Pineal Regulation: Molecular basis of development

松果体调节：发育的分子基础

• 批准号: 7734834
• 负责人: David Klein
• 金额: $ 44.9万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/7734834
• 关键词: Address; Adrenergic Agents; Adrenergic Agonists; Adult; Arylalkylamine N-Acetyltransferase; Attention; Binding Sites; Boxing; Brain; Brain region; Cells; Cerebellum; Cervical; Chronobiology Disorders; Circadian Rhythms; Cyclic AMP; Cyclic AMP Response Element; Daily; Development; Disease; Drosophila genus; E-Box Elements; Ectopic Expression; Elements; Embryo; Embryonic Development; Endocrine; Enzymes; Event; Evolution; Excision; Exhibits; Family; Family member; Fetus; Foundations; Ganglionectomy; Gene Cluster; Gene Expression; Gene Expression Profiling; Gene Targeting; Genes; Gland; Glutamate Decarboxylase; Goals; Hand; Heart; Homeobox; Homeobox Genes; Homologous Gene; In Vitro; Introns; Kinesin; Knock-out; Knockout Mice; Knowledge; Learning; Light; Lighting; Localized; Mediating; Melatonin; Messenger RNA; Modeling; Molecular; Monitor; Mutation; N-acetylserotonin; NIH 3T3 Cells; Neonatal; Nerve; Neurons; Norepinephrine; Organism; Pancreas; Pathway interactions; Pattern; Phenotype; Photoreceptors; Phototransduction; Physiological; Pineal gland; Pinealocyte; Play; Polymerase Chain Reaction; Preparation; Production; Proteins; Psyche structure; Quantitative Reverse Transcriptase PCR; RNA Interference; Rattus; Regulation; Regulatory Element; Reporting; Research; Retina; Retinal; Retinal Diseases; Retinal Photoreceptors; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Role; Schedule; Sea Bream; Sequence Analysis; Serotonin; Signal Transduction; Sleep; Staging; Sympathectomy; System; Testing; Time; Tissues; Transcript; Transcriptional Regulation; Transgenes; Transgenic Organisms; Undifferentiated; Variant; Vertebrate Biology; Vertebrate Photoreceptors; Vertebrates; Work; Zebrafish; adrenergic; base; circadian pacemaker; day; driving force; immune function; in vivo; interest; member; mutant; neurodevelopment; postnatal; prevent; promoter; relating to nervous system; trafficking; transcription factor; trend

