Development And Regulation Of The Luteinizing Hormone Releasing Hormone System

黄体生成素释放激素系统的发育与调控

• 批准号: 7594664
• 负责人: SUSAN WRAY
• 金额: $ 252.59万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594664
• 关键词: Address; Adult; Alteplase; Anterior Pituitary Gland; Anterior Pituitary Hormones; Attenuated; Axon; BHLH Protein; Biochemical; Brain; Calcium; Cells; Characteristics; Cholecystokinin; Cholecystokinin A Receptor; Cholecystokinin Receptor; Complement; Coupled; Cues; Data; Destinations; Development; Disruption; Electrophysiology (science); Embryo; Estrogen Receptors; Estrogens; Exhibits; Female; Fiber; Functional disorder; Gene Expression; Genetic; Goals; Gonadotropin Hormone Releasing Hormone; Growth Factor; Hepatocyte Growth Factor; Hypothalamic structure; Image; In Situ; In Vitro; Incisor; Knockout Mice; Label; Libraries; Life; Link; Localized; Location; Maintenance; Measures; Microscopy; Modeling; Monitor; Movement; Mus; Mutant Strains Mice; Neuronal Differentiation; Neurons; Neuropeptides; Neurosecretory Systems; Nose; Numbers; Pacemakers; Patients; Pattern; Peptide Synthesis; Peptides; Phenotype; Pituitary Gland; Play; Population; Process; Prosencephalon; Puberty; Receptor Activation; Regulation; Reproduction; Reproductive Behavior; Role; Route; Screening procedure; Signal Transduction; Slice; Synapses; Syndrome; System; Time; Tooth structure; Transgenic Animals; Treatment Protocols; Vertebrates; Work; Zebrafish; biomineralization; cell type; cytokine; electrical property; hepatocyte growth factor activator; hypothalamic pituitary gonadal axis; immunocytochemistry; in vitro Assay; in vivo; member; migration; molecular array; negative elongation factor; neutralizing antibody; pituitary gonadal axis; progenitor; promoter; receptor; reproductive; reproductive function; research study; tool

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. 1) Over the past year we have investigated the role of the brain-gut peptide, CCK, as a modulator of GnRH-1 neurnal activity. Functional analysis indicated that CCK inhibits GnRH-1 neuronal activity via CCK-1 receptors. CCK is a neuropeptide abundantly expressed in the brain, which is implicated in activation of female reproductive behaviors and release of anterior pituitary hormones. Using dual-label immunocytochemistry coupled to confocal analysis, GnRH-1 neurons in adult mouse brain were found to express CCK-1 receptors (CCK-1R) and CCK fibers were detected contacting GnRH-1 axons. To address the function of CCK on GnRH-1 neurons, calcium imaging was used to monitor patterns of activity of GnRH-1 neurons maintained in an in vitro system known to retain many characteristics of GnRH-1 cells in vivo. Endogenous receptors for CCK (CCK-1R and CCK-2R) were blocked with selective antagonists. Results indicate that CCK-1R but not CCK-2R antagonist treatment increased the number of calcium peaks/GnRH-1 cell, mean peak amplitude and percentage of GnRH-1 cells displaying high activity. The increased activity in GnRH-1 neurons observed after application of CCK-1R antagonist was blocked by co-incubation with exogenous CCK. This study provides evidence that CCK acts directly on GnRH-1 neurons to attenuate GnRH-1 neuronal activity via CCK-1R activation. 