Circadian clock drives novel functions through transcriptional cascade

Using larval zebrafish as a model system, we applied a genome-wide transcriptome approach that allowed us to investigate circadian gene expression that can be associated with various tissues and cell types. Our analysis of circadian gene regulatory network revealed a general principle: circadian clock controls diverse aspects of circadian physiology through transcriptional cascade of transcription factors (TFs). As a proof of this principle, we focused on microphthalmia-associated transcription factor a (mitfa), a dark-induced TF controlling melanogenesis in melanocytes. We demonstrated experimentally that there is a circadian rhythm of melanin synthesis mediated by mitfa. The circadian rhythm of mitfa is in turn driven by both endogenous clock and external light/dark cycle. The circadian rhythm of melanin synthesis may play an important role in zebrafish’s adaptation to daily cycle of lighting condition in the environment. Total RNA of larval sample was extracted using Trizol (Invitrogen) according to the manufacturer’s instruction. The quantity and quality of the RNA samples were assessed with a NanoDrop ND-1000 spectrometer (NanoDrop Technologies) and an Agilent 2100 bioanalyzer (Agilent). 12 LD RNA samples and 12 DD RNA samples were used for Agilent whole zebrafish 4x44K microarray, which contains 43,603 probes for a whole-genome transcriptional profile. Morpholinos oligonucleotides (MOs) were purchased from Gene Tools. All MOs except standard control were designed to target the start codon region of the genes. MOs were used at the following final doses: clock MO, 2.5 ng; mitfa MO, 9 ng or 13ng (microarray analysis). MOs were pressure-injected into 1- to 2-cell stage embryos at a volume of 1nl using Picospritzer II injectors as previously described (Janik et al. 2000). Each MO sample contained 40 individually treated larvae. Then the sample are collected and microarray are conducted just as before.

以斑马鱼幼体作为模型系统，本研究采用全基因组转录组学分析策略，对可与多种组织和细胞类型相关联的昼夜节律基因表达展开系统性探究。我们对昼夜节律基因调控网络的分析揭示了一条通用原则：昼夜节律钟通过转录因子（transcription factors, TFs）的转录级联反应，调控昼夜生理活动的多个维度。为验证该原则，我们聚焦于小眼畸形相关转录因子a（microphthalmia-associated transcription factor a, mitfa）——一种在黑素细胞中调控黑色素生成的黑暗诱导型转录因子。本研究通过实验证实，mitfa介导了黑色素合成的昼夜节律；而mitfa自身的昼夜节律，则由内源生物钟与外界光暗周期共同驱动。黑色素合成的昼夜节律可能在斑马鱼适应环境每日光照周期的过程中发挥关键作用。按照制造商操作说明，使用Trizol（Invitrogen）提取幼体样本的总RNA；通过NanoDrop ND-1000分光光度计（NanoDrop Technologies）与Agilent 2100生物分析仪（Agilent）评估RNA样本的浓度与质量。我们选取12份光暗周期（LD）RNA样本与12份持续黑暗（DD）RNA样本，开展安捷伦全斑马鱼4x44K基因芯片实验，该芯片搭载43603个探针，用于全基因组转录谱分析。吗啉代寡核苷酸（Morpholinos oligonucleotides, MOs）购自Gene Tools公司；除标准对照MO外，其余MO均靶向基因的起始密码子区域。MO的最终使用浓度设置如下：clock MO为2.5 ng，mitfa MO为9 ng或13 ng（用于基因芯片分析）。参照此前报道的方法（Janik等，2000年），使用Picospritzer II显微注射仪，以1 nl的体积将MO注射至1至2细胞期的胚胎中；每组MO样本包含40条经单独处理的幼体。随后按照前述方法收集样本并完成基因芯片实验。