Polyphasic circadian neural circuits drive differential activities in multiple downstream rhythmic centers.

Circadian clocks align various behaviors such as locomotor activity, sleep/wake, feeding, and mating to times of day that are most adaptive. How rhythmic information in pacemaker circuits is translated to neuronal outputs is not well understood. Here, we used brain-wide, 24-h in vivo calcium imaging in the Drosophila brain and searched for circadian rhythmic activity among identified clusters of dopaminergic (DA) and peptidergic neurosecretory (NS) neurons.

The incidence of candidate binding sites for β-arrestin in Drosophila neuropeptide GPCRs.

To support studies of neuropeptide neuromodulation, I have studied beta-arrestin binding sites (BBS's) by evaluating the incidence of BBS sequences among the C terminal tails (CTs) of each of the 49 Drosophila melanogaster neuropeptide GPCRs. BBS were identified by matches with a prediction derived from structural analysis of rhodopsin:arrestin and vasopressin receptor: arrestin complexes [1]. To increase the rigor of the identification, I determined the conservation of BBS sequences between two long-diverged species D.

Regulation of PDF receptor signaling controlling daily locomotor rhythms in Drosophila.

Each day and in conjunction with ambient daylight conditions, neuropeptide PDF regulates the phase and amplitude of locomotor activity rhythms in Drosophila through its receptor, PDFR, a Family B G protein-coupled receptor (GPCR). We studied the in vivo process by which PDFR signaling turns off, by converting as many as half of the 28 potential sites of phosphorylation in its C terminal tail to a non-phosphorylatable residue (alanine). We report that many such sites are conserved evolutionarily, and their conversion creates a specific behavioral syndrome opposite to loss-of-function phenotypes previously described for pdfr.

Circadian pacemaker neurons display cophasic rhythms in basal calcium level and in fast calcium fluctuations.

Circadian pacemaker neurons in the Drosophila brain display daily rhythms in the levels of intracellular calcium. These calcium rhythms are driven by molecular clocks and are required for normal circadian behavior. To study their biological basis, we employed genetic manipulations in conjunction with improved methods of in vivo light-sheet microscopy to measure calcium dynamics in individual pacemaker neurons over complete 24-h durations at sampling frequencies as high as 5 Hz.

Spatiotemporal expression of regulatory kinases directs the transition from mitosis to cellular morphogenesis in Drosophila.

Embryogenesis depends on a tightly regulated balance between mitosis, differentiation, and morphogenesis. Understanding how the embryo uses a relatively small number of proteins to transition between growth and morphogenesis is a central question of developmental biology, but the mechanisms controlling mitosis and differentiation are considered to be fundamentally distinct. Here we show the mitotic kinase Polo, which regulates all steps of mitosis in Drosophila, also directs cellular morphogenesis after cell cycle exit.

Morning and Evening Circadian Pacemakers Independently Drive Premotor Centers via a Specific Dopamine Relay.

Many animals exhibit morning and evening peaks of locomotor behavior. In Drosophila, two corresponding circadian neural oscillators-M (morning) cells and E (evening) cells-exhibit a corresponding morning or evening neural activity peak. Yet we know little of the neural circuitry by which distinct circadian oscillators produce specific outputs to precisely control behavioral episodes.

A Series of Suppressive Signals within the Drosophila Circadian Neural Circuit Generates Sequential Daily Outputs.

We studied the Drosophila circadian neural circuit using whole-brain imaging in vivo. Five major groups of pacemaker neurons display synchronized molecular clocks, yet each exhibits a distinct phase of daily Ca activation. Light and neuropeptide pigment dispersing factor (PDF) from morning cells (s-LNv) together delay the phase of the evening (LNd) group by ∼12 hr; PDF alone delays the phase of the DN3 group by ∼17 hr.

Functional PDF Signaling in the Drosophila Circadian Neural Circuit Is Gated by Ral A-Dependent Modulation.

