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Circadian rhythms of fetal liver transcription persist in the absence of canonical circadian clock gene expression rhythms in vivo.

Li C, Yu S, Zhong X, Wu J, Li X - PLoS ONE (2012)

Bottom Line: In the fetal liver we did not observe circadian rhythms of clock gene expression or many other transcripts known to be rhythmically expressed in the adult liver.Upon data filtering by coefficient of variation, the expression levels for transcripts related to pancreatic exocrine enzymes and zymogen secretion were found to undergo synchronized daily fluctuations at high amplitudes.These results suggest that maternal cues influence the fetal liver, despite the fact that we did not detect circadian rhythms of canonical clock gene expression in the fetal liver.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei Province, People's Republic of China.

ABSTRACT
The cellular circadian clock and systemic cues drive rhythmicity in the transcriptome of adult peripheral tissues. However, the oscillating status of the circadian clocks in fetal tissues, and their response to maternal cues, are less clear. Most clock genes do not cycle in fetal livers from mice and rats, although tissue level rhythms rapidly emerge when fetal mouse liver explants are cultured in vitro. Thus, in the fetal mouse liver, the circadian clock does not oscillate at the cellular level (but is induced to oscillate in culture). To gain a comprehensive overview of the clock status in the fetal liver during late gestation, we performed microarray analyses on fetal liver tissues. In the fetal liver we did not observe circadian rhythms of clock gene expression or many other transcripts known to be rhythmically expressed in the adult liver. Nevertheless, JTK_CYCLE analysis identified some transcripts in the fetal liver that were rhythmically expressed, albeit at low amplitudes. Upon data filtering by coefficient of variation, the expression levels for transcripts related to pancreatic exocrine enzymes and zymogen secretion were found to undergo synchronized daily fluctuations at high amplitudes. These results suggest that maternal cues influence the fetal liver, despite the fact that we did not detect circadian rhythms of canonical clock gene expression in the fetal liver. These results raise important questions on the role of the circadian clock, or lack thereof, during ontogeny.

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Heatmap of pancreatic exocrine enzyme-related probe sets in series 2 data.Normalized log2 scale expression values for the probe sets representing transcripts related to pancreatic exocrine enzymes and zymogen secretion from series 2 data were plotted. Log2 scale data were used to facilitate viewing of all expression peaks. More dramatic variations in expression peaks in natural scale data led to improper viewing of those peaks on heatmap.
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pone-0030781-g003: Heatmap of pancreatic exocrine enzyme-related probe sets in series 2 data.Normalized log2 scale expression values for the probe sets representing transcripts related to pancreatic exocrine enzymes and zymogen secretion from series 2 data were plotted. Log2 scale data were used to facilitate viewing of all expression peaks. More dramatic variations in expression peaks in natural scale data led to improper viewing of those peaks on heatmap.

