The Glial Regenerative Response to Central Nervous System Injury Is Enabled by Pros-Notch and Pros-NFκB Feedback

Organisms are structurally robust, as cells accommodate changes preserving structural integrity and function. The molecular mechanisms underlying structural robustness and plasticity are poorly understood, but can be investigated by probing how cells respond to injury. Injury to the CNS induces proliferation of enwrapping glia, leading to axonal re-enwrapment and partial functional recovery. This glial regenerative response is found across species, and may reflect a common underlying genetic mechanism. Here, we show that injury to the Drosophila larval CNS induces glial proliferation, and we uncover a gene network controlling this response. It consists of the mutual maintenance between the cell cycle inhibitor Prospero (Pros) and the cell cycle activators Notch and NFκB. Together they maintain glia in the brink of dividing, they enable glial proliferation following injury, and subsequently they exert negative feedback on cell division restoring cell cycle arrest. Pros also promotes glial differentiation, resolving vacuolization, enabling debris clearance and axonal enwrapment. Disruption of this gene network prevents repair and induces tumourigenesis. Using wound area measurements across genotypes and time-lapse recordings we show that when glial proliferation and glial differentiation are abolished, both the size of the glial wound and neuropile vacuolization increase. When glial proliferation and differentiation are enabled, glial wound size decreases and injury-induced apoptosis and vacuolization are prevented. The uncovered gene network promotes regeneration of the glial lesion and neuropile repair. In the unharmed animal, it is most likely a homeostatic mechanism for structural robustness. This gene network may be of relevance to mammalian glia to promote repair upon CNS injury or disease

Representations of Reward and Movement in Drosophila Dopaminergic Neurons

The neuromodulator dopamine is known to influence both immediate and future behavior, motivating and invigorating an animal’s ongoing movement but also serving as a reinforcement signal to instruct learning. Yet it remains unclear whether this dual role of dopamine involves the same dopaminergic pathways. Although reward-responsive dopaminergic neurons display movement-related activity, debate continues as to what features of an individual’s experience these motor-correlates correspond and how they influence concurrent behavior. The mushroom body, a prominent neuropil in the brain of the fruit fly Drosophila melanogaster, is richly innervated by dopaminergic neurons that play an essential role in the formation of olfactory associations. While dopaminergic neurons respond to reward and punishment to drive associative learning, they have also been implicated in a number of adaptive behaviors and their activity correlates with the behavioral state of an animal and its coarse motor actions. Here, we take advantage of the concise circuit architecture of the Drosophila mushroom body to investigate the nature of motor-related signals in dopaminergic neurons that drive associative learning. In vivo functional imaging during naturalistic tethered locomotion reveals that the activity of different subsets of mushroom body dopaminergic neurons reflects distinct aspects of movement. To gain insight into what facets of an animal’s experience are represented by these movement-related signals, we employed a closed loop virtual reality paradigm to monitor neural activity as animals track an olfactory stimulus and are actively engaged in a goal-directed and sensory-motivated behavior. We discover that odor responses in dopaminergic neurons correlate with the extent to which an animal tracks upwind towards the fictive odor source. In different experimental contexts where distinct motor actions were required to track the odor, dopaminergic neurons become emergently linked to the behavioral metric most relevant for effective olfactory navigation. Subsets of dopaminergic neurons were correlated with the strength of upwind tracking regardless of the identity of the odor and remained so even after the satiety state of an animal was altered. We proceed to demonstrate that transient inhibition of dopaminergic neurons that are positively correlated with upwind tracking significantly diminishes the normal approach responses to an appetitive olfactory cue. Accordingly, activation of those same dopaminergic neurons enhances approach to an odor and even drives upwind tracking in clean air alone. Together, these results reveal that the same dopaminergic pathways that convey reinforcements to instruct learning also carry representations of an animal’s moment-by-moment movements and actively influence behavior. The complex activity patterns of mushroom body dopaminergic neurons therefore represent neither purely sensory nor motor variables but rather reflect the goal or motivation underlying an animal’s movements. Our data suggest a fundamental coupling between reinforcement signals and motivation-related locomotor representations within dopaminergic circuitry, drawing a striking parallel between the mushroom body dopaminergic neurons described here and the emerging understanding of mammalian dopaminergic pathways. The apparent conservation in dopaminergic neuromodulatory networks between mammals and insects suggests a shared logic for how neural circuits assign meaning to both sensory stimuli and motor actions to generate flexible and adaptive behavior

