This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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). Because RA is a direct inducer of Hoxa1 gene expression and is also an inducer of ES cell differentiation, we hypothesize that the role of Hoxa1 is to promote neuronal cell differentiation by repressing endodermal and/or mesodermal cell lineages. We also hypothesize that the specific activation of RARb may lead to endodermal differentiation and repression of neurogenesis in mouse ES cells. To test this hypothesis, we first examined the effect of the RARb2 agonist AC55649 on protein expression by employing antibody microarrays. As a consequence of RARb2 treatment, we observed that about 40 (out of 224) proteins examined with the array were increased by 2-fold or more, and of these, most were found to be related to the Ap1/c-Jun pathway after Pathway Architect examination. The results obtained from these studies may result in novel differentiation strategies for the generation in culture of homogeneous cell populations.
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