Project description:A challenge in systems biology is to understand the gene regulatory networks that connect early cellular specification to terminal differentiation of specific cell types. In neurogenesis, neural specification has been well studied, but the link between the proneural transcriptional regulators of specification and the genes that must be activated to construct differentiated neurons is obscure. High resolution temporal profiling of gene expression reveals the events downstream of Atonal (Ato) proneural gene function in Drosophila sensory neurons. Unexpectedly, many differentiation genes are activated soon after specification, even before cell cycle exit and overt neuronal differentiation. Prominent among them are genes required for construction of the ciliary dendrite. Ato activates differentiation both directly and indirectly via several intermediate transcriptional regulators, including Rfx and a new Forkhead family factor. Our analysis of these factors and their regulation provides insight into how proneural factors regulate neuronal subtype differentiation. Investigating the molecular mechanisms of peripheral nervous sytem development in Drosophila melanogaster. Affymetrix Drosophila version 2.0 chips used to measure gene expression from GFP+ and GFP- cells from embryos expressing GFP under the control of the atonal gene enhancer in both wild type and mutant embryos. Data generated for three developmental time-points in quadruplicate.
Project description:A challenge in systems biology is to understand the gene regulatory networks that connect early cellular specification to terminal differentiation of specific cell types. In neurogenesis, neural specification has been well studied, but the link between the proneural transcriptional regulators of specification and the genes that must be activated to construct differentiated neurons is obscure. High resolution temporal profiling of gene expression reveals the events downstream of Atonal (Ato) proneural gene function in Drosophila sensory neurons. Unexpectedly, many differentiation genes are activated soon after specification, even before cell cycle exit and overt neuronal differentiation. Prominent among them are genes required for construction of the ciliary dendrite. Ato activates differentiation both directly and indirectly via several intermediate transcriptional regulators, including Rfx and a new Forkhead family factor. Our analysis of these factors and their regulation provides insight into how proneural factors regulate neuronal subtype differentiation. Investigating the molecular mechanisms of peripheral nervous sytem development in Drosophila melanogaster.
Project description:<p>Chronic sleep loss profoundly impacts metabolic health and shortens lifespan, but studies of the mechanisms involved have focused largely on acute sleep deprivation. To identify metabolic consequences of chronically reduced sleep, we conducted unbiased metabolomics on heads of three adult Drosophila short-sleeping mutants with very different mechanisms of sleep loss: fumin (fmn), redeye (rye), and sleepless (sss). Common features included elevated ornithine and polyamines, with lipid, acyl-carnitine, and TCA cycle changes suggesting mitochondrial dysfunction. Studies of excretion demonstrate inefficient nitrogen elimination in adult sleep mutants, likely contributing to their polyamine accumulation. Increasing levels of polyamines, particularly putrescine, promote sleep in control flies but poison sleep mutants. This parallels the broadly enhanced toxicity of high dietary nitrogen load from protein in chronically sleep-restricted Drosophila, including both sleep mutants and flies with hyper-activated wake-promoting neurons. Together, our results implicate nitrogen stress as a novel mechanism linking chronic sleep loss to adverse health outcomes-and perhaps for linking food and sleep homeostasis at the cellular level in healthy organisms.</p>
Project description:This project’s aim was to compare the transcriptional profiles of olfactory sensory neurons in Drosophila melanogaster in order to identify novel genes that specify neuron-specific functions/phenotypes or may otherwise be involved in the development of the olfactory system. The isolation of sufficient numbers of intact olfactory sensory neurons (OSN) from the antenna of Drosophila melanogaster has so far limited single-cell transcriptomic approaches being applied to the adult fly antenna. Targeted DamID (TaDa) provides an alternative approach for profiling transcriptional activity in a cell-specific manor that bypasses the need for isolating OSN. Using the Gal4/UAS system, we applied TaDa to seven OSN populations and compared differences in Pol II occupancy for genes across these datasets.
