Cell-Type Specific Transcriptomic Profiling to Dissect Mechanisms of Differential Dendritogenesis

The establishment, maintenance and modulation of cell-type specific neural architectures are critically important to the formation of functional neural networks. At the neuroanatomical level, differential patterns of dendritic arborization directly impact neural function and connectivity, however the molecular mechanisms underlying the specification of distinct dendrite morphologies remain incompletely understood. To address this question, we analyzed global gene expression from purified populations of wild-type class I and class IV Drosophila melanogaster dendritic arborization (da) sensory neurons compared to wild-type whole larval RNA using oligo DNA microarray expression profiling. Herein we present detailed experimental methods and bioinformatic anal- yses to correspond with our data reported in the Gene Expression Omnibus under accession number GSE46154. We further provide R code to facilitate data accession, perform quality controls, and conduct bioinformatic analyses relevant to this dataset. Our cell-type specific gene expression datasets provide a valuable resource for guiding further investigations designed to explore the molecular mechanisms underlying differential patterns of neuronal patterning

The RhoGEF Trio Functions in Sculpting Class Specific Dendrite Morphogenesis in Drosophila Sensory Neurons

As the primary sites of synaptic or sensory input in the nervous system, dendrites play an essential role in processing neuronal and sensory information. Moreover, the specification of class specific dendrite arborization is critically important in establishing neural connectivity and the formation of functional networks. Cytoskeletal modulation provides a key mechanism for establishing, as well as reorganizing, dendritic morphology among distinct neuronal subtypes. While previous studies have established differential roles for the small GTPases Rac and Rho in mediating dendrite morphogenesis, little is known regarding the direct regulators of these genes in mediating distinct dendritic architectures.Here we demonstrate that the RhoGEF Trio is required for the specification of class specific dendritic morphology in dendritic arborization (da) sensory neurons of the Drosophila peripheral nervous system (PNS). Trio is expressed in all da neuron subclasses and loss-of-function analyses indicate that Trio functions cell-autonomously in promoting dendritic branching, field coverage, and refining dendritic outgrowth in various da neuron subtypes. Moreover, overexpression studies demonstrate that Trio acts to promote higher order dendritic branching, including the formation of dendritic filopodia, through Trio GEF1-dependent interactions with Rac1, whereas Trio GEF-2-dependent interactions with Rho1 serve to restrict dendritic extension and higher order branching in da neurons. Finally, we show that de novo dendritic branching, induced by the homeodomain transcription factor Cut, requires Trio activity suggesting these molecules may act in a pathway to mediate dendrite morphogenesis.Collectively, our analyses implicate Trio as an important regulator of class specific da neuron dendrite morphogenesis via interactions with Rac1 and Rho1 and indicate that Trio is required as downstream effector in Cut-mediated regulation of dendrite branching and filopodia formation

Turtle functions downstream of Cut in differentially regulating class specific dendrite morphogenesis in Drosophila.

BACKGROUND:Dendritic morphology largely determines patterns of synaptic connectivity and electrochemical properties of a neuron. Neurons display a myriad diversity of dendritic geometries which serve as a basis for functional classification. Several types of molecules have recently been identified which regulate dendrite morphology by acting at the levels of transcriptional regulation, direct interactions with the cytoskeleton and organelles, and cell surface interactions. Although there has been substantial progress in understanding the molecular mechanisms of dendrite morphogenesis, the specification of class-specific dendritic arbors remains largely unexplained. Furthermore, the presence of numerous regulators suggests that they must work in concert. However, presently, few genetic pathways regulating dendrite development have been defined. METHODOLOGY/PRINCIPAL FINDINGS:The Drosophila gene turtle belongs to an evolutionarily conserved class of immunoglobulin superfamily members found in the nervous systems of diverse organisms. We demonstrate that Turtle is differentially expressed in Drosophila da neurons. Moreover, MARCM analyses reveal Turtle acts cell autonomously to exert class specific effects on dendritic growth and/or branching in da neuron subclasses. Using transgenic overexpression of different Turtle isoforms, we find context-dependent, isoform-specific effects on mediating dendritic branching in class II, III and IV da neurons. Finally, we demonstrate via chromatin immunoprecipitation, qPCR, and immunohistochemistry analyses that Turtle expression is positively regulated by the Cut homeodomain transcription factor and via genetic interaction studies that Turtle is downstream effector of Cut-mediated regulation of da neuron dendrite morphology. CONCLUSIONS/SIGNIFICANCE:Our findings reveal that Turtle proteins differentially regulate the acquisition of class-specific dendrite morphologies. In addition, we have established a transcriptional regulatory interaction between Cut and Turtle, representing a novel pathway for mediating class specific dendrite development

