Photoreceptor Axon Guidance Research Articles

Glial insulin regulates cooperative or antagonistic Golden goal/Flamingo interactions during photoreceptor axon guidance.

Transmembrane protein Golden goal (Gogo) interacts with atypical cadherin Flamingo (Fmi) to direct R8 photoreceptor axons in the Drosophila visual system. However, the precise mechanisms underlying Gogo regulation during columnar- and layer-specific R8 axon targeting are unknown. Our studies demonstrated that the insulin secreted from surface and cortex glia switches the phosphorylation status of Gogo, thereby regulating its two distinct functions. Non-phosphorylated Gogo mediates the initial recognition of the glial protrusion in the center of the medulla column, whereas phosphorylated Gogo suppresses radial filopodia extension by counteracting Flamingo to maintain a one axon-to-one column ratio. Later, Gogo expression ceases during the midpupal stage, thus allowing R8 filopodia to extend vertically into the M3 layer. These results demonstrate that the long- and short-range signaling between the glia and R8 axon growth cones regulates growth cone dynamics in a stepwise manner, and thus shapes the entire organization of the visual system.

Drosophila ClC-a is required in glia of the stem cell niche for proper neurogenesis and wiring of neural circuits.

Glial cells form part of the neural stem cell niche and express a wide variety of ion channels; however, the contribution of these channels to nervous system development is poorly understood. We explored the function of the Drosophila ClC‐a chloride channel, since its mammalian ortholog CLCN2 is expressed in glial cells, and defective channel function results in leukodystrophies, which in humans are accompanied by cognitive impairment. We found that ClC‐a was expressed in the niche in cortex glia, which are closely associated with neurogenic tissues. Characterization of loss‐of‐function ClC‐a mutants revealed that these animals had smaller brains and widespread wiring defects. We showed that ClC‐a is required in cortex glia for neurogenesis in neuroepithelia and neuroblasts, and identified defects in a neuroblast lineage that generates guidepost glial cells essential for photoreceptor axon guidance. We propose that glia‐mediated ionic homeostasis could nonautonomously affect neurogenesis, and consequently, the correct assembly of neural circuits.

Retinal Axon Guidance Requires Integration of Eya and the Jak/Stat Pathway into Phosphotyrosine-Based Signaling Circuitries in Drosophila.

The transcriptional coactivator and phosphatase eyes absent (Eya) is dynamically compartmentalized between the nucleus and cytoplasm. Although the nuclear transcriptional circuits within which Eya operates have been extensively characterized, understanding of its cytoplasmic functions and interactions remains limited. Our previous work showed that phosphorylation of Drosophila Eya by the Abelson tyrosine kinase can recruit Eya to the cytoplasm and that eya-abelson interactions are required for photoreceptor axons to project to correct layers in the brain. Based on these observations, we postulated that photoreceptor axon targeting might provide a suitable context for identifying the cytoplasmic signaling cascades with which Eya interacts. Using a dose-sensitive eya misexpression background, we performed an RNA interference-based genetic screen to identify suppressors. Included among the top 10 hits were nonreceptor tyrosine kinases and multiple members of the Jak/Stat signaling network (hop, Stat92E, Socs36E, and Socs44A), a pathway not previously implicated in axon targeting. Individual loss-of-function phenotypes combined with analysis of axonal projections in Stat92E null clones confirmed the importance of photoreceptor autonomous Jak/Stat signaling. Experiments in cultured cells detected cytoplasmic complexes between Eya and Hop, Socs36E and Socs44A; the latter interaction required both the Src homology 2 motif in Socs44A and tyrosine phosphorylated Eya, suggesting direct binding and validating the premise of the screen. Taken together, our data provide new insight into the cytoplasmic phosphotyrosine signaling networks that operate during photoreceptor axon guidance and suggest specific points of interaction with Eya.