Mentions: We also observed that the expression values for some transcripts dramatically fluctuated in fetal liver and had high peak to trough ratios (sometimes with fold differences in the hundreds). We performed data filtering (criteria: coefficient of variation ≥0.5) on the expression values from the 12 time points examined on E18 and E19 for all probe sets in our two data series. One-hundred-sixty-six common probe sets (representing 132 unique DAVID IDs) were found between our two series of data (Table S4). Within those highly variable transcripts, 26 showed regular daily expression peaks that aligned together (Figures 3 and S3) on heatmaps, suggesting coordinated transcription. Twenty-two of the probe sets were matched to known pancreatic exocrine enzymes involved in food digestion [31]: Amy2a (amylase 2a2/5), Cela (chymotrypsin-like elastase 2A and 3B), Cel (carboxyl ester lipase), Clps (colipase), Cpa (carboxypeptidase A1 and A2), Cpb (carboxypeptidase B1), Ctrb1 (chymotrypsinogen B1), Ctrl (chymotrypsin-like), Pnliprp (pancreatic lipase related protein 1 and 2), Prss (protease, serine, 2; also known as Try2), and Try (trypsin 4/5). Four additional probe sets representing two unique transcripts, Zg16 (zymogen granule protein 16) and Sync (syncollin) were related to zymogen secretion [31], [32]. We performed semi-quantitative RT-PCRs using fetal liver tissues to confirm some of these transcripts. The abundance of PCR products also varied across time points (Figures 4 and S4), with patterns similar to that on the heatmaps. It was known that high sequence similarities existed between some of the transcripts, such as had been shown by alignment between Try and Prss proteases, and gene duplications between amylases [33]. Sequencing of the PCR products revealed most amplicons represented the desired products. Amplicons for Try and Prss, however, matched to related sequences, due to close sequence similarities. Amplicons for Amy2a also matched to multiple amylases 2a transcripts, due to their close sequence homologies that have resulted from gene duplication events throughout evolution. It was considered likely that transcripts with high sequence similarities were also expressed similarly, but we did not further pursue this complex issue by gene-specific RT-PCRs. Among the pancreatic exocrine enzymes identified here, Clps and Pnliprp2, had been previously characterized as rhythmically expressed in liver when adult mice were under constant darkness (but not under LD conditions) [34]. We thus took advantage of the available microarray data on circadian liver transcriptome analyses that had been deposited in the public database [10], [30] and searched for those pancreatic exocrine enzymes-related transcripts to determine their expression patterns under various conditions in adult mice. Most of those transcripts showed regular expression spikes, spaced about 21 hours apart, according to an adult mouse liver circadian transcriptome analyses at one-hour resolution (GSE 11923 of [30]; Table S5 and Figure S5). We then performed semi-quantitative RT-PCR using RNAs from adult male mouse livers that were collected under constant darkness (DD). Expression peaks occurred at about CT18 for those transcripts (Figure S6). One previous study had comprehensively addressed the contributions made by the liver clock and feeding on the hepatic transcriptome [10]. Our analyses of the microarray data (GSE13093 in [10] and GSE11923 in [30]) indicated that, in the liver, those pancreatic exocrine enzymes-related transcripts were regulated by feeding patterns regardless of the oscillation status of the liver clock in adult mice (Table S5 and Figure S6; also see Discussion). Moreover, the expression values for those transcripts were significantly rhythmic in our series 2 fetal liver data, but not rhythmic in our series 1 data (by JTK_CYCLE analysis; Table S4). We also observed differences in those transcripts' expression profiles on heatmaps (Figures 3 and S3) and by semi-quantitative RT-PCR, particularly on late E19 (Figures 4 and S4). These differences were not readily explicable, but might be attributable to variations in maternal feeding (see Discussion).


Circadian rhythms of fetal liver transcription persist in the absence of canonical circadian clock gene expression rhythms in vivo.

Li C, Yu S, Zhong X, Wu J, Li X - PLoS ONE (2012)

Heatmap of pancreatic exocrine enzyme-related probe sets in series 2 data.Normalized log2 scale expression values for the probe sets representing transcripts related to pancreatic exocrine enzymes and zymogen secretion from series 2 data were plotted. Log2 scale data were used to facilitate viewing of all expression peaks. More dramatic variations in expression peaks in natural scale data led to improper viewing of those peaks on heatmap.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285613&req=5

