Cadherin-N


REGULATION

Transcriptional Regulation

Mapping Dmef2-binding regulatory modules by using a ChIP-enriched in silico targets approach

Mapping the regulatory modules to which transcription factors bind in vivo is a key step toward understanding of global gene expression programs. A chromatin immunoprecipitation (ChIP)-chip strategy has been developed for identifying factor-specific regulatory regions acting in vivo. This method, called the ChIP-enriched in silico targets (ChEST) approach, combines immunoprecipitation of cross-linked protein-DNA complexes (X-ChIP) with in silico prediction of targets and generation of computed DNA microarrays. Use of ChEST in Drosophila is described to identify several previously unknown targets of myocyte enhancer factor 2 (MEF2), a key regulator of myogenic differentiation. The approach was validated by demonstrating that the identified sequences act as enhancers in vivo and are able to drive reporter gene expression specifically in MEF2-positive muscle cells. Presented here, the ChEST strategy was originally designed to identify regulatory modules in Drosophila, but it can be adapted for any sequenced and annotated genome (Junion, 2005).

To predict Dmef2-dependent CRMs, the Drosophila genome was scanned for modules containing one of the three previously described in vivo-acting Dmef2 binding sites. Because muscle differentiation events are controlled by the synergistic action of MADS-box (Mef2 family) and E-box (Twist and MyoD bHLH family) factors modules were sought containing Mef2- and E-box-binding sites. This scanning procedure led to the identification of ~1,243 potential Dmef2-binding CRMs, from which 99 modules were selected, amplified, and spotted to produce a computed Dmef2-CRM array. Three of four previously described Dmef2-CRMs, located in the vicinity of Paramyosin, Act57B, and Dmef2, were identified during genome scanning and selected for spotting as positive control. The fourth in vivo-acting Dmef2-CRM located close to b3-tubulin did not come out from the screen because of the high number of E-box sites used as the scanning criterion. The CRMs located in the vicinity of genes that are not expressed in the mesoderm or genes of unknown expression and function (some of CGs) were rejected. In parallel, X-ChIP was used to isolate DNA fragments to which Dmef2 binds in vivo. ChIP-DNA immunoprecipitated either with Dmef2 antibody or with nonimmune serum was then labeled and used to probe the Dmef2-CRM array. The three previously described in vivo-acting CRMs were found enriched in ChIP-DNA immunoprecipitated with Dmef2 antibody. Importantly, numerous other in silico-identified Dmef2-CRMs were enriched in ChIP-DNA, thus demonstrating the efficacy of the ChEST method (Junion, 2005).

Of the 99 in silico-predicted Dmef2-CRMs, 62 were enriched in the DNA immunoprecipitated with anti-Dmef2 antibody. The CRM-associated Dmef2 targets included genes expressed in all muscle cell types, in which Dmef2 has previously been reported to function. In addition to expected candidates encoding fusion (Lmd, Hibris) and structural (Ket, Pod1) muscle proteins, a large number of CRM-associated Dmef2 target genes coded for TFs and signal transduction proteins. For example, CRMs upstream of Fz2 and within the introns of Ci and Pan indicate a potential role of Dmef2 in transcriptional regulation of genes transducing to the mesodermal cells ectodermal Wg and Hh signals, whereas CRMs within the introns of If and Pka-C3 suggest that by regulating transcription of these genes Dmef2 is involved in the attachment of muscle fibers and in fiber contraction, respectively. In some cases (e.g., Kettin, NetB, N-cad), several Dmef2-dependent CRMs were mapped in the vicinity of the same gene, highlighting the complexity of transcriptional regulation in which Dmef2 is involved. Analysis of the position of CRMs in relation to adjacent genes revealed that the majority of ChIP-enriched modules are located upstream (42%) or within the introns (39%) of target genes. In these two categories, the most frequent positions of Dmef2-CRMs appear between 1 and 5 kb upstream of the gene and within the first intron (Junion, 2005).

