Pineal Regulation: Molecular basis of development

CRX：锥杆同源盒(CRX)编码CRX，一种转录因子，选择性地在视网膜光感受器和松果体细胞表达，松果体的主要细胞类型。在这项研究中，通过光镜和电子显微镜以及利用微阵列和qRTPCR技术研究了CRX对哺乳动物松果体的影响，从而扩展了先前对选定基因的研究(Furukawa等人)。1999年)。没有发现CRX的缺失改变松果体的形态，但发现广泛地调节了小鼠松果体的转录组，其特征是543个基因的2倍下调和745个基因的2倍上调(p#8195；&lt；&#8195；0.05)。其中，Hoxc4基因上调程度最高(18倍)，它是Hox基因家族的成员，已知其成员控制基因表达级联反应。在24小时的时间里，51个基因在野生型动物的松果体中表现出不同的昼夜表达；其中只有8个基因在CRX(-/-)松果体中也有昼夜表达。然而，在CRX(-/-)松果体中，41个基因表现出夜间/白天的差异表达，这在野生动物中是看不到的。这些发现表明，CRX广泛调节松果体转录组，并影响该组织中不同的夜间/白天基因表达。CRX缺失对松果体转录组的某些影响可能是通过上调Hoxc4来实现的。(摘自Rovsing等人，2011年) RAX：视网膜和前神经折叠同源框(RAX)基因编码一种对脊椎动物眼睛发育至关重要的转录因子。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，在大鼠视网膜发育过程中，Rax的表达水平发生了显著变化，高峰出现在胚胎第18天，而松果体中Rax的表达相对滞后，直到胚胎第20天才能检测到。在这两个组织中，Rax在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了2倍，高峰出现在黄昏。这些发现与Rax在眼睛发育中的功能重要性的证据是一致的，并提示Rax在松果体发育中的作用。此外，Rax似乎可能有助于成熟视网膜和松果体的表型维持，并可能促进松果体转录组的24小时变化(摘自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。为了确定这些组织中NeuroD1基因敲除的效果，采用Cre/loxP重组策略靶向NeuroD1基因并产生NeuroD1条件性基因敲除(CKO)小鼠。在CRX启动子的控制下表达的Cre重组酶被赋予组织特异性，它选择性地在松果体和视网膜中表达。在2个月龄时，NeuroD1CKO视网膜的视杆细胞和视锥细胞驱动的视网膜电信号显著减少，并且含有缩短且排列紊乱的外节；到4月龄时，NeuroD1CKO视网膜上没有光感受器。相反，神经D1CKO松果体在组织学上看起来是正常的。对2个月大的NeuroD1CKO视网膜和松果体进行的微阵列分析发现，部分基因受到2-100倍的影响；此外，一小部分基因显示出夜间/白天差异表达的改变。下调的基因包括Aipl1和Ankrd33，Aipl1是防止视网膜退化所必需的，Ankrd33的蛋白产物选择性地在外节表达。这些发现表明，NeuroD1可能通过Aipl1和其他基因来维持光感受器的动态平衡。 分子进化： 芳基烷基胺N-乙酰基转移酶(AANAT)催化乙酰辅酶A(AcCoA)中的乙酰基转移到芳基烷基胺，包括吲哚乙胺和苯乙胺。由于全基因组和基因复制，硬骨鱼中存在多个AANAT。鱼类aanat1a和aanat2paralog表现出不同的组织表达模式，编码具有不同底物偏好的蛋白质：aanat1a在视网膜表达，乙酰化吲哚乙胺和苯乙胺；而aanat2在松果体表达，优先乙酰化吲哚乙胺。因此，这两种酶被认为起着不同的作用。本文通过研究银鱼(Sperus Aurata)中AANAT的结构与功能的关系，研究了导致其专化的分子变化。相互突变的酶的乙酰化活性指向有助于酶的底物专一性的特定残基。抑制试验和对预测的酶三维模型的补充分析表明，苯乙胺和吲哚乙胺都与两种酶的催化口袋结合。这些结果表明，AANAT1a和AANAT2的底物选择性取决于底物在催化口袋中的位置以及它对催化的可及性。这说明了由复制基因编码的酶获得不同的活性和发挥不同的生物作用的进化过程。(摘自齐尔伯曼-佩莱德等人，2011年)。

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).