DEVELOPMENT AND REGULATION OF THE LUTEINIZING HORMONE RELEASING HORMONE SYSTEM

LHRH神经元，生殖的关键，来自嗅基板和迁移到大脑，在那里他们成为下丘脑-垂体-性腺轴的组成部分。我们在正常/转基因动物和鼻外植体中研究LHRH神经元分化和迁移的机制。胚胎LHRH神经元LHRH基因表达、肽合成和分泌的内在和跨突触调节（CNS外）与出生后LHRH神经元本实验室以前的工作使用鼻外植体1）表明，嗅轴突的生长和LHRH神经元的迁移发生在分离的嗅窝组织中，2）揭示了定位于中线鼻软骨组织的嗅觉轴突引导线索和3）鉴定了LHRH神经元中的两个发育基因; AP-2（一种发育转录因子）和克隆18（一种新基因）。在过去的一年中，我们已经解决了AP-2的作用，在LHRH的发展使用AP-2突变小鼠。这些研究表明，AP-2是重要的LHRH基因的表达，因为这些神经元进入中枢神经系统的维护。由于LHRH启动子不包含AP-2共有序列，AP-2必须通过间接途径起作用。关于我们的新基因，克隆18，我们能够产生对编码蛋白质特异的抗体。克隆18的mRNA转录物和蛋白质的作图已经完成。虽然在多种CNS区域中发现表达，但与我们的工作最相关的是，编码的蛋白质在嗅觉系统和迁移的LHRH神经元中表达。体外初步研究表明，该蛋白质与细胞膜的细胞外部分相关，使其成为迁移的候选分子。为了了解LHRH神经元活性的调节，我们已经完成了小鼠LHRH肽含量的发育变化的工作。这些研究表明，LHRH肽含量/细胞在体内LHRH神经元中以及在鼻外植体中维持的LHRH神经元（体外胚胎LHRH神经元）中增加。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。我们还证明了体外培养的LHRH神经元可以释放LHRH肽。正在进行的工作集中在：1）分离影响嗅觉轴突生长的中线线索; 2）克隆18在LHRH迁移中的作用，3）LHRH神经元在鼻外植体中是否维持（没有大脑的影响）显示出有节奏的活动模式，如果是这样，这种节奏的机制，和4）在LHRH神经元中差异表达的基因作为发育和生殖状态的函数。- 促性腺激素释放激素，神经元迁移，轴突导向，搏动，鼻移植，单细胞聚合酶链反应。

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.