To determine whether the ChEST-identified DNA fragments are able to act as regulatory modules in vivo, ten Dmef2-CRMs by reporter gene transgenesis were tested. Nine of 10 CRMs were found to drive reporter gene expression in Dmef2-positive muscle cells. In all transgenic Drosophila lines, lacZ reporter expression at least partially reproduced endogenous gene expression, indicating that the identified CRMs were bona fide enhancers of adjacent genes. For example, the Ket-1, Ket-2, and Ket-3 CRMs laying in the vicinity of the kettin gene, which encodes a giant muscle protein required for the formation and maintenance of normal sarcomere structure, were found to drive lacZ expression in distinct subsets of differentiating body wall muscles. These data indicate that the pan-muscular expression of kettin is regulated in a muscle-type-specific manner, and by multiple Dmef2-binding enhancers. Interestingly, four other analyzed CRMs located within the introns of N-cad and acon and upstream of fz2, sfl, and Meso18E also drive lacZ expression in discrete subsets of somatic muscle precursors. The muscle-type-restricted activity of these modules suggests that both CRM regulators (Dmef2, E-box factors) and their target genes are involved in different aspects of muscle precursors diversification, including muscle fiber shape and axial positioning. Alternatively, the observed muscle-type-specific expression of lacZ may result from the limited size (250-300 bp) of genomic sequences tested. In embryos carrying a Dmef2-dependent CRM found in the intron of If, lacZ expression is detected in a group of ventrolateral muscles. This lacZ pattern correlates with distribution of endogenous If, which accumulates at the extremities of ventrolateral muscle fibers and is required for their correct epidermal insertion. The reduced level of target gene expression in Dmef2 mutant embryos provides an additional support for Dmef2-dependent in vivo regulation of ChEST-identified CRMs (Junion, 2005).

Dorsal-ventral (DV) patterning of the Drosophila embryo is initiated by Dorsal, a sequence-specific transcription factor distributed in a broad nuclear gradient in the precellular embryo. Previous studies have identified as many as 70 protein-coding genes and one microRNA (miRNA) gene that are directly or indirectly regulated by this gradient. A gene regulation network, or circuit diagram, including the functional interconnections among 40 Dorsal (Dl) target genes and 20 associated tissue-specific enhancers, has been determined for the initial stages of gastrulation. This study attempts to extend this analysis by identifying additional DV patterning genes using a recently developed whole-genome tiling array. This analysis led to the identification of another 30 protein-coding genes, including the Drosophila homolog of Idax, an inhibitor of Wnt signaling. In addition, remote 5' exons were identified for at least 10 of the ~100 protein-coding genes that were missed in earlier annotations. As many as nine intergenic uncharacterized transcription units (TUs) were identified, including two that contain known microRNAs, miR-1 and -9a. The potential functions of these recently identified genes are discussed and it is suggested that intronic enhancers are a common feature of the DV gene network (Biemar, 2006).

The Dl nuclear gradient differentially regulates a variety of target genes in a concentration-dependent manner. The gradient generates as many as five different thresholds of gene activity, which define distinct cell types within the presumptive mesoderm, neuroectoderm, and dorsal ectoderm. Total RNA was extracted from embryos produced by three different maternal mutants: pipe/pipe, Tollrm9/Tollrm10, and Toll10B. pipe/pipe mutants completely lack Dl nuclear protein and, as a result, overexpress genes that are normally repressed by Dl and restricted to the dorsal ectoderm. For example, the decapentaplegic (dpp) TU is strongly "lit up" by total RNA extracted from pipe/pipe mutant embryos. The intron-exon structure of the transcribed region is clearly delineated by the hybridization signal, most likely because the processed mRNA sequences are more stable than the intronic sequences present in the primary transcript. There is little or no signal detected with RNAs extracted from Tollrm9/Tollrm10 (neuroectoderm) and Toll10B (mesoderm) mutants. Instead, these other mutants overexpress different subsets of the Dl target genes. For example, Tollrm9/Tollrm10 mutants contain low levels of Dl protein in all nuclei in ventral, lateral, and dorsal regions. These low levels are sufficient to activate target genes such as intermediate neuroblasts defective (ind), ventral neuroblasts defective (vnd), rhomboid (rho), and short gastrulation (sog) but insufficient to activate snail (sna). In contrast, Toll10B mutants overexpress genes (e.g., sna) normally activated by peak levels of the Dl gradient in ventral regions constituting the presumptive mesoderm (Biemar, 2006).

