DEVELOPMENT AND REGULATION OF THE LUTEINIZING HORMONE RELEASING HORMONE SYSTEM

对生殖至关重要的LHRH神经元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LHRH神经元在正常/转基因动物和鼻外植体中的分化、迁移和轴突靶向的机制。研究了胚胎LHRH神经元（在中枢神经系统外）和出生后LHRH神经元（在中枢神经系统内）对LHRH基因表达、肽合成和分泌的内在和跨突触调控。本实验室前期的研究表明：1)嗅轴突的生长和LHRH神经元的迁移发生在离体嗅窝组织中；2)发现了定位于鼻中线软骨组织的嗅轴突“引导线索”；3)在LHRH神经元中发现了两个“发育基因”；AP-2（一种发育转录因子）和一种称为NELF的新基因。在过去的一年里，我们利用正常和AP-2突变小鼠研究了AP-2在LHRH发展中的作用。这些研究表明，AP-2限制了呼吸上皮细胞中LHRH的表达，并在LHRH细胞进入中枢神经系统时维持LHRH基因的表达。在过去的一年里，我们也发现了NELF在发育过程中的表达模式。NELF主要局限于PNS和CNS组织。与我们的工作相关，编码蛋白在嗅觉系统和迁移的LHRH神经元中表达。反义实验表明，NELF的敲除减少了嗅轴突的生长和随后的LHRH神经元迁移。我们假设NELF通过亲同性相互作用起作用。未来的研究旨在了解NELF在发育过程中的作用，以及由该分子激活的下游信号导致轴突生长和细胞运动。为了了解LHRH神经元的调控及其细胞反应，我们在鼻外植体中检测了LHRH神经元的活性。我们已经发现，这些细胞的内在特性中有一些重要的成熟事件。我们还证明，体外维持的LHRH神经元可以释放LHRH肽，并根据分泌事件快速合成新的肽。正在进行的工作主要集中在：1)分离影响嗅轴突生长的中线线索；2) NELF和其他分子在LHRH迁移中的作用，3)LHRH神经元在鼻腔外植体（不受大脑影响）中是否表现出节律性活动模式，如果是，这种节律的机制，以及4)LHRH神经元中基因的差异表达作为发育和生殖状态的功能。

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 LHRH neuronal differentiation,migration and axonal targeting in normal/transgenic animals, and nasal explants. Intrinsic and trans-synaptic regulation of LHRH gene expression, peptide synthesis and secretion in embryonic LHRH neurons (outside the CNS) versus postnatal LHRH neurons (in the CNS) is studied using nasal explants and organotypic cultures, respectively. Previous work in our lab using nasal explants 1) showed that outgrowth of olfactory axons and migration of LHRH neurons occur in isolated olfactory pit tissue, 2) revealed olfactory axon 'guidance cues' localized to midline nasal cartilagenous tissue and 3) identified two 'developmental genes' in LHRH neurons; AP-2 (a developmental transcription factor) and a novel gene termed NELF. Over the past year, we have addressed the role of AP-2 in LHRH development using normal and AP-2 mutant mice. These studies demonstrate that AP-2 restricts LHRH expression from cells in the respiratory epithelium and is important for maintenance of LHRH gene expression as LHRH cells enter the CNS. Over the past year, we also characterized the expression pattern of NELF during development NELF is primarily limited to PNS and CNS tissues. Pertinent to our work, the encoded protein is expressed in the olfactory system and migrating LHRH neurons. Antisense experiments demonstrated that knock-down of NELF decreased olfactory axon outgrowth and subsequent LHRH neuronal migration. We hypothesize NELF acts via a homophilic interaction. Future studies are directed at understanding the role of NELF during development and the downstream signals activated by this molecule that result in axon outgrowth and cell movement. To understand the regulation of LHRH neurons and their cellular responses, we are examining the activity of LHRH neurons in nasal explants. We have found that there are important maturational events that are intrinsic properties of these cells. We have also demonstrated that LHRH neurons maintained in vitro can release LHRH peptide and rapidly synthesize new peptide in response to a secretory event. Work in progress is focused on: 1) isolation of the midline cues which influence olfactory axon outgrowth; 2) the role of NELF and other molecules in LHRH migration, 3) whether LHRH neurons maintained in nasal explants (devoid of brain influences) show rhythmic activity patterns and if so the mechanisms underlying this rhythm, and 4) genes differentially expressed in LHRH neurons as a function of developmental and reproductive state.