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Successful pregnancies rely on sufficient energy and nutrient supply, which require the mother to metabolically adapt to support fetal needs. The placenta has a critical role in this process, as this specialized organ produces hormones and peptides that regulate fetal and maternal metabolism. The ability for the mother to metabolically adapt to support the fetus depends on maternal prepregnancy health. Two-thirds of pregnancies in the United States involve obese or overweight women at the time of conception. This poses significant risks for the infant and mother by disrupting metabolic changes that would normally occur during pregnancy. Despite well characterized functions of placental hormones, there is scarce knowledge surrounding placental endocrine regulation of maternal metabolic trends in pathological pregnancies. In this review, we discuss current efforts to close this gap of knowledge and highlight areas where more research is needed. As the intrauterine environment predetermines the health and wellbeing of the offspring in later life, adequate metabolic control is essential for a successful pregnancy outcome. Understanding how placental hormones contribute to aberrant metabolic adaptations in pathological pregnancies may unveil disease mechanisms and provide methods for better identification and treatment. Studies discussed in this review were identified through PubMed searches between the years of 1966 to the present. We investigated studies of normal pregnancy and metabolic disorders in pregnancy that focused on energy requirements during pregnancy, endocrine regulation of glucose metabolism and insulin resistance, cholesterol and lipid metabolism, and placental hormone regulation.

Significance Determining the state of an individual’s internal physiological clock has important implications for precision medicine, from diagnosing neurological disorders to optimizing drug delivery. To be useful, such a test must be accurate, minimally burdensome to the patient, and robust to differences in patient protocols, sample collection, and assay technologies. TimeSignature is a machine-learning approach to predict physiological time based on gene expression in human blood. A powerful feature is TimeSignature’s generalizability, enabling it to be applied to samples from disparate studies and yield highly accurate results despite systematic differences between the studies. This quality is unique among expression-based predictors and addresses a major challenge in the development of reliable and clinically useful biomarker tests., Circadian clocks play a key role in regulating a vast array of biological processes, with significant implications for human health. Accurate assessment of physiological time using transcriptional biomarkers found in human blood can significantly improve diagnosis of circadian disorders and optimize the delivery time of therapeutic treatments. To be useful, such a test must be accurate, minimally burdensome to the patient, and readily generalizable to new data. A major obstacle in development of gene expression biomarker tests is the diversity of measurement platforms and the inherent variability of the data, often resulting in predictors that perform well in the original datasets but cannot be universally applied to new samples collected in other settings. Here, we introduce TimeSignature, an algorithm that robustly infers circadian time from gene expression. We demonstrate its application in data from three independent studies using distinct microarrays and further validate it against a new set of samples profiled by RNA-sequencing. Our results show that TimeSignature is more accurate and efficient than competing methods, estimating circadian time to within 2 h for the majority of samples. Importantly, we demonstrate that once trained on data from a single study, the resulting predictor can be universally applied to yield highly accurate results in new data from other studies independent of differences in study population, patient protocol, or assay platform without renormalizing the data or retraining. This feature is unique among expression-based predictors and addresses a major challenge in the development of generalizable, clinically useful tests.

TimeSignature’s power is that it is highly accurate across transcriptomic platforms and experimental protocols. TimeSignature can be trained using data from a single platform/study and be applied to independent data without additional processing. Demonstrating this robustness and generalizability required applying TimeSignature to as diverse a set of studies as possible (i.e., refs. 1⇓⇓–4). Because not all studies had melatonin data, we chose to use draw time as a proxy for melatonin phase. Laing et al. (5) criticize this methodology; however, each of the studies (1⇓⇓–4) selected subjects for whom circadian phase was well aligned with local time. We have confirmed that dim-light melatonin onset (DLMO)25%, the time at which 25% of maximum blood melatonin is reached, is strongly correlated with local time in studies with available melatonin data (1⇓–3) (Fig. 1). As expected, training on local time thus accurately predicts melatonin phase (Fig. 2, oTS). Fig. 1. Comparison of circadian and clock … [↵][1]1To whom correspondence should be addressed. Email: rbraun{at}northwestern.edu. [1]: #xref-corresp-1-1

