Project description:Gilthead sea bream larvae were sampled at 30 dph during a 24 h cycle taking 10 individuals every 3 hours (00:00, 03:00, 06:00, 09:00, 12:00, 15:00, 18:00, 21:00 and 24:00 h Zeitgeber time) and their expression profile was analyzed by means of a specific oligo-microarray. One-way ANOVA showed that 2,229 genes present in the microarray were differentially expressed among the nine temporal groups. Principal component analysis of these differentially expressed genes clearly showed a cyclic distribution of the groups along the two components that accounted for most of the total variation (48.3% and 32.8%, respectively). The k-means clustering of differentially expressed genes identified four major clusters with a sequential expression profile. Cluster 1 comprised the lowest number of genes (132), although their magnitude of response was higher than in the other clusters, with an early peak of expression at 0h zeitgeber time and the minimum after 12 h. Clusters termed as 2, 3 and 4 contained 675, 758 and 650 genes, respectively, and they also showed a circadian rhythmicity of expression with intensity peaks at 3h (cluster 2), 9-15h (cluster 3) and 18-21h zeitgeber time. For functional analysis of differentially expressed genes, the 92.6 % (2,052 genes) was eligible for pathway analysis in the IPA software. Regarding molecular and cellular functions, the most significant for cluster 1 were molecular transport, lipid metabolism, amino acid metabolism, nucleic acid metabolism, carbohydrate metabolism, and vitamin and mineral metabolism. Of note, the most significant canonical pathway in this cluster was thyroid hormone receptor and retinoid X receptor (TR/RXR) activation, represented by collagen alpha-3(VI) chain, cytochrome P450 7A1, phosphoenolpyruvate carboxykinase 1, and the three core mitochondrial isoforms of uncoupling proteins (UCP1, UCP2, and UCP3). Secondly, phototransduction was significantly over-represented by five genes: arrestin 3, cyclic nucleotide-gated channel alpha 1, cyclic nucleotide-gated channel alpha 1, phosducin, and guanylate cyclase activator 1B. In cluster 2, the molecular functions “RNA post-transcriptional modification” and “DNA replication, recombination, and repair” were clearly the most significant. Overlapping analysis of associated canonical pathways identified for cluster 2 a group of 30 related genes, which were mainly involved with repair response to DNA damage and also cell cycle regulation. “Cell cycle” and “DNA replication, recombination, and repair” were also among the top molecular and cellular functions of cluster 3, together with “cellular assembly and organization”. Significant overlapping of canonical pathways from cluster 3 identified 72 genes mostly related to unfolded protein responses, oxidative stress, and regulation of cell cycle. By contrast, there were no clearly prominent molecular and cellular functions among the cluster 4 genes, and overlapping analysis only resulted in six related pathways (26 genes), including cell cycle and signaling processes related to embryogenesis (neuregulin signaling) and apoptosis (TWEAK -TNF-like weak inducer of apoptosis- signaling). Seven genes in cluster 1-3 were unequivocally identified as classic activators of circadian rhythms (“clock genes”). These were the circadian protein homolog 3 (PER3) in cluster 1, PER1, PER2 and chryptophores 1 and 2 (CRYP1/CRYP2) in cluster 2, and aryl hydrocarbon receptor nuclear translocator-like (BMAL1) and circadian locomotor output cycles kaput (CLOCK) in cluster 3. Interestingly, correlation analysis showed that a number of genes in each of these three clusters shared the expression dynamics of the identified clock genes. Figure 7 shows the number of related genes for each one of the clock genes and their average expression profiles. The entire list of significantly correlated genes to these reference clock genes and their normalized intensity values is provided as supplemental material (Supplemental Table 2). Expression values of CRYP1, BMAL1 and CLOCK were also assessed by qPCR and results were quite consistent (r=0.93) with those of the microarray analysis.
2017-10-31 | GSE64481 | GEO