Evolution of pineal gland: "The defining feature of the pineal gland is the capacity to function as a melatonin factory that operates on a approximately 24 h schedule, reflecting the unique synthetic capacities of the pinealocyte. Melatonin synthesis is typically elevated at night and serves to provide the organism with a signal of nighttime. Melatonin levels can be viewed as hands of the clock. Issues relating to the evolutionary events leading up to the immergence of this system have not received significant attention. When did melatonin synthesis appear in the evolutionary line leading to vertebrates? When did a distinct pineal gland first appear? What were the forces driving this evolutionary trend? As more knowledge has grown about the pinealocyte and the relationship it has to retinal photoreceptors, it has become possible to generate a plausible hypothesis to explain how the pineal gland and the melatonin rhythm evolved. At the heart of the hypothesis is the melatonin rhythm enzyme arylalkylamine N-acetyltransferase (AANAT). The advances supporting the hypothesis will be reviewed here and expanded beyond the original foundation; the hypothesis and its implications will be addressed." From (1) AANAT E-Box: "Arylalkylamine N-acetyltransferase (Aanat) is the penultimate enzyme in the serotonin-N-acetylserotonin-melatonin pathway. It is nearly exclusively expressed in the pineal gland and the retina. A marked rhythm of Aanat gene expression in the rat pineal is mediated by cyclic AMP response elements located in the promoter and first intron. Intron 1 also contains E-box elements, which mediate circadian gene expression in other cells. Here we examined whether these elements contribute to rhythmic Aanat expression in the pineal gland. This was done using transgenic rats carrying Aanat transgenes with mutant E-box elements. Circadian expression of Aanat transgenes was not altered by these mutations. However, these mutations enhanced ectopic expression establishing that the intronic Aanat E-box elements contribute to the gene's pineal specific expression. A similar role of the Aanat E-box has been reported in zebrafish, indicating that Aanat E-box mediated silencing is a conserved feature of vertebrate biology." From (2). "In all ..... species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2 In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms." (From 3) NeuroD: "NeuroD1/BETA2, a member of the bHLH transcription factor family, is known to influence the fate of specific neuronal, endocrine and retinal cells. We report here that NeuroD1 mRNA is highly abundant in the developing and adult rat pineal gland. Pineal expression begins in the 17-day embryo at which time it is also detectable in other brain regions. Expression in the pineal gland increases during the embryonic period and is maintained thereafter at levels equivalent to those found in the cerebellum and retina. In contrast, NeuroD1 mRNA decreases markedly in non-cerebellar brain regions during development. Pineal NeuroD1 levels are similar during the day and night, and do not appear to be influenced by sympathetic neural input. Gene expression analysis of the pineal glands from neonatal NeuroD1 knockout mice identifies 127 transcripts that are down-regulated (>twofold, p < 0.05) and 16 that are up-regulated (>twofold, p < 0.05). According to quantitative RT-PCR, the most dramatically down-regulated gene is kinesin family member 5C ( approximately 100-fold) and the most dramatically up-regulated gene is glutamic acid decarboxylase 1 ( approximately fourfold). Other impacted transcripts encode proteins involved in differentiation, development, signal transduction and trafficking. These findings represent the first step toward elucidating the role of NeuroD1 in the rodent pinealocyte." From (4). Crx/Otx: "Otx2 is a vertebrate homeobox gene, which has been found to be essential for the development of rostral brain regions and appears to play a role in the development of retinal photoreceptor cells and pinealocytes. In this study, the temporal expression pattern of Otx2 was revealed in the rat brain, with special emphasis on the pineal gland throughout late embryonic and postnatal stages. Widespread high expression of Otx2 in the embryonic brain becomes progressively restricted in the adult to the pineal gland. Crx (cone-rod homeobox), a downstream target gene of Otx2, showed a pineal expression pattern similar to that of Otx2, although there was a distinct lag in time of onset. Otx2 protein was identified in pineal extracts and found to be localized in pinealocytes. Total pineal Otx2 mRNA did not show day-night variation, nor was it influenced by removal of the sympathetic input, indicating that the level of Otx2 mRNA appears to be independent of the photoneural input to the gland. Our results are consistent with the view that pineal expression of Otx2 is required for development and we hypothesize that it plays a role in the adult in controlling the expression of the cluster of genes associated with phototransduction and melatonin synthesis." From (5) Pax4: "Pax 4 is a homeobox gene encoding a transcription factor that is essential for embryonic development of the endocrine pancreas. In the pancreas, Pax4 counters the effects of the related Pax6 protein, which is also involved in development of the retina and the pineal gland. In this study, we report that Pax4 is strongly expressed in the pineal gland and retinal photoreceptors of the rat. Pineal and retinal Pax4 mRNA is low in the fetus and increases postnatally; Pax6 exhibits an inverse pattern of expression, being very strongly expressed in both tissues in the fetus. In the adult, the abundance of Pax4 mRNA exhibits a diurnal rhythm in both the pineal gland and retina with maximal levels occurring late in the day. Sympathetic denervation of the pineal gland by superior cervical ganglionectomy prevents the nocturnal decrease in pineal Pax4 mRNA but does not alter the pattern of retinal Pax4 expression. Norepinephrine is released by sympathetic nerves in the pineal gland at night; in the present study, we found that treatment with adrenergic agonists suppresses pineal Pax4 expression in vivo and in vitro. Norepinephrine is known to elevate pineal cyclic AMP; here it was found that treatment with a cyclic AMP mimic reduces pineal Pax4 mRNA. These findings suggest that the nocturnal decrease in pineal Pax4 mRNA is controlled by an adrenergic-cyclic AMP mechanism and that Pax4 may function to mediate adrenergic control of circadian gene expression in the pineal gland." (Rath et al., in preparation). Eya2. Eya2, The homolog of the Drosophila gene eyeless has been identified in the pineal gland and retina. Efforts are underway to characterize the gene products, expression pattern and function.