Technologies and Approaches to Elucidate and Model the Virulence Program of Salmonella

Salmonella is a primary cause of enteric diseases in a variety of animals. During its evolution into a pathogenic bacterium, Salmonella acquired an elaborate regulatory network that responds to multiple environmental stimuli within host animals and integrates them resulting in fine regulation of the virulence program. The coordinated action by this regulatory network involves numerous virulence regulators, necessitating genome-wide profiling analysis to assess and combine efforts from multiple regulons. In this review we discuss recent high-throughput analytic approaches used to understand the regulatory network of Salmonella that controls virulence processes. Application of high-throughput analyses have generated large amounts of data and necessitated the development of computational approaches for data integration. Therefore, we also cover computer-aided network analyses to infer regulatory networks, and demonstrate how genome-scale data can be used to construct regulatory and metabolic systems models of Salmonella pathogenesis. Genes that are coordinately controlled by multiple virulence regulators under infectious conditions are more likely to be important for pathogenesis. Thus, reconstructing the global regulatory network during infection or, at the very least, under conditions that mimic the host cellular environment not only provides a bird's eye view of Salmonella survival strategy in response to hostile host environments but also serves as an efficient means to identify novel virulence factors that are essential for Salmonella to accomplish systemic infection in the host

Transcriptional control of somatosensory neuron diversification in Drosophila

Primary sensory neurons deliver information from the periphery to specific circuits in the central nervous system. It is vital that each sensory neuron detects the appropriate type of stimulus and conveys that information to appropriate regions of the sensory neuropil to target second-order neurons. Molecular programs that coordinate sensory morphology in the periphery with axon projection patterns centrally are poorly understood. I have used the multidendritic (md) sensory neurons of the Drosophila melanogaster peripheral nervous system to identify genetic and molecular programs that coordinate dendrite and axonal morphogenesis in individual sensory neurons. The homeodomain transcription factor Cut is expressed in neurons with complex dendrite morphologies that innervate the epidermis and ventral axon projections in the CNS, and is absent from putative proprioceptive neurons that have simpler dendrites and target to more dorsal CNS regions. In this thesis I demonstrate that, in defined subsets of sensory neurons, loss of Cut leads to dendritic transformation to a proprioceptive-type arbor that is accompanied by a dorsal shift in the termination of their axons in the CNS. Mechanistically, I show that Cut functions at least in part by repressing the expression of the POU domain transcription factors Pdm1 and Pdm2 (Pdm1/2), which are normally expressed only in proprioceptive neurons. Gain and loss of function studies further suggest instructive roles for Pdm1/2 in the development of proprioceptive dendritic arborization and axonal targeting. Together these results identify a transcriptional program that coordinately specifies proprioceptive dendrite morphology and sensory axon targeting to modality-specific domains of the CNS. Using a candidate based approached I have identified three molecular regulators of proprioceptive neuron dendrite morphology. In addition, gene profiling of sensory neurons forced to express Pdm2 has identified over 600 genes that show changes in expression when Pdm2 is misexpressed and that may mediate the effects of Pdm1/2 in directing proprioceptive dendrite and axon development. These profiling experiments pave the way for the identification of novel regulators of dendrite and axon morphogenesis that link transcriptional programs to specific morphologies with consequences for sensory circuit function

E Unibus Plurum: Genomic Analysis of an Experimentally Evolved Polymorphism in Escherichia coli

