Light-dependent development of circadian gene expression in transgenic zebrafish

The roles of environmental stimuli in initiation and synchronization of circadian oscillation during development appear to vary among different rhythmic processes. In zebrafish, a variety of rhythms emerge in larvae only after exposure to light-dark (LD) cycles, whereas zebrafish period3 (per3) mRNA has been reported to be rhythmic from day 1 of development in constant conditions. We generated transgenic zebrafish in which expression of the firefly luciferase (luc) gene is driven by the zebrafish per3 promoter. Live larvae from these lines are rhythmically bioluminescent, providing the first vertebrate system for high-throughput measurement of circadian gene expression in vivo. Circadian rhythmicity in constant conditions was observed only after 5–6 d of development, and only if the fish were exposed to LD signals after day 4. Regardless of light exposure, a novel developmental profile was observed, with low expression during the first few days and a rapid increase when active swimming begins. Ambient temperature affected the developmental profile and overall levels of per3 and luc mRNA, as well as the critical days in which LD cycles were needed for robust bioluminescence rhythms. In summary, per3-luc zebrafish has revealed complex interactions among developmental events, light, and temperature in the expression of a clock gene.     

A novel pineal-specific product of the oligopeptide transporter PepT1 gene: circadian expression mediated by cAMP activation of an intronic promoter

The oligopeptide transporter 1, PepT1, is a member of the Slc15 family of 12 membrane-spanning domain transporters; PepT1 has proton/peptide cotransport activity and is selectively expressed in intestinal epithelial cells, where it is responsible for the nutritional absorption of di- and tri-peptides. Here, a novel PepT1 gene product has been identified in the rat pineal gland, termed pgPepT1. It encodes a 150-amino acid protein encompassing the C-terminal 3 membrane-spanning domains of intestinal PepT1 protein, with 3 additional N-terminal residues. Expression of pgPepT1 appears to be restricted to the pineal gland and follows a marked circadian pattern with >100-fold higher levels of mRNA occurring at night; this is accompanied by an accumulation of membrane-associated pgPepT1 protein ( approximately 16 kDa). The daily rhythm in pgPepT1 mRNA is regulated by the well described neural pathway that controls pineal melatonin production. This includes the retina, the circadian clock in the suprachiasmatic nucleus, central structures, and projections from the superior cervical ganglia; activation of this pathway results in the release of norepinephrine. Here it was found that pgPepT1 expression is mediated by a norepinephrine-->cyclic AMP mechanism that activates an alternative promoter located in intron 20 of the gene. pgPepT1 protein was found to have transporter-modulator activity; it could contribute to circadian changes in pineal function through this mechanism.     

A chromatin-dependent mechanism regulates gene expression at the core of the Arabidopsis circadian clock

The mechanisms of circadian clock function in Arabidopsis rely on the complex relationships among core clock components. The current model of the Arabidopsis oscillator comprises a myriad of repressors but the mechanisms responsible for activation remain largely unknown. In our recent studies, we have demonstrated that the rhythms in H3 acetylation (H3ac) and H3K4 trimethylation (H3K4me3) are a key mechanism at the positive arm of the oscillator. H3K4me3 rhythmic accumulation is delayed compared to that of H3ac, which opens the possibility for separate roles for each mark. Indeed, the use of inhibitors that block H3K4me3 accumulation was concomitant with increased clock repressor binding, suggesting that H3K4me3 might control the timing from activation to repression. Plants mis-expressing the histone methyltransferase SET DOMAIN GROUP 2 (SDG2/ATXR3) displayed altered H3K4me3 accumulation, oscillator gene expression and clock repressor binding, suggesting that SDG2/ATXR3 is a key component contributing to proper circadian expression.     

A circadian oscillator in Aspergillus spp. regulates daily development and gene expression

We have established the presence of a circadian clock in Aspergillus flavus and Aspergillus nidulans by morphological and molecular assays, respectively. In A. flavus, the clock regulates an easily assayable rhythm in the development of sclerotia, which are large survival structures produced by many fungi. This developmental rhythm exhibits all of the principal clock properties. The rhythm is maintained in constant environmental conditions with a period of 33 h at 30°C, it can be entrained by environmental signals, and it is temperature compensated. This endogenous 33-h period is one of the longest natural circadian rhythms reported for any organism, and this likely contributes to some unique responses of the clock to environmental signals. In A. nidulans, no obvious rhythms in development are apparent. However, a free running and entrainable rhythm in the accumulation of gpdA mRNA (encoding glyceraldehyde-3-phosphate dehydrogenase) is observed, suggesting the presence of a circadian clock in this species. We are unable to identify an Aspergillus ortholog of frequency, a gene required for normal circadian rhythmicity in Neurospora crassa. Together, our data indicate the existence of an Aspergillus circadian clock, which has properties that differ from that of the well-described clock of N. crassa.     