松果体的进化：“松果体的决定性特征是作为褪黑激素工厂的功能，大约 24 小时运行，反映了松果体细胞独特的合成能力。褪黑激素的合成通常在夜间升高，并为生物体提供夜间信号。褪黑激素水平可以被视为时钟的指针。与导致松果体细胞的进化事件相关的问题 该系统的沉浸并没有受到足够的关注。褪黑素合成何时出现在脊椎动物的进化谱系中？独特的松果体何时首次出现？推动这一演变趋势的力量是什么？随着对松果体细胞及其与视网膜光感受器的关系的了解越来越多，有可能产生一个合理的假设来解释松果体和褪黑激素节律如何 进化了。该假说的核心是褪黑激素节律酶芳基烷基胺 N-乙酰转移酶 (AANAT)。支持该假设的进展将在这里进行回顾，并扩展到原始基础之外；该假设及其影响将得到解决。”来自 (1) AANAT E-Box：“芳烷基胺 N-乙酰转移酶 (Aanat) 是血清素-N-乙酰血清素-褪黑素途径中的倒数第二个酶。它几乎只在松果体和视网膜中表达。大鼠松果体中 Aanat 基因表达的显着节律是由位于启动子和第一个的环 AMP 响应元件介导的。 内含子。内含子 1 还包含 E-box 元件，可介导其他细胞中的昼夜节律基因表达。在这里，我们检查了这些元素是否有助于松果体中 Aanat 的节律表达。这是使用携带带有突变 E-box 元件的 Aanat 转基因的转基因大鼠完成的。 Aanat 转基因的昼夜节律表达并未因这些突变而改变。然而，这些突变增强了异位表达，表明 内含子 Aanat E-box 元件有助于该基因的松果体特异性表达。 Aanat E-box 在斑马鱼中也有类似的作用，表明 Aanat E-box 介导的沉默是脊椎动物生物学的一个保守特征。”来自(2)。 “在所有......物种中，AANAT 活性在翻译后水平上受到调节，并且在不同程度上也在转录水平上受到调节。这里，研究了金头海鲷（Sparus aurata）的松果体 aanat (aanat2) 的转录调节。对整个 24 小时周期中收集的松果体中 aanat2 mRNA 水平进行实时聚合酶链反应定量，揭示了 有节奏的表达模式。在培养的松果体中，振幅降低，但在恒定光照下维持日常节律性表达模式，表明鲷鱼 aanat2 的昼夜节律控制调节。在 NIH-3T3 细胞中，鲷鱼 aanat2 启动子通过 BMAL/CLOCK 和正牙齿同源盒 5 (OTX5) 的协同作用被激活。 启动子序列分析揭示了光感受器保守元件和扩展E-box的存在（即先前与松果体特异性和节律性基因表达相关的BMAL/CLOCK和OTX5的结合位点）。这些结果表明，鲷鱼 aanat2 是一个受保守机制调节的时钟控制基因。” （从 3） NeuroD：“NeuroD1/BETA2 是 bHLH 转录因子家族的成员，已知会影响特定神经元、内分泌和视网膜细胞的命运。我们在此报告，NeuroD1 mRNA 在发育中和成年大鼠松果体中含量丰富。松果体表达在胚胎 17 天时开始，此时在其他大脑区域也可检测到。松果体中的表达在胚胎期和成年期增加。 此后维持在与小脑和视网膜中发现的水平相当的水平。相比之下，在发育过程中，非小脑脑区域的 NeuroD1 mRNA 显着减少。白天和晚上松果体 NeuroD1 水平相似，并且似乎不受交感神经输入的影响。对新生 NeuroD1 敲除小鼠松果体的基因表达分析确定了 127 个转录本 下调（>两倍，p < 0.05）和 16 上调（>两倍，p < 0.05）。根据定量RT-PCR，下调最显着的基因是驱动蛋白家族成员5C（约100倍），上调最显着的基因是谷氨酸脱羧酶1（约4倍）。其他受影响的转录本编码 参与分化、发育、信号转导和运输的蛋白质。这些发现代表了阐明 NeuroD1 在啮齿动物松果体细胞中的作用的第一步。” 来自(4)。 Crx/Otx：“Otx2 是一种脊椎动物同源框基因，已被发现对于大脑头端区域的发育至关重要，并且似乎在视网膜感光细胞和松果体细胞的发育中发挥作用。