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

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

Neurometric quantitative analyses of C-IV TF phenotypes.

<p>(<b>A-F</b>) TFs that resulted in dendritic changes that were significantly different from controls in at least two independent RNAi lines have been shown as bar graphs where the average N = 7 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 length is shown for ddaC (<b>A</b>), v’ada (<b>B</b>) and vdaB (<b>C</b>). (<b>D-F</b>) Quantitative analyses of total dendritic branches is shown for ddaC (<b>D</b>), v’ada (<b>E</b>) and vdaB (<b>F</b>). (<b>G</b>) Venn diagram distribution of TF-induced phenotypes among the three C-IV subtypes.</p

Microarray analysis of two morphologically distinct classes of <i>Drosophila</i> sensory neurons.

<p>To access differences in gene expression, RNA from enriched C-I and C-IV neuronal populations were compared with that of whole larvae in microarray experiments. Live confocal images of neuronal populations labeled by (<b>A</b>) <i>GAL4²²¹</i> (C-I) and (<b>B</b>) <i>GAL4^ppk1.9</i> (C- IV) visualized by the trans-membrane fusion construct mCD8::GFP. Neurons have been pseudo-colored to distinguish individual subtypes and dendritic territories. (<b>C</b>) <i>GAL4²²¹</i> strongly labels C-I da neurons along with weakly labelling of C-IV neurons in the background (dotted red trace). (<b>D</b>) <i>GAL80</i> driven by a <i>ppk</i> promoter was combined in the background of <i>GAL4²²¹(ppk-GAL80; GAL4²²¹</i>) that results in highly class I specific <i>GAL4</i> expression. (<b>E</b>) A representative whole larval image of <i>GAL4^ppk1.9</i> driving the expression of <i>UAS-mCD8::GFP</i> (gut is auto-fluorescent). (<b>F-J</b>) Strategy of class-specific neuronal isolation. Larvae expressing mCD8::GFP under the control of either <i>ppk-GAL80; GAL4²²¹</i> or <i>GAL4^ppk1.9</i> (<b>F</b>) were dissociated (<b>G</b>) filtered and incubated with superparamagnetic beads coated with anti-mCD8 antibody (<b>H</b>). The C-I/C-IV neurons bound to the magnetic beads were purified using a strong magnet (<b>I</b>), washed several time and used to perform microarray gene expression profiling (<b>J</b>). An identical region from the C-I and C-IV microarray are represented to show their dramatic qualitative differences (<b>J</b>). The microarray replicates were highly correlated, as represented in the correlation map (<b>K</b>). Principle component analysis revealed the three microarray samples from C-I, C-IV and whole larval lysate cluster into three distinct and well-defined clusters (<b>L</b>).</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

Trio overexpression in class IV da neurons.

<p>(<b>A–F</b>) Live confocal images of third instar larval dorsal ddaC class IV da neurons labeled with <i>GAL4⁴⁷⁷,UAS-mCD8::GFP</i>. As compared to control (<b>A</b>), Trio overexpression results in decreased dendritic branching (<b>B</b>). (<b>C</b>) In contrast, Trio-GEF1 overexpression leads to an increase in dendritic branching, whereas Trio-GEF2 overexpression results in a reduction in dendritic branching (<b>D</b>). (<b>E</b>) RNAi knockdown of Rac1 in a Trio-GEF1 overexpression background suppresses defects in dendritic development as compared to Trio-GEF1 overexpression alone. (<b>F</b>) RNAi knockdown of Rho1 in a Trio-GEF2 overexpression background suppresses defects in dendrite morphogenesis as compared to Trio-GEF2 overexpression alone. (<b>G</b>) Analyses of the number of dendritic terminals reveals a decrease in dendritic branching with Trio and Trio-GEF2 overexpression whereas Trio-GEF1 overexpression leads to an increase in dendritic branching relative to wild-type controls. Knockdown of Rac1 via RNAi or by co-expression of the dominant negative Rac1.N17 suppresses defects in dendritic branching relative to Trio-GEF1 overexpression alone, whereas knockdown of Rho1 via RNAi suppresses defects in branching as compared to Trio-GEF2 overexpression alone. (<b>H</b>) Quantitation of total dendritic length reveals a mild to moderate reduction with Trio, Trio-GEF1 and Trio-GEF2 overexpression as compared to wild-type controls. Consistent with dendritic branching, disrupting Rac1 or Rho1 function suppresses defects in dendritic length as compared to Trio-GEF1 or Trio-GEF2 overexpression, respectively. (<b>I</b>) Relative to control (<i>n</i> = 6), dendritic branch order analyses reveal a proximal shift in the percentage of lower order branching with Trio (<i>n</i> = 8) and Trio-GEF2 (<i>n</i> = 8) overexpression, whereas Trio-GEF1 (<i>n</i> = 8) overexpression results in a distal shift towards higher order branching in class IV ddaC neurons. Images were taken at 20× magnification and size bar represents 50 microns. The total <i>n</i> value for each neuron and genotype quantified is reported on the bar graph. Statistically significant <i>p</i> values are reported on the graphs as follows (* = <i>p</i><0.05; ** = <i>p</i><0.01; *** = <i>p</i><0.001). Genotypes: <b>WT</b>: <i>GAL4⁴⁷⁷,UAS-mCD8::GFP</i>/+. <b>TRIO</b>: <i>UAS-trio/+</i>;<i>GAL4⁴⁷⁷,UAS-mCD8::GFP/+</i>. <b>GEF1</b>: <i>UAS-trio-GEF1-myc/GAL4⁴⁷⁷,UAS-mCD8::GFP. </i><b>GEF2</b>: <i>GAL4⁴⁷⁷,UAS-mCD8::GFP/+;UAS-trio-GEF2-myc/+</i>. <b>GEF1+Rac1-RNAi</b>: <i>UAS-trio-GEF1-myc/GAL4⁴⁷⁷,UAS-mCD8::GFP;UAS-Rac1^JF02813</i>/+. <b>GEF1+Rac1.N17</b>: <i>UAS-trio-GEF1-myc/GAL4⁴⁷⁷,UAS-mCD8::GFP;UAS-Rac1.N17</i>/+ <b>GEF2+Rho1-RNAi</b>: <i>GAL4⁴⁷⁷,UAS-mCD8::GFP/+;UAS-trio-GEF2-myc/UAS-Rho1-dsRNA</i>.</p