Microbial populations founded by a single clone and propagated under resource limitation can become polymorphic. We sought to elucidate genetic mechanisms whereby a polymorphism evolved in Escherichia coli under glucose limitation and persisted because of cross-feeding among multiple adaptive clones. Apart from a 29 kb deletion in the dominant clone, no large-scale genomic changes distinguished evolved clones from their common ancestor. Using transcriptional profiling on co-evolved clones cultured separately under glucose-limitation we identified 180 genes significantly altered in expression relative to the common ancestor grown under similar conditions. Ninety of these were similarly expressed in all clones, and many of the genes affected (e.g., mglBAC, mglD, and lamB) are in operons coordinately regulated by CRP and/or rpoS. While the remaining significant expression differences were clone-specific, 93% were exhibited by the majority clone, many of which are controlled by global regulators, CRP and CpxR. When transcriptional profiling was performed on adaptive clones cultured together, many expression differences that distinguished the majority clone cultured in isolation were absent, suggesting that CpxR may be activated by overflow metabolites removed by cross-feeding strains in co-culture. Relative to their common ancestor, shared expression differences among adaptive clones were partly attributable to early-arising shared mutations in the trans-acting global regulator, rpoS, and the cis-acting regulator, mglO. Gene expression differences that distinguished clones may in part be explained by mutations in trans-acting regulators malT and glpK, and in cis-acting sequences of acs. In the founder, a cis-regulatory mutation in acs (acetyl CoA synthetase) and a structural mutation in glpR (glycerol-3-phosphate repressor) likely favored evolution of specialists that thrive on overflow metabolites. Later-arising mutations that led to specialization emphasize the importance of compensatory rather than gain-of-function mutations in this system. Taken together, these findings underscore the importance of regulatory change, founder genotype, and the biotic environment in the adaptive evolution of microbes

Acetaldehyde as a drug of abuse: Involvement of endocannabinoid- and dopamine neurotransmission

Acetaldehyde (ACD), the first metabolite of ethanol, directly enhances dopamine neurotransmission (1) and has rewarding and motivational properties in paradigms tailored for studying addictive-like behaviours (2, 3). The endocannabinoid system affects distinct drug-related behaviours, since it may in turn fine-tune dopamine cell activity (4, 5). In light of this, the present study aimed at investigating the effects of a direct manipulation of the DAergic synapse, and the contribution of the endocannabinoid system on oral ACD self-administration in rats. ACD drinking-behaviour was evaluated in an operant paradigm consisting of acquisition and maintenance; extinction; deprivation and relapse; conflict. D2-receptor agonists, quinpirole (0.03 mg/Kg, i.p.) and ropinirole (0.03 mg/Kg, i.p.), and CB1-receptor antagonist, AM281 (1 mg/Kg, i.p.), were administered during different phases of the experiment. Our results show that oral ACD readily induced the acquisition and maintenance of an operant drinking-behaviour, even during reinstatement and conflict. Quinpirole decreased lever presses for ACD during extinction (p<0.05) and relapse (p<0.01; p<0.001) Ropinirole, administered during abstinence, reduced ACD intake during reinstatement (p<0.001). AM281 significantly decreased lever presses for ACD during extinction (p<0.001), relapse (p<0.001) and conflict (p<0.001). These data suggest that whereas the direct modulation of the dopaminergic synapse influences drug-seeking and relapse behaviour, the endocannabinoid system may also play a role in shock-paired compulsive ACD intake. These findings highlight the mandatory need for further investigation on the therapeutical potential played by the endocannabinoid system taking into account its crucial role in alcohol, and ACD neuropharmacology

Genome-Wide Screening of Genes Whose Enhanced Expression Affects Glycogen Accumulation in Escherichia coli

Using a systematic and comprehensive gene expression library (the ASKA library), we have carried out a genome-wide screening of the genes whose increased plasmid-directed expression affected glycogen metabolism in Escherichia coli. Of the 4123 clones of the collection, 28 displayed a glycogen-excess phenotype, whereas 58 displayed a glycogen-deficient phenotype. The genes whose enhanced expression affected glycogen accumulation were classified into various functional categories including carbon sensing, transport and metabolism, general stress and stringent responses, factors determining intercellular communication, aggregative and social behaviour, nitrogen metabolism and energy status. Noteworthy, one-third of them were genes about which little or nothing is known. We propose an integrated metabolic model wherein E. coli glycogen metabolism is highly interconnected with a wide variety of cellular processes and is tightly adjusted to the nutritional and energetic status of the cell. Furthermore, we provide clues about possible biological roles of genes of still unknown functions