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

Trio overexpression in class III da neurons.

<p>(<b>A–D</b>) Live confocal images of class III and IV dorsal cluster da neurons at the third larval instar labeled with the F-actin reporter <i>UAS-GMA</i> driven by the <i>ppk-GAL4</i> transgene. Class III ddaA and ddaF neuron cell bodies are indicated by arrows and for clarity the class III neuron cell bodies and dendrites have been highlighted by a magenta pseudo-color trace overlay. (<b>A</b>) Representative image of wild-type class III da neuron displaying regularly distributed, unbranched, actin-rich dendritic filopodia (dashed line circles and high magnification image (<b>A′</b>)) projecting from the primary dendritic branches. (<b>B</b>) Full length Trio overexpression dramatically altered dendritic filopodia producing a hyper-branched, splintered morphology (dashed line circles and high magnification image (<b>B′</b>)). In addition, the filopodia displayed a more clustered distribution as compared to control. (<b>C</b>) Overexpression of the Trio-GEF1 domain increased dendritic branching overall and produced a highly similar splintered morphology on dendritic filopodia. (<b>D</b>) Overexpression of the Trio-GEF2 domain produces the opposite effect by reducing dendritic branching overall and leading to a dramatic decrease in the number of dendritic filopodia. (<b>E</b>) Quantitative analyses of the number of dendritic terminals as a measure of dendritic branching reveals no significant change with full length Trio overexpression, whereas upregulation of Trio-GEF1 dramatically leads to a significant increase in terminals both proximal and distal to the cell body, while upregulation of Trio-GEF2 leads to a significant decrease in terminals both proximal and distal to the cell body. 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 graph as follows (n.s. = not significant; *** = <i>p</i><0.001). Genotypes: <b>WT</b>: <i>UAS-GMA/+;ppk-GAL4</i>/+;+. <b>TRIO</b>: <i>UAS-GMA/UAS-trio;ppk-GAL4/+</i>;+. <b>GEF1</b>: <i>UAS-GMA/+;ppk-GAL4</i>/<i>UAS-trio-GEF1-myc</i>;+. <b>GEF2</b>: <i>UAS-GMA/+;ppk-GAL4</i>/<i>+</i>;<i>UAS-trio-GEF2-myc/</i>+.</p

Trio promotes class I da neuron dendritic branching and field coverage.

<p>(<b>A–D</b>) Live confocal images of third instar larval dorsal (ddaD/E) and ventral (vpda) class I da neurons labeled with <i>GAL4²²¹,UAS-mCD8::GFP</i>. As compared to controls (<b>A,C</b>), <i>UAS-trio^RNAi</i> knockdown results in reduced dendritic branching in ddaD/E (<b>B)</b> and vpda (<b>D</b>) class I neurons. (<b>E–H</b>) Quantitative analyses of dendritic branching, extension, and area in <i>trio^RNAi</i> relative to controls. (<b>E</b>) <i>trio</i> knockdown leads to a significant reduction in the average number of dendritic terminals reflecting an overall reduction in dendritic branching in all class I neurons. (<b>F</b>) Disruption of <i>trio</i> results in an overall reduction in total dendritic length in all class I neurons. (<b>G</b>) The average length per dendritic branch is increased in all class I neurons following <i>trio</i> knockdown. (<b>H</b>) Relative to control, <i>trio^RNAi</i> knockdown reduces total dendritic area of vpda 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>GAL4²²¹,UAS-mCD8::GFP</i>/+. <b><i>trio^RNAi</i></b>: <i>GAL4²²¹,UAS-mCD8::GFP/UAS-trio^RNAi</i>.</p

Trio is required for dendritic branching and the formation of filopodia in class III da neurons.