The neuropeptide PDF promotes the normal sequencing of circadian behavioral rhythms in Drosophila, but its signaling mechanisms are not well understood. We report daily rhythmicity in responsiveness to PDF in critical pacemakers called small LNvs. There is a daily change in potency, as great as 10-fold higher, around dawn.

Synchronous Drosophila circadian pacemakers display nonsynchronous Ca²⁺ rhythms in vivo.

In Drosophila, molecular clocks control circadian rhythmic behavior through a network of ~150 pacemaker neurons. To explain how the network's neuronal properties encode time, we performed brainwide calcium imaging of groups of pacemaker neurons in vivo for 24 hours. Pacemakers exhibited daily rhythmic changes in intracellular Ca(2+) that were entrained by environmental cues and timed by molecular clocks.

Exploring the Biology of G Protein-Coupled Receptors from In Vitro to In Vivo.

In August 2014, an international group of researchers gathered for 5 days at the Lorentz Center in Leiden, The Netherlands, to explore the technical and conceptual issues associated with the analysis of G protein-coupled receptor functions utilizing information from crystal structure models to the use of model organisms. This collection of review articles evolved from the 5-day meeting, with brief presentations and structured discussion periods that were designed to identify key questions remaining in understanding G protein-coupled receptor function and to propose novel strategies by integrating scientific disciplines to guide future research.

The Drosophila Prosecretory Transcription Factor dimmed Is Dynamically Regulated in Adult Enteroendocrine Cells and Protects Against Gram-Negative Infection.

The endocrine system employs peptide hormone signals to translate environmental changes into physiological responses. The diffuse endocrine system embedded in the gastrointestinal barrier epithelium is one of the largest and most diverse endocrine tissues. Furthermore, it is the only endocrine tissue in direct physical contact with the microbial environment of the gut lumen.

Model Organisms in G Protein-Coupled Receptor Research.

The study of G protein-coupled receptors (GPCRs) has benefited greatly from experimental approaches that interrogate their functions in controlled, artificial environments. Working in vitro, GPCR receptorologists discovered the basic biologic mechanisms by which GPCRs operate, including their eponymous capacity to couple to G proteins; their molecular makeup, including the famed serpentine transmembrane unit; and ultimately, their three-dimensional structure. Although the insights gained from working outside the native environments of GPCRs have allowed for the collection of low-noise data, such approaches cannot directly address a receptor's native (in vivo) functions.

Genome-wide features of neuroendocrine regulation in Drosophila by the basic helix-loop-helix transcription factor DIMMED.

Neuroendocrine (NE) cells use large dense core vesicles (LDCVs) to traffic, process, store and secrete neuropeptide hormones through the regulated secretory pathway. The dimmed (DIMM) basic helix-loop-helix transcription factor of Drosophila controls the level of regulated secretory activity in NE cells. To pursue its mechanisms, we have performed two independent genome-wide analyses of DIMM's activities: (i) in vivo chromatin immunoprecipitation (ChIP) to define genomic sites of DIMM occupancy and (ii) deep sequencing of purified DIMM neurons to characterize their transcriptional profile.

Peptidergic cell-specific synaptotagmins in Drosophila: localization to dense-core granules and regulation by the bHLH protein DIMMED.

Bioactive peptides are packaged in large dense-core secretory vesicles, which mediate regulated secretion by exocytosis. In a variety of tissues, the regulated release of neurotransmitters and hormones is dependent on calcium levels and controlled by vesicle-associated synaptotagmin (SYT) proteins. Drosophila express seven SYT isoforms, of which two (SYT-α and SYT-β) were previously found to be enriched in neuroendocrine cells.

E and M circadian pacemaker neurons use different PDF receptor signalosome components in drosophila.

We used real-time imaging to detect cAMP levels in neurons of intact fly brains to study the mechanisms of circadian pacemaker synchronization by the neuropeptide pigment dispersing factor (PDF) in Drosophila. PDF receptor (PDF-R) is expressed by both M (sLNv) and E (LNd) pacemaker subclasses and is coupled to G(sα) in both cases. We previously reported that PDF-R in M pacemakers elevates cAMP levels by activating the ortholog of mammalian adenylate cyclase 3 (AC3) but that AC3 disruptions had no effect on E pacemaker sensitivity to PDF.

