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:Class I and IV Drosophila dendritic arborization sensory neurons were isolated via magnetic bead sorting and total RNA isolated from the samples was used for gene expression profiling. Elucidating the molecular mechanisms controlling dendrite development is key to understanding the pivotal role these structures play in influencing synaptic integration and neural function. Despite significant advances in this field, genetic pleiotropy remains a significant impediment to investigating such complex developmental processes. To circumvent this problem, we have applied class specific neuron transcriptional expression profiling coupled to an in vivo RNAi functional validation screen in order to dissect the molecular bases of Drosophila class I and class IV dendritic arborization (da) neuron dendritogenesis.
Project description:Class I and IV Drosophila dendritic arborization sensory neurons were isolated via magnetic bead sorting and total RNA isolated from the samples was used for gene expression profiling. Elucidating the molecular mechanisms controlling dendrite development is key to understanding the pivotal role these structures play in influencing synaptic integration and neural function. Despite significant advances in this field, genetic pleiotropy remains a significant impediment to investigating such complex developmental processes. To circumvent this problem, we have applied class specific neuron transcriptional expression profiling coupled to an in vivo RNAi functional validation screen in order to dissect the molecular bases of Drosophila class I and class IV dendritic arborization (da) neuron dendritogenesis. Gene expression profiling of class I and IV da neurons, isolated via magnetic bead sorting (using protocols as described in Iyer et al., 2009), was performed at the third instar larval stage of development from independent cell isolations from 40-50 age-matched third instar larvae expressing mCD8::GFP under the control of either GAL4ppk.1.9 driver or Gal80ppk; GAL4221. For controls, age matched whole larval homogenate RNA was used.
Project description:Class III Drosophila dendritic arborization sensory neurons were isolated via magnetic bead sorting and total RNA isolated from the samples was used for gene expression profiling to explore molecular functions of this sensory neuron subtype.
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. da neurons were isolated by magnetic bead sorting from larvae of the following three genotypes and obtained genome-wide expression profiles: larvae expressing a cell-surface marker mCD8:GFP alone (Control), and larvae ectopically expressing Abrupt (Ab ME) or Knot (Kn ME) together with mCD8:GFP in all classes of da neurons. 3 biological replicates were collected.
Project description:Elucidating the molecular mechanisms controlling dendrite development is key to understanding the pivotal role these structures play in influencing synaptic integration and neural function. Despite significant advances in this field, genetic pleiotropy remains a significant impediment to investigating such complex developmental processes. To circumvent this problem, we have applied class specific neuron transcriptional expression profiling coupled to an in vivo RNAi functional validation screen in order to dissect the molecular bases of Drosophila class IV dendritic arborization (da) neuron dendritogenesis. Microarray analyses reveal transcriptional regulation as one highly enriched biological and functional category with 420 transcription factors significantly expressed in class IV neurons. Among these, we identify roles for 268 genes in mediating a broad spectrum of functions including dendritic field coverage, branching, routing, and tiling. Collectively, our analyses provide a more comprehensive framework of the role complex transcriptional networks play in directing distinct aspects of class specific dendrite morphogenesis. Gene expression profiling of class IV da neurons performed at the third instar larval stage of development from two independent cell isolations from 40-50 age-matched third instar larvae expressing mCD8::GFP under the control of the GAL4ppk.1.9 driver