To identify potential Dl targets, ranking scores were assigned for the six possible comparisons of the various mutant backgrounds, pipe vs. Tollrm9/Tollrm10, pipe vs. Toll10B, Tollrm9/Tollrm10 vs. Toll10B, Tollrm9/Tollrm10 vs. pipe, Toll10B vs. Tollrm9/Tollrm10, and Toll10B vs. pipe, using the TiMAT software package. As a first approximation, only hits with a median fold difference of 1.5 and above were considered. For further analysis, the top 100 TUs were selected for each of the comparisons, with the exception of Tollrm9/Tollrm10 vs. pipe for which the TiMAT analysis returned only 43 hits that meet the cutoff. To refine the search for TUs specifically expressed in the mesoderm, where levels of nuclear Dl are highest, only those present in the Toll10B vs. Tollrm9/Tollrm10 and Toll10B vs. pipe, but not pipe vs. Tollrm9/Tollrm10 comparisons were selected. For TUs induced by intermediate and low levels of nuclear Dl in the neuroectoderm, those present in both the Tollrm9/Tollrm10 vs. Toll10B and Tollrm9/Tollrm10 vs. pipe, but not pipe vs. Toll10B comparisons were selected. For TUs restricted to the dorsal ectoderm, only those present in the pipe vs. Tollrm9/Tollrm10 and pipe vs. Toll10B, but not Tollrm9/Tollrm10 vs. Toll10B, were selected. Finally, the TUs corresponding to annotated genes already identified in the previous screen were eliminated to focus on annotated genes not previously considered as potential Dorsal targets, as well as transcribed fragments (transfrags) not previously characterized. Using these criteria, 45 previously annotated protein-coding genes were identified, along with 23 uncharacterized transfrags. Of the 45 protein-coding genes, 29 exhibited localized patterns of gene expression across the DV axis, whereas the remaining 16 were not tested (Biemar, 2006).

In addition to protein-coding genes, the tiling array also identified uncharacterized TUs not previously annotated. Some of them are associated with ESTs, providing independent evidence for transcriptional activity in these regions. For 14 of these transfrags (61%), visual inspection of neighboring loci using the Integrated Genome Browser suggested coordinate expression of a neighboring protein-coding region (i.e., overexpressed in the same mutant background). The N-Cadherin gene (CadN) has a complex intron-exon structure consisting of ~20 different exons. The strongest hybridization signals are detected within the limits of exons, but an unexpected signal was detected ~10 kb upstream of the 5'-most exon. It is specifically expressed in the mesoderm, suggesting that it represents a previously unidentified 5' exon of the CadN gene. Support for this contention stems from two lines of evidence: (1) in situ hybridization using a probe against the 5' exon detects transcription in the presumptive mesoderm, the initial site of CadN expression; (2) using primers anchored in the 5' transfrag as well as the first exon of CadN, confirmation was obtained by RT-PCR that the recently identified TU is part of the CadN transcript. This recently identified 5' exon appears to contribute to the 5' leader of the CadN mRNA. It is possible that this extended leader sequence influences translational efficiency as seen in yeast. Because there seems to be a considerable lag between the time when CadN is first transcribed and the first appearance of the protein, it is suggested that this extended leader sequence might inhibit translation. An interesting possibility is that it does so through short upstream ORFs, as has been shown for several oncogenes in vertebrates (Biemar, 2006).

The variable transmembrane domain of Drosophila N-cadherin regulates adhesive activity

