Development And Regulation Of The Luteinizing Hormone Releasing Hormone System

GnRH-1（也被称为LHRH）神经元，对生殖至关重要，来源于鼻位并迁移到大脑，在那里它们成为下丘脑-垂体-性腺轴的组成部分。我们研究了GnRH-1神经元在正常/转基因动物和鼻腔外植体中的分化、迁移和轴突靶向的机制。使用这些相同的模型，我们的工作还解决了GnRH-1神经元中调节（内在和跨突触）GnRH基因表达、肽合成和分泌的机制。多种方法用于识别和理解在引导GnRH-1神经元到达其在中枢神经系统中的最终位置中发挥作用的众多分子和因素。这些包括从迁移和非迁移细胞中获得的文库的差异筛选，在迁移路线的关键位置检查差异表达的分子，敲除小鼠中GnRH-1系统发育的形态学检查，以及体外分子扰动和随后监测GnRH-1神经元运动。随着GnRH-1神经元的迁移，它们也成熟了，这两个过程实际上可能是相关联的。为了研究GnRH-1神经元的成熟，我们采用钙显像、电生理和生化方法检测了GnRH-1神经元的活性和肽分泌。

GnRH-1 (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-1 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-1 neurons. Multiple approaches are used to identify and understand the multitude of molecules and factors which play a role in directing the GnRH-1 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-1 system in knockout mice, and perturbation of molecules in vitro and subsequent monitoring of GnRH-1 neuronal movement. As GnRH-1 neurons migrate they also mature and the two processes may in fact be linked. To investigate the maturation of GnRH-1 neurons we use calcium imaging, electrophysiology and biochemical measures to examine GnRH-1 neuronal activity and peptide secretion. Over the past year several studies were finished : 1) Cyclic nucleotide-gated (CNG) channels have been proposed as a mechanism to integrate the cAMP signal evoked by many neurotransmitters. We documented expression of theCNGA2 subunit in GnRH-1 neurons and showed the ability of GnRH-1 neurons to increase their activity in response to forskolin (activator of adenylyl cyclases), or 3-isobutyl-1-methylxanthine (inhibitor of phosphodiesterases) even after removal of GABA-ergic input. However we subsequently found that CNGA2 channels are not involved in either the response of GnRH-1 neurons to cAMP increases or the basal rhythmic activity of GnRH-1 neurons. 2) Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are involved in neuronal pacemaking a characteristic of GnRH-1 cells. HCN channels can integrate cAMP signals. cAMP dependent protein kinase (PKA) is also activated by cAMP signals and PKA-dependent phosphorylation modulates voltage-activated channels. GnRH-1 neurons express HCN channel protein. We examined documented that PKA-dependent phosphorylation is involved in the FSK-induced stimulation of GnRH-1 neurons rather than HCN channels, but that HCN channels integrate the FSK-induced stimulation on GABAergic neurons. In addition, blockade of HCN channels did not modify basal GnRH-1 neuronal activity when GABAergic input was intact or removed, negating a role for these channels in basal GABAergic or GnRH-1 neuronal activity. 3) Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 influence neuronal migration and are in nasal regions. We examined the role of SDF-1/CXCR4 in development of the GnRH-1/olfactory systems. Migrating GnRH-1 neurons were CXCR4 immunopositive as were the fibers along which they migrate and SDF-1 highlighted the GnRH-1 migratory pathway. CXCR4-deficient mice showed a decrease in GnRH-1 cells at the nasal forebrain junction and in brain, but the overall migratory pathway remained intact, no ectopic GnRH-1 cells were detected and olfactory axons reached the olfactory bulb. To further characterize the influence of SDF-1/CXCR4 in the GnRH-1 system, nasal explants were used. Treatment of explants with a CXCR4 antagonist attenuated GnRH-1 neuronal migration and sensory axon outgrowth. Moreover, the number of GnRH-1 neurons in the explant periphery was reduced. The effects were blocked by coincubation with SDF-1. Removal of midline SDF-1 cells did not alter directional outgrowth of olfactory axons. These results indicate that SDF-1/CXCR4 signaling in not necessary for olfactory axon guidance but rather influences sensory axon extension and GnRH-1 neuronal migration, and maintains GnRH-1 neuronal expression as the cells move away from nasal pit regions. 4) Cholecystokinin (CCK) is a peptide found in both gut and brain. Although numerous studies address the role of brain CCK postnatally, relatively little is known about the ontogeny of CCK expression in the central nervous system (CNS). Recent work revealed that CCK modulates olfactory axon outgrowth and gonadotropin-releasing hormone-1 (GnRH-1) neuronal migration, suggesting that CCKmay be an important factor during CNS development. To further characterize the developmental expression of CCK in the nervous system, in situ hybridization experiments were performed. CCK mRNA expression was widely distributed in the developing mouse brain. As early as E12.5, robust CCK expression is detected in the thalamus and spinal cord. By E17.5, cells in the cortex, hippocampus, thalamus and hypothalamus express CCK. In addition, CCK mRNA was also detected in the external zone of the median eminence where axons of the neuroendocrine hypophysiotropic systems terminate. Our study demonstrates that CCK mRNA is expressed prenatally in multiple areas of the CNS, many of which maintain CCK mRNA expression postnatally into adult life. In addition, we provide evidence that regions of the CNS known to integrate hormonal and sensory information associated with reproduction and the GnRH-1 system, expressed CCK already during prenatal development. 5) Gonadotropin releasing-hormone-1 (GnRH-1) is expressed in mouse incisors during development. In this paper, we identify (1) cell type(s) that express GnRH-1 throughout tooth development, (2) the GnRH-1 receptor, and (3) the role of GnRH- 1/GnRH-1 receptor signaling in tooth maturation. Results show that GnRH-1-positive cells in dental epithelium differentiate and populate multiple tooth structures including ameloblast and papillary layers that are involved in enamel formation and mineralization. The GnRH-1 receptor was present; and in vitro, a GnRH-1 antagonist attenuated incisor GnRH-1 cell expression. In vivo, in mice lacking GnRH-1 (-/-), the incisors were discolored, longer, and more curved compared to wildtype. Elemental analysis of calcium, phosphorus and iron revealed changes in -/- incisors consistent with GnRH-1 affecting movement of minerals into the dental matrix. In sum, in tooth development a signal transduction pathway exists for GnRH-1 via the GnRH-1 receptor and that disruption of such signaling affects incisor growth and biomineralization. Studies in progress center on the role of NELF (a migrational molecule), cytokines, and growth factors in GnRH-1 development as well as in situ characterization of the migration of LHRH neurons (real time microscopy). We are examining the early development of the GnRH-1 neurons and the location of their progenitor cells in relation to nasal and anterior pituitary placodal cells. In addition, we continue to study the role of estrogen on GnRH-1 neuronal activity and have recently start monitoring GnRH-1 neuronal activity in nasal explants generated from estrogen receptor knockout mice. Other studies include examining/identifying 1) the electrical properties associated with GnRH-1 neuronal activity (combining electrical recording and calcium imaging), 2) molecules that modulate GnRH-1 neuronal activity that participate in reproductive functions such as NPY and Kisspeptin, 3) midline cues which influence olfactory axon outgrowth and 4) GABAergic signals during development of the GnRH-1 system