Gonadotropin releasing-hormone (GnRH) is released from the GnRH neurons of the basal hypothalamus and represents the primary neural signal regulating the hypothalmic-pituitary-gonadal axis. GnRH neurons originate early in murine development (10.75 days post conception [pc]) and migrate across the nasal cavity and into the basal forebrain between days 11.5 and 16.5 pc. The final anatomical distribution of the 1000 or so GnRH neurons in the murine brain consists of scattered neurons from the olfactory bulbs to the rostral aspect of the median eminence with the highest concentration of GnRH neurons located at the level of the OVLT and the POA. A failure of GnRH neuronal migration from the olfactory placode results in a form of X-linked hypogonadotropic hypogonadism, associated with anosmia, Kallmann's Syndrome. The study of the molecular elements important for the regulation of neuron migration, and GnRH gene expression in the GnRH neuron has been hampered by both their scattered distribution and their low number. The Otx2 homeodomain protein has been shown to be expressed in GnRH neurons at day 13pc when GnRH neurons are migratory. Otx2 plays an important role in the organization of the forebrain, including the GnRH neuron containing regions partly through regulation of a variety of adhesion factor genes. Therefore, Otx2 is a candidate as a regulator of GnRH neuronal migration. Interestingly Otx2 consensus binding sites have been observed in the promoters of both the human GnRH gene and the mouse GnRH gene. So Otx2 may additionally exist in the GnRH neurons to regulate GnRH gene expression.
The aims of these studies are to identify a role for the Otx2 protein in coordinating the migration of GnRH neurons or for the regulation of GnRH gene expression.
In aim 1, further studies will be undertaken to determine the developmental stages that Otx2, and a related protein, Otx1, are expressed in GnRH neurons. These studies will be performed to provide an anatomical substrate for Otx regulation of the GnRH neuron.
In aim 2, I will examine the structural, using gel mobility shift assays, and functional, using transient transfection studies, interactions of Otx2 and the GnRH promoter in vitro. The studies outlined in aim 3 are designed to construct a GnRH neuron specific targeted knock out of the Otx2 gene. Anatomical and physiological examination of these mice will provide important insights into the role that Otx2 serves in both directing appropriate migration of GnRH neurons, and in regulating GnRH gene expression.