2) Studies continued to identify the role of GnRH-1 in developing incisor. Morphological changes in the incisor of GnRH-1 mutant mice were analyzed and indicate GnRH-1 participated in tooth maturation and biomineralization. 3) A collaborative study examined the development of the GnRH systems in zebrafish to verify that GnRH-1 cells arise in association with the nasal placode. Using morphological studies as well as tagged GnRH-1 cells in live embryos, GnRH-1 cells were observed migration from the nasal placode region into the developing forebrain. 4) Another collaborative study examined the role hepatocyte growth factor (HGF) and its receptor Met during development of the GnRH-1 system. GnRH-1 cells express Met during their migration and downregulate its expression once they complete this process. Tissue-type plasminogen activator (tPA), a known HGF activator, is also detected in migratory GnRH-1 neurons. Consistent with in vivo expression, HGF is present in nasal explants and GnRH-1 neurons express Met. HGF neutralizing antibody was applied to explants to examine the role of the endogenous growth factor. Migration of GnRH-1 cells and olfactory axon outgrowth was significantly reduced, in line with disruption of a guidance gradient. Exogenous application of HGF to explants increased the distance that GnRH-1 cells migrated suggesting that HGF also acts as a motogen to GnRH-1 neurons. Functional experiments, performed on organotypic slice cultures, show that creation of an opposing HGF gradient inhibits GnRH-1 neuronal migration. Finally, tPA-/- : uPA-/- knock-out mice exhibit strong reduction of the GnRH-1 cell population. Together, these data indicate that HGF signaling via Met receptor influences the development of GnRH-1. 5) Pubertal development is impaired in mice lacking the basic helix-loop-helix transcription factor Nhlh2. The mechanisms underlying changes in reproduction in Nhlh2-/- mice were unclear. In this study, we show that hypothalamic GnRH-1 content is reduced in adult Nhlh2-/- mice as is the number of GnRH-1 neurons localized to mid- and caudal hypothalamic regions. This reduction was detected postnatally after normal migration of GnRH-1 neurons within nasal regions had occurred. Phenotype rescue experiments showed that female Nhlh2-/- mice were responsive to estrogen treatment. In contrast, puberty could not be primed in female Nhlh2-/- mice with a GnRH-1 regimen. The adenohypophysis of Nhlh2-/- mice was hypoplastic although it contained a full complement of the five anterior pituitary cell types. GnRH-1 receptors (GnRHRs) were reduced in Nhlh2-/- pituitary gonadotropes as compared to wild type. In vitro assays indicated that Nhlh2 expression is regulated in parallel with GnRHR expression. However, direct transcriptional activity of Nhlh2 on the GnRHR promoter was not found. These results indicate that Nhlh2 plays a role in the development and functional maintenance of the hypothalamic-pituitarygonadal axis at least at two levels: 1) in the hypothalamus by regulating the number and distribution of GnRH-1 neurons and, 2) in the developing and mature adenohypophysis. 