<p>(<b>A,B</b>) Live confocal images of third instar larval dorsal class III and IV da neurons labeled by the F-actin reporter, <i>UAS-GMA</i>, and driven by the <i>ppk-GAL4</i> transgene. Class III da neurons are distinguished by the presence of short, actin-rich dendritic filopodia emanating from the primary branches. The class III ddaA and ddaF neuron cell bodies are indicated by the arrows and for clarity the class III neuron cell bodies and dendrites have been highlighted by a magenta pseudo-color trace overlay. As compared to wild-type controls (<b>A</b>), <i>UAS-trio^RNAi</i> knockdown results in a strong reduction in dendritic branching particularly with respect to the characteristic dendritic filopodia in class III neurons (<b>B</b>). (<b>C</b>) Quantitative analyses of the average number of dendritic terminals reveal significant reductions in branching proximal and distal to the cell body. For these analyses, 100×100 micron boxes were drawn in parallel areas proximal and distal to the cell body in both wild-type and <i>trio^RNAi</i> and the average number of dendritic terminals quantified. 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 graph as follows (** = <i>p</i><0.01; *** = <i>p</i><0.001). Genotypes: <b>WT</b>: <i>UAS-GMA/+;ppk-GAL4</i>/+;+. <b><i>trio^RNAi</i></b>: <i>UAS-GMA/+;ppk-GAL4/+;UAS-trio^RNAi/+</i>.</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

Trio regulates higher order dendritic branching and field coverage in class IV da neurons.

<p>(<b>A,B</b>) Confocal live images of the dorsal cluster class IV ddaC neuron labeled by the class IV specific reporter <i>GAL4⁴⁷⁷,UAS-GFP</i> at the the third instar larval stage of development. (<b>A</b>) Wild-type class IV ddaC neuron characterized by full coverage of dendritic field and highly complex dendrite branching pattern particularly at dendritic termini. (<b>B</b>) Class IV ddaC neuron expressing a <i>UAS-trio-RNAi</i> transgene. The loss-of-function <i>trio</i> phenotype is characterized by a dramatic reduction in dendrite branching complexity at both proximal primary branches and distal dendritic terminal branches. (<b>C–G</b>) Quantitative analyses of dendritic branching, length, and field coverage in <i>trio^RNAi</i> relative to controls. <i>trio</i> knockdown results in a significant reduction in the number of dendritic terminals reflecting a decrease in overall branching (<b>C</b>) and a reduction in total dendritic length (<b>D</b>). (<b>E</b>) Disruption of <i>trio</i> function also leads to a reduction in the average dendritic length per branch. (<b>F</b>) The percentage of dendritic field coverage is significantly reduced with <i>trio</i> knockdown (71%) as compared to controls (95%) reflecting defects in branching and growth. (<b>G</b>) Analyses of dendritic branch order reveal defects in the specification of higher order branching resulting in a proximal shift in branch order distribution in <i>trio</i> knockdown (<i>n</i> = 7) relative to controls (<i>n</i> = 6). 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 graph as follows (** = <i>p</i><0.01; *** = <i>p</i><0.001). Genotypes: <b>WT</b>: <i>GAL4⁴⁷⁷,UAS-mCD8::GFP/+</i>. <b><i>trio^RNAi</i></b>: <i>GAL4⁴⁷⁷,UAS-mCD8::GFP/+;UAS-trio^RNAi/+</i>.</p

Trio overexpression in class I da neurons.

