Rhythmic motor patterns underlying many types of locomotion are thought to be produced by central pattern generators (CPGs). Our knowledge of how CPG networks generate motor patterns in complex nervous systems remains incomplete, despite decades of work in a variety of model organisms. Substrate borne locomotion in Drosophila larvae is driven by waves of muscular contraction that propagate through multiple body segments. We use the motor circuitry underlying crawling in larval Drosophila as a model to try to understand how segmentally coordinated rhythmic motor patterns are generated. Whereas muscles, motoneurons and sensory neurons have been well investigated in this system, far less is known about the identities and function of interneurons. Our recent study identified a class of glutamatergic premotor interneurons, PMSIs (period-positive median segmental interneurons), that regulate the speed of locomotion. Here, we report on the identification of a distinct class of glutamatergic premotor interneurons called Glutamatergic Ventro-Lateral Interneurons (GVLIs). We used calcium imaging to search for interneurons that show rhythmic activity and identified GVLIs as interneurons showing wave-like activity during peristalsis. Paired GVLIs were present in each abdominal segment A1-A7 and locally extended an axon towards a dorsal neuropile region, where they formed GRASP-positive putative synaptic contacts with motoneurons. The interneurons expressed vesicular glutamate transporter (vGluT) and thus likely secrete glutamate, a neurotransmitter known to inhibit motoneurons. These anatomical results suggest that GVLIs are premotor interneurons that locally inhibit motoneurons in the same segment. Consistent with this, optogenetic activation of GVLIs with the red-shifted channelrhodopsin, CsChrimson ceased ongoing peristalsis in crawling larvae. Simultaneous calcium imaging of the activity of GVLIs and motoneurons showed that GVLIs' wave-like activity lagged behind that of motoneurons by several segments. Thus, GVLIs are activated when the front of a forward motor wave reaches the second or third anterior segment. We propose that GVLIs are part of the feedback inhibition system that terminates motor activity once the front of the motor wave proceeds to anterior segments.

Hunger is a complex motivational state that drives multiple behaviors. The sensation of hunger is caused by an imbalance between energy intake and expenditure. One immediate response to hunger is increased food consumption. Hunger also modulates behaviors related to food seeking such as increased locomotion and enhanced sensory sensitivity in both insects [1-5] and vertebrates [6, 7]. In addition, hunger can promote the expression of food-associated memory [8, 9]. Although progress is being made [10], how hunger is represented in the brain and how it coordinates these behavioral responses is not fully understood in any system. Here, we use Drosophila melanogaster to identify neurons encoding hunger. We found a small group of neurons that, when activated, induced a fed fly to eat as though it were starved, suggesting that these neurons are downstream of the metabolic regulation of hunger. Artificially activating these neurons also promotes appetitive memory performance in sated flies, indicating that these neurons are not simply feeding command neurons but likely play a more general role in encoding hunger. We determined that the neurons relevant for the feeding effect are serotonergic and project broadly within the brain, suggesting a possible mechanism for how various responses to hunger are coordinated. These findings extend our understanding of the neural circuitry that drives feeding and enable future exploration of how state influences neural activity within this circuit.

Molecular and cellular processes in neurons are critical for sensing and responding to energy deficit states, such as during weight-loss. AGRP neurons are a key hypothalamic population that is activated during energy deficit and increases appetite and weight-gain. Cell type-specific transcriptomics can be used to identify pathways that counteract weight-loss, and here we report high-quality gene expression profiles of AGRP neurons from well-fed and food-deprived young adult mice. For comparison, we also analyzed POMC neurons, an intermingled population that suppresses appetite and body weight. We find that AGRP neurons are considerably more sensitive to energy deficit than POMC neurons. Furthermore, we identify cell type-specific pathways involving endoplasmic reticulum-stress, circadian signaling, ion channels, neuropeptides, and receptors. Combined with methods to validate and manipulate these pathways, this resource greatly expands molecular insight into neuronal regulation of body weight, and may be useful for devising therapeutic strategies for obesity and eating disorders.

Programmed genome rearrangements in the unicellular eukaryote Oxytricha trifallax produce a transcriptionally active somatic nucleus from a copy of its germline nucleus during development. This process eliminates noncoding sequences that interrupt coding regions in the germline genome, and joins over 225,000 remaining DNA segments, some of which require inversion or complex permutation to build functional genes. This dynamic genomic organization permits some single DNA segments in the germline to contribute to multiple, distinct somatic genes via alternative processing. Like alternative mRNA splicing, the combinatorial assembly of DNA segments contributes to genetic variation and facilitates the evolution of new genes. In this study, we use comparative genomic analysis to demonstrate that the emergence of alternative DNA splicing is associated with the origin of new genes. Short duplications give rise to alternative gene segments that are spliced to the shared gene segments. Alternative gene segments evolve faster than shared, constitutive segments. Genes with shared segments frequently have different expression profiles, permitting functional divergence. This study reports alternative DNA splicing as a mechanism of new gene origination, illustrating how the process of programmed genome rearrangement gives rise to evolutionary innovation.

Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

Intrinsically disordered protein regions (IDRs) are peptide segments that fail to form stable 3-dimensional structures in the absence of partner proteins. They are abundant in eukaryotic proteomes and are often associated with human diseases, but their biological functions have been elusive to study. Here we report the identification of a tin(IV) oxochloride-derived cluster that binds an evolutionarily conserved IDR within the metazoan TFIID transcription complex. Binding arrests an isomerization of promoter-bound TFIID that is required for the engagement of Pol II during the first (de novo) round of transcription initiation. However, the specific chemical probe does not affect reinitiation, which requires the re-entry of Pol II, thus, mechanistically distinguishing these two modes of transcription initiation. This work also suggests a new avenue for targeting the elusive IDRs by harnessing certain features of metal-based complexes for mechanistic studies, and for the development of novel pharmaceutical interventions.

The K2 Summit camera was initially the only commercially available direct electron detection camera that was optimized for high-speed counting of primary electrons and was also the only one that implemented centroiding so that the resolution of the camera can be extended beyond the Nyquist limit set by the physical pixel size. In this study, we used well-characterized two-dimensional crystals of the membrane protein aquaporin-0 to characterize the performance of the camera below and beyond the physical Nyquist limit and to measure the influence of electron dose rate on image amplitudes and phases.

Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.

To facilitate large scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently comprised of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated web site, the RNAi Stock Validation and Phenotypes Project (RSVP; www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (USA), National Institute of Genetics (Japan), and TsingHua Fly Center (China).