Circadian profiling of the transcriptome in NIH/3T3 fibroblasts: comparison with rhythmic gene expression in SCN2.2 cells and the rat SCN.

To screen for output signals that may distinguish the pacemaker in the mammalian suprachiasmatic nucleus (SCN) from peripheral-type oscillators in which the canonical clockworks are similarly regulated in a circadian manner, the rhythmic behavior of the transcriptome in forskolin-stimulated NIH/3T3 fibroblasts was analyzed and compared relative to SCN2.2 cells in vitro and the rat SCN. Similar to the circadian profiling of the SCN2.2 and rat SCN transcriptomes, NIH/3T3 fibroblasts exhibited circadian fluctuations in the expression of the core clock genes, Per2, Cry1, and Bmal1, and 323 functionally diverse transcripts, many of which regulate cellular communication. Overlap in rhythmic transcripts among NIH/3T3 fibroblasts, SCN2.2 cells, and the rat SCN was limited to these clock genes and four other genes that mediate fatty acid and lipid metabolism or function as nuclear factors. Compared with NIH/3T3 cells, circadian gene expression in SCN oscillators was more prevalent among genes mediating glucose metabolism and neurotransmission. Coupled with evidence for the rhythmic regulation of the inducible isoform of nitric oxide synthase (iNos) in SCN2.2 cells and the rat SCN but not in fibroblasts, studies examining the effects of a NOS inhibitor on metabolic rhythms in cocultures containing SCN2.2 cells and untreated NIH/3T3 cells suggest that the gaseous neurotransmitter nitric oxide may play a key role in SCN pacemaker function. This comparative analysis of circadian gene expression in SCN and non-SCN cells may have important implications in the selective analysis of circadian signals involved in the coupling of SCN oscillators and regulation of rhythmicity in downstream cells.

Regeneration and asexual reproduction share common molecular changes: upregulation of a neural glycoepitope during morphallaxis in Lumbriculus.

Neural morphallaxis is a regenerative process characterized by wide-spread anatomical and physiological changes in an adult nervous system. During segmental regeneration of the annelid worm, Lumbriculus variegatus, neural morphallaxis involved a reorganization of sensory, interneuronal, and motor systems as posterior fragments gained a more anterior body position. A monoclonal antibody, Lan 3-2, which labels a neural glyco-domain in the leech, was reactive in Lumbriculus. In the worm, this antibody labeled neural structures, particularly axonal tracts and giant fiber pathways of the central nervous system. A 60kDa protein, possessing a lumbriculid mannose-rich glycoepitope, was upregulated during neural morphallaxis, peaking in its expression at 3 weeks post-amputation. Peak upregulation of the Lan 3-2 epitope, or the protein possessing it, corresponded to the time of major neurobehavioral plasticity during regeneration. Analyses of asexually reproducing animals also revealed induction of the Lan 3-2 epitope. In this developmental context, Lan 3-2 epitope upregulation was also confined to segments expressing both changes in positional identity and neurobehavioral plasticity, but these molecular and behavioral changes occurred prior to body fragmentation. These results suggest that the lumbriculid Lan 3-2 glycoepitope and proteins that bear them have been co-opted for neural morphallactic programs, induced both in anticipation of reproductive fragmentation and in compensation for injury-induced fragmentation.

Circadian profiling of the transcriptome in immortalized rat SCN cells.

Endogenous oscillations in gene expression are a prevalent feature of the circadian clock in the mammalian suprachiasmatic nucleus (SCN) and similar timekeeping systems in other organisms. To determine whether immortalized cells derived from the rat SCN (SCN2.2) retain these intrinsic rhythm-generating properties, oscillatory behavior of the SCN2.2 transcriptome was analyzed and compared with that found in the rat SCN in vivo using rat U34A Affymetrix GeneChips. In SCN2.2 cells, 116 unique genes and 46 ESTs or genes of unknown function exhibited circadian fluctuations with a 1.5-fold or greater difference in their mRNA abundance for two cycles. Many (35%) of these rhythmically regulated genes in SCN2.2 cells also exhibited circadian profiles of mRNA expression in the rat SCN in vivo. Functional analyses and cartography indicate that a diverse set of cellular pathways are strategically regulated by the circadian clock in SCN2.2 cells and that the largest categories of rhythmic genes are those involved in cellular and systems-level communication or in metabolic processes like cellular respiration, fatty acid recycling, and steroid synthesis. Because many of the same genes or nodes within these functional categories were rhythmically expressed in both SCN2.2 cells and the rat SCN, the circadian regulation of these pathways may be important in modulating input to or output from the SCN clock mechanism. In summary, global expression and circadian regulation of the SCN2.2 transcriptome retain many SCN-like properties, suggesting that genes displaying rhythmic profiles in both experimental models may be integral to their function as both circadian oscillators and pacemakers.

Diurnal and circadian retinomotor movements in zebrafish.

Key indicators of circadian regulation include the persistence of physiological rhythmicity in the absence of environmental time cues and entrainment of this rhythmicity by the ambient light cycle. In some teleosts, the inner segments of rod and cone photoreceptors contract and elongate according to changes in ambient lighting and the circadian cycle. Pigment granules in the retinal pigment epithelium (RPE) disperse and aggregate in a similar manner. Collectively, these movements are known as retinomotor movements. We report the histological characterization of diurnal and circadian retinomotor movements in zebrafish, Danio rerio. Adult fish subjected to a 14:10 light:dark (LD) cycle, constant darkness (DD), or constant light (LL) were sacrificed at 1-13 h intervals and processed for semithin sectioning of the retina. Using bright-field microscopy, 15 measurements of pigment granule position and the inner segment lengths of 30 rods and 30-45 cones were collected from the central third of the dorso-optic retina per time point. In LD, rods and cones followed a clear diurnal rhythm in their inner segment movements. Short-single, UV-sensitive cones were found to contract significantly 1 h before light onset in LD conditions. In DD conditions, the inner segments movements of short-single and double cones displayed statistically significant rhythms. RPE pigment granule movements are rhythmically regulated in both LD and DD although fluctuations are damped in the absence of photic cues. No significant retinomotor movements were observed in LL. These findings indicate retinomotor movements in zebrafish are differentially regulated by an endogenous oscillator and by light-dependent mechanisms.