Retinal axon target selection in Drosophila is regulated by a receptor protein tyrosine phosphatase

Different Drosophila photoreceptors (R cells) connect to neurons in different optic lobe layers. R1-R6 axons project to the lamina; R7 and R8 axons project to separate layers of the medulla. We show a receptor tyrosine phosphatase, PTP69D, is required for lamina target specificity. In Ptp69D mutants, R1-R6 project through the lamina, terminating in the medulla. Genetic mosaics, transgene rescue, and immunolocalization indicate PTP69D functions in R1-R6 growth cones. PTP69D overexpression in R7 and R8 does not respecify their connections, suggesting PTP69D acts in combination with other factors to determine target specificity. Structure-function analysis indicates the extracellular fibronectin type III domains and intracellular phosphatase activity are required for targeting. We propose PTP69D promotes R1-R6 targeting in response to extracellular signals by dephosphorylating substrate(s) in R1-R6 growth cones.     

Local N-cadherin interactions mediate distinct steps in the targeting of lamina neurons

The organization of neuronal processes into a series of layers is a hallmark of many brain regions. Homophilic cell adhesion molecules of the cadherin family have been implicated in layer choice. How they contribute to the targeting of neurons to distinct layers remains unclear. Here we systematically explore the role of a classical cadherin, Drosophila N-cadherin (CadN), in the targeting of five classes of related neurons to a series of consecutive layers in the fly visual system. We show that CadN is required in lamina neurons at discrete developmental steps but not used in a layer-specific fashion. Local CadN expression patterns correlate with specific growth cone movements, and CadN expression on one growth cone in a specific layer is essential for the targeting of processes of another neuron to this layer. We propose that dynamic regulation of CadN enables this widely expressed protein to mediate specific local interactions during neural circuit assembly.     

Drosophila N-cadherin functions in the first stage of the two-stage layer-selection process of R7 photoreceptor afferents

Visual information received from the three types of photoreceptor neurons (R1-R6, R7 and R8) in the fly compound eyes converges to the external part of the medulla neuropil (M1-M6 layers) in a layer-specific fashion: R7 and R8 axons terminate at the M6 and M3 layers, respectively, whereas lamina neurons (L1-L5) relay R1-R6 to multiple medulla layers (M1-M5). Here, we show that during development, R7 and R8 neurons establish layer-specific projections in two separate stages: during the first stage, R7 and R8 axons sequentially target to the R7- and R8-temporary layers, respectively; and at the second stage, R7 and R8 growth cones progress synchronously to their destined layers. Using a set of mutations that delete different afferent subsets or alter R7 connectivity, we defined the mechanism of layer selection. We observed that R8, R7 and L1-L5 afferents target to their temporary layers independently, suggesting that afferent-target, but not afferent-afferent, interactions dictate the targeting specificity. N-cadherin is required in the first stage for R7 growth cones to reach and remain in the R7-temporary layer. The Ncad gene contains three pairs of alternatively spliced exons and encodes 12 isoforms. However, expressing a single Ncad isoform in Ncad mutant R7s is sufficient to rescue mistargeting phenotypes. Furthermore, Ncad isoforms mediate promiscuous heterophilic interactions in an in vitro cell-aggregation assay. We propose that Ncad isoforms do not form an adhesion code; rather, they provide permissive adhesion between R7 growth cones and their temporary targets.     

Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1-EC5). Studies on cadherin specificity have implicated the NH(2)-terminal EC1 domain in the homophilic binding interaction, but the roles of the other extracellular cadherin (EC) domains have not been evaluated. We have undertaken a systematic analysis of the binding properties of the entire cadherin extracellular domain and the contributions of the other EC domains to homophilic binding. Lateral (cis) dimerization of the extracellular domain is thought to be required for adhesive function. Sedimentation analysis of the soluble extracellular segment of C-cadherin revealed that it exists in a monomer-dimer equilibrium with an affinity constant of approximately 64 microm. No higher order oligomers were detected, indicating that homophilic binding between cis-dimers is of significantly lower affinity. The homophilic binding properties of a series of deletion constructs, lacking successive or individual EC domains fused at the COOH terminus to an Fc domain, were analyzed using a bead aggregation assay and a cell attachment-based adhesion assay. A protein with only the first two NH(2)-terminal EC domains (CEC1-2Fc) exhibited very low activity compared with the entire extracellular domain (CEC1-5Fc), demonstrating that EC1 alone is not sufficient for effective homophilic binding. CEC1-3Fc exhibited high activity, but not as much as CEC1-4Fc or CEC1-5Fc. EC3 is not required for homophilic binding, however, since CEC1-2-4Fc and CEC1-2-4-5Fc exhibited high activity in both assays. These and experiments using additional EC combinations show that many, if not all, the EC domains contribute to the formation of the cadherin homophilic bond, and specific one-to-one interaction between particular EC domains may not be required. These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions (Sivasankar, S., W. Brieher, N. Lavrik, B. Gumbiner, and D. Leckband. 1999. PROC: Natl. Acad. Sci. USA. 96:11820-11824; Sivasankar, S., B. Gumbiner, and D. Leckband. 2001. Biophys J. 80:1758-68). We propose new models for how the cadherin extracellular repeats may contribute to adhesive specificity and function.     

