Analyzing Synaptic Modulation of <em>Drosophila melanogaster</em> Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Tokyo Institute of Technology (Titech)

Rac GTPases regulate the actin cytoskeleton to control changes in cell shape. To date, the analysis of Rac function during development has relied heavily on the use of dominant mutant isoforms. Here, we use loss-of-function mutations to show that the three Drosophila Rac genes, Rac1, Rac2 and Mtl, have overlapping functions in the control of epithelial morphogenesis, myoblast fusion, and axon growth and guidance. They are not required for the establishment of planar cell polarity, as had been suggested on the basis of studies using dominant mutant isoforms. The guanine nucleotide exchange factor, Trio, is essential for Rac function in axon growth and guidance, but not for epithelial morphogenesis or myoblast fusion. Different Rac activators thus act in different developmental processes. The specific cellular response to Rac activation may be determined more by the upstream activator than the specific Rac protein involved.

Rac function and regulation during Drosophila development.

During Drosophila visual system development, photoreceptor (R) axons choose their correct paths and targets in a step-wise fashion. R axons with different identities make specific pathfinding decisions at different stages during development. We show here that the transmembrane protein Golden goal (Gogo), which is dynamically expressed in all R neurons and localizes predominantly to growth cones, is required in two distinct steps of R8 photoreceptor axon pathfinding: Gogo regulates axon-axon interactions and axon-target interactions in R8 photoreceptor axons. gogo loss-of-function and gain-of-function phenotypes suggest that Gogo mediates repulsive axon-axon interaction between R8 axons to maintain their proper spacing, and it promotes axon-target recognition at the temporary layer to enable R8 axons to enter their correct target columns in the medulla. From detailed structure-function experiments, we propose that Gogo functions as a receptor that binds an unidentified ligand through its conserved extracellular domain.

The transmembrane protein Golden goal regulates R8 photoreceptor axon-axon and axon-target interactions.

Neurons steer their axons towards their proper targets during development. Molecularly, a number of guidance receptors have been identified. The transmembrane protein Golden goal (Gogo) was reported previously to guide photoreceptor (R) axons in the Drosophila visual system. Here, we show that Hts, the Drosophila homologue of Adducin, physically interacts with Gogo's cytoplasmic domain via its head-neck domain. hts null mutants show similar defects in R axon guidance as do gogo mutants. Rescue experiments suggest that the C-terminal tail but not the MARCKS homology domain of Hts is required. Overexpression of either gogo or hts causes abnormally thick swellings of R8 axons in the medulla, but if both are co-overexpressed, R8 axons appear normal and the amount of excessive Hts is reduced. Our results fit with a model where Gogo both positively and negatively regulates Hts that affects the Actin-Spectrin cytoskeleton in growth cone filopodia, thereby guiding R axons.

Hts, the Drosophila homologue of Adducin, physically interacts with the transmembrane receptor Golden goal to guide photoreceptor axons.

Neuronal connections are often organized in layers that contain synapses between neurons that have similar functions. In Drosophila, R7 and R8 photoreceptors, which detect different wavelengths, form synapses in distinct medulla layers. The mechanisms underlying the specificity of synaptic-layer selection remain unclear. We found that Golden Goal (Gogo) and Flamingo (Fmi), two cell-surface proteins involved in photoreceptor targeting, functionally interact in R8 photoreceptor axons. Our results indicate that Gogo promotes R8 photoreceptor axon adhesion to the temporary layer M1, whereas Gogo and Fmi collaborate to mediate axon targeting to the final layer M3. Structure-function analysis suggested that Gogo and Fmi interact with intracellular components through the Gogo cytoplasmic domain. Moreover, Fmi was also required in target cells for R8 photoreceptor axon targeting. We propose that Gogo acts as a functional partner of Fmi for R8 photoreceptor axon targeting and that the dynamic regulation of their interaction specifies synaptic-layer selection of photoreceptors.

Golden Goal collaborates with Flamingo in conferring synaptic-layer specificity in the visual system.

Golden goal (Gogo) is a cell surface protein that is crucial for proper synaptic layer targeting of photoreceptors (R cells) in the Drosophila visual system. In collaboration with the seven-transmembrane cadherin Flamingo (Fmi), Gogo mediates both temporary and final layer targeting of R-cell axons through its cytoplasmic activity. However, it is not known how Gogo activity is regulated. Here, we show that a conserved Tyr-Tyr-Asp (YYD) tripeptide motif in the Gogo cytoplasmic domain is required for photoreceptor axon targeting. Deleting the YYD motif is sufficient to abolish Gogo function. We demonstrate that the YYD motif is a phosphorylation site and that mutations in the YYD tripeptide impair synaptic layer targeting. Gogo phosphorylation results in axon stopping at the temporary targeting layer, and dephosphorylation is crucial for final layer targeting in collaboration with Fmi. Therefore, both temporary and final layer targeting strongly depend on the Gogo phosphorylation status. Drosophila Insulin-like receptor (DInR) has been reported to regulate the wiring of photoreceptors. We show that insulin signaling is a positive regulator, directly or indirectly, of YYD motif phosphorylation. Our findings indicate a novel mechanism for the regulation of Gogo activity by insulin signaling-mediated phosphorylation. We propose the model that a constant phosphorylation signal is antagonized by a presumably temporal dephosphorylation signal, which creates a permissive signal that controls developmental timing in axon targeting.

