A Computational Approach towards a Gene Regulatory Network for the Developing Nematostella vectensis Gut

Background

The starlet sea anemone Nematostella vectensis is a diploblastic cnidarian that expresses a set of conserved genes for gut formation during its early development. During the last decade, the spatial distribution of many of these genes has been visualized with RNA hybridization or protein immunolocalization techniques. However, due to N. vectensis'' curved and changing morphology, quantification of these spatial data is problematic. A method is developed for two-dimensional gene expression quantification, which enables a numerical analysis and dynamic modeling of these spatial patterns.

Methods/Result

In this work, first standardized gene expression profiles are generated from publicly available N. vectensis embryo images that display mRNA and/or protein distributions. Then, genes expressed during gut formation are clustered based on their expression profiles, and further grouped based on temporal appearance of their gene products in embryonic development. Representative expression profiles are manually selected from these clusters, and used as input for a simulation-based optimization scheme. This scheme iteratively fits simulated profiles to the selected profiles, leading to an optimized estimation of the model parameters. Finally, a preliminary gene regulatory network is derived from the optimized model parameters.

Outlook

While the focus of this study is N. vectensis, the approach outlined here is suitable for inferring gene regulatory networks in the embryonic development of any animal, thus allowing to comparatively study gene regulation of gut formation in silico across various species.     

Function of the Shaw potassium channel within the Drosophila circadian clock

Background

In addition to the molecular feedback loops, electrical activity has been shown to be important for the function of networks of clock neurons in generating rhythmic behavior. Most studies have used over-expression of foreign channels or pharmacological manipulations that alter membrane excitability. In order to determine the cellular mechanisms that regulate resting membrane potential (RMP) in the native clock of Drosophila we modulated the function of Shaw, a widely expressed neuronal potassium (K⁺) channel known to regulate RMP in Drosophila central neurons.

Methodology/Principal Findings

We show that Shaw is endogenously expressed in clock neurons. Differential use of clock gene promoters was employed to express a range of transgenes that either increase or decrease Shaw function in different clusters of clock neurons. Under LD conditions, increasing Shaw levels in all clock neurons (LNv, LNd, DN₁, DN₂ and DN₃), or in subsets of clock neurons (LNd and DNs or DNs alone) increases locomotor activity at night. In free-running conditions these manipulations result in arrhythmic locomotor activity without disruption of the molecular clock. Reducing Shaw in the DN alone caused a dramatic lengthening of the behavioral period. Changing Shaw levels in all clock neurons also disrupts the rhythmic accumulation and levels of Pigment Dispersing Factor (PDF) in the dorsal projections of LNv neurons. However, changing Shaw levels solely in LNv neurons had little effect on locomotor activity or rhythmic accumulation of PDF.

Conclusions/Significance

Based on our results it is likely that Shaw modulates pacemaker and output neuronal electrical activity that controls circadian locomotor behavior by affecting rhythmic release of PDF. The results support an important role of the DN clock neurons in Shaw-mediated control of circadian behavior. In conclusion, we have demonstrated a central role of Shaw for coordinated and rhythmic output from clock neurons.     

Disrupting Circadian Homeostasis of Sympathetic Signaling Promotes Tumor Development in Mice

Background

Cell proliferation in all rapidly renewing mammalian tissues follows a circadian rhythm that is often disrupted in advanced-stage tumors. Epidemiologic studies have revealed a clear link between disruption of circadian rhythms and cancer development in humans. Mice lacking the circadian genes Period1 and 2 (Per) or Cryptochrome1 and 2 (Cry) are deficient in cell cycle regulation and Per2 mutant mice are cancer-prone. However, it remains unclear how circadian rhythm in cell proliferation is generated in vivo and why disruption of circadian rhythm may lead to tumorigenesis.