The timing of behavior under natural light-dark conditions is a function of circadian clocks and photic input pathways. Yet a mechanistic understanding of how these pathways collaborate in animals is lacking. Here we demonstrate in Drosophila that the Phosphatase of Regenerating Liver-1 (PRL-1) sets period length and behavioral phase gated by photic signals. PRL-1 knockdown in PDF clock neurons dramatically lengthens circadian period. PRL-1 mutants exhibit allele-specific interactions with the light- and clock-regulated gene timeless (tim). Moreover, we show that PRL-1 promotes TIM accumulation and dephosphorylation. Interestingly, the PRL-1 mutant period lengthening is suppressed in constant light and PRL-1 mutants display a delayed phase under short, but not long, photoperiod conditions. Thus, our studies reveal that PRL-1 dependent dephosphorylation of TIM is a core mechanism of the clock that sets period length and phase in darkness, enabling the behavioral adjustment to changing day-night cycles.

The Drosophila wing imaginal disc is subdivided along the proximodistal axis into the distal pouch, the hinge, the surrounding pleura, and the notum. While the genetic pathways that specify the identity of each of these domains have been well studied, the mechanisms that coordinate the relative expansion of these domains are not well understood. Here we investigated the role of the stat92E signal transducer and activator of transcription in wing proximodistal development. We find that Stat92E is active ubiquitously in early wing imaginal discs, where it acts to inhibit the induction of ectopic wing fields. Subsequently, Stat92E activity is down regulated in the notum and distal pouch. These dynamics coincide with and contribute to the proportional subdivision and expansion of these primordia. As development proceeds, Stat92E activity becomes restricted to the hinge, where it promotes normal expansion of the hinge, and restricts expansion of the notum. We also find that stat92E is required autonomously to specify dorsal pleura identity and inhibit notum identity to properly subdivide the body wall. Our data suggest that Stat92E activity is regulated along the proximodistal axis to pattern this axis and control the relative expansion of the pouch, hinge, and notum.

To compare circadian gene expression within highly discrete neuronal populations, we separately purified and characterized two adjacent but distinct groups of Drosophila adult circadian neurons: the 8 small and 10 large PDF-expressing ventral lateral neurons (s-LNvs and l-LNvs, respectively). The s-LNvs are the principal circadian pacemaker cells, whereas recent evidence indicates that the l-LNvs are involved in sleep and light-mediated arousal. Although half of the l-LNv–enriched mRNA population, including core clock mRNAs, is shared between the l-LNvs and s-LNvs, the other half is l-LNv– and s-LNv–specific. The distribution of four specific mRNAs is consistent with prior characterization of the four encoded proteins, and therefore indicates successful purification of the two neuronal types. Moreover, an octopamine receptor mRNA is selectively enriched in l-LNvs, and only these neurons respond to in vitro application of octopamine. Dissection and purification of l-LNvs from flies collected at different times indicate that these neurons contain cycling clock mRNAs with higher circadian amplitudes as well as at least a 10-fold higher fraction of oscillating mRNAs than all previous analyses of head RNA. Many of these cycling l-LNv mRNAs are well expressed but do not cycle or cycle much less well elsewhere in heads. The results suggest that RNA cycling is much more prominent in circadian neurons than elsewhere in heads and may be particularly important for the functioning of these neurons.

SummaryCircadian clocks regulate membrane excitability in master pacemaker neurons to control daily rhythms of sleep and wake. Here, we find that two distinctly timed electrical drives collaborate to impose rhythmicity on Drosophila clock neurons. In the morning, a voltage-independent sodium conductance via the NA/NALCN ion channel depolarizes these neurons. This current is driven by the rhythmic expression of NCA localization factor-1, linking the molecular clock to ion channel function. In the evening, basal potassium currents peak to silence clock neurons. Remarkably, daily antiphase cycles of sodium and potassium currents also drive mouse clock neuron rhythms. Thus, we reveal an evolutionarily ancient strategy for the neural mechanisms that govern daily sleep and wake.