Drosophila N-cadherin (CadN) is an evolutionarily conserved classic cadherin which has a large, complex extracellular domain and a catenin-binding cytoplasmic domain. The CadN locus contains three modules of alternative exons (7a/b, 13a/b, and 18a/b) and undergoes alternative splicing to generate multiple isoforms. Using quantitative transcript analyses and green fluorescent protein-based cell sorting, it was found that during development CadN alternative splicing is regulated in a temporal but not cell-type-specific fashion. In particular, exon 18b is predominantly expressed during early developmental stages, while exon 18a is prevalent at the late developmental and adult stages. All CadN isoforms share the same molecular architecture but have different sequences in their extracellular and transmembrane domains, suggesting functional diversity. In vitro quantitative cell aggregation assays revealed that all CadN isoforms mediate homophilic interactions, but the isoforms encoded by exon 18b have a higher adhesive activity than those by its alternative, 18a. Domain-swapping experiments further revealed that the different sequences in the transmembrane domains of isoforms are responsible for their differential adhesive activities. CadN alternative splicing might provide a novel mechanism to fine-tune its adhesive activity at different developmental stages or to restrict the use of high-affinity 18b-type isoforms at the adult stage (Yonekura, 2006; full text of article)

Protein Interactions

Immunoprecipitation of Cadherin-N shows that it binds not only to alpha-Catenin but also to beta-Catenin/Armadillo (Arm). Alternative splicing generates two Arm isoforms: the 105 kDa ubiquitous form and the 82 kDa neural form (Loureiro and Peifer, personal communication to Iwai, 1997). Cadherin-N associates predominantly with the 82 kDa Arm, whereas Shotgun preferentially binds to the 105 kDa isoform. Transfection of Drosophila cultured cells with Cadherin-N cDNA induces cells to form aggregates. The activity of Cadherin-N is comparable to that of Shotgun (Iwai, 1997).

Cadherin-N seems to be the major cadherin that assembles catenins in axons. Mutants were isolated that produce only a small amount of Cadherin-N. Alpha-Catenin expression was compared between these mutant and wild-type embryos. Axonal expression of alpha-Catenin is greatly down-regulated in mutants although neuronal cell bodies retain a low level of alpha-catenin signals. In contrast, even in these mutants, alpha-Catenin is normally present in the midline glial cells and epithelia that synthesize Shotgun. The level of 82 kDa Arm, as opposed to that of the 105 kDa ubiquitous form, is preferentially reduced in the mutants (Iwai, 1997).

The Arf-GEF Schizo/Loner regulates N-cadherin to induce fusion competence of Drosophila myoblasts

Myoblast fusion is a key process in multinucleated muscle formation. Prior to fusion, myoblasts recognize and adhere to each other with the aid of cell-adhesion proteins integrated into the membrane. Their intracellular domains participate in signal transduction by binding to cytoplasmic proteins. This study identified the calcium-dependent cell-adhesion protein N-cadherin as the binding partner of the guanine-nucleotide exchange factor Schizo/Loner in Drosophila melanogaster. N-cadherin was expressed in founder cells and fusion-competent myoblasts of Drosophila during the first fusion phase. The genetic analyses demonstrated that the myoblast fusion defect of schizo/loner mutants is rescued in part by the loss-of-function mutation of N-cadherin, which suggests that Schizo/Loner is a negative regulator of N-cadherin. Based on these findings, a model is proposed where N-cadherin must be removed from the myoblast membrane to induce a protein-free zone at the cell-cell contact point to permit fusion (Dottermusch-Heidel, 2012).

This study has identified the calcium-dependent cell-adhesion protein N-cadherin as a Schizo/Loner interaction partner. N-cadherin null mutants do not phenocopy the schizo/loner mutant phenotype. Instead, it was found that the schizo/loner loss-of-function phenotype can be partially rescued when N-cadherin is removed. The data suggested that regulation of N-cadherin from the myoblast membrane of schizo/loner mutants is important for the fusion process to proceed (Dottermusch-Heidel, 2012).

Drosophila and mammalian myoblast fusion is based on the ability of myoblasts to recognize and adhere. In Drosophila, members of the immunoglobulin superfamily (IgSF) are involved in this process. Duf, Rst and Sns are expressed in a ring-like manner and serve as a platform for the formation of a signalling centre called FuRMAS (Fusion-Restricted Myogenic-Adhesive Structure). The diameter of the cell-adhesion ring ranges from 1 to 5μm. FCMs with 5μm cell-adhesion rings at cell-cell contact points are flattened and closely attached to the FC/growing myotube (Kesper, 2007). In some fusion mutants, the adhesion ring fails to expand up to 5μm (Kesper, 2007). This suggests that cell-adhesion proteins move away from the contact zone. One prerequisite for the fusion of lipid bilayers is that proteins must be cleared from the site of fusion. Therefore, the alteration of the ring size may be due to the redistribution of the cell-adhesion proteins of the IgSF class at the myoblast membrane. IgSF proteins and cadherins have also been implicated in mediating myoblast adhesion during skeletal muscle development (Pavlath, 2010). A requirement for the removal of M-cadherins from the site of fusion has also been suggested by studies on rat Ric10 satellite cells (Dottermusch-Heidel, 2012).

