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Analysis of a gene regulatory cascade mediating circadian rhythm in zebrafish.

Li Y, Li G, Wang H, Du J, Yan J - PLoS Comput. Biol. (2013)

Bottom Line: Our computational analysis of the circadian gene regulatory network revealed several transcription factors (TFs) involved in diverse aspects of circadian physiology through transcriptional cascade.Of these, microphthalmia-associated transcription factor a (mitfa), a dark-induced TF, mediates a circadian rhythm of melanin synthesis, which may be involved in zebrafish's adaptation to daily light cycling.Our study describes a systematic method to discover previously unidentified TFs involved in circadian physiology in complex organisms.

View Article: PubMed Central - PubMed

Affiliation: CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT
In the study of circadian rhythms, it has been a puzzle how a limited number of circadian clock genes can control diverse aspects of physiology. Here we investigate circadian gene expression genome-wide using larval zebrafish as a model system. We made use of a spatial gene expression atlas to investigate the expression of circadian genes in various tissues and cell types. Comparison of genome-wide circadian gene expression data between zebrafish and mouse revealed a nearly anti-phase relationship and allowed us to detect novel evolutionarily conserved circadian genes in vertebrates. We identified three groups of zebrafish genes with distinct responses to light entrainment: fast light-induced genes, slow light-induced genes, and dark-induced genes. Our computational analysis of the circadian gene regulatory network revealed several transcription factors (TFs) involved in diverse aspects of circadian physiology through transcriptional cascade. Of these, microphthalmia-associated transcription factor a (mitfa), a dark-induced TF, mediates a circadian rhythm of melanin synthesis, which may be involved in zebrafish's adaptation to daily light cycling. Our study describes a systematic method to discover previously unidentified TFs involved in circadian physiology in complex organisms.

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The circadian rhythm of melanogenesis in larval zebrafish.(A) Images of 5 dpf WT larval melanocytes in 4 hour intervals over 24 hours under LD conditions. (B) The area of melanocytes in WT larvae showed robust circadian rhythm in LD starting at 4 dpf (p<0.001, Fisher's g test). (C) Melanin concentrations of WT larvae showed robust circadian rhythm in LD while increasing with time (p<0.002, Fisher's g test after detrend). (D) The rhythm of melanin concentration was abolished in clock morphants in LD conditions, while the rhythm in control morphants remained robust. (E) Under DD conditions, the circadian rhythms of melanin concentrations in clock morphants and control morphants were not significant.
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pcbi-1002940-g006: The circadian rhythm of melanogenesis in larval zebrafish.(A) Images of 5 dpf WT larval melanocytes in 4 hour intervals over 24 hours under LD conditions. (B) The area of melanocytes in WT larvae showed robust circadian rhythm in LD starting at 4 dpf (p<0.001, Fisher's g test). (C) Melanin concentrations of WT larvae showed robust circadian rhythm in LD while increasing with time (p<0.002, Fisher's g test after detrend). (D) The rhythm of melanin concentration was abolished in clock morphants in LD conditions, while the rhythm in control morphants remained robust. (E) Under DD conditions, the circadian rhythms of melanin concentrations in clock morphants and control morphants were not significant.

Mentions: We reasoned that the circadian oscillation of mitfa expression implies that melanin synthesis also oscillates on a daily basis. To experimentally demonstrate the circadian oscillation of melanin synthesis, we first measured the areas of melanocytes in the head regions of larval zebrafish under LD beginning at 4 dpf (Figure 6A, Materials and Methods) and found a significant circadian rhythm in pigmentation with a peak around CT0 (p<0.001, Fisher's g test, Figure 6B). Then we quantified total melanin concentration using a melanin assay in whole larval zebrafish. The melanin concentration showed a strong circadian rhythm with a circadian phase also around CT0 in LD (p<0.002, Fisher's g test after detrend, Figure 6C) starting at 4 dpf, while the absolute amount of melanin increased during development. To determine the effect of clock on pigment synthesis, we examined the melanin concentration rhythm in clock morphants. The melanin concentration showed a significantly reduced circadian rhythm in LD compared to control morphants (Figure 6D). Melanin oscillations were not significant in DD in either clock morphants or controls (Figure 6E). Similarly, the amplitude of mitfa expression was significantly reduced by over 30% in clock morphants in LD compared to WT and controls, and oscillations are nearly absent in DD. From this we conclude that the circadian control of melanogenesis is mediated by mitfa in zebrafish larvae.