The Zinc-BED Transcription Factor Bedwarfed Promotes Proportional Dendritic Growth and Branching through Transcriptional and Translational Regulation in <i>Drosophila</i>

Dendrites are the primary points of sensory or synaptic input to a neuron and play an essential role in synaptic integration and neural function. Despite the functional importance of dendrites, relatively less is known about the underlying mechanisms regulating cell type-specific dendritic patterning. Herein, we have dissected the functional roles of a previously uncharacterized gene, CG3995, in cell type-specific dendritic development in Drosophila melanogaster. CG3995, which we have named bedwarfed (bdwf), encodes a zinc-finger BED-type protein that is required for proportional growth and branching of dendritic arbors. It also exhibits nucleocytoplasmic expression and functions in both transcriptional and translational cellular pathways. At the transcriptional level, we demonstrate a reciprocal regulatory relationship between Bdwf and the homeodomain transcription factor (TF) Cut. We show that Cut positively regulates Bdwf expression and that Bdwf acts as a downstream effector of Cut-mediated dendritic development, whereas overexpression of Bdwf negatively regulates Cut expression in multidendritic sensory neurons. Proteomic analyses revealed that Bdwf interacts with ribosomal proteins and disruption of these proteins resulted in phenotypically similar dendritic hypotrophy defects as observed in bdwf mutant neurons. We further demonstrate that Bdwf and its ribosomal protein interactors are required for normal microtubule and F-actin cytoskeletal architecture. Finally, our findings reveal that Bdwf is required to promote protein translation and ribosome trafficking along the dendritic arbor. These findings shed light on the complex, combinatorial, and multi-functional roles of transcription factors (TFs) in directing the diversification of cell type-specific dendritic development

Functional Genomic Analyses of Two Morphologically Distinct Classes of <i>Drosophila</i> Sensory Neurons: Post-Mitotic Roles of Transcription Factors in Dendritic Patterning

<div><p>Background</p><p>Neurons are one of the most structurally and functionally diverse cell types found in nature, owing in large part to their unique class specific dendritic architectures. Dendrites, being highly specialized in receiving and processing neuronal signals, play a key role in the formation of functional neural circuits. Hence, in order to understand the emergence and assembly of a complex nervous system, it is critical to understand the molecular mechanisms that direct class specific dendritogenesis.</p><p>Methodology/Principal Findings</p><p>We have used the <i>Drosophila</i> dendritic arborization (da) neurons to gain systems-level insight into dendritogenesis by a comparative study of the morphologically distinct Class-I (C-I) and Class-IV (C-IV) da neurons. We have used a combination of cell-type specific transcriptional expression profiling coupled to a targeted and systematic <i>in vivo</i> RNAi functional validation screen. Our comparative transcriptomic analyses have revealed a large number of differentially enriched/depleted gene-sets between C-I and C-IV neurons, including a broad range of molecular factors and biological processes such as proteolytic and metabolic pathways. Further, using this data, we have identified and validated the role of 37 transcription factors in regulating class specific dendrite development using <i>in vivo</i> class-specific RNAi knockdowns followed by rigorous and quantitative neurometric analysis.</p><p>Conclusions/Significance</p><p>This study reports the first global gene-expression profiles from purified <i>Drosophila</i> C-I and C-IV da neurons. We also report the first large-scale semi-automated reconstruction of over 4,900 da neurons, which were used to quantitatively validate the RNAi screen phenotypes. Overall, these analyses shed global and unbiased novel insights into the molecular differences that underlie the morphological diversity of distinct neuronal cell-types. Furthermore, our class-specific gene expression datasets should prove a valuable community resource in guiding further investigations designed to explore the molecular mechanisms underlying class specific neuronal patterning.</p></div

Hierarchical clustering representation of RNAi phenotypic screen data.