The intracellular domains of the IgSF cell adhesion molecules are essential for binding intracellular proteins, which are involved in signal transduction during myoblast fusion (reviewed by Abmayr, 2012) or might promote the redistribution of the cell adhesion molecules. One important consequence of cell recognition and adhesion is the formation of dynamic F-actin at the site of fusion (reviewed by Abmayr, 2012; Dottermusch-Heidel, 2012).

Schizo/Loner is an essential intracellular protein during Drosophila myoblast fusion. However, the function of Schizo/Loner during myoblast fusion remained unclear. Biochemical data from Chen (2003) and Bulchand (2010) imply that Schizo/Loner binds to the intracellular domain of the cell-adhesion molecule Duf. Furthermore, Chen (2003) has shown that schizo/loner mutants display an aberrant Rac localization, which indicates that Schizo/Loner is involved in actin regulation during myoblast fusion. In accordance with these findings, Richardson (2007) found that the number of F-actin foci is increased in schizo/loner mutants. Even so, actin foci do not colocalize with Schizo/Loner and it was suggested that Schizo/Loner regulates fusion independently of actin foci (Dottermusch-Heidel, 2012).

This study identifies the cell-adhesion molecule N-cadherin as a Schizo/Loner binding partner, and proposes an additional new function of Schizo/Loner during myoblast fusion. N-cadherin is present at the membrane of FCs and FCMs during the first phase of myoblast fusion. Similarly, the mammalian classical cadherins N-cadherin and M-cadherin are also expressed at the plasma membrane of myoblasts and satellite cells. However, the loss of N-cadherin and M-cadherin does not impair myoblast fusion in mammals, which indicates that N-cadherin can compensate for the loss of M-cadherin or vice versa. As found in mammals, this study found that the loss of Drosophila N-cadherin does not disturb myoblast fusion and a quantitative analysis revealed that fusion is neither decreased nor increased in N-cadherin loss-of-function mutants. Since Drosophila lacks M-cadherin, whether N-cadherin is functionally redundant with the classical cadherins N-cadherin2 or E-cadherin during fusion was investigated. No evidence was found that either N-cadherin2 or E-cadherin are able to compensate for the loss of N-cadherin in Drosophila (Dottermusch-Heidel, 2012).

At the transcriptional level, N-cadherin expression seems to be controlled by the transcription factor Dmef2. Genetic data indicated that Schizo/Loner is involved in the post-translational regulation of N-cadherin. The severe fusion phenotype of schizo/loner mutants was partially rescued by the loss of N-cadherin. Protein-interaction data in yeasts suggested that the N-terminal region of Schizo/Loner (amino acids 116-323 according to SchizoP2) directly binds to the intracellular domain of N-cadherin. Since the recognition and adhesion of FCs and FCMs in schizo/loner mutants still occurs, it is proposed that N-cadherin must be removed from the site of fusion in these mutants to permit fusion. However, why N-cadherin is expressed initially at the myoblast membrane remains to be elucidated. At CNS synapses, the surface concentration of cadherins is critical in determining the strength of adhesion. The trans-interaction of cadherin molecules results in weak interactions, whereas cis-clustering of two or more cadherins results in the formation of strong adhesion complexes. It is possible that N-cadherin first initiates a loose adhesion between FCs and FCMs and that the IgSF proteins stabilize cell adhesion and restrict the area of membrane fusion. That muscle formation in CadNM19; sizC128/U112 double mutants does not proceed completely may be an indication that the negative regulation of N-cadherin is not the only function of Schizo/Loner during myoblast fusion (Dottermusch-Heidel, 2012).

