Differential Expression of the Circadian Clock in Maternal and Embryonic Tissues of Mice

BACKGROUND: Molecular feedback loops involving transcription and translation and several key genes are at the core of circadian regulatory cycles affecting cellular pathways and metabolism. These cycles are active in most adult animal cells but little is known about their expression or influence during development. METHODOLOGY/PRINCIPAL FINDINGS: To determine if circadian cycles are active during mammalian development we measured the expression of key circadian genes during embryogenesis in mice using quantitative real-time RT-PCR. All of the genes examined were expressed in whole embryos beginning at the earliest age examined, embryonic day 10. In contrast to adult tissues, circadian variation was absent for all genes at all of the embryonic ages examined in either whole embryos or individual tissues. Using a bioluminescent fusion protein that tracks translation of the circadian gene, per2, we also analyzed protein levels. Similar to mRNA, a protein rhythm was observed in adult tissue but not in embryonic tissues collected in-vivo. In contrast, when tissues were placed in culture for the continuous assay of bioluminescence, rhythms were observed in embryonic (E18) tissues. We found that placing embryonic tissues in culture set the timing (phase) of these rhythms, suggesting the importance of a synchronizing signal for the expression of circadian cycles in developing tissues. CONCLUSIONS/SIGNIFICANCE: These results show that embryonic tissues express key circadian genes and have the capacity to express active circadian regulatory cycles. In vivo, circadian cycles are not expressed in embryonic tissues as they are in adult tissues. Individual cells might express oscillations, but are not synchronized until later in development

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations.

SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology

A Fear-Inducing Odor Alters PER2 and c-Fos Expression in Brain Regions Involved in Fear Memory

Evidence demonstrates that rodents learn to associate a foot shock with time of day, indicating the formation of a fear related time-stamp memory, even in the absence of a functioning SCN. In addition, mice acquire and retain fear memory better during the early day compared to the early night. This type of memory may be regulated by circadian pacemakers outside of the SCN. As a first step in testing the hypothesis that clock genes are involved in the formation of a time-stamp fear memory, we exposed one group of mice to fox feces derived odor (TMT) at ZT 0 and one group at ZT 12 for 4 successive days. A separate group with no exposure to TMT was also included as a control. Animals were sacrificed one day after the last exposure to TMT, and PER2 and c-Fos protein were quantified in the SCN, amygdala, hippocampus, and piriform cortex. Exposure to TMT had a strong effect at ZT 0, decreasing PER2 expression at this time point in most regions except the SCN, and reversing the normal rhythm of PER2 expression in the amygdala and piriform cortex. These changes were accompanied by increased c-Fos expression at ZT0. In contrast, exposure to TMT at ZT 12 abolished the rhythm of PER2 expression in the amygdala. In addition, increased c-Fos expression at ZT 12 was only detected in the central nucleus of the amygdala in the TMT12 group. TMT exposure at either time point did not affect PER2 or c-Fos in the SCN, indicating that under a light-dark cycle, the SCN rhythm is stable in the presence of repeated exposure to a fear-inducing stimulus. Taken together, these results indicate that entrainment to a fear-inducing stimulus leads to changes in PER2 and c-Fos expression that are detected 24 hours following the last exposure to TMT, indicating entrainment of endogenous oscillators in these regions. The observed effects on PER2 expression and c-Fos were stronger during the early day than during the early night, possibly to prepare appropriate systems at ZT 0 to respond to a fear-inducing stimulus

Chronic stimulation of the hypothalamic vasoactive intestinal peptide receptor lengthens circadian period in mice and hamsters

Evidence suggests that circadian rhythms are regulated through diffusible signals generated by the suprachiasmatic nucleus (SCN). Vasoactive intestinal peptide (VIP) is located in SCN neurons positioned to receive photic input from the retinohypothalamic tract and transmit information to other SCN cells and adjacent hypothalamic areas. Studies using knockout mice indicate that VIP is essential for synchrony among SCN cells and for the expression of normal circadian rhythms. To test the hypothesis that VIP is also an SCN output signal, we recorded wheel-running activity rhythms in hamsters and continuously infused the VIP receptor agonist BAY 55-9837 in the third ventricle for 28 days. Unlike other candidate output signals, infusion of BAY 55-9837 did not affect activity levels. Instead, BAY 55-9837 lengthened the circadian period by 0.69 ± 0.04 h (P < 0.0002 compared with controls). Period returned to baseline after infusions. We analyzed the effect of BAY 55-9837 on cultured SCN from PER2::LUC mice to determine if lengthening of the period by BAY 55-9837 is a direct effect on the SCN. Application of 10 μM BAY 55-9837 to SCN in culture lengthened the period of PER2 luciferase expression (24.73 ± 0.24 h) compared with control SCN (23.57 ± 0.26, P = 0.01). In addition, rhythm amplitude was significantly increased, consistent with increased synchronization of SCN neurons. The effect of BAY 55-9837 in vivo on period is similar to the effect of constant light. The present results suggest that VIP-VPAC2 signaling in the SCN may play two roles, synchronizing SCN neurons and setting the period of the SCN as a whole

TMT exposure alters expression of PER2 and c-Fos in the BLA.

<p>TMT altered the rhythm and amount of expression of PER2 in the BLA resulting in significantly lower expression at ZT 0 for the TMT0 group and significantly higher expression at ZT 6 and ZT 12 for both experimental groups (A). TMT exposure also resulted in an increase of c-Fos expression at ZT 0 for both groups, with the increase for the TMT0 group significantly higher than the TMT12 group (B). Photomicrographs showing low amount of PER2 immunoreactivity in the BLA of a TMT 0 animal at ZT 0 (C) and normal amount of immunoreactivity the same timepoint for a TMT12 animal (D). Photomicrographs showing increased c-Fos expression at ZT 0 in the BLA of a TMT0 animal (E) and lower level of expression at ZT 0 in a TMT12 animal (F). *significantly different from control group at that time point (p<0.05) **significantly different from control and experimental group at that timepoint (p<0.05). The values for ZT 0 are repeated as ZT 24. Error bars represent standard errors.</p

One-Way ANOVA (main effect of ZT).

<p>One-Way ANOVA (main effect of ZT).</p

Schedule of TMT exposure.

<p>After 10 days of baseline wheel running activity in a LD cycle, one group of mice were exposed to TMT for 4 days at ZT 0 and to a control KimWipe with no odor at ZT 12 (shaded gray area) (A). Animals were then given one full 24 hour cycle without exposure before being sacrificed at 6 hour intervals on the following cycle. A second group of mice were exposed to a control KimWipe at ZT 0 and to TMT at ZT 12 (B).</p