Gonadotropin-releasing hormone (GnRH) is the central regulator of the hypothalamic-pituitary-gonadal axis, controlling sexual maturation and fertility in diverse species from fish to humans. GnRH gene expression is limited to a discrete population of neurons that migrate through the nasal region into the hypothalamus during embryonic development. The GnRH regulatory region contains four conserved homeodomain binding sites (ATTA) that are essential for basal promoter activity and cell-specific expression of the GnRH gene. MSX and DLX are members of the Antennapedia class of non-Hox homeodomain transcription factors that regulate gene expression and influence development of the craniofacial structures and anterior forebrain. Here, we report that expression patterns of the Msx and Dlx families of homeodomain transcription factors largely coincide with the migratory route of GnRH neurons and co-express with GnRH in neurons during embryonic development. In addition, MSX and DLX family members bind directly to the ATTA consensus sequences and regulate transcriptional activity of the GnRH promoter. Finally, mice lacking MSX1 or DLX1 and 2 show altered numbers of GnRH-expressing cells in regions where these factors likely function. These findings strongly support a role for MSX and DLX in contributing to spatiotemporal regulation of GnRH transcription during development.Proper sexual maturation and fertility are dependent upon the correct function of the hypothalamic-pituitary-gonadal axis, initiated by a small, yet critical population of gonadotropin-releasing hormone (GnRH) 1 neurons. The GnRH gene is expressed in a complex spatiotemporal manner during embryonic development and into postnatal life with several populations of GnRH-expressing neurons originating at different developmental stages and locations. These populations include the classical, septohypothalamic neurons, as well as populations in the lateral septum, posterior bed nucleus stria terminalis (pBNST), and tectum (1, 2). Although the role of each of these populations of GnRH-producing neurons remains to be elucidated, the contribution of the septohypothalamic population is required for maturation of the hypothalamic-pituitarygonadal axis and fertility (3).The precursor cells of the septohypothalamic GnRH neurons have been reported to originate within the olfactory placode (4 -6) or the neural crest (7) and begin to express the GnRH transcript in a discrete population of cells located in close proximity to the olfactory placode of the embryonic mouse by 11.5-days postcoitum (11.5 dpc). By 12.5 dpc, the full complement of septohypothalamic GnRH neurons (ϳ800) in the adult population is established (6), and over the course of the next several days, these neurons migrate toward the CNS, closely associated with the established position of the peripherin-positive nerve bundle of the olfactory nerve (8, 9). By 16.5 dpc the majority of the septohypothalamic neurons have reached their destination, scattered throughout the preoptic area, the diagonal band of Broc...