在这项研究中，揭示了 Otx2 在大鼠大脑中的时间表达模式，特别是在胚胎晚期和出生后阶段的松果体中。广泛的高表达 Otx2 在胚胎大脑中的表达在成人中逐渐受限于松果体。 Crx (cone-rod homeobox) 是 Otx2 的下游靶基因，显示出与 Otx2 相似的松果体表达模式，尽管在发病时间上存在明显的滞后。在松果体提取物中鉴定出 Otx2 蛋白，并发现其位于松果体细胞中。总松果体 Otx2 mRNA 不显示昼夜变化，也不受交感神经输入去除的影响，表明 Otx2 mRNA 的水平似乎独立于腺体的光神经输入。我们的结果与松果体表达 Otx2 是发育所必需的观点一致，我们假设它在成人中发挥着控制与光转导和褪黑激素合成相关的基因簇表达的作用。” 来自 (5) Pax4：“Pax 4 是一种同源框基因，编码对内分泌胰腺胚胎发育至关重要的转录因子。在胰腺中，Pax4 对抗相关 Pax6 蛋白的作用，Pax6 蛋白也参与视网膜和松果体的发育。在这项研究中，我们报告 Pax4 在大鼠的松果体和视网膜感光器中强烈表达。松果体和视网膜 Pax4 mRNA 在胎儿中较低，出生后会增加； Pax6 表现出相反的表达模式，在胎儿的两种组织中都强烈表达。在成人中，Pax4 mRNA 的丰度在松果体和视网膜中表现出昼夜节律，最高水平出现在当天晚些时候。通过颈上神经节切除术去除松果体交感神经可以预防 夜间松果体 Pax4 mRNA 减少，但不改变视网膜 Pax4 表达模式。去甲肾上腺素在夜间由松果体的交感神经释放；在本研究中，我们发现肾上腺素能激动剂治疗可抑制体内和体外松果体 Pax4 的表达。已知去甲肾上腺素可提高松果体环 AMP；在这里发现用循环治疗 AMP 模拟物可减少松果体 Pax4 mRNA。这些发现表明松果体 Pax4 mRNA 的夜间减少是由肾上腺素环 AMP 机制控制的，并且 Pax4 可能发挥调节肾上腺素对松果体昼夜节律基因表达的控制作用。” （Rath 等人，正在准备中）。 埃亚2。 Eya2，果蝇基因 eyeless 的同源物已在松果体和视网膜中被发现。 目前正在努力表征基因产物、表达模式和功能。

项目成果

NeuroD1: developmental expression and regulated genes in the rodent pineal gland.

NeuroD1：啮齿动物松果体中的发育表达和调节基因。

• DOI: 10.1111/j.1471-4159.2007.04605.x
• 发表时间: 2007
• 期刊: Journal of neurochemistry
• 影响因子: 4.7
• 作者: Munoz,EstelaM;Bailey,MichaelJ;Rath,MartinF;Shi,Qiong;Morin,Fabrice;Coon,StevenL;Moller,Morten;Klein,DavidC
• 通讯作者: Klein,DavidC

Expression of the Otx2 homeobox gene in the developing mammalian brain: embryonic and adult expression in the pineal gland.

Otx2 同源盒基因在发育中的哺乳动物大脑中的表达：胚胎和成人在松果体中的表达。

• DOI: 10.1111/j.1471-4159.2006.03773.x
• 发表时间: 2006
• 期刊: Journal of neurochemistry
• 影响因子: 4.7
• 作者: Rath,MartinF;Munoz,Estela;Ganguly,Surajit;Morin,Fabrice;Shi,Qiong;Klein,DavidC;Moller,Morten
• 通讯作者: Moller,Morten