BIOLOGICAL OVERVIEW

The visual system of Drosophila provides a powerful genetic system to analyze the cellular and molecular mechanisms that regulate axon target selection. The compound eye comprises ~750 ommatidia, each containing eight photoreceptor neurons (R cells; R1-R8). Each class of R cells forms specific connections with neurons in two different ganglia in the optic lobe, the lamina and medulla. R1-R6 axons terminate in the lamina, while R7 and R8 axons pass through the lamina and stop in the medulla. As R cell axons enter the lamina, they encounter both glial cells and neurons. non-stop (encoding a ubiquitin-specific protease) was isolated in a genetic screen for R cell projection defects (Martin, 1995). non-stop is required for glial cell development and hedgehog for neuronal development. Removal of glial cells but not neurons disrupts R1-R6 targeting. It is proposed that glial cells provide the initial stop signal promoting growth cone termination in the lamina. These findings uncover a novel function for neuron-glial interactions in regulating target specificity (Poeck, 2001).

Target layer selection occurs during larval development. At this stage, the lamina target area consists of glial cells and neurons. R1-R6 growth cones terminate between rows of epithelial and marginal glial cells. The cell bodies of lamina neurons form columns above the lamina plexus. These neurons represent the future synaptic partners of R1-R6 axons in the adult. Although R cell growth cones terminate within the lamina plexus in the larva, they do not form synapses until some four days later, during the second half of pupal development (Poeck, 2001).

The formation of the R cell projection pattern relies on complex bidirectional interactions between R cell axons and different populations of cells in the target. R cell axons provide anterograde signals (including the ligand Hedgehog and Spitz) to induce the proliferation and differentiation of lamina neurons as well as the differentiation and migration of glial cells. In turn, lamina neurons, glial cells, or both cell types may then provide retrograde signals acting as guidance cues for R cell axons. Genes encoding receptors, signaling molecules, and nuclear factors that act within R cells control target selection. Neither the targeting signals nor the cells that produce them in the lamina have been identified. While glial cells have been proposed to act as intermediate targets for R1-R6 growth cones, based on their characteristic positions in the lamina, this hypothesis has not been critically addressed. It has been shown that loss of glial cells but not of neurons at an early stage of lamina development results in R1-R6 mistargeting. These findings provide evidence that glial cells can regulate the target specificity of neuronal connections (Poeck, 2001).

Non-stop is expressed in the cytoplasm of cells in the optic lobe and central brain. Higher levels of expression, however, are observed in lamina precursor cells but not in differentiated lamina neurons. Non-stop is also expressed at higher levels in marginal, epithelial, and medulla glial cells adjacent to the lamina plexus. Based on the expression pattern, non-stop could function in the LPCs to control both R1-R6 targeting and glial cell migration. Alternatively, non-stop could function directly in lamina glial cells to promote their migration; failure to migrate leads to loss of intermediate targets and R1-R6 mistargeting. One research goal was to distinguish between these possibilities; two different genetic approaches were used. First, the ability of targeted expression of non-stop to glial cells was assessed, using the GAL4/UAS system to rescue the mutant phenotypes. Since basal expression of UAS-non-stop (i.e., in the absence of GAL4 driven in lamina glial cells) is sufficient to rescue both the glial cell migration and targeting defects, this approach to resolving the issue is not possible. Therefore the FLP/FRT system was used to generate clones of lamina glial cells or neurons (and their precursors) homozygous mutant for non-stop in an otherwise wild-type (not¹/+) background. A heat shock-FLP (hsFLP) transgene provided recombinase in mitotically active cells in the target area. Animals carried a gene expressed in all cells, encoding the green fluorescent protein under the control of a ubiquitin promoter (Ub-GFP). As a result of mitotic recombination, non-stop homozygous mutant cells did not express GFP; heterozygous cells expressed moderate levels of GFP, since they carry a single copy of the marker gene; and wild-type clones, carrying two copies of Ub-GFP, were identified by their higher expression levels. non-stop homozygous mutant clones were compared to control clones created in the same manner by using a wild-type FRT in place of a not¹ FRT chromosome (Poeck, 2001).