The cadherin-catenin complex as a focal point of cell adhesion and signalling: new insights from three-dimensional structures

Cadherins are a large family of single-pass transmembrane proteins principally involved in Ca2+-dependent homotypic cell adhesion. The cadherin molecules comprise three domains, the intracellular domain, the transmembrane domain and the extracellular domain, and form large complexes with a vast array of binding partners (including cadherin molecules of the same type in homophilic interactions and cellular protein catenins), orchestrating biologically essential extracellular and intracellular signalling processes. While current, contrasting models for classic cadherin homophilic interaction involve varying numbers of specific repeats found in the extracellular domain, the structure of the domain itself clearly remains the main determinant of cell stability and binding specificity. Through intracellular interactions, cadherin enhances its adhesive properties binding the cytoskeleton via cytoplasmic associated factors alpha- catenin, beta-catenin and p120ctn. Recent structural studies on classic cadherins and these catenin molecules have provided new insight into the essential mechanisms underlying cadherin-mediated cell interaction and catenin-mediated cellular signalling. Remarkable structural diversity has been observed in beta-catenin recognition of other cellular factors including APC, Tcf and ICAT, proteins that contribute to or compete with cadherin/catenin functioning.     

Transplantation of photoreceptor precursor cells into the retina of an adult Drosophila

Blindness caused by the disconnection between photoreceptor cells and the brain can be cured by restoring this connection through the transplantation of retinal precursor neurons. However, even after transplanting these cells, it is still unclear how to guide the axons over the long distance from the retina to the brain. To establish a method of guiding the axons of transplanted neurons, we used the Drosophila visual system. By testing different conditions, including the dissociation and preincubation length, we have successfully established a method to transplant photoreceptor precursor cells isolated from the developing eye discs of third‐instar larvae into the adult retina. Moreover, we overexpressed N‐cadherin (CadN) in the transplant, since it is known to be broadly expressed in the optic lobe well after developmental stages, continuing through adult stages. We found that promoting the cell adhesive properties using CadN enhances the axonal length of the grafted photoreceptor neurons and therefore is useful for future transplantation. We tested the overexpression of a CadN::Frazzled chimeric receptor and found that there was no difference in axonal length from our wild‐type transplants, suggesting that the intracellular domain of CadN is necessary for axonal elongation. Altogether, using the Drosophila visual system, we have established an excellent platform for exploring the molecules required for proper axon extension of transplanted neuronal cells. Future studies building from this platform will be useful for regenerative therapy of the human nervous system based on transplantation.     

Drosophila LAR regulates R1-R6 and R7 target specificity in the visual system.

Different classes of photoreceptor neurons (R cells) in the Drosophila compound eye connect to specific targets in the optic lobe. Using a behavioral screen, we identified LAR, a receptor tyrosine phosphatase, as being required for R cell target specificity. In LAR mutant mosaic eyes, R1-R6 cells target to the lamina correctly, but fail to choose the correct pattern of target neurons. Although mutant R7 axons initially project to the correct layer of the medulla, they retract into inappropriate layers. Using single cell mosaics, we demonstrate that LAR controls targeting of R1-R6 and R7 in a cell-autonomous fashion. The phenotypes of LAR mutant R cells are strikingly similar to those seen in N-cadherin mutants.     

