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

A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the <i>Drosophila</i> Neuromuscular Junction

<div><p>At the <i>Drosophila</i> NMJ, BMP signaling is critical for synapse growth and homeostasis. Signaling by the BMP7 homolog, Gbb, in motor neurons triggers a canonical pathway—which modulates transcription of BMP target genes, and a noncanonical pathway—which connects local BMP/BMP receptor complexes with the cytoskeleton. Here we describe a novel noncanonical BMP pathway characterized by the accumulation of the pathway effector, the phosphorylated Smad (pMad), at synaptic sites. Using genetic epistasis, histology, super resolution microscopy, and electrophysiology approaches we demonstrate that this novel pathway is genetically distinguishable from all other known BMP signaling cascades. This novel pathway does not require Gbb, but depends on presynaptic BMP receptors and specific postsynaptic glutamate receptor subtypes, the type-A receptors. Synaptic pMad is coordinated to BMP’s role in the transcriptional control of target genes by shared pathway components, but it has no role in the regulation of NMJ growth. Instead, selective disruption of presynaptic pMad accumulation reduces the postsynaptic levels of type-A receptors, revealing a positive feedback loop which appears to function to stabilize active type-A receptors at synaptic sites. Thus, BMP pathway may monitor synapse activity then function to adjust synapse growth and maturation during development.</p></div

Complex genetic control for synaptic pMad accumulation.

<p>(A-C) Confocal images of NMJ4 boutons from larvae of indicated genotypes labeled for pMad (red), HRP (blue) and GluRIIA (green). The accumulation of pMad at <i>gbb</i> mutant NMJs is reduced by postsynaptic <i>GluRIIA</i> knockdown (A) or loss of Wit (B). Knockdown of Mav in the glia or Put in the striated muscle (C) diminished the synaptic pMad accumulation (quantified in D). Genotypes: control (<i>w</i>^<i>1118</i>), <i>gbb</i> (<i>gbb</i>^<i>1/2</i>), <i>gbb; IIA</i>^<i>RNAi</i> (<i>gbb</i>^<i>1/2</i>; <i>UAS-GluRIIA</i>^<i>RNAi</i>/<i>24B-Gal4</i>), <i>gbb; wit</i> (<i>gbb</i>^<i>1/2</i>; <i>wit</i>^{<i>A12/Df</i>}), <i>G>mav</i>^<i>RNAi</i> <i>(repo-Gal4/UAS-mav</i>^<i>RNAi</i><i>)</i>, <i>gbb; G>mav</i>^<i>RNAi</i> <i>(gbb</i>^<i>1/2</i><i>; repo-Gal4/UAS-mav</i>^<i>RNAi</i>, <i>M>put</i>^<i>RNAi</i> <i>(G14-Gal4/UAS-put</i>^<i>RNAi</i><i>)</i>. Error bars indicate SEM. ***; p<0.001, *; p<0.05. Scale bars: 5 μm.</p