Eye-brain complexes were stained with mAb24B10 to visualize R cell axons and with anti-Repo to identify glial cells. Approximately 10% of all animals examined contained clones in the target area. In mosaic animals with clones in lamina precursors and lamina neurons, the R cell projection pattern appeared normal. Furthermore, normal rows of wild-type epithelial, marginal, and medulla glial cells formed beneath these clones. This indicates that non-stop is not required in lamina neurons to regulate either R1-R6 targeting or glial cell development (Poeck, 2001).

Glial cells are generated in the GPC areas and migrate to their positions adjacent to the lamina plexus. In about half of the wild-type control samples, glial cell clones were large, containing at least five epithelial or marginal glial cells. The GPC areas showed clones of variable sizes, with unlabeled cells next to groups of marked cells carrying one or two copies of GFP. The region adjacent to the dorsoventral midline close to the optic lobe surface appearsto give rise to especially large clones. Furthermore, it was observed that 21 of 23 clones consisted of both epithelial and marginal glial cells; two small clones contained epithelial glial cells only (Poeck, 2001).

The number of non-stop mutant epithelial and marginal glial cells bordering the lamina plexus was markedly reduced compared to control clones. In wild-type control clones, 203 unlabeled cells were counted in 23 clones. In contrast, in non-stop mutant clones, there were 125 cells in 39 clones examined. This represents about a 3-fold difference. Moreover, for marginal glial cells, this difference was 10-fold; in wild-type clones there were 71 cells in 23 clones, whereas there were 11 cells in 39 non-stop mutant clones. The non-stop mutant and control clones in the GPC area were similar in both size and frequency. In six cases, although homozygous non-stop clones were present in the GPC areas, there were no mutant glial cells in the target. In contrast, unlabeled epithelial and marginal glial cells were observed along the lamina plexus in all wild-type clones examined. Blocks of non-stop mutant glial cells were never observed adjacent to the lamina plexus. Presumably, in mosaic animals, wild-type cells migrate into these regions, effectively replacing the mutant glial cells and rescuing the R1-R6 targeting defect seen in non-stop mutants. These data support a model in which non-stop is required in glial cells, their precursors, or other cell types within the GPC area for the migration of epithelial and marginal glial cells from the GPC to the target area (Poeck, 2001).

While genetic mosaic studies established that non-stop is required in glial cells for their migration, it remained formally possible that non-stop was required in lamina neurons to mediate R1-R6 termination in the lamina. To critically assess this issue, attempts were made to remove lamina neurons from the target region. If non-stop were required in lamina neurons, then removing lamina neurons entirely should lead to R1-R6 mistargeting. hedgehog¹ (hh¹) is a regulatory mutation that specifically affects the visual system. In hh¹, ~12 rows of R cell clusters are formed; R cell axons, however, lack Hh and fail to induce lamina neurons. Conversely, the migration and differentiation of glial cells do not depend on Hh signaling. A subset of R1-R6 axons was visualized in hh¹ mutants, using the marker Ro-taulacZ. These axons stopped in the lamina, despite the absence of lamina neurons, as detected using an antibody to Dachshund. Labeling with mAb24B10 to visualize all R cell axons revealed that the array of R7 and R8 growth cones in the medulla was indistinguishable from wild type. These findings demonstrate that initial targeting of R1-R6 axons does not require lamina neurons (Poeck, 2001).

The mechanisms controlling glial cell migration in the lamina and the molecular pathways involved are not known. That non-stop encodes a ubiquitin-specific protease suggests that protein degradation pathways may play an important role in this process. In the ubiquitin-proteasome pathway, proteins are targeted for degradation to the 26S proteasome after being 'tagged' with ubiquitin. Ubiquitin modification is reversible. Deubiquitination is catalyzed by two families of specific proteases, ubiquitin-C-terminal hydrolases and ubiquitin-specific proteases (UBPs). While these families are structurally distinct, they have overlapping functions. Non-stop is related to the second family because of two conserved consensus sequences within the catalytic domain, the Cys and His domains. UBPs have been shown to play diverse roles by either inhibiting or stimulating protein degradation. They can prevent protein degradation and reverse ubiquitin modification to 'proofread' mistakenly ubiquitinated proteins or to regulate protein stability by antagonizing proteasome activity. UBPs also have been found to stimulate protein degradation by editing the size of polyubiquitin chains, releasing ubiquitin after the protein has been targeted to the proteasome, or disassembling polyubiquitin chains to restore the cellular pool of free ubiquitin (Poeck, 2001).