pone-0030781-g003: Heatmap of pancreatic exocrine enzyme-related probe sets in series 2 data.Normalized log2 scale expression values for the probe sets representing transcripts related to pancreatic exocrine enzymes and zymogen secretion from series 2 data were plotted. Log2 scale data were used to facilitate viewing of all expression peaks. More dramatic variations in expression peaks in natural scale data led to improper viewing of those peaks on heatmap.
Mentions: We also observed that the expression values for some transcripts dramatically fluctuated in fetal liver and had high peak to trough ratios (sometimes with fold differences in the hundreds). We performed data filtering (criteria: coefficient of variation ≥0.5) on the expression values from the 12 time points examined on E18 and E19 for all probe sets in our two data series. One-hundred-sixty-six common probe sets (representing 132 unique DAVID IDs) were found between our two series of data (Table S4). Within those highly variable transcripts, 26 showed regular daily expression peaks that aligned together (Figures 3 and S3) on heatmaps, suggesting coordinated transcription. Twenty-two of the probe sets were matched to known pancreatic exocrine enzymes involved in food digestion [31]: Amy2a (amylase 2a2/5), Cela (chymotrypsin-like elastase 2A and 3B), Cel (carboxyl ester lipase), Clps (colipase), Cpa (carboxypeptidase A1 and A2), Cpb (carboxypeptidase B1), Ctrb1 (chymotrypsinogen B1), Ctrl (chymotrypsin-like), Pnliprp (pancreatic lipase related protein 1 and 2), Prss (protease, serine, 2; also known as Try2), and Try (trypsin 4/5). Four additional probe sets representing two unique transcripts, Zg16 (zymogen granule protein 16) and Sync (syncollin) were related to zymogen secretion [31], [32]. We performed semi-quantitative RT-PCRs using fetal liver tissues to confirm some of these transcripts. The abundance of PCR products also varied across time points (Figures 4 and S4), with patterns similar to that on the heatmaps. It was known that high sequence similarities existed between some of the transcripts, such as had been shown by alignment between Try and Prss proteases, and gene duplications between amylases [33]. Sequencing of the PCR products revealed most amplicons represented the desired products. Amplicons for Try and Prss, however, matched to related sequences, due to close sequence similarities. Amplicons for Amy2a also matched to multiple amylases 2a transcripts, due to their close sequence homologies that have resulted from gene duplication events throughout evolution. It was considered likely that transcripts with high sequence similarities were also expressed similarly, but we did not further pursue this complex issue by gene-specific RT-PCRs. Among the pancreatic exocrine enzymes identified here, Clps and Pnliprp2, had been previously characterized as rhythmically expressed in liver when adult mice were under constant darkness (but not under LD conditions) [34]. We thus took advantage of the available microarray data on circadian liver transcriptome analyses that had been deposited in the public database [10], [30] and searched for those pancreatic exocrine enzymes-related transcripts to determine their expression patterns under various conditions in adult mice. Most of those transcripts showed regular expression spikes, spaced about 21 hours apart, according to an adult mouse liver circadian transcriptome analyses at one-hour resolution (GSE 11923 of [30]; Table S5 and Figure S5). We then performed semi-quantitative RT-PCR using RNAs from adult male mouse livers that were collected under constant darkness (DD). Expression peaks occurred at about CT18 for those transcripts (Figure S6). One previous study had comprehensively addressed the contributions made by the liver clock and feeding on the hepatic transcriptome [10]. Our analyses of the microarray data (GSE13093 in [10] and GSE11923 in [30]) indicated that, in the liver, those pancreatic exocrine enzymes-related transcripts were regulated by feeding patterns regardless of the oscillation status of the liver clock in adult mice (Table S5 and Figure S6; also see Discussion). Moreover, the expression values for those transcripts were significantly rhythmic in our series 2 fetal liver data, but not rhythmic in our series 1 data (by JTK_CYCLE analysis; Table S4). We also observed differences in those transcripts' expression profiles on heatmaps (Figures 3 and S3) and by semi-quantitative RT-PCR, particularly on late E19 (Figures 4 and S4). These differences were not readily explicable, but might be attributable to variations in maternal feeding (see Discussion).

Bottom Line: In the fetal liver we did not observe circadian rhythms of clock gene expression or many other transcripts known to be rhythmically expressed in the adult liver.Upon data filtering by coefficient of variation, the expression levels for transcripts related to pancreatic exocrine enzymes and zymogen secretion were found to undergo synchronized daily fluctuations at high amplitudes.These results suggest that maternal cues influence the fetal liver, despite the fact that we did not detect circadian rhythms of canonical clock gene expression in the fetal liver.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei Province, People's Republic of China.

ABSTRACT
The cellular circadian clock and systemic cues drive rhythmicity in the transcriptome of adult peripheral tissues. However, the oscillating status of the circadian clocks in fetal tissues, and their response to maternal cues, are less clear. Most clock genes do not cycle in fetal livers from mice and rats, although tissue level rhythms rapidly emerge when fetal mouse liver explants are cultured in vitro. Thus, in the fetal mouse liver, the circadian clock does not oscillate at the cellular level (but is induced to oscillate in culture). To gain a comprehensive overview of the clock status in the fetal liver during late gestation, we performed microarray analyses on fetal liver tissues. In the fetal liver we did not observe circadian rhythms of clock gene expression or many other transcripts known to be rhythmically expressed in the adult liver. Nevertheless, JTK_CYCLE analysis identified some transcripts in the fetal liver that were rhythmically expressed, albeit at low amplitudes. Upon data filtering by coefficient of variation, the expression levels for transcripts related to pancreatic exocrine enzymes and zymogen secretion were found to undergo synchronized daily fluctuations at high amplitudes. These results suggest that maternal cues influence the fetal liver, despite the fact that we did not detect circadian rhythms of canonical clock gene expression in the fetal liver. These results raise important questions on the role of the circadian clock, or lack thereof, during ontogeny.

Show MeSH
Related in: MedlinePlus