Studies in Drosophila circadian neurons reveal a bifurcation in the Pigment Dispersing Factor (PDF) neuropeptide signaling pathway, independently synchronizing circadian clocks via PKA or acutely controlling neuronal excitability via cAMP., Molecular circadian clocks are interconnected via neural networks. In Drosophila, PIGMENT-DISPERSING FACTOR (PDF) acts as a master network regulator with dual functions in synchronizing molecular oscillations between disparate PDF(+) and PDF(−) circadian pacemaker neurons and controlling pacemaker neuron output. Yet the mechanisms by which PDF functions are not clear. We demonstrate that genetic inhibition of protein kinase A (PKA) in PDF(−) clock neurons can phenocopy PDF mutants while activated PKA can partially rescue PDF receptor mutants. PKA subunit transcripts are also under clock control in non-PDF DN1p neurons. To address the core clock target of PDF, we rescued per in PDF neurons of arrhythmic per01 mutants. PDF neuron rescue induced high amplitude rhythms in the clock component TIMELESS (TIM) in per-less DN1p neurons. Complete loss of PDF or PKA inhibition also results in reduced TIM levels in non-PDF neurons of per01 flies. To address how PDF impacts pacemaker neuron output, we focally applied PDF to DN1p neurons and found that it acutely depolarizes and increases firing rates of DN1p neurons. Surprisingly, these effects are reduced in the presence of an adenylate cyclase inhibitor, yet persist in the presence of PKA inhibition. We have provided evidence for a signaling mechanism (PKA) and a molecular target (TIM) by which PDF resets and synchronizes clocks and demonstrates an acute direct excitatory effect of PDF on target neurons to control neuronal output. The identification of TIM as a target of PDF signaling suggests it is a multimodal integrator of cell autonomous clock, environmental light, and neural network signaling. Moreover, these data reveal a bifurcation of PKA-dependent clock effects and PKA-independent output effects. Taken together, our results provide a molecular and cellular basis for the dual functions of PDF in clock resetting and pacemaker output., Author Summary Circadian clocks provide a mechanism for predicting and adapting behavioral and physiological processes to 24-hour rhythms in the environment. In animal nervous systems, cell-autonomous molecular oscillators are coupled via neural networks that control daily patterns of activity. A major neuropeptide synchronizing neural oscillators in the Drosophila clock network is PIGMENT DISPERSING FACTOR (PDF). Here we identify a fork in the processing of the PDF signal in circadian neurons to independently reset the molecular clock and regulate neuronal activity. We show that the cAMP-activated protein kinase A (PKA) in circadian neurons is necessary and sufficient for many PDF-dependent behaviors. In addition, we find that a PDF>PDF receptor>PKA pathway targets the clock component TIMELESS to control molecular oscillators, and that this process may be influenced by rhythmic expression of PKA. We show that this pathway splits at the level of cAMP generation, with PDF and cAMP acutely increasing the activity of clock neurons in a PKA-independent manner. Thus, PDF operates via dual signaling pathways: one via PKA to reset clocks and the other via cAMP to acutely control activity. These results have broad implications given the conserved involvement of neuropeptide signaling in synchronizing clocks in circadian neural networks.

BackgroundIn the first optic neuropil (lamina) of the fly's visual system, monopolar cells L1 and L2 and glia show circadian rhythms in morphological plasticity. They change their size and shape during the day and night. The most pronounced changes have been detected in circadian size of the L2 axons. Looking for a functional significance of the circadian plasticity observed in axons, we examined the morphological plasticity of the L2 dendrites. They extend from axons and harbor postsynaptic sites of tetrad synaptic contacts from the photoreceptor terminals.Methodology/principal findingsThe plasticity of L2 dendrites was evaluated by measuring an outline of the L2 dendritic trees. These were from confocal images of cross sections of L2 cells labeled with GFP. They were in wild-type and clock mutant flies held under different light conditions and sacrified at different time points. We found that the L2 dendrites are longest at the beginning of the day in both males and females. This rhythm observed under a day/night regime (LD) was maintained in constant darkness (DD) but not in continuous light (LL). This rhythm was not present in the arrhythmic per(01) mutant in LD or in DD. In the clock photoreceptor cry(b) mutant the rhythm was maintained but its pattern was different than that observed in wild-type flies.Conclusions/significanceThe results obtained showed that the L2 dendrites exhibit circadian structural plasticity. Their morphology is controlled by the per gene-dependent circadian clock. The L2 dendrites are longest at the beginning of the day when the daytime tetrad presynaptic sites are most numerous and L2 axons are swollen. The presence of the rhythm, but with a different pattern in cry(b) mutants in LD and DD indicates a new role of cry in the visual system. The new role is in maintaining the circadian pattern of changes of the L2 dendrite length and shape.