Peptide neuromodulation in invertebrate model systems.

Neuropeptides modulate neural circuits controlling adaptive animal behaviors and physiological processes, such as feeding/metabolism, reproductive behaviors, circadian rhythms, central pattern generation, and sensorimotor integration. Invertebrate model systems have enabled detailed experimental analysis using combined genetic, behavioral, and physiological approaches. Here we review selected examples of neuropeptide modulation in crustaceans, mollusks, insects, and nematodes, with a particular emphasis on the genetic model organisms Drosophila melanogaster and Caenorhabditis elegans, where remarkable progress has been made.

The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila.

The neuropeptide Pigment Dispersing Factor (PDF) is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R) is present in both M and subsets of E cells.

Therapeutic peptide production in Drosophila.

Bioactive peptides are important therapeutic drugs, yet conventional methods of peptide synthesis are challenged to meet increasing demand. We developed a novel and efficient means of metabolic engineering: therapeutic peptide production in Drosophila and as a proof of concept, we demonstrate production of fully matured human insulin. This in vivo system offers an innovative means to produce valuable bioactive peptides for therapies, its inherent flexibility facilitates drug development, and its ease of producing fully processed peptides simplifies metabolic engineering of new peptide products.

Circadian pacemakers: how clock properties relate to their cellular properties.

Circadian (~24 hour) pacemaking mechanisms exist within single cells. Which cellular properties contrive to produce a precise clockworks, and which cell properties are downstream of it? The literature is conflicted as to whether membrane excitability contributes to the mechanism. Now, a new conditional genetic strategy argues excitability is largely dispensable.

Scaling factors: transcription factors regulating subcellular domains.

Developing cells acquire mature fates in part by selective (i.e. qualitatively different) expression of a few cell-specific genes.

Molecular organization of Drosophila neuroendocrine cells by Dimmed.

Background: In Drosophila, the basic-helix-loop-helix protein DIMM coordinates the molecular and cellular properties of all major neuroendocrine cells, irrespective of the secretory peptides they produce. When expressed by nonneuroendocrine neurons, DIMM confers the major properties of the regulated secretory pathway and converts such cells away from fast neurotransmission and toward a neuroendocrine state.

Results: We first identified 134 transcripts upregulated by DIMM in embryos and then evaluated them systematically using diverse assays (including embryo in situ hybridization, in vivo chromatin immunoprecipitation, and cell-based transactivation assays).

PDFR and CRY signaling converge in a subset of clock neurons to modulate the amplitude and phase of circadian behavior in Drosophila.

Background: To synchronize their molecular rhythms, circadian pacemaker neurons must input both external and internal timing cues and, therefore, signal integration between sensory information and internal clock status is fundamental to normal circadian physiology.

Methodology/principal Findings: We demonstrate the specific convergence of clock-derived neuropeptide signaling with that of a deep brain photoreceptor. We report that the neuropeptide PDF receptor and the circadian photoreceptor CRYPTOCROME (CRY) are precisely co-expressed in a subset of pacemakers, and that these pathways together provide a requisite drive for circadian control of daily locomotor rhythms.

Circadian rhythms: biological clocks work in phospho-time.

The 24 hour molecular oscillator requires precisely calibrated degradation of core clock proteins, like PERIOD. New studies shed light on a sequential series of PERIOD phosphorylation events that first inhibits, then accelerates PERIOD degradation.

PDF receptor expression reveals direct interactions between circadian oscillators in Drosophila.

Daily rhythms of behavior are controlled by a circuit of circadian pacemaking neurons. In Drosophila, 150 pacemakers participate in this network, and recent observations suggest that the network is divisible into M and E oscillators, which normally interact and synchronize. Sixteen oscillator neurons (the small and large lateral neurons [LNvs]) express a neuropeptide called pigment-dispersing factor (PDF) whose signaling is often equated with M oscillator output.