Methodology/Principal Findings

Mice lacking Per1 and 2, Cry1 and 2, or one copy of Bmal1, all show increased spontaneous and radiation-induced tumor development. The neoplastic growth of Per-mutant somatic cells is not controlled cell-autonomously but is dependent upon extracellular mitogenic signals. Among the circadian output pathways, the rhythmic sympathetic signaling plays a key role in the central-peripheral timing mechanism that simultaneously activates the cell cycle clock via AP1-controlled Myc induction and p53 via peripheral clock-controlled ATM activation. Jet-lag promptly desynchronizes the central clock-SNS-peripheral clock axis, abolishes the peripheral clock-dependent ATM activation, and activates myc oncogenic potential, leading to tumor development in the same organ systems in wild-type and circadian gene-mutant mice.

Conclusions/Significance

Tumor suppression in vivo is a clock-controlled physiological function. The central circadian clock paces extracellular mitogenic signals that drive peripheral clock-controlled expression of key cell cycle and tumor suppressor genes to generate a circadian rhythm in cell proliferation. Frequent disruption of circadian rhythm is an important tumor promoting factor.     

Identification and In Vivo Characterization of NvFP-7R,a Developmentally Regulated Red Fluorescent Protein of Nematostella vectensis

Background

In recent years, the sea anemone Nematostella vectensis has emerged as a critical model organism for comparative genomics and developmental biology. Although Nematostella is a member of the anthozoan cnidarians (known for producing an abundance of diverse fluorescent proteins (FPs)), endogenous patterns of Nematostella fluorescence have not been described and putative FPs encoded by the genome have not been characterized.

Methodology/Principal Findings

We described the spatiotemporal expression of endogenous red fluorescence during Nematostella development. Spatially, there are two patterns of red fluorescence, both restricted to the oral endoderm in developing polyps. One pattern is found in long fluorescent domains associated with the eight mesenteries and the other is found in short fluorescent domains situated between tentacles. Temporally, the long domains appear simultaneously at the 12-tentacle stage. In contrast, the short domains arise progressively between the 12- and 16-tentacle stage. To determine the source of the red fluorescence, we used bioinformatic approaches to identify all possible putative Nematostella FPs and a Drosophila S2 cell culture assay to validate NvFP-7R, a novel red fluorescent protein. We report that both the mRNA expression pattern and spectral signature of purified NvFP-7R closely match that of the endogenous red fluorescence. Strikingly, the red fluorescent pattern of NvFP-7R exhibits asymmetric expression along the directive axis, indicating that the nvfp-7r locus senses the positional information of the body plan. At the tissue level, NvFP-7R exhibits an unexpected subcellular localization and a complex complementary expression pattern in apposed epithelia sheets comprising each endodermal mesentery.

Conclusions/Significance

These experiments not only identify NvFP-7R as a novel red fluorescent protein that could be employed as a research tool; they also uncover an unexpected spatio-temporal complexity of gene expression in an adult cnidarian. Perhaps most importantly, our results define Nematostella as a new model organism for understanding the biological function of fluorescent proteins in vivo.     

Dengue infection increases the locomotor activity of Aedes aegypti females

Background

Aedes aegypti is the main vector of the virus causing Dengue fever, a disease that has increased dramatically in importance in recent decades, affecting many tropical and sub-tropical areas of the globe. It is known that viruses and other parasites can potentially alter vector behavior. We investigated whether infection with Dengue virus modifies the behavior of Aedes aegypti females with respect to their activity level.

Methods/Principal Findings

We carried out intrathoracic Dengue 2 virus (DENV-2) infections in Aedes aegypti females and recorded their locomotor activity behavior. We observed an increase of up to ∼50% in the activity of infected mosquitoes compared to the uninfected controls.

Conclusions

Dengue infection alters mosquito locomotor activity behavior. We speculate that the higher levels of activity observed in infected Aedes aegypti females might involve the circadian clock. Further studies are needed to assess whether this behavioral change could have implications for the dynamics of Dengue virus transmission.     