Analysis of a gene regulatory cascade mediating circadian rhythm in zebrafish.

Li Y, Li G, Wang H, Du J, Yan J - PLoS Comput. Biol. (2013)

The circadian rhythm of melanogenesis in larval zebrafish.(A) Images of 5 dpf WT larval melanocytes in 4 hour intervals over 24 hours under LD conditions. (B) The area of melanocytes in WT larvae showed robust circadian rhythm in LD starting at 4 dpf (p<0.001, Fisher's g test). (C) Melanin concentrations of WT larvae showed robust circadian rhythm in LD while increasing with time (p<0.002, Fisher's g test after detrend). (D) The rhythm of melanin concentration was abolished in clock morphants in LD conditions, while the rhythm in control morphants remained robust. (E) Under DD conditions, the circadian rhythms of melanin concentrations in clock morphants and control morphants were not significant.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3585402&req=5

pcbi-1002940-g006: The circadian rhythm of melanogenesis in larval zebrafish.(A) Images of 5 dpf WT larval melanocytes in 4 hour intervals over 24 hours under LD conditions. (B) The area of melanocytes in WT larvae showed robust circadian rhythm in LD starting at 4 dpf (p<0.001, Fisher's g test). (C) Melanin concentrations of WT larvae showed robust circadian rhythm in LD while increasing with time (p<0.002, Fisher's g test after detrend). (D) The rhythm of melanin concentration was abolished in clock morphants in LD conditions, while the rhythm in control morphants remained robust. (E) Under DD conditions, the circadian rhythms of melanin concentrations in clock morphants and control morphants were not significant.
Mentions: We reasoned that the circadian oscillation of mitfa expression implies that melanin synthesis also oscillates on a daily basis. To experimentally demonstrate the circadian oscillation of melanin synthesis, we first measured the areas of melanocytes in the head regions of larval zebrafish under LD beginning at 4 dpf (Figure 6A, Materials and Methods) and found a significant circadian rhythm in pigmentation with a peak around CT0 (p<0.001, Fisher's g test, Figure 6B). Then we quantified total melanin concentration using a melanin assay in whole larval zebrafish. The melanin concentration showed a strong circadian rhythm with a circadian phase also around CT0 in LD (p<0.002, Fisher's g test after detrend, Figure 6C) starting at 4 dpf, while the absolute amount of melanin increased during development. To determine the effect of clock on pigment synthesis, we examined the melanin concentration rhythm in clock morphants. The melanin concentration showed a significantly reduced circadian rhythm in LD compared to control morphants (Figure 6D). Melanin oscillations were not significant in DD in either clock morphants or controls (Figure 6E). Similarly, the amplitude of mitfa expression was significantly reduced by over 30% in clock morphants in LD compared to WT and controls, and oscillations are nearly absent in DD. From this we conclude that the circadian control of melanogenesis is mediated by mitfa in zebrafish larvae.

Bottom Line: Our computational analysis of the circadian gene regulatory network revealed several transcription factors (TFs) involved in diverse aspects of circadian physiology through transcriptional cascade.Of these, microphthalmia-associated transcription factor a (mitfa), a dark-induced TF, mediates a circadian rhythm of melanin synthesis, which may be involved in zebrafish's adaptation to daily light cycling.Our study describes a systematic method to discover previously unidentified TFs involved in circadian physiology in complex organisms.

View Article: PubMed Central - PubMed

Affiliation: CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT
In the study of circadian rhythms, it has been a puzzle how a limited number of circadian clock genes can control diverse aspects of physiology. Here we investigate circadian gene expression genome-wide using larval zebrafish as a model system. We made use of a spatial gene expression atlas to investigate the expression of circadian genes in various tissues and cell types. Comparison of genome-wide circadian gene expression data between zebrafish and mouse revealed a nearly anti-phase relationship and allowed us to detect novel evolutionarily conserved circadian genes in vertebrates. We identified three groups of zebrafish genes with distinct responses to light entrainment: fast light-induced genes, slow light-induced genes, and dark-induced genes. Our computational analysis of the circadian gene regulatory network revealed several transcription factors (TFs) involved in diverse aspects of circadian physiology through transcriptional cascade. Of these, microphthalmia-associated transcription factor a (mitfa), a dark-induced TF, mediates a circadian rhythm of melanin synthesis, which may be involved in zebrafish's adaptation to daily light cycling. Our study describes a systematic method to discover previously unidentified TFs involved in circadian physiology in complex organisms.

Show MeSH
Related in: MedlinePlus