Karaganis Stephen P, Kumar Vinod, Beremand Phillip D, Bailey Michael J, Thomas Terry L, Cassone Vincent M
Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA.
BMC Genomics. 2008 May 3;9:206. doi: 10.1186/1471-2164-9-206.
Chick pinealocytes exhibit all the characteristics of a complete circadian system, comprising photoreceptive inputs, molecular clockworks and an easily measured rhythmic output, melatonin biosynthesis. These properties make the in vitro pineal a particularly useful model for exploring circadian control of gene transcription in a pacemaker tissue, as well as regulation of the transcriptome by primary inputs to the clock (both photic and noradrenergic).
We used microarray analysis to investigate the expression of approximately 8000 genes within cultured pinealocytes subjected to both LD and DD. We report that a reduced subset of genes was rhythmically expressed in vitro compared to those previously published in vivo, and that gene expression rhythms were lower in amplitude, although the functional distribution of the rhythmic transcriptome was largely similar. We also investigated the effects of 6-hour pulses of light or of norepinephrine on gene expression in free-running cultures during both subjective day and night. As expected, both light and norepinephrine inhibited melatonin production; however, the two treatments differentially enhanced or suppressed specific sets of genes in a fashion that was dependent upon time of day.
Our combined approach of utilizing a temporal, photic and pharmacological microarray experiment allowed us to identify novel genes linking clock input to clock function within the pineal. We identified approximately 30 rhythmic, light-responsive, NE-insensitive genes with no previously known clock function, which may play a role in circadian regulation of the pineal. These are candidates for future functional genomics experiments to elucidate their potential role in circadian physiology. Further, we hypothesize that the pineal circadian transcriptome is reduced but functionally conserved in vitro, and supports an endogenous role for the pineal in regulating local rhythms in metabolism, immune function, and other conserved pathways.
鸡松果体细胞展现出完整昼夜节律系统的所有特征，包括光感受输入、分子时钟机制以及易于测量的节律性输出——褪黑素生物合成。这些特性使得体外培养的松果体成为探索起搏器组织中基因转录的昼夜节律控制以及时钟的主要输入（光和去甲肾上腺素能）对转录组调控的特别有用的模型。
我们使用微阵列分析来研究在光暗循环（LD）和持续黑暗（DD）条件下培养的松果体细胞中约8000个基因的表达。我们报告称，与先前体内研究发表的基因相比，体外节律性表达的基因子集有所减少，并且基因表达节律的幅度较低，尽管节律性转录组的功能分布在很大程度上是相似的。我们还研究了在主观白天和黑夜期间，6小时的光脉冲或去甲肾上腺素脉冲对自由运转培养物中基因表达的影响。正如预期的那样，光和去甲肾上腺素都抑制褪黑素的产生；然而，这两种处理以依赖于一天中时间的方式差异性地增强或抑制特定的基因集。
我们利用时间、光和药理学微阵列实验的综合方法使我们能够识别出将时钟输入与松果体中的时钟功能联系起来的新基因。我们鉴定出大约30个具有节律性、光反应性、对去甲肾上腺素不敏感且以前未知时钟功能的基因，它们可能在松果体的昼夜节律调节中发挥作用。这些是未来功能基因组学实验的候选对象，以阐明它们在昼夜生理中的潜在作用。此外，我们假设松果体昼夜节律转录组在体外有所减少但功能保守，并支持松果体在调节代谢、免疫功能和其他保守途径中的局部节律方面的内源性作用。