<p>Hierarchically clustered heat-maps of the quantified total dendritic length (TDL) and total dendritic branches (B) are represented by a color-code (red and blue), where red represent a significant increase in the phenotypic value and blue represents a significant decrease in phenotypic value according to the designated scale. All non-significant values were thresholded to zero (2 tailed Students t-test, p≤0.05). Here the quantified neurometric parameters from each experiment have been converted to a percentage change from control for normalization and to reflect both positive and negative changes from the controls when compared to the experimentals.</p

Identification of differentially enriched transcription factors in C-I and IV neurons.

<p>(<b>A</b>) A set of 40 TFs were found to be specifically enriched within C-I and C-IV da neurons in comparison to whole-larvae controls (p≤0.01, ANOVA and Tukeys HSD test), of which 9 were enriched uniquely within C-I neurons, 17 in C-IV neurons and 14 in both C-I and C-IV’s as represented in the Venn diagram (<b>B</b>). (<b>C</b>) The relative microarray fold-change expression values for the differentially expressed TFs in C-I and C-IV neurons, in comparison to whole larval control samples, are represented as hierarchically clustered heat map and the relative values are represented as a rainbow color scheme according to the designated scale (p≤0.01, ANOVA and Tukey’s HSD test).</p

C-I TF screen quantitative phenotypic analyses.

<p>All TFs that resulted in C-I dendritic changes that were significantly different from controls in at least two independent RNAi lines have been shown as bar graphs where the N = 9 for each RNAi line tested per gene (p≤0.05, Student’s t-test). The bars have been color-coded to represent increase (red) or decrease (green) in values relative to controls (black). (<b>A-C)</b> Quantitative analyses of total dendritic branches is shown for ddaD (<b>A</b>), ddaE (<b>B</b>) and vpda (<b>C</b>). (<b>D-F</b>) Quantitative analyses of total dendritic length is shown for ddaD (<b>D</b>), ddaE (<b>E</b>) and vpda (<b>F</b>). (<b>G</b>) Venn diagram distribution of TF-induced phenotypes among the three C-I subtypes. (<b>H-S</b>) Representative images of selected RNAi-induced phenotypes observed in C-I da neurons. Live confocal images of wild-type (wt) and RNAi (<i>UAS-IR</i>) phenotypes in the three C-I subtypes labelled using <i>UAS-mCD8::GFP</i> driven by C-I <i>GAL4</i>. Compared to wild-type C-I neurons (<b>H, N, Q</b>), phenotypes of <i>UAS-lola-IR</i> (<b>I, P</b>), <i>UAS-cwo-IR</i> (<b>J</b>), <i>UAS-Gnf1-IR</i> (<b>K</b>), <i>UAS-dom-IR</i> (<b>L</b>), <i>UAS-kay-IR</i> (<b>O</b>) and <i>UAS-cnc-IR</i> (<b>R</b>) are shown. Panels (<b>M</b>) and (<b>S</b>) represent phenotypes of <i>UAS-gcm2-IR</i> which was used as a positive control. Phenotypic information for each image is represented by color-coded arrows, where red arrows represent dendritic branching and blue arrows represent total dendritic length. The direction of arrow represents increase (up), decrease (down) or no change (hyphen). Size bar corresponds to 50 microns.</p

Functional characterization of differentially expressed gene-sets in C-I and C-IV neurons.

<p>(<b>A</b>) Venn diagram representing the number of genes differentially upregulated/downregulated either uniquely or commonly in C-I and C-IV da neurons with respect to whole larvae controls. (<b>B-D</b>) Analysis of gene ontology (GO) categories for genes that are enriched/depleted in C-I and C-IV neurons. The graph represents GO categories that are significantly over-represented (P≤0.01) in the population of differentially expressed genes that are uniquely regulated in C-I neurons (<b>B</b>), uniquely regulated in C-IV neurons (<b>C</b>) or commonly regulated in both C-I and C-IV neurons (<b>D</b>) when compared to whole larval controls. Bars indicate the fold enrichment (top X axis) of the genes belonging to a given GO term in the population of regulated genes in comparison to the total population of genes in the Agilent 4×44k array. Diamonds indicate the Modified Fisher’s Exact p-value (EASE score, bottom X axis) for each category.</p