Independent signaling by Drosophila insulin receptor for axon guidance and growth

The Drosophila insulin receptor (DInR) regulates a diverse array of biological processes including growth, axon guidance, and sugar homeostasis. Growth regulation by DInR is mediated by Chico, the Drosophila homolog of vertebrate insulin receptor substrate proteins IRS1–4. In contrast, DInR regulation of photoreceptor axon guidance in the developing visual system is mediated by the SH2-SH3 domain adaptor protein Dreadlocks (Dock). In vitro studies by others identified five NPXY motifs, one in the juxtamembrane region and four in the signaling C-terminal tail (C-tail), important for interaction with Chico. Here we used yeast two-hybrid assays to identify regions in the DInR C-tail that interact with Dock. These Dock binding sites were in separate portions of the C-tail from the previously identified Chico binding sites. To test whether these sites are required for growth or axon guidance in whole animals, a panel of DInR proteins, in which the putative Chico and Dock interaction sites had been mutated individually or in combination, were tested for their ability to rescue viability, growth and axon guidance defects of dinr mutant flies. Sites required for viability were identified. Unexpectedly, mutation of both putative Dock binding sites, either individually or in combination, did not lead to defects in photoreceptor axon guidance. Thus, either sites also required for viability are necessary for DInR function in axon guidance and/or there is redundancy built into the DInR/Dock interaction such that Dock is able to interact with multiple regions of DInR. We also found that simultaneous mutation of all five NPXY motifs implicated in Chico interaction drastically decreased growth in both male and female adult flies. These animals resembled chico mutants, supporting the notion that DInR interacts directly with Chico in vivo to control body size. Mutation of these five NPXY motifs did not affect photoreceptor axon guidance, segregating the roles of DInR in the processes of growth and axon guidance.

Localized Netrins Act as Positional Cues to Control Layer-Specific Targeting of Photoreceptor Axons in Drosophila

SummaryA shared feature of many neural circuits is their organization into synaptic layers. However, the mechanisms that direct neurites to distinct layers remain poorly understood. We identified a central role for Netrins and their receptor Frazzled in mediating layer-specific axon targeting in the Drosophila visual system. Frazzled is expressed and cell autonomously required in R8 photoreceptors for directing their axons to the medulla-neuropil layer M3. Netrin-B is specifically localized in this layer owing to axonal release by lamina neurons L3 and capture by target neuron-associated Frazzled. Ligand expression in L3 is sufficient to rescue R8 axon-targeting defects of Netrin mutants. R8 axons target normally despite replacement of diffusible Netrin-B by membrane-tethered ligands. Finally, Netrin localization is instructive because expression in ectopic layers can retarget R8 axons. We propose that provision of localized chemoattractants by intermediate target neurons represents a highly precise strategy to direct axons to a positionally defined layer.

Patterning Axon Targeting of Olfactory Receptor Neurons by Coupled Hedgehog Signaling at Two Distinct Steps

We present evidence for a coupled two-step action of Hedgehog signaling in patterning axon targeting of Drosophila olfactory receptor neurons (ORNs). In the first step, differential Hedgehog pathway activity in peripheral sensory organ precursors creates ORN populations with different levels of the Patched receptor. Different Patched levels in ORNs then determine axonal responsiveness to target-derived Hedgehog in the brain: only ORN axons that do not express high levels of Patched are responsive to and require a second step of Hedgehog signaling for target selection. Hedgehog signaling in the imaginal sensory organ precursors thus confers differential ORN responsiveness to Hedgehog-mediated axon targeting in the brain. This mechanism contributes to the spatial coordination of ORN cell bodies in the periphery and their glomerular targets in the brain. Such coupled two-step signaling may be more generally used to coordinate other spatially and temporally segregated developmental events.

Tiling of R7 Axons in the Drosophila Visual System Is Mediated Both by Transduction of an Activin Signal to the Nucleus and by Mutual Repulsion

The organization of neuronal wiring into layers and columns is a common feature of both vertebrate and invertebrate brains. In the Drosophila visual system, each R7 photoreceptor axon projects within a single column to a specific layer of the optic lobe. We refer to the restriction of terminals to single columns as tiling. In a genetic screen based on an R7-dependent behavior, we identified the Activin receptor Baboon and the nuclear import adaptor Importin-alpha3 as being required to prevent R7 axon terminals from overlapping with the terminals of R7s in neighboring columns. This tiling function requires the Baboon ligand, dActivin, the transcription factor, dSmad2, and retrograde transport from the growth cone to the R7 nucleus. We propose that dActivin is an autocrine signal that restricts R7 growth cone motility, and we demonstrate that it acts in parallel with a paracrine signal that mediates repulsion between R7 terminals.