Locomotor Activity of the Fiddler Crab,Uca subcylindrica (Stimpson), under Artificial Illumination

Specimens of the fiddler crab Uca subcylindrica (Stimpson) were captured in south Texas (USA) for locomotor rhythm studies. Actographic data were analyzed using Tau? sofware. Under constant illumination (LL) and darkness (DD), the semiterrestrial crabs express a circadian rhythm of locomotion. When exposed to illumination/darkness cycles (LD_12:12 or LD_14:10), their bouts of activity are entrained to the photoperiod. In LD, activity is generally bimodal with an initial burst about 0.5 h after illumination. A second burst of activity begins 1 to 2 h before the end of illumination. When transferred from LD to LL, a locomotor rhythm with an average period of 24.6 ± 1.0 h (n = 19) is expressed in 89 percent of the crabs. On the other hand, when placed in DD after LD (n = 8), the crabs are either arrythmic or weakly rhythmic (period = 23.8 ± 0.2 h; n = 2). If the onset of illumination is advanced by 6 h, a period of less than 24.0 h is detected in the actogram. If the onset of illumination is delayed by 6 h, a locomotor rhythm with a period greater than 24.0 h appears. The locomotor behavior of this species of fiddler crab, Uca subcylindrica, is not related to the tidal rhythmicities seen in other members of the genus Uca. Rather, it has strong circadian components.     

AMPK regulates circadian rhythms in a tissue- and isoform-specific manner

Background

AMP protein kinase (AMPK) plays an important role in food intake and energy metabolism, which are synchronized to the light-dark cycle. In vitro, AMPK affects the circadian rhythm by regulating at least two clock components, CKIα and CRY1, via direct phosphorylation. However, it is not known whether the catalytic activity of AMPK actually regulates circadian rhythm in vivo.

Methodology/Principal Finding

The catalytic subunit of AMPK has two isoforms: α1 and α2. We investigate the circadian rhythm of behavior, physiology and gene expression in AMPKα1−/− and AMPKα2−/− mice. We found that both α1−/− and α2−/− mice are able to maintain a circadian rhythm of activity in dark-dark (DD) cycle, but α1−/− mice have a shorter circadian period whereas α2−/− mice showed a tendency toward a slightly longer circadian period. Furthermore, the circadian rhythm of body temperature was dampened in α1−/− mice, but not in α2−/− mice. The circadian pattern of core clock gene expression was severely disrupted in fat in α1−/− mice, but it was severely disrupted in the heart and skeletal muscle of α2−/− mice. Interestingly, other genes that showed circadian pattern of expression were dysreguated in both α1−/− and α2−/− mice. The circadian rhythm of nicotinamide phosphoryl-transferase (NAMPT) activity, which converts nicotinamide (NAM) to NAD⁺, is an important regulator of the circadian clock. We found that the NAMPT rhythm was absent in AMPK-deficient tissues and cells.

Conclusion/Significance

This study demonstrates that the catalytic activity of AMPK regulates circadian rhythm of behavior, energy metabolism and gene expression in isoform- and tissue-specific manners.     

Genome Sequence Analysis Indicates that the Model Eukaryote Nematostella vectensis Harbors Bacterial Consorts

Analysis of the genome sequence of the starlet sea anemone, Nematostella vectensis, reveals many genes whose products are phylogenetically closer to proteins encoded by bacteria or bacteriophages than to any metazoan homologs. One explanation for such sequence affinities could be that these genes have been horizontally transferred from bacteria to the Nematostella lineage. We show, however, that bacterium-like and phage-like genes sequenced by the N. vectensis genome project tend to cluster on separate scaffolds, which typically do not include eukaryotic genes and differ from the latter in their GC contents. Moreover, most of the bacterium-like genes in N. vectensis either lack introns or the introns annotated in such genes are false predictions that, when translated, often restore the missing portions of their predicted protein products. In a freshwater cnidarian, Hydra, for which a proteobacterial endosymbiont is known, these gene features have been used to delineate the DNA of that endosymbiont sampled by the genome sequencing project. We predict that a large fraction of bacterium-like genes identified in the N. vectensis genome similarly are drawn from the contemporary bacterial consorts of the starlet sea anemone. These uncharacterized bacteria associated with N. vectensis are a proteobacterium and a representative of the phylum Bacteroidetes, each represented in the database by an apparently random sample of informational and operational genes. A substantial portion of a putative bacteriophage genome was also detected, which would be especially unlikely to have been transferred to a eukaryote.     

