Project Summary Puberty is a transformative developmental process resulting in sexual and reproductive maturity. Both the onset of puberty and fertility in adult life depend on the activity of neurons expressing gonadotropin-releasing hormone- 1 (hereafter referred to as GnRH-1ns). Originating in the nasal placode during embryonic development, GnRH- 1ns migrate along axons to the hypothalamus, where they take up residence in the brain and regulate the release of gonadotropins from the pituitary gland throughout life. Defective GnRH-1 neuronal development can cause congenital hypogonadotropic hypogonadism (HH), and sex hormone deficiencies that can cause delays or the absence of puberty, resulting in infertility. HH can manifest clinically as either (i) Kallmann syndrome (KS), wherein HH is associated with different levels of olfactory deficits, or (ii) normosmic idiopathic hypogonadotropic hypogonadism (nIHH), wherein HH occurs with a normal sense of smell. The migration of GnRH-1ns proceeds along the axons of the terminal nerve and is known to depend on an additional cell type, the olfactory ensheathing cells (OECs). However, what cellular and molecular mechanisms underlie the development of the olfactory ensheathing cells, and the terminal nerve of mammals is still largely unknown. The long-term goal of our research program is to discover the molecular mechanisms leading to Kallmann syndrome and normosmic forms of niHH. By screening a cohort of human patients, we recently discovered that a loss-of-function mutation in the gene encoding the transcriptional regulator Gli3 was associated with KS. Moreover, our preliminary data show that Gli3 loss-of-function affects both OECs and terminal nerve development in mice. Our central hypothesis states that Gli3 controls the development of the terminal nerve and the development of the OECs in a dose-dependent fashion. Moreover, we postulate that and that the expression of inductive factors in the developing olfactory system controls the formation of the OECs. Guided by our strong preliminary data, we will here test our hypothesis through three specific aims: 1) To determine the dose- dependent effects of Gli3 on terminal nerve development and gene expression. 2) To identify Gli3 gene targets in the developing nose. 3) To discover the molecular mechanisms controlling the development of OECs. Our innovative approach will exploit advanced single-cell transcriptomics, mouse genetics, imaging, CUT&RUN, and bioinformatics to discover mechanisms underlying the development of the terminal nerve and OECs and the formation of a functional GnRH-1 system. The proposed research is significant to human health since it will advance and expand our understanding of KS and nIHH disease mechanisms 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.