<p>(<b>A–H</b>) Live confocal images of third instar larval dorsal (ddaD/E) and ventral (vpda) class I da neurons labeled with <i>GAL4²²¹,UAS-mCD8::GFP</i>. As compared to controls (<b>A,B</b>), Trio overexpression results in increased dendritic branching in ddaD/E (<b>C</b>) and vpda (<b>D</b>) class I neurons. Trio-GEF1 overexpression likewise leads to a dramatic increase in dendritic branching in ddaD/E (<b>E</b>) and vpda (<b>F</b>) neurons. In contrast, Trio-GEF2 overexpression results in a mild reduction in dendritic branching in ddaD/E (<b>G</b>) and (<b>H</b>) vpda neurons. (<b>I–M</b>) Quantitative analyses of dendritic branching, length, and field coverage in Trio, Trio-GEF1, and Trio-GEF overexpression relative to controls. (<b>I</b>) Analyses of the number of dendritic terminals reveals an increase in dendritic branching in all class I neurons following Trio and Trio-GEF1 overexpression, whereas Trio-GEF2 overexpression leads to a reduction in branching in ddaE and vpda neurons. (<b>J</b>) Quantitation of total dendritic length reveals no significant change following Trio overexpression, however with Trio-GEF1 overexpression there is an increase in length as a function of higher levels of dendritic branching, whereas there is a decrease in length with Trio-GEF2 overexpression. (<b>K</b>) The average length per dendritic branch is significantly reduced in both Trio and Trio-GEF1 overexpression, whereas Trio-GEF2 overexpression leads to an increase in ddaE neurons. (<b>L</b>) Overexpression of Trio, Trio-GEF1, and Trio-GEF reduces total dendritic area of vpda neurons as compared to control. (<b>M</b>) Relative to control (<i>n</i> = 12), dendritic branch order analyses in vpda neurons reveals a distal shift in the percentage of higher order branching with Trio (<i>n</i> = 16) and Trio-GEF1 (<i>n = </i>16) overexpression, whereas a slight proximal shift towards lower order branching, with a steep decline in higher order branching, is observed with Trio-GEF2 overexpression (<i>n</i> = 12). 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; n.s. = not significant). Genotypes: <b>WT</b>: <i>GAL4²²¹,UAS-mCD8::GFP</i>/+. <b>TRIO</b>: <i>UAS-trio/+</i>;+; <i>GAL4²²¹,UAS-mCD8::GFP/+</i>. <b>GEF1</b>: <i>UAS-trio-GEF1-myc/+</i>;<i>GAL4²²¹,UAS-mCD8::GFP/</i>+. <b>GEF2</b>: <i>UAS-trio-GEF2-myc/GAL4²²¹,UAS-mCD8::GFP</i>.</p

Cut-induced <i>de novo</i> dendritic branching and filopodia formation requires Trio activity.

<p>(<b>A–D</b>) Live confocal images of class I da neurons (ddaD, ddaE, vpda) at the third larval instar. (<b>G,H</b>) Live confocal images of class IV ddaC neurons. Images were collected at 20× magnification and size bars represents 50 microns. (<b>A,B</b>) Representative images of dorsal ddaD/E neurons (<b>A</b>) and ventral vpda neuron (<b>B</b>) ectopically overexpressing Cut. Cut ectopic overexpression results in class I neurons displaying increased dendritic branching complexity characterized by a high incidence of dendritic filopodia emanating from the primary branches. (<b>C,D</b>) Representative images of dorsal ddaD/E neurons (<b>C</b>) and ventral vpda neuron (<b>D</b>) in which Cut ectopic overexpression is combined with knockdown of <i>trio</i> via <i>UAS-trio^RNAi</i>. As compared to Cut overexpression, <i>trio</i> knockdown results in strong suppression of the Cut phenotype, particularly with respect to dendritic filopodia. (<b>E</b>) Quantitative analyses reveal a significant reduction in the total number of dendritic terminals in neurons expressing <i>trio^RNAi</i> relative to Cut ectopic overexpression alone. (<b>F</b>) Quantitative analyses reveal no statistically significant difference in total dendritic length. (<b>G</b>) Representative image of class IV ddaC neuron expressing <i>UAS-cut^RNAi</i>. (<b>H</b>) Representative image of ddaC neuron simultaneously expressing <i>UAS-cut^RNAi</i> and <i>UAS-trio</i> transgenes reveals partial rescue of <i>cut</i> mutant defects in dendritic branching. (<b>I</b>) Quantitative analyses reveal knockdown of <i>cut</i> via RNAi significantly reduces the total number of dendritic terminals relative to wild-type (WT) controls, whereas Trio overexpression in a <i>cut^RNAi</i> background partially rescues the <i>cut</i> mutant phenotype. 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 graph as follows (* = <i>p</i><0.05; ** = <i>p</i><0.01; *** = <i>p</i><0.001). Genotypes: (<b>A,B</b>) <i>UAS-cut/+; GAL4²²¹,UASmCD8::GFP/+</i>; (<b>C,D</b>) <i>UAS-cut/+; GAL4²²¹,UASmCD8::GFP/UAS-trio^RNAi</i>; (<b>G</b>) <i>GAL4⁴⁷⁷,UASmCD8::GFP/+;UAS-cut^RNAi/+</i>; (<b>H</b>) <i>UAS-trio/+; GAL4⁴⁷⁷,UASmCD8::GFP/+; UAS-cut^RNAi/+</i>.</p