Mechanisms of TSC-mediated Control of Synapse Assembly and Axon Guidance

Tuberous sclerosis complex is a dominant genetic disorder produced by mutations in either of two tumor suppressor genes, TSC1 and TSC2; it is characterized by hamartomatous tumors, and is associated with severe neurological and behavioral disturbances. Mutations in TSC1 or TSC2 deregulate a conserved growth control pathway that includes Ras homolog enriched in brain (Rheb) and Target of Rapamycin (TOR). To understand the function of this pathway in neural development, we have examined the contributions of multiple components of this pathway in both neuromuscular junction assembly and photoreceptor axon guidance in Drosophila. Expression of Rheb in the motoneuron, but not the muscle of the larval neuromuscular junction produced synaptic overgrowth and enhanced synaptic function, while reductions in Rheb function compromised synapse development. Synapse growth produced by Rheb is insensitive to rapamycin, an inhibitor of Tor complex 1, and requires wishful thinking, a bone morphogenetic protein receptor critical for functional synapse expansion. In the visual system, loss of Tsc1 in the developing retina disrupted axon guidance independently of cellular growth. Inhibiting Tor complex 1 with rapamycin or eliminating the Tor complex 1 effector, S6 kinase (S6k), did not rescue axon guidance abnormalities of Tsc1 mosaics, while reductions in Tor function suppressed those phenotypes. These findings show that Tsc-mediated control of axon guidance and synapse assembly occurs via growth-independent signaling mechanisms, and suggest that Tor complex 2, a regulator of actin organization, is critical in these aspects of neuronal development.

Activity-Independent Prespecification of Synaptic Partners in the Visual Map of Drosophila

Specifying synaptic partners and regulating synaptic numbers are at least partly activity-dependent processes during visual map formation in all systems investigated to date . In Drosophila, six photoreceptors that view the same point in visual space have to be sorted into synaptic modules called cartridges in order to form a visuotopically correct map . Synapse numbers per photoreceptor terminal and cartridge are both precisely regulated . However, it is unknown whether an activity-dependent mechanism or a genetically encoded developmental program regulates synapse numbers. We performed a large-scale quantitative ultrastructural analysis of photoreceptor synapses in mutants affecting the generation of electrical potentials (norpA, trp;trpl), neurotransmitter release (hdc, syt), vesicle endocytosis (synj), the trafficking of specific guidance molecules during photoreceptor targeting (sec15), a specific guidance receptor required for visual map formation (Dlar), and 57 other novel synaptic mutants affecting 43 genes. Remarkably, in all these mutants, individual photoreceptors form the correct number of synapses per presynaptic terminal independently of cartridge composition. Hence, our data show that each photoreceptor forms a precise and constant number of afferent synapses independently of neuronal activity and partner accuracy. Our data suggest cell-autonomous control of synapse numbers as part of a developmental program of activity-independent steps that lead to a "hard-wired" visual map in the fly brain.

De Novo GMP Synthesis Is Required for Axon Guidance in Drosophila

Guanine nucleotides are key players in mediating growth-cone signaling during neural development. The supply of cellular guanine nucleotides in animals can be achieved via the de novo synthesis and salvage pathways. The de novo synthesis of guanine nucleotides is required for lymphocyte proliferation in animals. Whether the de novo synthesis pathway is essential for any other cellular processes, however, remains unknown. In a search for genes required for the establishment of neuronal connectivity in the fly visual system, we identify the burgundy (bur) gene as an essential player in photoreceptor axon guidance. The bur gene encodes the only GMP synthetase in Drosophila that catalyzes the final reaction of de novo GMP synthesis. Loss of bur causes severe defects in axonal fasciculation, retinotopy, and growth-cone morphology, but does not affect photoreceptor differentiation or retinal patterning. Similar defects were observed when the raspberry (ras) gene, encoding for inosine monophosphate dehydrogenase catalyzing the IMP-to-XMP conversion in GMP de novo synthesis, was mutated. Our study thus provides the first in vivo evidence to support an essential and specific role for de novo synthesis of guanine nucleotides in axon guidance.

