by repressing endodermal and/or mesodermal cell lineages. We will test this hypothesis with the following specific aims: Aim 1 will employ retinoic acid receptor (RAR) agonists to determine the mechanism of RA-induced expression of Hoxa1 in EBs; Aim 2 will characterize the role of Hoxa1 as a repressor of endodermal differentiation and as an inducer of neuronal gene expression; Aim 3 will characterize the role of RA isomers during the differentiation of ES cells into neurons. The results obtained from these studies may result in novel differentiation strategies for the generation in culture of homogeneous neuronal cell populations. Because the results obtained using our murine system can be applied to human cells, the knowledge obtained from this project will have an important vertical impact on the treatment of spinal cord or brain injuries in humans.