The Juxtamembrane Region of the Cadherin Cytoplasmic Tail Supports Lateral Clustering, Adhesive Strengthening, and Interaction with p120ctn

Cadherin cell–cell adhesion molecules form membrane-spanning molecular complexes that couple homophilic binding by the cadherin ectodomain to the actin cytoskeleton. A fundamental issue in cadherin biology is how this complex converts the weak intrinsic binding activity of the ectodomain into strong adhesion. Recently we demonstrated that cellular cadherins cluster in a ligand-dependent fashion when cells attached to substrata coated with the adhesive ectodomain of Xenopus C-cadherin (CEC1-5). Moreover, forced clustering of the ectodomain alone significantly strengthened adhesiveness (Yap, A.S., W.M. Brieher, M. Pruschy, and B.M. Gumbiner. Curr. Biol. 7:308–315). In this study we sought to identify the determinants of the cadherin cytoplasmic tail responsible for clustering activity. A deletion mutant of C-cadherin (CT669) that retained the juxtamembrane 94–amino acid region of the cytoplasmic tail, but not the β-catenin–binding domain, clustered upon attachment to substrata coated with CEC1-5. Like wild-type C-cadherin, this clustering was ligand dependent. In contrast, mutant molecules lacking either the complete cytoplasmic tail or just the juxtamembrane region did not cluster. The juxtamembrane region was itself sufficient to induce clustering when fused to a heterologous membrane-anchored protein, albeit in a ligand-independent fashion. The CT669 cadherin mutant also displayed significant adhesive activity when tested in laminar flow detachment assays and aggregation assays. Purification of proteins binding to the juxtamembrane region revealed that the major associated protein is p120^ctn. These findings identify the juxtamembrane region of the cadherin cytoplasmic tail as a functionally active region supporting cadherin clustering and adhesive strength and raise the possibility that p120^ctn is involved in clustering and cell adhesion.     

The cadherin Flamingo mediates level-dependent interactions that guide photoreceptor target choice in Drosophila

Quantitative differences in cadherin activity have been proposed to play important roles in patterning connections between pre- and postsynaptic neurons. However, no examples of such a function have yet been described, and the mechanisms that would allow such differences to direct growth cones to specific synaptic targets are unknown. In the Drosophila visual system, photoreceptors are genetically programmed to make a complex, stereotypic set of synaptic connections. Here we show that the atypical cadherin Flamingo functions as a short-range, homophilic signal, passing between specific R cell growth cones to influence their choice of postsynaptic partners. We find that individual growth cones are sensitive to differences in Flamingo activity through opposing interactions between neighboring cells and require these interactions to be balanced in order to extend along the appropriate trajectory.     

Transmembrane control of cadherin-mediated cell adhesion: a 94 kDa protein functionally associated with a specific region of the cytoplasmic domain of E-cadherin.

Cadherins are a family of transmembrane glycoproteins which play a key role in Ca(2+)-dependent cell-cell adhesion. Cytoplasmic domains of these molecules are anchored to the cell cytoskeleton and are required for cadherin function. To elucidate how the function of cadherins is controlled through their cytoplasmic domains, we deleted five different regions in the cytoplasmic domain of E-cadherin. After transfecting L cells with cDNA encoding the mutant polypeptides, we assayed aggregating activity of these transfectants; all these mutant proteins were shown to have an extracellular domain with normal Ca(2+)-sensitivity and molecular weight. Two mutant polypeptides with deletions in the carboxy half of the cytoplasmic domain, however, did not promote cell-cell adhesion and had also lost the ability to bind to the cytoskeleton, whereas the mutant molecules with deletions of other regions retained the ability to promote cell adhesion and to anchor to the cytoskeleton. Thus, the cytoplasmic domain contains a subdomain which was involved in the cell adhesion and cytoskeleton-binding functions. When E-cadherin in F9 cells or in L cells transfected with wild-type or functional mutant cadherin polypeptides was solubilized with nonionic detergents and immunoprecipitated, two additional 94 and 102 kDa components were coprecipitated. The 94 kDa component, however, was not detected in the immunoprecipitates from cells expressing the mutant cadherins which had lost the adhesive function. These results suggest that the interaction of the carboxy half of the cytoplasmic domain with the 94 kDa component regulates the cell binding function of the extracellular domain of E-cadherin.     