Mutations in Drosophila sec15 Reveal a Function in Neuronal Targeting for a Subset of Exocyst Components

The exocyst is a complex of proteins originally identified in yeast that has been implicated in polarized secretion. Components of the exocyst have been implicated in neurite outgrowth, cell polarity, and cell viability. We have isolated an exocyst component, sec15, in a screen for genes required for synaptic specificity. Loss of sec15 causes a targeting defect of photoreceptors that coincides with mislocalization of specific cell adhesion and signaling molecules. Additionally, sec15 mutant neurons fail to localize other exocyst members like Sec5 and Sec8, but not Sec6, to neuronal terminals. However, loss of sec15 does not cause cell lethality in contrast to loss of sec5 or sec6. Our data suggest a role of Sec15 in an exocyst-like subcomplex for the targeting and subcellular distribution of specific proteins. The data also show that functions of other exocyst components persist in the absence of sec15, suggesting that different exocyst components have separable functions.

An axon scaffold induced by retinal axons directs glia to destinations in the Drosophila optic lobe.

In the developing Drosophila visual system, glia migrate into stereotyped positions within the photoreceptor axon target fields and provide positional information for photoreceptor axon guidance. Glial migration conversely depends on photoreceptor axons, as glia precursors stall in their progenitor zones when retinal innervation is eliminated. Our results support the view that this requirement for retinal innervation reflects a role of photoreceptor axons in the establishment of an axonal scaffold that guides glial cell migration. Optic lobe cortical axons extend from dorsal and ventral positions towards incoming photoreceptor axons and establish at least four separate pathways that direct glia to proper destinations in the optic lobe neuropiles. Photoreceptor axons induce the outgrowth of these scaffold axons. Most glia do not migrate when the scaffold axons are missing. Moreover, glia follow the aberrant pathways of scaffold axons that project aberrantly, as occurs in the mutant dachsous. The local absence of glia is accompanied by extensive apoptosis of optic lobe cortical neurons. These observations reveal a mechanism for coordinating photoreceptor axon arrival in the brain with the distribution of glia to multiple target destinations, where they are required for axon guidance and neuronal survival.

Big effects of Runt on Drosophila photoreceptor axon guidance

The Drosophila visual system has provided many important insights into the genetic and molecular mechanisms of axon guidance. Much of this research has focussed on cell surface receptors in growth cones, and signals downstream of them. It is therefore refreshing to see this work by Kaminker et al. [1], in which they define a new level of genetic control over photoreceptor targeting decisions.

Trio Combines with Dock to Regulate Pak Activity during Photoreceptor Axon Pathfinding in Drosophila

Correct pathfinding by Drosophila photoreceptor axons requires recruitment of p21-activated kinase (Pak) to the membrane by the SH2-SH3 adaptor Dock. Here, we identify the guanine nucleotide exchange factor (GEF) Trio as another essential component in photoreceptor axon guidance. Regulated exchange activity of one of the two Trio GEF domains is critical for accurate pathfinding. This GEF domain activates Rac, which in turn activates Pak. Mutations in trio result in projection defects similar to those observed in both Pak and dock mutants, and trio interacts genetically with Rac, Pak, and dock. These data define a signaling pathway from Trio to Rac to Pak that links guidance receptors to the growth cone cytoskeleton. We propose that distinct signals transduced via Trio and Dock act combinatorially to activate Pak in spatially restricted domains within the growth cone, thereby controlling the direction of axon extension.

Analysis of Drosophila photoreceptor axon guidance in eye-specific mosaics.

During development of the adult Drosophila visual system, axons of the eight photoreceptors in each ommatidium fasciculate together and project as a single bundle towards the optic lobes of the brain. Within the brain, individual photoreceptor axons from each bundle then seek specific targets in distinct layers of the optic lobes. The axons of photoreceptors R1-R6 terminate in the lamina, while R7 and R8 axons pass through the lamina to terminate in separate layers of the medulla. To identify genes required for photoreceptor axon guidance, including those with essential functions during early development, we have devised a strategy for the simple and efficient generation of genetic mosaics in which mutant photoreceptor axons innervate a predominantly wild-type brain. In a large-scale saturation mutagenesis performed using this system, we recovered new alleles of the gene encoding the receptor tyrosine phosphatase PTP69D. PTP69D has previously been shown to function in the correct targeting of motor axons in the embryo and R1-R6 axons in the visual system. Here, we show that PTP69D is also required for correct targeting of R7 axons. Whereas mutant R1-R6 axons occasionally extend beyond their normal targets in the lamina, mutant R7 axons often fail to reach their targets in the medulla, stopping instead at the same level as the R8 axon. These targeting errors are difficult to reconcile with models in which PTP69D plays an instructive role in photoreceptor axon targeting, as previously proposed. Rather, we suggest that PTP69D plays a permissive role, perhaps reducing the adhesion of R1-R6 and R7 growth cones to the pioneer R8 axon so that they can respond independently to their specific targeting cues.