A hybrid SOM-SVM approach for the zebrafish gene expression analysis

Microarray technology can be employed to quantitatively measure the expression of thousands of genes in a single experiment. It has become one of the main tools for global gene expression analysis in molecular biology research in recent years. The large amount of expression data generated by this technology makes the study of certain complex biological problems possible, and machine learning methods are expected to play a crucial role in the analysis process. In this paper, we present our results from integrating the self-organizing map （SOM） and the support vector machine （SVM） for the analysis of the various functions of zebrafish genes based on their expression. The most distinctive characteristic of our zebrafish gene expression is that the number of samples of different classes is imbalanced. We discuss how SOM can be used as a data-filtering tool to improve the classification performance of the SVM on this data set.     

The HMGB protein gene family in zebrafish: Evolution and embryonic expression patterns

The High-Mobility Group Box (HMGB) proteins are highly abundant proteins with both nuclear and extracellular roles in key biological processes. In mammals, three family members are present: HMGB1, HMGB2 and HMGB3. We characterized the HMGB family in zebrafish and report a detailed phylogenetic analysis of HMGB proteins. The B1, B2, and B3 subfamilies are present in cartilaginous fish, bony fish, and tetrapods, while jawless fish sequences emerge as basal to the gene family expansion. Two co-orthologs of each mammalian HMGB gene are present in zebrafish. All six zebrafish hmgb genes are maternally expressed, but huge differences in expression levels exist during embryonic development. The hmgb2a/hmgb2b genes are the most highly expressed, while hmgb3b is expressed at the lowest level. Remarkably, hmgb3 genes are not present in fugu, medaka, Tetraodon and stickleback. Our analysis highlights substantial overlaps, but also subtle differences and specificities in the expression patterns of the zebrafish hmgb genes.     

Discovering molecular pathways from protein interaction and gene expression data

In this paper, we describe an approach for identifying 'pathways' from gene expression and protein interaction data. Our approach is based on the assumption that many pathways exhibit two properties: their genes exhibit a similar gene expression profile, and the protein products of the genes often interact. Our approach is based on a unified probabilistic model, which is learned from the data using the EM algorithm. We present results on two Saccharomyces cerevisiae gene expression data sets, combined with a binary protein interaction data set. Our results show that our approach is much more successful than other approaches at discovering both coherent functional groups and entire protein complexes.     

Vasa protein expression and localization in the zebrafish

Primordial germ cell (PGC) development in the zebrafish is poorly understood. The expression of vasa RNA, the only molecular marker so far found to be expressed in fish PGCs, suggests its function in the establishment of the germline. The protein product of vasa is present throughout the life cycle in the germline of Drosophila, Caenorhabditis and Xenopus. The expression pattern of the Vasa protein in zebrafish, is still unresolved. We generated an anti-Vasa polyclonal antibody and show that it is maternally expressed initially throughout the embryo. Interestingly, from the two-cell- to the 1000-cell stage the protein is highly concentrated in two 'dots' near the center of the blastomeres and as such remains longest detectable in the animal pole blastomeres. The first distinct cell-specific expression occurs at 60% epiboly on one side of the blastoderm margin. The Vasa protein in the PGCs is organized in a subcellular granular-like conformation which is dynamic throughout development.     

Functional modules by relating protein interaction networks and gene expression

Genes and proteins are organized on the basis of their particular mutual relations or according to their interactions in cellular and genetic networks. These include metabolic or signaling pathways and protein interaction, regulatory or co-expression networks. Integrating the information from the different types of networks may lead to the notion of a functional network and functional modules. To find these modules, we propose a new technique which is based on collective, multi-body correlations in a genetic network. We calculated the correlation strength of a group of genes (e.g. in the co-expression network) which were identified as members of a module in a different network (e.g. in the protein interaction network) and estimated the probability that this correlation strength was found by chance. Groups of genes with a significant correlation strength in different networks have a high probability that they perform the same function. Here, we propose evaluating the multi-body correlations by applying the superparamagnetic approach. We compare our method to the presently applied mean Pearson correlations and show that our method is more sensitive in revealing functional relationships.     

Environmental and genetic effects on circadian clock-regulated gene expression in Arabidopsis.

Expression patterns of the cold-circadian rhythm-RNA binding (CCR) and chlorophyll a/b binding (CAB) protein genes have circadian rhythms with phases that are different from each other and are affected differently by cold (4 degrees C) treatment. Cycling of CCR and CAB RNA levels was observed in Arabidopsis seedlings grown for 5 days at 4 degrees C under a light/ dark photoperiod, although the cycling had reduced amplitude compared with normal growth conditions (20 degrees C). CCR RNA levels were elevated in the cold, whereas CAB RNA levels were reduced in the cold relative to levels in control seedlings. Cold pulses (4 degrees C for 12 or 20 hr) under continuous light affected the rhythms of CCR and CAB RNA levels in similar ways. The 12-hr cold pulse caused a 4-hr phase delay in both rhythms, whereas the 20-hr cold pulse resulted in a 12-hr phase delay in both rhythms. The timing of CAB expression 1 (toc1) mutation shortened the period of the CCR rhythm, matching previous results for the regulation of the CAB-luciferase (CAB-luc) transgene in this mutant. The results suggest that CCR and CAB share clock machinery but are regulated by downstream components that are affected differently by the cold. Also, the circadian clock regulating these genes in Arabidopsis has a cold-sensitive phase under continuous light conditions.