GnRH neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain where they become integral members of the hypothalamic-pituitary-gonadal axis. We study mechanism(s) underlying GnRH neuronal differentiation, migration and axonal targeting in normal/transgenic animals, and nasal explants. Using these same models, our work also addresses the mechanisms regulating (intrinsic and trans-synaptic) GnRH gene expression, peptide synthesis and secretion in GnRH neurons. Multiple approaches are used to identify and understand the multitude of molecules and factors which play a role in directing the GnRH neurons to their final location in the CNS. These include differential screening of libraries obtained from migrating versus non-migrating cells, examination of molecules differentially expressed at key locations along the migratory route, morphological examination of the development of the GnRH system in knockout mice, and perturbation of molecules in vitro and subsequent monitoring of GnRH neuronal movement. As GnRH neurons migrate they also mature and the two processes may in fact be linked. To investigate the maturation of GnRH neurons we use calcium imaging, electrophysiology and biochemical measures to examine GnRH neuronal activity and peptide secretion. In addition, we collaborate with labs performing human genetic screening of Kallman patients. Once a mutation is identified, we analyze the expression pattern in mice and perform biological assays to determine the outcome of the mutated gene on GnRH development. Over the past year, four studies were finished: 1) Bone morphogenic protein-4 (BMP4) and fibroblast growth factor-8 (FGF8) were thought to have opposite roles in defining epithelial versus neurogenic fate in the developing olfactory/vomeronasal system. In particular, FGF8 was implicated in specification of olfactory and GnRH neurons, as well as in controlling olfactory stem cell survival. Using different mouse lines and lineage tracing, Fgf8 expression and cell lineage was analyzed in the developing nose in relation to expression of Bmp4 and its antagonist Noggin (Nog). FGF8 was expressed by cells that acquire an epidermal, respiratory cell fate and not by stem cells that acquired neuronal olfactory or vomeronasal cell fate. Ectodermal and mesenchymal sources of BMP4 controlled the expression of BMP/TGFb antagonist Nog, whereas mesenchymal sources of Nog defined the neurogenic borders of the olfactory pit. Fgf8 hypomorph mouse models displayed severe craniofacial defects together with overlapping defects in the olfactory pit including (1) lack of neuronal formation ventrally, where GnRH neurons normally form, and (2) altered expression of Bmp4 and Nog, with Nog ectopically expressed in the nasal mesenchyme and no longer defining the GnRH and vomeronasal neurogenic border. Together our data showed that (1) FGF8 is not sufficient to induce ectodermal progenitors of the olfactory pit to acquire neural fate and (2) altered neurogenesis and lack of GnRH neuron specification after chronically reduced Fgf8 expression reflected dysgenesis of the nasal region and loss of a specific neurogenic permissive milieu that was defined by mesenchymal signals. 2) Temporal and spatial localization of nerve growth factor receptor (p75NGFR) in the developing olfactory system and GnRH system was characterized and its role analyzed using p75NGFR null mice and nasal explants. Prenatally, p75NGFR was expressed by GnRH neurons and olfactory ensheathing cells (OECs). In p75NGFR null mice, no change in the number of GnRH cells was detected as compared to wild-type. However, in null mice, a shift in the distribution of GnRH neurons was found, with a small population of GnRH cells migrating further caudally toward the median eminence. Additionally, a reduction of both GAD67 positive olfactory axons and GFAP positive OEC fibers ooccurred. Acute administration of a p75NGFR blocker to GnRH cells maintained in vitro increased migration rate, consistent with the change in distribution detected in p75NGFR null mice. Chronic inhibition of p75NGFR caused an attenuation of olfactory axon fasciculation and a decrease in OEC density, again mimicking the changes detected in null mice. However, a reduction in GnRH cell number was found after chronic treatment that was not observed in KO animals suggesting indirect changes occur during chronic treatment in vitro and/or a compensatory mechanism occurs in vivo that prevents loss of GnRH neurons in the absence of p75NGFR. 3) Metabolic dysfunctions are often linked to reproductive abnormalities. Adiponectin, a peripheral hormone secreted by white adipose tissue, is important in energy homeostasis and appetite regulation. Our work examined whether adiponectin can directly act on GnRH neurons. We found that a subpopulation of GnRH neurons express AdipoR2. GnRH/AdipoR2+ cells were distributed throughout the forebrain. To determine the influence of adiponectin on GnRH neuronal activity and the signal transduction pathway of AdipoR2, GnRH neurons maintained in explants were assayed using whole cell patch clamping and calcium imaging. Single cell PCR analysis and immunocytochemistry confirmed the presence of AdipoR2 in GnRH neurons in explants. Functional analysis revealed 20% of the total GnRH population responded to adiponectin, exhibiting hyperpolarization or decreased calcium oscillations. Pertubation studies revealed that adiponection activates AMP-Kinase via the PKCzeta;/LKB1 pathway. The modulation of GnRH neuronal activity by adiponectin directly links energy balance to neurons controlling reproduction. 4) Disruption of GnRH migration results in Kallmann syndrome (KS), which is characterized by anosmia and pubertal failure due to hypogonadotropic hypogonadism. Candidate-gene screening, autozygosity mapping, and whole-exome sequencing in a cohort of 30 individuals with KS, identified homozygous loss-of-function mutations in FEZF1 in two independent consanguineous families each with two affected siblings. We show that the mutation causes partial loss of function using a mouse model.The normal FEZF1 product is known to enable axons of olfactory receptor neurons to penetrate the CNS basal lamina in mice. Because a subset of axons in these tracks is the migratory pathway for GnRH neurons, in FEZF1 deficiency, GnRH neurons also fail to enter the brain. These results indicate that FEZF1 is required for establishment of the central component of the HPG axis in humans.
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