In transfected SL2 cells, Schizo/Loner and N-cadherin are associated with small vesicles, which suggested that Schizo/Loner regulates N-cadherin by controlling vesicle trafficking. The first experiments using the mesodermal transcription factor twist as GAL4 driver line to express dominant-negative Dynamin suggested that Dynamin might be involved in myoblast fusion. However, a block of Dynamin-dependent endocytosis specifically in FCs or FCMs did not disturb myoblast fusion. The finding that the number of FCs is higher in embryos expressing dominant-negative Shibire with twist-GAL4 led to a proposal that more FCs are determined and that the observed failure in myoblast fusion is due to a secondary defect, namely in cell-fate specification (Dottermusch-Heidel, 2012).

Interestingly, it was discovered that expression of activated Drosophila d-Arf1 in FCs and FCMs of schizo/loner mutants leads to a partial rescue, as similarly observed in CadNM19; sizC128/U112 double mutants, and that d-Arf1 interacts genetically with N-cadherin. This suggests that regulation of N-cadherin is dependent on Schizo/Loner and d-Arf1. Arf1 has been extensively studied in mammals and has been proposed to be involved Cdc42-dependent endocytosis, which does not require Shibire function. This might explain why the expression of dominant-negative Shibire does not result in a schizo-like phenotype during myoblast fusion (Dottermusch-Heidel, 2012).


DEVELOPMENTAL BIOLOGY

Embryonic

Cadherin-N mRNA is first seen within nuclei of presumptive mesodermal cells prior to gastrulation at stage 5. mRNA transport to the cytoplasm starts at about stage 6-7, and the messengers are distributed throughout the cytoplasm by stage 8. Cadherin-N protein first appears at intercellular contacts in the mesoderm at stage 9, and then the protein is detected at boundaries of mesodermally derived cells that inititate transcription of the Shotgun gene at stage 13. Cadherin-N also appears in developing neural cells, presumably at their postmitotic stage; subsequently, Cadherin-N accumulates in axons of the entire CNS. At the subcellular level, neuronal processes including growth cones are labeled. Gastrulation and neurulation coincide with a switch of cadherin expression from Shotgut to Cadherin-N. Glial cells do not express Cadherin-N (Iwai, 1997).

During Drosophila gastrulation, morphogenesis occurs as a series of cell shape changes and cell movements that probably involve adhesive interactions between cells. The dynamic aspects of cadherin-based cell-cell adhesion were examined in the morphogenetic events to assess the contribution of such activity to morphogenesis. Shotgun and Cadherin-N show complementary expression patterns in the presumptive ectoderm and mesoderm at the mRNA level. Switching of cadherin expression from the Shotgun to the CadN type in the mesodermal germ layer occurs downstream of the mesoderm-determination genes twist and snail. In contrast to twi and sna mutations, folded gastrulation mutants show normal replacement of Shotgun with CadN in cells corresponding to the mesoderm. Shotgun mRNA is present uniformly in the embryo until late stage 5, but it begins to disappear in the presumptive mesoderm shortly before the onset of ventral furrow formation. After stage 7 Cadherin-N mRNA is visible in the mesoderm. However, examination of cadherin protein expression patterns shows that considerable amounts of Shotgun remains on the surfaces of mesodermal cells during invagination, while CadN does not appear on the cell surfaces at this stage. Further immunocytochemical analysis of the localizations of Shotgun and its associated proteins Armadillo (beta-catenin) and Dalpha-catenin reveals dynamic changes in their distributions that are accompanied by changes in cell morphology in the neuroectoderm and mesoderm. Shotgun, together with Armadillo and Dalpha-catenin, most strongly accumulate at apical contacts of neuroectodermal cells, at the same time that large apical junctions (AJs) are observed at the corresponding sites. As soon as the germ band starts to elongate (stage 8), the apical accumulation along lateral cell surfaces becomes disordered or obscure. Adherens junctions, based on the cadherin-catenin system, change their location, size, and morphology. At this time large AJs are rarely found. During mesodermal invagination, as invaginating mesodermal cells are converted from wedge-shaped to round cells, Shotgun is gradually redistributed from AJs to a uniform distribution over the entire cell surface, including the cell contact-free areas in rounded mesodermal cells at stage 8. After this stage, Shotgun is completely eliminated from the mesoderm, and Arm and Dalpha-catenin are reduced to undetectable levels. These dynamic aspects of cadherin-based cell-cell adhesion appear to be associated with the following: (1) initial establishment of the blastoderm epithelium; (2) acquisition of cell motility in the neuroectoderm; (3) cell sheet folding, and (4) epithelial to mesenchymal conversion of the mesoderm. These observations suggest that the behavior of the Shotgun-catenin adhesion system may be regulated in a stepwise manner during gastrulation to perform successive cell-morphology conversions. Also discussed are the processes responsible for loss of epithelial cell polarity and elimination of preexisting Shotgun-based epithelial junctions during early mesodermal morphogenesis (Oda, 1998).