A putative tyrosine phosphorylation site of the cell surface receptor Golden goal is involved in synaptic layer selection in the visual system.

A hallmark of the central nervous system is its spatial and functional organization in synaptic layers. During neuronal development, axons form transient contacts with potential post-synaptic elements and establish synapses with appropriate partners at specific layers. These processes are regulated by synaptic cell-adhesion molecules. In the Drosophila visual system, R7 and R8 photoreceptor subtypes target distinct layers and form en passant pre-synaptic terminals at stereotypic loci of the axonal shaft. A leucine-rich repeat transmembrane protein, Capricious (Caps), is known to be selectively expressed in R8 axons and their recipient layer, which led to the attractive hypothesis that Caps mediates R8 synaptic specificity by homophilic adhesion. Contradicting this assumption, our results indicate that Caps does not have a prominent role in synaptic-layer targeting and synapse formation in Drosophila photoreceptors, and that the specific recognition of the R8 target layer does not involve Caps homophilic axon-target interactions. We generated flies that express a tagged synaptic marker to evaluate the presence and localization of synapses in R7 and R8 photoreceptors. These genetic tools were used to assess how the synaptic profile is affected when axons are forced to target abnormal layers by expressing axon guidance molecules. When R7 axons were mistargeted to the R8-recipient layer, R7s either maintained an R7-like synaptic profile or acquired a similar profile to r8s depending on the overexpressed protein. When R7 axons were redirected to a more superficial medulla layer, the number of presynaptic terminals was reduced. These results indicate that cell-surface molecules are able to dictate synapse loci by changing the axon terminal identity in a partially cell-autonomous manner, but that presynapse formation at specific sites also requires complex interactions between pre- and post-synaptic elements.

Assessing the role of cell-surface molecules in central synaptogenesis in the Drosophila visual system.

To achieve the precise wiring of axons in the brain required to form a fine architecture, a molecular level interaction between axons and their targets is necessary. The Drosophila visual system has a layered and columnar structure which is often found in the brain of vertebrates. With powerful genetic tools for its analysis, the Drosophila visual system provides a useful framework to examine the molecular mechanisms of axon targeting specificity. The medulla is the second optic ganglion in the Drosophila optic lobe, and is subdivided into ten layers. Among the eight photoreceptor types, R7 and R8 pass through the first optic ganglion lamina and innervate the medulla. In the medulla, R7 and R8 axons grow in a distinct manner to reach their final target layers: M6 and M3, respectively. The axons from R7 and R8 take characteristic steps to extend toward their target layer. In this review, we discuss the formation of the Drosophila optic lobe and the molecular mechanisms of layer specific targeting of R8 axons in the medulla. Fundamental and comprehensive understanding of the crosstalk of growing axons and target regions in the Drosophila optic lobe will elucidate the general principles applicable to more complex nervous systems.

Cell surface control of the layer specific targeting in the Drosophila visual system.

Neural activity contributes to the regulation of the properties of synapses in sensory systems, allowing for adjustment to a changing environment. Little is known about how synaptic molecular components are regulated to achieve activity-dependent plasticity at central synapses. Here, we found that after prolonged exposure to natural ambient light the presynaptic active zone in Drosophila photoreceptors undergoes reversible remodeling, including loss of Bruchpilot, DLiprin-α, and DRBP, but not of DSyd-1 or Cacophony. The level of depolarization of the postsynaptic neurons is critical for the light-induced changes in active zone composition in the photoreceptors, indicating the existence of a feedback signal. In search of this signal, we have identified a crucial role of microtubule meshwork organization downstream of the divergent canonical Wnt pathway, potentially via Kinesin-3 Imac. These data reveal that active zone composition can be regulated in vivo and identify the underlying molecular machinery.

Satoko Hakeda-Suzuki

Graduate School of Life Science and Technology

Tokyo Institute of Technology (Titech)

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Satoko Hakeda-Suzuki

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Graduate School of Life Science and Technology

Tokyo Institute of Technology (Titech)

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Graduate School of Life Science and Technology