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). 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) midline cues which influence olfactory axon outgrowth, 3) pacemaker molecules in GnRH-1 neurons that participate in establishment/maintenance of rhythmic activity as well as regulator molecules such as Kisspeptin, and 4) GABAergic signals during development of the GnRH-1 system.

GnRH-1（也称为 LHRH）神经元对生殖至关重要，源自鼻基板并迁移到大脑中，成为下丘脑-垂体-性腺轴的组成部分。我们研究正常/转基因动物和鼻外植体中 GnRH-1 神经元分化、迁移和轴突靶向的潜在机制。使用这些相同的模型，我们的工作还解决了 GnRH-1 神经元中（内在和跨突触）GnRH 基因表达、肽合成和分泌的调节机制。使用多种方法来识别和理解在引导 GnRH-1 神经元到达中枢神经系统最终位置方面发挥作用的多种分子和因子。 这些包括对从迁移细胞和非迁移细胞获得的文库进行差异筛选，检查迁移路线上关键位置差异表达的分子，对基因敲除小鼠中 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。 当 GnRH-1 神经元迁移时，它们也会成熟，这两个过程实际上可能是相关的。 为了研究 GnRH-1 神经元的成熟，我们使用钙成像、电生理学和生化措施来检查 GnRH-1 神经元活性和肽分泌。 1) 在过去的一年里，我们研究了脑肠肽 CCK 作为 GnRH-1 神经活动调节剂的作用。功能分析表明CCK 通过CCK-1 受体抑制GnRH-1 神经元活性。 CCK 是一种在大脑中大量表达的神经肽，与女性生殖行为的激活和垂体前叶激素的释放有关。使用双标记免疫细胞化学结合共聚焦分析，发现成年小鼠大脑中的 GnRH-1 神经元表达 CCK-1 受体 (CCK-1R)，并且检测到接触 GnRH-1 轴突的 CCK 纤维。为了研究 CCK 对 GnRH-1 神经元的功能，使用钙成像来监测体外系统中维持的 GnRH-1 神经元的活动模式，该系统已知在体内保留了 GnRH-1 细胞的许多特征。 CCK 的内源性受体（CCK-1R 和 CCK-2R）被选择性拮抗剂阻断。结果表明CCK-1R而非CCK-2R拮抗剂处理增加了钙峰的数量/GnRH-1细胞、平均峰振幅和表现出高活性的GnRH-1细胞的百分比。应用CCK-1R拮抗剂后观察到的GnRH-1神经元活性增加被与外源CCK共孵育所阻断。这项研究提供了证据，证明 CCK 直接作用于 GnRH-1 神经元，通过 CCK-1R 激活来减弱 GnRH-1 神经元活性。 2) 研究继续确定 GnRH-1 在门牙发育中的作用。分析 GnRH-1 突变小鼠门牙的形态变化，表明 GnRH-1 参与牙齿成熟和生物矿化。 3) 一项合作研究检查了斑马鱼 GnRH 系统的发育，以验证 GnRH-1 细胞的产生与鼻基板相关。通过形态学研究以及活胚胎中标记的 GnRH-1 细胞，观察到 GnRH-1 细胞从鼻基板区域迁移到发育中的前脑。 4) 另一项合作研究考察了肝细胞生长因子 (HGF) 及其受体 Met 在 GnRH-1 系统发育过程中的作用。 GnRH-1 细胞在迁移过程中表达 Met，并在完成此过程后下调其表达。组织型纤溶酶原激活剂 (tPA) 是一种已知的 HGF 激活剂，也在迁移的 GnRH-1 神经元中检测到。与体内表达一致，HGF 存在于鼻外植体中，并且 GnRH-1 神经元表达 Met。将 HGF 中和抗体应用于外植体以检查内源生长因子的作用。 GnRH-1 细胞的迁移和嗅觉轴突的生长显着减少，与引导梯度的破坏一致。外植体上应用 HGF 增加了 GnRH-1 细胞迁移的距离，表明 HGF 也可作为 GnRH-1 神经元的运动原。在器官切片培养物上进行的功能实验表明，反向 HGF 梯度的产生会抑制 GnRH-1 神经元迁移。 最后，tPA-/- : uPA-/- 敲除小鼠表现出 GnRH-1 细胞群的强烈减少。总之，这些数据表明 HGF 信号通过 Met 受体影响 GnRH-1 的发育。 5)缺乏碱性螺旋-环-螺旋转录因子Nhlh2的小鼠青春期发育受到损害。 Nhlh2-/- 小鼠生殖变化的机制尚不清楚。在这项研究中，我们发现成年 Nhlh2-/- 小鼠下丘脑 GnRH-1 含量减少，定位于下丘脑中部和尾部区域的 GnRH-1 神经元数量也减少。这种减少是在出生后鼻区 GnRH-1 神经元发生正常迁移后检测到的。表型拯救实验表明雌性 Nhlh2-/- 小鼠对雌激素治疗有反应。相比之下，使用 GnRH-1 方案无法启动雌性 Nhlh2-/- 小鼠的青春期。 Nhlh2-/- 小鼠的腺垂体发育不全，尽管它含有完整的五种垂体前叶细胞类型。与野生型相比，Nhlh2-/- 垂体促性腺激素中的 GnRH-1 受体 (GnRHR) 减少。体外测定表明 Nhlh2 表达与 GnRHR 表达平行调节。然而，没有发现 Nhlh2 对 GnRHR 启动子的直接转录活性。这些结果表明，Nhlh2 至少在两个水平上在下丘脑-垂体性腺轴的发育和功能维持中发挥作用：1) 在下丘脑中通过调节 GnRH-1 神经元的数量和分布，2) 在发育和成熟的腺垂体中。 正在进行的研究集中在 NELF（一种迁移分子）、细胞因子和生长因子在 GnRH-1 发育中的作用以及 LHRH 神经元迁移的原位表征（实时显微镜）。此外，我们继续研究雌激素对 GnRH-1 神经元活性的作用，并最近开始监测雌激素受体敲除小鼠鼻外植体中的 GnRH-1 神经元活性。 其他研究包括检查/识别 1) 与 GnRH-1 神经元活动相关的电特性（结合电记录和钙成像），2) 影响嗅觉轴突生长的中线线索，3) GnRH-1 神经元中参与节律活动建立/维持的起搏分子以及 Kisspeptin 等调节分子，以及 4) GnRH-1 系统发育过程中的 GABA 信号。