Microsatellite abundance across the Anthozoa and Hydrozoa in the phylum Cnidaria

Background

Microsatellite loci have high mutation rates and thus are indicative of mutational processes within the genome. By concentrating on the symbiotic and aposymbiotic cnidarians, we investigated if microsatellite abundances follow a phylogenetic or ecological pattern. Individuals from eight species were shotgun sequenced using 454 GS-FLX Titanium technology. Sequences from the three available cnidarian genomes (Nematostella vectensis, Hydra magnipapillata and Acropora digitifera) were added to the analysis for a total of eleven species representing two classes, three subclasses and eight orders within the phylum Cnidaria.

Results

Trinucleotide and tetranucleotide repeats were the most abundant motifs, followed by hexa- and dinucleotides. Pentanucleotides were the least abundant motif in the data set. Hierarchical clustering and log likelihood ratio tests revealed a weak relationship between phylogeny and microsatellite content. Further, comparisons between cnidaria harboring intracellular dinoflagellates and those that do not, show microsatellite coverage is higher in the latter group.

Conclusions

Our results support previous studies that found tri- and tetranucleotides to be the most abundant motifs in invertebrates. Differences in microsatellite coverage and composition between symbiotic and non-symbiotic cnidaria suggest the presence/absence of dinoflagellates might place restrictions on the host genome.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-939) contains supplementary material, which is available to authorized users.     

Circadian Timekeeping Is Disturbed in Rheumatoid Arthritis at Molecular Level

Introduction

Patients with rheumatoid arthritis (RA) have disturbances in the hypothalamic-pituitary-adrenal (HPA) axis. These are reflected in altered circadian rhythm of circulating serum cortisol, melatonin and IL-6 levels and in chronic fatigue. We hypothesized that the molecular machinery responsible for the circadian timekeeping is perturbed in RA. The aim of this study was to investigate the expression of circadian clock in RA.

Methods

Gene expression of thirteen clock genes was analyzed in the synovial membrane of RA and control osteoarthritis (OA) patients. BMAL1 protein was detected using immunohistochemistry. Cell autonomous clock oscillation was started in RA and OA synovial fibroblasts using serum shock. The effect of pro-inflammatory stimulus on clock gene expression in synovial fibroblasts was studied using IL-6 and TNF-α.

Results

Gene expression analysis disclosed disconcerted circadian timekeeping and immunohistochemistry revealed strong cytoplasmic localization of BMAL1 in RA patients. Perturbed circadian timekeeping is at least in part inflammation independent and cell autonomous, because RA synovial fibroblasts display altered circadian expression of several clock components, and perturbed circadian production of IL-6 and IL-1β after clock resetting. However, inflammatory stimulus disturbs the rhythm in cultured fibroblasts. Throughout the experiments ARNTL2 and NPAS2 appeared to be the most affected clock genes in human immune-inflammatory conditions.

Conclusion

We conclude that the molecular machinery controlling the circadian rhythm is disturbed in RA patients.     

A genome-wide survey of photoreceptor and circadian genes in the coral, Acropora digitifera

Corals exhibit circadian behaviors, but little is known about the molecular mechanisms underlying the regulation of these behaviors. We surveyed the recently decoded genome of the coral, Acropora digitifera, for photoreceptor and circadian genes, using molecular phylogenetic analyses. Our search for photoreceptor genes yielded seven opsin and three cryptochrome genes. Two genes from each family likely underwent tandem duplication in the coral lineage. We also found the following A. digitifera orthologs to Drosophila and mammalian circadian clock genes: four clock, one bmal/cycle, three pdp1-like, one creb/atf, one sgg/zw3, two ck2alpha, one dco (csnk1d/cnsk1e), one slim/BTRC, and one grinl. No vrille, rev-ervα/nr1d1, bhlh2, vpac2, adcyap1, or adcyaplr1 orthologs were found. Intriguingly, in spite of an extensive survey, we also failed to find homologs of period and timeless, although we did find one timeout gene. In addition, the coral genes were compared to orthologous genes in the sea anemone, Nematostella vectensis. Thus, the coral and sea anemone genomes share a similar repertoire of circadian clock genes, although A. digitifera contains more clock genes and fewer photoreceptor genes than N. vectensis. This suggests that the circadian clock system was established in a common ancestor of corals and sea anemones, and was diversified by tandem gene duplications and the loss of paralogous genes in each lineage. It will be interesting to determine how the coral circadian clock functions without period.     