The observation that additional ubiquitinated proteins accumulate in mutant larvae is consistent with Non-stop acting as a UBP. Furthermore, enhancement of targeting defects in non-stop mutants resulting from removing a single copy of a gene encoding a proteosome subunit suggests that Non-stop promotes protein degradation. Since the loss of non-stop results in a specific defect in the developing visual system, it may regulate the levels of specific substrates necessary for glial cell migration. Indeed, another Drosophila UBP, Ubiquitin-63E, controls border cell migration during oogenesis indirectly by stabilizing the transcription factor C/EBP (Rørth, 2000). There is also precedent for ubiquitin-dependent regulation of signaling proteins, such as cell surface receptors and cytoskeletal regulators, which may be directly involved in cell migration (Poeck, 2001).

non-stop mutations provide a key reagent for addressing the cellular requirement for R1-R6 targeting. Loss-of-function non-stop mutations selectively disrupt glial cell development at an early stage. During the time glial cells are generated in the precursor region, migration into the developing lamina is disrupted. As such, large regions of the lamina target are depleted of glial cells. Genetic mosaic analyses argue that R1-R6 mistargeting results from a loss of glial cells in the lamina target and not from a requirement for non-stop in R cell axons or lamina neurons. That lamina neurons are dispensable for R1-R6 targeting is further supported by the analysis of hedgehog¹ mutants in which layer selection is normal in the absence of lamina neurons. The genetic analysis, however, does not allow for the exclusion of the formal (albeit unlikely) possibility that non-stop functions in glial cells in two distinct processes to regulate migration and R1-R6 targeting separately (Poeck, 2001).

The relative contributions of epithelial, marginal, and medulla glial cells in R1-R6 targeting could not be determined, since all three appear affected in non-stop mutants. Their morphology, however, suggests that they have different functions. R1-R6 growth cones in the lamina plexus are in intimate contact with a dense fringe of glial cell processes from both epithelial and marginal glia but not with processes from the medulla glia. It is likely that marginal glial cells issue the stop signals, since R1-R6 axons grow past epithelial glial cells but terminate along the distal face of the marginal glial cells. In addition, both epithelial and marginal glial cells may provide signals to R1-R6 growth cones, 'holding' them in place. Nitric oxide (see Drosophila Nitric oxide synthase) plays an important role in maintaining the position of R7 and R8 axons within medulla neuropil during early pupal development and this raises the intriguing possibility that a similar mechanism may keep R1-R6 neurons within the lamina (Poeck, 2001).

In summary, the requirement for non-stop in glial cell development, the failure of R1-R6 growth cones to terminate in the lamina in non-stop mutants, and the close association between lamina glial cells and R1-R6 growth cones in the lamina plexus argue that epithelial and marginal glial cells provide targeting signals for R1-R6 neurons (Poeck, 2001)

The existence of intermediate targets is essential for generating specific patterns of R cell connections. These targets provide a means of delaying the formation of neuronal connections until the future synaptic partners of R cells, the lamina neurons, have formed. R1-R6 axons project from the eye primordium in a sequential fashion to their target layer during larval development. Growth cones from R cells within the same ommatidium form a tight cluster nestled between the epithelial and marginal glial cells and pause within this region through early pupal development. Early arriving R cell axons wait for about 70 hr, while later arriving axons pause for about 36 hr. During this period, lamina neurons assemble into columns, adopt specific cell fates (i.e., L1-L5), and project axons through the lamina plexus and into the medulla. Maturation of lamina neurons is reflected by the expression of the molecular marker Elav. This marker is seen initially in single cells within columns closest to the lamina furrow and accumulates in all lamina neurons in the more mature columns at the posterior edge of the developing lamina. Thus, the precise array of lamina neurons develops long after R cell growth cones enter the target region (Poeck, 2001).