Migration and function of glia in the developing Drosophila eye.

Although glial cells have been implicated widely in the formation of axon tracts in both insects and vertebrates, their specific function appears to be context-dependent, ranging from providing essential guidance cues to playing a merely facilitory role. Here we examine the role of the retinal basal glia (RBG) in photoreceptor axon guidance in Drosophila. The RBG originate in the optic stalk and have been thought to migrate into the eye disc along photoreceptor axons, thus precluding any role in axon guidance. Here we show the following. (1) The RBG can, in fact, migrate into the eye disc even in the absence of photoreceptor axons in the optic stalk; they also migrate to ectopic patches of differentiating photoreceptors without axons providing a continuous physical substratum. This suggests that glial cells are attracted into the eye disc not through haptotaxis along established axons, but through another mechanism, possibly chemotaxis. (2) If no glial cells are present in the eye disc, photoreceptor axons are able to grow and direct their growth posteriorly as in wild type, but are unable to enter the optic stalk. This indicates that the RBG have a crucial role in axon guidance, but not in axonal outgrowth per se. (3) A few glia close to the entry of the optic stalk suffice to guide the axons into the stalk, suggesting that glia instruct axons by local interaction.

Pak Functions Downstream of Dock to Regulate Photoreceptor Axon Guidance in Drosophila

The SH2/SH3 adaptor protein Dock has been proposed to transduce signals from guidance receptors to the actin cytoskeleton in Drosophila photoreceptor (R cell) growth cones. Here, we demonstrate that Drosophila p 21-a ctivated k inase (Pak) is required in a Dock pathway regulating R cell axon guidance and targeting. Dock and Pak colocalize to R cell axons and growth cones, physically interact, and their loss-of-function phenotypes are indistinguishable. Normal patterns of R cell connectivity require Pak’s kinase activity and binding sites for both Dock and Cdc42/Rac. A membrane-tethered form of Pak (Pakmyr) acts as a dominant gain-of-function protein. Retinal expression of Pakmyr rescues the R cell connectivity phenotype in dock mutants. These data establish Pak as a critical regulator of axon guidance and a downstream effector of Dock in vivo.

The Drosophila giant lens gene plays a dual role in eye and optic lobe development: Inhibition of differentiation of ommatidial cells and interference in photoreceptor axon guidance

The Drosophila giant lens ( gil) gene encodes a secreted molecule which if absent leads to the recruitment of additiional ommatidial cells normally eliminated by apoptosis. Heat induced ectopic gil expression leads to a reduction of ommatidial cells suggesting that gil is secreted by differentiating cells to prevent the development of an excess of cells of a given ommatidial cell type. A second important defect is the misrouting of photoreceptor axons in gil mutants. However, gil function is not required in photoreceptor axons for the establishment of proper connections. We propose that gil acts on the development of lamina cells preventing the correct differentiation of the target of photoreceptor axons and therefore leading to an axon guidance phenotype.

Giant lens, a gene involved in cell determination and axon guidance in the visual system of Drosophila melanogaster.

Mutations in the Drosophila gene giant lens (gil) affect ommatidial development, photoreceptor axon guidance and optic lobe development. We have cloned the gene using an enhancer trap line. Molecular analysis of gil suggests that it encodes a secreted protein with an epidermal-growth-factor-like motif. We have generated mutations at the gil locus by imprecise excision of the enhancer trap P-element. In the absence of gil, additional photoreceptors develop at the expense of pigment cells, suggesting an involvement of gil in cell determination during eye development. In addition, gil mutants show drastic effects on photoreceptor axon guidance and optic lobe development. In wildtype flies, photoreceptor axons grow from the eye disc through the optic stalk into the larval brain hemisphere, where retinal innervation is required for the normal development of the lamina and distal medulla. The projection pattern of these axons in the developing lamina and medulla is highly regular and reproducible. In gil, photoreceptor axons enter the larval brain but fail to establish proper connections in the lamina or medulla. We propose that gil encodes a new type of signalling molecule involved in the process of axon pathfinding and cell determination in the visual system of Drosophila.