The Relationship between Thermoregulation and REM Sleep Behaviour Disorder in Parkinson’s Disease

Background

This study explored the relationship between symptoms of rapid eye movement sleep behaviour disorder, thermoregulation and sleep in Parkinson’s Disease.

Methods

The study group comprised 12 patients with Parkinson’s Disease and 11 healthy age-matched controls. We investigated markers of thermoregulation (core-body temperature profile), circadian rhythm (locomotor actigraphy) and sleep (polysomnography).

Results

The mesor (the mean value around which the core temperature rhythm oscillates) of the core-body temperature in patients with Parkinson’s Disease was significantly lower than that of controls. In addition, the nocturnal fall in CBT (the difference between the mesor and the nadir temperature) was also significantly reduced in PD patients relative to controls. Furthermore, in patients the reduction in the amplitude of their core-body temperature profile was strongly correlated with the severity of self-reported rapid eye movement sleep behaviour disorder symptom, reduction in the percentage of REM sleep and prolonged sleep latency. By contrast, these disturbances of thermoregulation and sleep architecture were not found in controls and were not related to other markers of circadian rhythm or times of sleep onset and offset.

Conclusions

These findings suggest that the brainstem pathology associated with disruption of thermoregulation in Parkinson’s disease may also contribute to rapid eye movement sleep behavioural disorder. It is possible that detailed analysis of the core-body temperature profile in at risk populations such as those patients with idiopathic rapid eye movement sleep behaviour disorder might help identify those who are at high risk of transitioning to Parkinson’s Disease.     

Perinatal asphyxia reduces dentate granule cells and exacerbates methamphetamine-induced hyperlocomotion in adulthood

Background

Obstetric complications have been regarded as a risk factor for schizophrenia later in life. One of the mechanisms underlying the association is postulated to be a hypoxic process in the brain in the offspring around the time of birth. Hippocampus is one of the brain regions implicated in the late-onset dopaminergic dysfunction associated with hypoxic obstetric complications.

Methodology/Principal Findings

We used an animal model of perinatal asphyxia, in which rat pups were exposed to 15 min of intrauterine anoxia during Cesarean section birth. At 6 and 12 weeks after birth, the behavior of the pups was assessed using a methamphetamine-induced locomotion test. In addition, the histopathology of the hippocampus was examined by means of stereology. At 6 weeks, there was no change in the methamphetamine-induced locomotion. However, at 12 weeks of age, we found an elevation in methamphetamine-induced locomotor activity, which was associated with an increase of dopamine release in the nucleus accumbens. At the same age, we also found a reduction of the dentate granule cells of the hippocampus.

Conclusions/Significance

These results suggest that the dopaminergic dysregulation after perinatal asphyxia is associated with a reduction in hippocampal dentate granule cells, and this may partly contribute to the pathogenesis of schizophrenia.     

Loss of Magel2, a Candidate Gene for Features of Prader-Willi Syndrome,Impairs Reproductive Function in Mice

Background

MAGEL2 is one of several genes typically inactivated in the developmental obesity disorder Prader-Willi syndrome (PWS). The physiological consequences of loss of MAGEL2, but without the concurrent loss of other PWS genes, are not well understood. Gene-targeted mutation of Magel2 in mice disrupts circadian rhythm and metabolism causing reduced total activity, reduced weight gain before weaning, and increased adiposity after weaning.

Principal Findings

We now show that loss of Magel2 in mice causes reduced fertility in both males and females through extended breeding intervals and early reproductive decline and termination. Female Magel2-null mice display extended and irregular estrous cycles, while males show decreased testosterone levels, and reduced olfactory preference for female odors.

Conclusions

Our results suggest that loss of MAGEL2 contributes to the reproductive deficits seen in people with PWS, and further highlights the role of normal circadian rhythm in the maintenance of fertility.