Pineal Regulation: Molecular basis of development

松果体调节：发育的分子基础

• 批准号: 8553950
• 负责人: David Klein
• 金额: $ 40.37万
• 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/8553950
• 关键词: Acetyl Coenzyme A; Acetylation; Adult; Affect; Age-Months; Animals; Anterior; Arylalkylamine N-Acetyltransferase; BHLH Protein; Binding; Biological; Brain; Catalysis; Cells; Chronobiology Disorders; Development; Disease; Down-Regulation; Electron Microscopy; Electroretinography; Elements; Embryo; Endocrine; Enzymes; Event; Evolution; Exhibits; Eye Development; Family member; Fishes; Gene Duplication; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Family; Genes; Genetic Recombination; Genome; Goals; Histologic; Homeobox; Homeostasis; Knock-out; Knockout Mice; Learning; Light; Maintenance; Mediating; Melatonin; Microarray Analysis; Modeling; Molecular; Molecular Evolution; Monitor; Morphology; Mus; Mutate; Neural Fold; Pattern; Phenethylamines; Phenotype; Photoreceptors; Physiological; Pineal gland; Pinealocyte; Play; Positioning Attribute; Process; Production; Proteins; Psyche structure; Quantitative Reverse Transcriptase PCR; RNA Interference; Rattus; Regulation; Regulatory Element; Research; Retina; Retinal; Retinal Degeneration; Retinal Diseases; Retinal Photoreceptors; Role; Sea Bream; Serotonin; Sleep; Specificity; Structure; Structure-Activity Relationship; Substrate Specificity; Technology; Testing; Tissues; Transcript; Undifferentiated; Up-Regulation; Vertebrate Photoreceptors; Vertebrates; Work; base; cell type; duplicate genes; immune function; interest; light microscopy; member; neurodevelopment; paralogous gene; postnatal; preference; prevent; promoter; recombinase; selective expression; teleost fish; three-dimensional modeling; transcription factor

Crx: Cone-rod homeobox (Crx) encodes Crx, a transcription factor expressed selectively in retinal photoreceptors and pinealocytes, the major cell type of the pineal gland. In this study, the influence of Crx on the mammalian pineal gland was studied by light and electron microscopy and by use of microarray and qRTPCR technology, thereby extending previous studies on selected genes (Furukawa et al. 1999). Deletion of Crx was not found to alter pineal morphology, but was found to broadly modulate the mouse pineal transcriptome, characterized by a&#8195;>&#8195;2-fold down-regulation of 543 genes and a&#8195;>&#8195;2-fold up-regulation of 745 genes (p&#8195;<&#8195;0.05). Of these, one of the most highly up-regulated (18-fold) was Hoxc4, a member of the Hox gene family, members of which are known to control gene expression cascades. During a 24-h period, a set of 51 genes exhibited differential day/night expression in pineal glands of wild-type animals; only eight of these were also day/night expressed in the Crx(-/-) pineal gland. However, in the Crx(-/-) pineal gland 41 genes exhibited differential night/day expression that was not seen in wild-type animals. These findings indicate that Crx broadly modulates the pineal transcriptome and also influences differential night/day gene expression in this tissue. Some effects of Crx deletion on the pineal transcriptome might be mediated by Hoxc4 up-regulation. (From Rovsing et al., 2011) Rax: Retina and anterior neural fold homeobox (Rax) gene encodes a transcription factor essential for vertebrate eye development. Recent microarray studies indicate that Rax is expressed in the adult rat pineal gland and retina. The present study reveals that Rax expression levels in the rat change significantly during retinal development with a peak occurring at embryonic day 18, whereas Rax expression in the pineal is relatively delayed and not detectable until embryonic day 20. In both tissues, Rax is expressed throughout postnatal development into adulthood. In the mature rat pineal gland, the abundance of Rax transcripts increases 2-fold during the light period with a peak occurring at dusk. These findings are consistent with the evidence that Rax is of functional importance in eye development and suggest a role of Rax in the developing pineal gland. In addition, it would appear possible that Rax contributes to phenotype maintenance in the mature retina and pineal gland and may facilitate 24-h changes in the pineal transcriptome.(From Rhode et al., 2011). NeuroD: NeuroD1 encodes a basic helix-loop-helix transcription factor involved in the development of neural and endocrine structures, including the retina and pineal gland. To determine the effect of NeuroD1 knockout in these tissues, a Cre/loxP recombination strategy was used to target a NeuroD1 floxed gene and generate NeuroD1 conditional knockout (cKO) mice. Tissue specificity was conferred using Cre recombinase expressed under the control of the promoter of Crx, which is selectively expressed in the pineal gland and retina. At 2&#8195;months of age, NeuroD1 cKO retinas have a dramatic reduction in rod- and cone-driven electroretinograms and contain shortened and disorganized outer segments; by 4&#8195;months, NeuroD1 cKO retinas are devoid of photoreceptors. In contrast, the NeuroD1 cKO pineal gland appears histologically normal. Microarray analysis of 2-month-old NeuroD1 cKO retina and pineal gland identified a subset of genes that were affected 2-100-fold; in addition, a small group of genes exhibit altered differential night/day expression. Included in the down-regulated genes are Aipl1, which is necessary to prevent retinal degeneration, and Ankrd33, whose protein product is selectively expressed in the outer segments. These findings suggest that NeuroD1 may act through Aipl1 and other genes to maintain photoreceptor homeostasis. Molecular Evolution: Arylalkylamine N-acetyltransferase (AANAT) catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to arylalkylamines, including indolethylamines and phenylethylamines. Multiple aanats are present in teleost fish as a result of whole genome and gene duplications. Fish aanat1a and aanat2 paralogs display different patterns of tissue expression and encode proteins with different substrate preference: AANAT1a is expressed in the retina, and acetylates both indolethylamines and phenylethylamines; while AANAT2 is expressed in the pineal gland, and preferentially acetylates indolethylamines. The two enzymes are therefore thought to serve different roles. Here, the molecular changes that led to their specialization were studied by investigating the structure-function relationships of AANATs in the gilthead seabream (sb, Sperus aurata). Acetylation activity of reciprocal mutated enzymes pointed to specific residues that contribute to substrate specificity of the enzymes. Inhibition tests followed by complementary analyses of the predicted three-dimensional models of the enzymes, suggested that both phenylethylamines and indolethylamines bind to the catalytic pocket of both enzymes. These results suggest that substrate selectivity of AANAT1a and AANAT2 is determined by the positioning of the substrate within the catalytic pocket, and its accessibility to catalysis. This illustrates the evolutionary process by which enzymes encoded by duplicated genes acquire different activities and play different biological roles. (From Zilberman-Peled et al., 2011).

