Summary The GnRH-1 neurons are cells in the brain that control pubertal onset, sexual competence and fertility of vertebrates. During development, the GnRH-1 neurons migrate from the embryonic nasal area into the brain, where they will eventually take up positions in the hypothalamus to control the release of gonadotropins from the pituitary gland. Defects in GnRH-1 migration induce various types of hypogonadotropic hypogonadism (HH) in humans, characterized by delayed pubertal onset, hypogonadism, and infertility. HH in humans manifests clinically as either Kallmann syndrome (KS) or normosmic idiopathic HH (nIHH). In KS, nIHH is associated with deficiencies in the sense of smell. This association led to the long-held, prevailing view that the GnRH-1 neurons migrate from the nose to the hypothalamus along the axons of olfactory and vomeronasal sensory neurons. However, we still do not know whether his assumption of GnRH-1 migration patterns is true. The overall objective of this application is to delineate the molecular mechanisms that guide GnRH-1 neurons and their migratory scaffold. Our central hypothesis states that GnRH-1 neurons migrate to the hypothalamus on the Terminal Nerve (TN), whose development occurs through molecular signals that only partially overlap with those controlling olfactory and vomeronasal neuronal development. The rationale for this hypothesis derives from understanding how specific genetic mutations linked to KS and nIHH can negatively affect TN development and induce GnRH-1 neuron mispositioning, independent from the olfactory system development. Our preliminary data indicate that transcriptional activator/repressor of the sonic hedgehog signaling pathway, Gli3, controls TN developments and GnRH-1 migration to the brain. Guided by our strong preliminary data, we will test our hypothesis through two specific aims: 1) Determine how the transcriptional regulator Gli3 regulates TN development and GnRH-1 neuronal migration and 2) Define the biologic role of GLI3 mutations in the etiology of KS/nIHH in humans. Our approach is innovative. We will exploit mouse genetics, advanced imaging techniques, human whole genome sequencing data and complementary in-vitro and in-vivo experiments to discover new mechanisms underlying TN development and formation of a functional GnRH-1 system in vertebrates. The proposed research is significant, since it will advance and expand vital clinical information for KS and nIHH in humans. The results from these studies will improve diagnostic criteria and stimulate the development of novel treatments and therapeutic strategies to improve the human condition.