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.