DEVELOPMENT AND REGULATION OF THE LUTEINIZING HORMONE RELEASING HORMONE SYSTEM

LHRH神经元对生殖至关重要，它们来自嗅觉区并迁移到大脑，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和迁移机制。研究了胚胎LHRH神经元（中枢神经系统外）和出生后LHRH神经元（中枢神经系统内）对LHRH基因表达、肽合成和分泌的内在和反突触调控。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)鉴定了LHRH神经元的两个发育基因；AP-2（一个发育转录因子）和克隆18（一个新基因）。在过去的一年里，我们利用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，当这些神经元进入中枢神经系统时，AP-2对于维持LHRH基因表达很重要。由于LHRH启动子不包含AP-2一致序列，AP-2必须通过间接途径起作用。至于我们的新基因，克隆18，我们能够产生一种针对编码蛋白的抗体。克隆18的mRNA转录物和蛋白图谱已经完成。尽管在各种中枢神经系统区域都发现了表达，但与我们的工作最相关的是，编码蛋白在嗅觉系统和迁移的LHRH神经元中表达。体外初步研究表明，该蛋白与细胞膜的胞外部分有关，使其成为迁移的候选分子。为了了解LHRH神经元活动的调控，我们完成了小鼠LHRH肽含量在发育过程中的变化。这些研究表明，体内LHRH神经元以及鼻腔外植体（体外胚胎LHRH神经元）中维持的LHRH神经元中LHRH肽/细胞含量增加。在体外，LHRH肽含量的增加与体内测量的相当，表明重要的成熟事件是这些细胞的内在特性。我们也证明了体外维持的LHRH神经元可以释放LHRH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)克隆18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；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。- GnRH，神经元迁移，轴突引导，脉搏，鼻外植体，单细胞pcr。

LHRH neurons, critical for reproduction, are derived from the olfactory 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 and migration 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 clone 18 (a novel gene). Over the past year, we have addressed the role of AP-2 in LHRH development using AP-2 mutant mice. These studies demonstrate that AP-2 is important for maintenance of LHRH gene expression as these neurons enter the CNS. Since the LHRH promoter does not contain an AP-2 consensus sequence, AP-2 must act via an indirect route. With regard to our novel gene, clone 18, we were able to generate an antibody specific for the encoded protein. Mapping of both the mRNA transcript and protein for clone 18 have been done. Although expression is found in a variety of CNS regions, most pertinent to our work, the encoded protein is expressed in the olfactory system and migrating LHRH neurons. Preliminary work in vitro suggests that the protein is associated with the extracellular portion of the cell membrane, making it a candidate molecule for migration. To understand the regulation of LHRH neuronal activity, we have completed work on the developmental changes in LHRH peptide content in mice. These studies demonstrate that LHRH peptide content/cell increases in LHRH neurons in vivo as well as LHRH neurons maintained in nasal explants (embryonic LHRH neurons in vitro). In vitro, the increase in LHRH peptide content is comparable to that measured in vivo, suggesting important maturational events are intrinsic properties of these cells. We have also demonstrated that LHRH neurons maintained in vitro can release LHRH peptide.Work in progress is focused on: 1) isolation of the midline cues which influence olfactory axon outgrowth; 2) the role of clone 18 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. - GnRH, neuronal migration, axon guidance, pulsatility, nasal explants, single cell pcr.