Lateral dimerization is required for the homophilic binding activity of C-cadherin

Regulation of cadherin-mediated adhesion can occur rapidly at the cell surface. To understand the mechanism underlying cadherin regulation, it is essential to elucidate the homophilic binding mechanism that underlies all cadherin-mediated functions. Therefore, we have investigated the structural and functional properties of the extracellular segment of Xenopus C-cadherin using a purified, recombinant protein (CEC 1-5). CEC 1-5 supported adhesion of CHO cells expressing C-cadherin. The extracellular segment was also capable of mediating aggregation of microspheres. Chemical cross-linking and gel filtration revealed that CEC 1-5 formed dimers in the presence as well as absence of calcium. Analysis of the functional activity of purified dimers and monomers demonstrated that dimers retained substantially greater homophilic binding activity than monomers. These results demonstrate that lateral dimerization is necessary for homophilic binding between cadherin extracellular segments and suggest multiple potential mechanisms for the regulation of cadherin activity. Since the extracellular segment alone possessed significant homophilic binding activity, the adhesive activity of the extracellular segment in a cellular context was analyzed. The adhesion of CHO cells expressing a truncated version of C-cadherin lacking the cytoplasmic tail was compared to cells expressing the wild-type C-cadherin using a laminar flow assay on substrates coated with CEC 1-5. CHO cells expressing the truncated C-cadherin were able to attach to CEC 1-5 and to resist detachment by low shear forces, demonstrating that tailless C-cadherin can mediate basic, weak adhesion of CHO cells. However, cells expressing the truncated C-cadherin did not exhibit the complete adhesive activity of cells expressing wild-type C-cadherin. Cells expressing wild-type C-cadherin remained attached to CEC 1-5 at high shear forces, while cells expressing the tailless C-cadherin did not adhere well at high shear forces. These results suggest that there may be two states of cadherin-mediated adhesion. The first, relatively weak state can be mediated through interactions between the extracellular segments alone. The second strong adhesive state is critically dependent on the cytoplasmic tail.     

Targeting of neuromodulin (GAP-43) fusion proteins to growth cones in cultured rat embryonic neurons.

Neuromodulin (GAP-43) is a membrane protein that is transported to neuronal growth cones. Zuber and co-workers have proposed that the N-terminal 10 amino acid sequence of neuromodulin is sufficient to target proteins to growth cones. We demonstrate that a neuromodulin-beta-galactosidase fusion protein is transported to growth cones of cultured rat neurons, whereas a fusion protein containing the N-terminal 10 amino acids of neuromodulin and beta-galactosidase is not. A mutant neuromodulin lacking cysteines 3 and 4, the palmitylation sites required for membrane attachment, does not target beta-galactosidase to growth cones. We conclude that membrane attachment is required for growth cone accumulation and that structural elements, in addition to the first 10 amino acids of neuromodulin, may be required for growth cone targeting.     

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.     

Regulation of layer-specific targeting by reciprocal expression of a cell adhesion molecule, capricious

Layer-specific innervation is a major form of synaptic targeting in the central nervous system. In the Drosophila visual system, photoreceptors R7 and R8 connect to targets in distinct layers of the medulla, a ganglion of the optic lobe. We show here that Capricious (CAPS), a transmembrane protein with leucine-rich repeats (LRRs), is a layer-specific cell adhesion molecule that regulates photoreceptor targeting in the medulla. During the period of photoreceptor targeting, caps is specifically expressed in R8 and its target layer but not in R7 or its recipient layer. caps loss-of-function mutations cause local targeting errors by R8 axons, including layer change. Conversely, ectopic expression of caps in R7 redirects R7 axons to terminate in the CAPS-positive R8 recipient layer. CAPS promotes homophilic cell adhesion in transfected S2 cells. These results suggest that CAPS regulates layer-specific targeting by mediating specific axon-target interaction.     

Regulation of dendritic maintenance and growth by a mammalian 7-pass transmembrane cadherin

Drosophila Flamingo is a 7-pass transmembrane cadherin that is necessary for dendritic patterning and axon guidance. How it works at the molecular level and whether homologs of Flamingo play similar roles in mammalian neurons or not have been unanswered questions. Here, we performed loss-of-function analysis using an RNAi system and organotypic brain slice cultures to address the role of a mammalian Flamingo homolog, Celsr2. Knocking down Celsr2 resulted in prominent simplification of dendritic arbors of cortical pyramidal neurons and Purkinje neurons, and this phenotype seemed to be due to branch retraction. Cadherin domain-mediated homophilic interaction appears to be required for the maintenance of dendritic branches. Furthermore, expression of various Celsr2 forms elicited distinct responses that were dependent on an extracellular subregion outside the cadherin domains and on a portion within the carboxyl intracellular tail. Based on these findings, we discuss how Celsr2 may regulate dendritic maintenance and growth.     