Moreover, the formation of connections between the retina and lamina requires the preassembly of a precisely organized target field. During pupal development, R1-R6 growth cones defasciculate from their original bundle. Each growth cone projects to different neighboring postsynaptic targets and develops into an extended terminal, forming many en passant synapses with a subset of lamina neurons. Synapse formation is complete by late pupal development. This complex reorganization of R cell axons within the target ensures that, in the adult, R cells 'looking' out from the eye at the same point in space connect to the same postsynaptic neurons in the lamina (Poeck, 2001).

The role of neurons as intermediate transient targets is well documented in the developing mammalian hippocampus and neocortex. As in the developing lamina, the final synaptic partners are not yet in place in these systems when the first afferent axons arrive. In this paper, it has been shown that glial cells also can act as intermediate targets. Indeed, this function for glial cells may be more widespread. In the developing olfactory system of the moth Manduca sexta, antennal axons interact with glial cells in the target area before establishing contacts with central target neurons. In animals rendered glial deficient by hydoxyurea treatment or gamma-irradiation, olfactory axons do not confine their projections to their normal targets, the olfactory glomeruli. This treatment also prevents the development of specific glial cells, which segregate axons into distinct fascicles as they enter the target region from the antennal nerve. Hence, it is not clear whether it is these glial cells or, alternatively, the neuropil-associated glial cells in the target area that are critical for regulating target specificity in this system. Glial cells also may function as intermediate targets in some regions of the mammalian nervous system. For instance, rodent olfactory axons interact with radial glial cells, prior to recruitment of mitral and periglomerular processes (e.g., the synaptic targets of olfactory neurons) into glomeruli. In the absence of mitral or periglomerular cells, olfactory axons select their appropriate glomerular targets. Based on these observations, it has been proposed that targeting of olfactory axons to specific glomeruli is regulated by glial cells. While the role for glial cells in target specification is new, glial cells have previously been shown to play key roles as 'guideposts' along axon trajectories to their targets. For instance, in the Drosophila embryonic central nervous system, midline glial cells provide a repellent signal, Slit, to prevent axons from inappropriate growth across the midline (Poeck, 2001 and references therein).

The identification of glial cells as intermediate targets for R1-R6 neurons provides an important step in the isolation of targeting signals regulating R1-R6 specificity. This may be achieved through molecular screens using GFP-labeled glial cells isolated from the developing optic lobe to identify genes encoding cell surface proteins selectively expressed in these cells. Alternatively, by targeting mitotic recombination to glial precursor cells using the FLP/FRT method, it may be possible to specifically isolate such genes required in glial cells to control target layer selection (Poeck, 2001).

GENE STRUCTURE

cDNA clone length - 2650 bases

Bases in 5' UTR - 242

Exons - 5

Bases in 3' UTR - 457

PROTEIN STRUCTURE

Amino Acids - 735

Structural Domains

Database searches reveal significant sequence identity between the Not protein and ubiquitin-specific proteases (UBPs) in yeast, C. elegans, mouse, and humans. Two stretches of amino acids, 18 and 55 amino acids in length, are conserved in the C-terminal half of Not. These conserved sequences include two catalytic signature sequences of UBP enzymes (the Cys domain and the His domain). A human cDNA, KIAA1063, isolated from a brain cDNA library, shows a high level of conservation. The human sequence is incomplete, and conceptual translation results in a polypeptide, which corresponds to ~80% of Drosophila Non-stop (aa 154-735), lacking the N-terminal regions. The Drosophila and human protein show high sequence identity (51%) throughout this region. The high degree of conservation between these two proteins compared to other UBPs suggests that KIAA1063 encodes the human Non-stop ortholog (Poeck, 2001).

date revised: 10 June 2001

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