Crx： 视锥-视杆同源异型盒（Crx）编码Crx，Crx是选择性地在视网膜光感受器和松果体细胞（松果体的主要细胞类型）中表达的转录因子。在这项研究中，Crx对哺乳动物松果体的影响是通过光学和电子显微镜以及使用微阵列和qRTPCR技术研究的，从而扩展了先前对选定基因的研究（Furukawa et al. 1999）。未发现Crx的缺失改变松果体形态，但发现广泛调节小鼠松果体转录组，其特征在于543个基因的> 2倍下调和745个基因的> 2倍上调（p <0.05）。&#8195;&#8195;&#8195;&#8195;&#8195;&#8195;其中，最高度上调（18倍）的是Hoxc4，Hox基因家族的成员，已知其成员控制基因表达级联。在24小时内，一组51个基因表现出不同的白天/晚上表达的野生型动物的松果体，只有其中8个也是白天/晚上表达的Crx（-/-）松果体。然而，在Crx（-/-）松果体41基因表现出差异的夜间/白天的表达，没有看到野生型动物。这些研究结果表明，Crx广泛地调节松果体转录组，也影响差异的夜间/白天在这个组织中的基因表达。Crx基因缺失对松果体转录组的某些影响可能是通过上调Hoxc4来介导的。(From Rovsing等人，（2011年） Rax：视网膜和前神经折叠同源框（Rax）基因编码脊椎动物眼睛发育所必需的转录因子。最近的基因芯片研究表明，Rax在成年大鼠的松果体和视网膜中表达。本研究表明，Rax在大鼠视网膜发育过程中的表达水平发生显着变化，在胚胎第18天出现峰值，而Rax在松果体的表达相对延迟，直到胚胎第20天才检测到。在这两种组织中，Rax在整个出生后发育到成年期都有表达。在成熟的大鼠松果体中，Rax转录本的丰度在光照期间增加2倍，在黄昏时出现峰值。这些发现与Rax在眼睛发育中具有重要功能的证据一致，并表明Rax在发育中的松果体中发挥作用。此外，它似乎可能是Rax有助于表型维持在成熟的视网膜和松果体，并可能促进24小时的变化，松果体转录组。(From Rhode等人，2011年）。 NeuroD： NeuroD1编码一种基本的螺旋-环-螺旋转录因子，参与神经和内分泌结构的发育，包括视网膜和松果体。为了确定NeuroD1敲除在这些组织中的作用，使用Cre/loxP重组策略靶向NeuroD1 floxed基因并产生NeuroD1条件性敲除（cKO）小鼠。组织特异性被赋予使用Cre重组酶的Crx，这是选择性地表达在松果体和视网膜的启动子的控制下表达。在2个月大时，NeuroD1 cKO视网膜的视杆细胞和视锥细胞驱动的视网膜电图显著减少，并且包含缩短和混乱的外段;到4个月大时，NeuroD1 cKO视网膜没有光感受器。&#8195;&#8195;相反，NeuroD1 cKO松果体在组织学上表现正常。2个月大的NeuroD1 cKO视网膜和松果体的微阵列分析确定了一个受影响2 - 100倍的基因子集;此外，一小组基因表现出改变的差异夜间/白天表达。下调的基因包括Aipl1和Ankrd33，Aipl1是防止视网膜变性所必需的，Ankrd33的蛋白质产物选择性地在外节中表达。这些发现表明NeuroD1可能通过Aipl1和其他基因来维持感光细胞的稳态。 分子进化： 芳烷基胺N-乙酰转移酶（AANAT）催化乙酰基从乙酰辅酶A（AcCoA）转移到芳烷基胺，包括吲哚乙胺和苯乙胺。硬骨鱼中存在多个aanats作为全基因组和基因复制的结果。鱼aanat1a和aanat2旁系同源物显示不同的组织表达模式，并编码具有不同底物偏好的蛋白质：AANAT 1a在视网膜中表达，并乙酰化吲哚乙胺和苯乙胺;而AANAT 2在松果体中表达，并优先乙酰化吲哚乙胺。因此，这两种酶被认为具有不同的作用。在这里，导致其专业化的分子变化进行了研究，通过调查的结构-功能关系的AANAT在金头鲷（SB，Sperus aurata）。相互突变的酶的乙酰化活性指向有助于酶的底物特异性的特定残基。抑制试验，然后通过互补分析的预测三维模型的酶，表明苯乙基胺和吲哚乙基胺绑定到催化口袋的两种酶。这些结果表明，AANAT 1a和AANAT 2的底物选择性是由催化口袋内的底物的定位，及其对催化的可及性决定的。这说明了进化过程中，重复基因编码的酶获得不同的活动，发挥不同的生物学作用。(From Zilberman-Peled等人，2011年）。