GnRH (also known as LHRH) 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. Over the past year, two studies were finished: 1) Stromal derived growth factor (SDF-1) and gamma-aminobutyric acid (GABA) are two extracellular cues that regulate neuronal migration during development and may act synergistically. The molecular mechanisms of this interaction were unclear. Both SDF-1 and GABA have been shown to regulate the rate of GnRH neuronal migration by accelerating and slowing migration, respectively. As such, this system was used to explore the mechanism by which these molecules act to produce coordinated cell movement during development. GABA and SDF-1 were shown to exert opposite effects on the speed of cell movement by activating depolarizing or hyperpolarizing signaling pathways, GABA via changes in chloride and SDF-1 via changes in potassium. GABA and SDF-1 were also found to act synergistically to promote linear rather than random movement. The simultaneous activation of these signaling pathways, therefore, results in tight control of cellular speed and improved directionality along the migratory pathway of GnRH neurons. 2) Proper assembly of neural circuits requires newly born neurons to migrate from their place of origin to their final location. Little is known about the mechanisms of axophilic neuronal migration, whereby neurons travel along axon pathways to navigate to their destinations. GnRH neurons migrate along olfactory axons from the nose into the forebrain during development, and were used as a model of axophilic migration. We investigated in mice the regulation of movement from calcium signals to cytoskeletal dynamics. Live imaging revealed robust calcium activity during axophilic migration, and calcium release through IP3 receptors was found to stimulate migration. This occurred through a signaling pathway involving the calcium sensor calcium/calmodulin protein kinase kinase (CaMKK), AMP-activated kinase (AMPK) and RhoA/ROCK. By imaging GnRH neurons expressing actin- GFP or Lifeact-RFP, calcium release was found to stimulate leading process actin flow away from the cell body. In contrast, actin contractions at the cell rear were unaffected by this calcium signaling pathway. These findings are the first to test the regulation of cytoskeletal dynamics in axophilic migration, and reveal mechanisms of movement that have broad implications for the migration of other CNS populations. New investigations are characterizing the role of Bone Morphogenic Protein-4 (Bmp4) and Fibroblast growth factor-8 (Fgf8) in defining epithelial versus neurogenic fate in the developing olfactory/vomeronasal system. Using different knock-in mouse lines and Cre-lox mediated lineage tracing, Fgf8 expression and cell lineage is being analyzed in relation to the expression of Bmp4 and its antagonist Noggin. These experiments will determine whether Fgf8 is sufficient to induce ectodermal progenitors of the olfactory pit to acquire neural fate as well as induce GnRH neuronal specification. In addition, to specifically remove molecules of interest from GnRH cells during development and examine migrational changes, Cre-lox-mice and/or pharmacological/molecular manipulations are being employed and in situ characterization of the migration of GnRH neurons done using real time microscopy. Specific studies in progress examine the role of growth factor receptors (VEGR1 and FGFR1). Lastly, we continue to study the physiology of GnRH neurons focusing on GABA and adiponectin. We are examining why GABA remains excitatory to GnRH neurons in the adult by evaluating chloride transporters and expression of GABA receptor subtypes over development. In humans and animal models, 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. We found that a subpopulation of GnRH neurons express AdipoR2. Thus, we will determine whether adiponectin can directly act on GnRH neurons using whole cell patch clamping and calcium imaging. Modulation of GnRH neuronal activity by adiponectin would demonstrate a direct link between energy balance and neurons controlling reproduction.

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Constantin, Stephanie; Wray, Susan (2016) Galanin Activates G Protein Gated Inwardly Rectifying Potassium Channels and Suppresses Kisspeptin-10 Activation of GnRH Neurons. Endocrinology 157:3197-212
Klenke, Ulrike; Constantin, Stephanie; Wray, Susan (2016) BPA Directly Decreases GnRH Neuronal Activity via Noncanonical Pathway. Endocrinology 157:1980-90
Hutchins, B Ian; Kotan, L Damla; Taylor-Burds, Carol et al. (2016) CCDC141 Mutation Identified in Anosmic Hypogonadotropic Hypogonadism (Kallmann Syndrome) Alters GnRH Neuronal Migration. Endocrinology 157:1956-66
Taylor-Burds, Carol; Cheng, Paul; Wray, Susan (2015) Chloride Accumulators NKCC1 and AE2 in Mouse GnRH Neurons: Implications for GABAA Mediated Excitation. PLoS One 10:e0131076
Forni, Paolo E; Wray, Susan (2015) GnRH, anosmia and hypogonadotropic hypogonadism--where are we? Front Neuroendocrinol 36:165-77
Hutchins, B Ian; Wray, Susan (2014) Capture of microtubule plus-ends at the actin cortex promotes axophilic neuronal migration by enhancing microtubule tension in the leading process. Front Cell Neurosci 8:400
Kotan, L Damla; Hutchins, B Ian; Ozkan, Yusuf et al. (2014) Mutations in FEZF1 cause Kallmann syndrome. Am J Hum Genet 95:326-31
Klenke, Ulrike; Taylor-Burds, Carol; Wray, Susan (2014) Metabolic influences on reproduction: adiponectin attenuates GnRH neuronal activity in female mice. Endocrinology 155:1851-63
Raucci, Franca; Tiong, Jean D; Wray, Susan (2013) P75 nerve growth factor receptors modulate development of GnRH neurons and olfactory ensheating cells. Front Neurosci 7:262
Hutchins, B Ian; Klenke, Ulrike; Wray, Susan (2013) Calcium release-dependent actin flow in the leading process mediates axophilic migration. J Neurosci 33:11361-71

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