Project description:While microRNAs (miRNAs) have recently emerged as critical post-transcriptional modulators of gene expression in neuronal development, very little is known regarding the roles of miRNA-mediated regulation in the specification of cell-type specific dendritic complexity. The dendritic arborization (da) sensory neurons of the Drosophila PNS offer an excellent model system for elucidating the molecular mechanisms governing class specific dendrite morphogenesis and for exploring miRNA-mediated control of this process. To facilitate functional analyses of miRNA regulation in da neurons, we have conducted whole-genome miRNA expression profiling as well as mRNA expression profiling of three distinct classes of da neurons, thereby generating a comprehensive molecular gene expression signature within these individual subclasses of da neurons. To further validate the role of these expressed miRNAs in directing dendritic architecture, we conducted a genome-wide UAS-miRNA phenotypic screen using live-image confocal microscopy followed by semi-automated neurometric quantification, to directly assess the effect of over/mis-expression of individual and clustered miRNAs on neurons of varying dendritic complexity. Through this approach, we have identified numerous miRNAs with previously unknown functions in dendritic development. Gain-of-function and loss-of-function analyses revealed an endogenous role miR-2b and miR-13b (members of the K-box family) and miR-12/283/304 in dendritic patterning in da neuron subclasses. Moreover, using an integrative bioinformatic analysis approach involving inverse correlation between miRNA and mRNA expression profiling data in combination with existing target prediction algorithms, we have identified putative target of these miRNAs in regulating da neuron dendritic development. Validation of these predicted miRNA-target relationships via phenotypic analyses as well as QPCR, revealed the regulatory effect of these molecules in restricting dendritic branching in da neurons.
Project description:Although DNA methylation plays a critical role in the development and function of mammalian central nervous system (CNS), its role in peripheral neurons has not been elucidated. To address this issue, we produced conditional knockout mice (CKO) specifically deleting the gene for maintenance DNA methyltransferase 1 (Dnmt1) during the development of neural crest cells. Despite global hypomethylation in the embryonic dorsal root ganglion (DRG) of the CKO mice, the number of sensory neurons was relatively unaffected. However, expression of many genes required for sensory neuron development was altered in embryonic mutant DRG, including down-regulation of Runx1 and TrkA genes as well as up-regulation of Id1 and Dtx1, two negative regulators for neurogenesis. Accompanied with the downregulation of an NGF receptor TrkA, the peripheral axonal projection and the branching of sensory neurons were impaired. Furthermore, the expression of the neuropeptide Galanin and several vanilloid receptors such as TrpV1 and TrpM8 were not detected in the DRG of the CKO mice during late embryonic and neonatal stages, suggesting that DNA methylation regulates the differentiation program for a subset of nociceptive sensory neurons. Taken together, our findings suggest that through transcriptional regulation of key developmental genes in sensory neurons, DNA methylation play a key role in the control of the axonal projection and fate specification of peripheral sensory neurons. We compared gene expression patterns in Wildtype and DNA methylation deficient (Wnt1-cre; Dnmt1 mutant) mouse dorsal cortex. We performed 4 replicates using different each individual mouse strain. The Sample GSM565172 table is the average log ratio for the 4 replicatesArrays were performed.
Project description:Paper abstract: The transcription factors Abrupt (Ab) and Knot (Kn) act as selectors of distinct dendritic arbor morphologies in two classes of Drosophila sensory neurons, termed class I and class IV, respectively. We performed binding-site mapping and transcriptional profiling of isolated these neurons. Their profiles were similarly enriched in cell-type-specific enhancers of genes implicated in neural development. We identified a total of 429 target genes, of which 56 were common to Ab and Kn; these targets included genes necessary to shape dendritic arbors in either or both of the two sensory subtypes. Furthermore, a common target gene, encoding the cell adhesion molecule Ten-m, was expressed more strongly in class I than IV, and this differential was critical to the class-selective directional control of dendritic branch sprouting or extension. Our analyses illustrate how differentiating neurons employ distinct and shared repertoires of gene expression to produce class-selective morphological traits. Each Dam-fusion-derived sample is compared to a control Dam-only sample. Four biological replicates were performed.
Project description:Dorsal root ganglia (DRG) play a crucial role in processing sensory information, making it essential to understand their development. Here, we construct a single-cell spatiotemporal transcriptomic atlas of human embryonic DRG. This atlas reveals the diversity of cell types and highlights the extrinsic signaling cascades and intrinsic regulatory hierarchies that guide cell fate decisions, including neuronal/glial lineage restriction, sensory neuron differentiation and specification, and the formation of neuron-satellite glial cell (SGC) unit. Additionally, we identify a human-enriched NTRK3+/DCC+ nociceptor subtype, which is involved in multimodal nociceptive processing. Mimicking the programmed activation of signaling pathways in vivo, we successfully establish functional human DRG organoids and underscore the critical roles of transcriptional regulators in the fate commitment of unspecialized sensory neurons (uSNs). Overall, our research elucidates the multilevel signaling pathways and transcription factor (TF) regulatory hierarchies that underpin the diversity of somatosensory neurons, emphasizing the phenotypic distinctions in human nociceptor subtypes.