Larval

In third instar larvae, Cadherin-N is expressed in CNS neuropil, photoreceptor axons, and precursors of adult muscles (Iwai, 1997).

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). 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, the mechanism of layer selection was defined. 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. It is proposed that Ncad isoforms do not form an adhesion code; rather, they provide permissive adhesion between R7 growth cones and their temporary targets (Ting, 2005).

Thus R7 layer-specific targeting occurs in two distinct stages: at the first stage, R7 afferents target to the R7-temporary layer where they remain for about one day until the mid-pupal stage (50% APF) when the R7 growth cones progress synchronously to their final target layer. The two-step target selection has been observed in the vertebrate hippocampus: during embryonic development, entorhinal axons and commissural and associational axons form transient synapses with Cajal-Retzius cells and GABAergic interneurons, respectively, before they synapse onto their postnatal targets, the pyramidal neurons. The two-stage R7 layer selection might serve to coordinate afferent innervation with target development, as in the hippocampus. Alternatively, it might function to reduce the number of potential targets among which R7 growth cones must choose (Ting, 2005).

Using the mutations that delete different afferent subsets or alter R7 connectivity, the mechanism of R7 layer selection has been defined. The genetic cell-ablation results suggest that R8, R7, and L1-L5 afferents target to their temporary layers independently. In addition, the wild-type R8 axons target correctly when the neighboring Ncad or LAR mutant R7s mistarget to the R8-recipient layer. Conversely, the removal of Ncad in single R8s disrupts R8 targeting without affecting the targeting of the neighboring R7s. Thus, the first stage of medulla layer-selection by R8, R7, and L1-L5 afferents probably involves primarily afferent-target interactions. In contrast, R1-R6 growth cone sorting to different lamina cartridges involves both afferent-afferent and afferent-target interactions, even though it required Ncad and LAR (Ting, 2005).

Developmental analyses of single Ncad mutant R7s revealed that Ncad is required for R7 axons to reach and to remain in the R7-temporary layer during the first layer-selection stage. On the basis of its homophilic activity and mutant phenotypes, it has been proposed that Ncad mediates the interaction between the R7 growth cones and the medulla target neurons. The medulla contains over 50 different types of neurons and many express Ncad during development. It is not technologically feasible at the current stage to remove Ncad activity in all or a large number of medulla neurons without affecting the pattern of the optic lobe. Thus, even though it was found that removing Ncad in small patches of medulla neurons does not affect R7 layer selection, the possibility cannot be ruled out that multiple medulla neurons provide redundant N-cadherin-mediated interactions for R7 growth cones in a similar fashion as L1-L5 neurons do for R1-R6 afferents. Alternatively, Ncad might function as a signaling receptor, rather than as a passive adhesive molecule in R7s. Recent studies demonstrated that the cytoplasmic domains of vertebrate classic cadherins can regulate the actin-cytoskeleton via catenins. The Drosophila Ncad contains the two conserved cytoplasmic regions that interact with catenins in vertebrate cadherins. It is conceivable that Ncad may regulate the actin-cytoskeleton in R7 growth cones in response to target-derived cues. Furthermore, although Ncad and LAR share the same adult phenotype, their differential onset of mutant phenotype and double mutant phenotype suggest that they probably regulate different aspects of the first R7 layer-selection stage. Ncad is required for R7 growth cones to initially target to, and remain in the R7-temporary layer throughout the first target-selection stage, while LAR is only required during the later phase (Ting, 2005).