Critical role of the fifth domain of E-cadherin for heterophilic adhesion with alpha E beta 7, but not for homophilic adhesion

The integrin alpha(E)beta(7) is expressed on intestinal intraepithelial T lymphocytes and CD8(+) T lymphocytes in inflammatory lesions near epithelial cells. Adhesion between alpha(E)beta(7)(+) T and epithelial cells is mediated by the adhesive interaction of alpha(E)beta(7) and E-cadherin; this interaction plays a key role in the damage of target epithelia. To explore the structure-function relationship of the heterophilic adhesive interaction between E-cadherin and alpha(E)beta(7), we performed cell aggregation assays using L cells transfected with an extracellular domain-deletion mutant of E-cadherin. In homophilic adhesion assays, L cells transfected with wild-type or a domain 5-deficient mutant formed aggregates, whereas transfectants with domain 1-, 2-, 3-, or 4-deficient mutants did not. These results indicate that not only domain 1, but domains 2, 3, and 4 are involved in homophilic adhesion. When alpha(E)beta(7)(+) K562 cells were incubated with L cells expressing the wild type, 23% of the resulting cell aggregates consisted of alpha(E)beta(7)(+) K562 cells. In contrast, the binding of alpha(E)beta(7)(+) K562 cells to L cells expressing a domain 5-deficient mutant was significantly decreased, with alpha(E)beta(7)(+) K562 cells accounting for only 4% of the cell aggregates, while homophilic adhesion was completely preserved. These results suggest that domain 5 is involved in heterophilic adhesion with alpha(E)beta(7), but not in homophilic adhesion, leading to the hypothesis that the fifth domain of E-cadherin may play a critical role in the regulation of heterophilic adhesion to alpha(E)beta(7) and may be a potential target for treatments altering the adhesion of alpha(E)beta(7)(+) T cells to epithelial cells in inflammatory epithelial diseases.     

The receptor tyrosine kinase Off-track is required for layer-specific neuronal connectivity in Drosophila

The nervous system in many species consists of multiple neuronal cell layers, each forming specific connections with neurons in other layers or other regions of the brain. How layer-specific connectivity is established during development remains largely unknown. In the Drosophila adult visual system, photoreceptor (R cell) axons innervate one of two optic ganglia layers; R1-R6 axons connect to the lamina layer, while R7 and R8 axons project through the lamina into the deeper medulla layer. Here, we show that the receptor tyrosine kinase Off-track (Otk) is specifically required for lamina-specific targeting of R1-R6 axons. Otk is highly expressed on R1-R6 growth cones. In the absence of otk, many R1-R6 axons connect abnormally to medulla instead of innervating lamina. We propose that Otk is a receptor or a component of a receptor complex that recognizes a target-derived signal for R1-R6 axons to innervate the lamina layer.     

Glial cells mediate target layer selection of retinal axons in the developing visual system of Drosophila

In the fly visual system, each class of photoreceptor neurons (R cells) projects to a different synaptic layer in the brain. 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. The cellular requirement for R1-R6 targeting was determined using loss-of-function mutations affecting different cell types in the lamina. nonstop (encoding a ubiquitin-specific protease) is required for glial cell development and hedgehog for neuronal development. Removal of glial cells but not neurons disrupts R1-R6 targeting. We propose 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.     

Intracellular domain of desmoglein 3 (pemphigus vulgaris antigen) confers adhesive function on the extracellular domain of E-cadherin without binding catenins

For the extracellular (EC) domain of E-cadherin to function in homophilic adhesion it is thought that its intracytoplasmic (IC) domain must bind alpha- and beta-catenins, which link it to the actin cytoskeleton. However, the IC domain of pemphigus vulgaris antigen (PVA or Dsg3), which is in the desmoglein subfamily of the cadherin gene superfamily, does not bind alpha- or beta-catenins. Because desmogleins have also been predicted to function in the cell adhesion of desmosomes, we speculated that the PVA IC domain might be able to act in a novel way in conferring adhesive function on the EC domain of cadherins. To test this hypothesis we studied aggregation of mouse fibroblast L cell clones that expressed chimeric cDNAs encoding the EC domain of E-cadherin with various IC domains. We show here that the full IC domain of PVA as well as an IC subdomain containing only 40 amino acids of the PVA intracellular anchor (IA) region confer adhesive function on the E-cadherin EC domain without catenin-like associations with cytoplasmic molecules or fractionation with the cell cytoskeleton. This IA region subdomain is evolutionarily conserved in desmogleins, but not classical cadherins. These findings suggest an important cell biologic function for the IA region of desmogleins and demonstrate that strong cytoplasmic interactions are not absolutely necessary for E- cadherin-mediated adhesion.