The comparison between the first and second stages of R7 layer selection reveals a glimpse of the underlying mechanism. (1) Targeting to the R7-temporary layer at the first stage appears to be critical for the R7 axons to reach their final destination. Ncad or LAR mutant R7 axons that mistarget to the R8-temporary layer at the first stage, later proceed to terminate incorrectly as well at the R8-recipient layer. (2) In contrast to the initial target selection which follows the axon outgrowth, all R7 and R8 axons enter the second layer-selection stage at approximately the same time, regardless of when they arrive at the medulla. Interestingly, centripetal growth of R1-R6 terminals and synaptogenesis in the lamina coincide with the second stage of R7 layer-selection. It is tempting to speculate that a global signal triggers the initiation of the second stage (Ting, 2005).

In this study, it is reported that the Ncad gene in Drosophila, and probably in other insects, undergoes alternative splicing to generate multiple isoforms. However, the lack of isoform-specificity, revealed by the transgene rescue, overexpression experiments, and heterophilic interaction assays, argues against the hypothesis that the Ncad isoforms constitute an adhesion code to direct targeting specificity. Instead, the idea is favored that Ncad plays a permissive role in R7 layer selection. Nevertheless, the remarkable conservation of the Ncad alternative splicing over 250 million years of evolution suggests an adaptive advantage for Ncad molecular diversity, whose function awaits further investigation (Ting, 2005).

Cooperative activities of Drosophila DE-cadherin and DN-cadherin regulate the cell motility process of ommatidial rotation

Ommatidial rotation is a cell motility read-out of planar cell polarity (PCP) signaling in the Drosophila eye. Although the signaling aspects of PCP establishment are beginning to be unraveled, the mechanistic aspects of the associated ommatidial rotation process remain unknown. This study demonstrates that the Drosophila DE- and DN-cadherins have opposing effects on rotation. DE-cadherin promotes rotation; DE-cad mutant ommatidia rotate less than wild type or not at all. By contrast, the two DN-cadherins act to restrict this movement, with ommatidia rotating too fast in the mutants. The opposing effects of DE- and DN-cadherins result in a coordinated cellular movement, enabling ommatidia of the same stage to rotate simultaneously. Genetic interactions, phenotypic analysis and localization studies indicate that EGF-receptor and Frizzled-PCP signaling feed into the regulation of cadherin activity and localization in this context. Thus, DE- and DN-cadherins integrate inputs from at least two signaling pathways, resulting in a coordinated cell movement (Mirkovic, 2006).

Although the role for DE-cad in tissues undergoing rearrangements during development is established, a direct role for DE-cad in cell and tissue movement has been more difficult to study in vivo owing to its essential role in maintenance of epithelial integrity. Analysis of adult eye phenotypes of a homozygous viable shg/DE-cad allele and a dominant-negative DE-cad construct (DE-cadDN), expressed in the R3/R4 and later R1/R6, R7 precursors, indicate that DE-cad is required throughout the rotation process. The ability of ommatidia to complete the precise 90° rotation directly depends on DE-cad activity. Both the extracellular domain, responsible for cell-cell adhesion, and the intracellular domain, linking DE-cad to the actin cytoskeleton, are required for rotation. DE-cad associates with the actin cytoskeleton primarily through interactions with Arm/ß-catenin. Although ß-catenin has a dual role in cell adhesion and Wg signaling (which can be separated), these data indicate that during ommatidial rotation ß-catenin acts through its role in cell adhesion (Mirkovic, 2006).

Ommatidial rotation represents the final step in establishing PCP during eye development. The direction of rotation depends on proper R3/R4 cell fate specification, which is determined by PCP signaling. The Egfr pathway and input by rotation-specific genes, e.g. nemo, are thought to function in parallel to Fz-PCP signaling. An enhancement of the sev>DE-cadDN rotation defects was observed by dose reduction in core regulatory PCP genes dgo and stbm; ommatidial under-rotation and the number of ommatidia that did not initiate rotation in sev>DE-cadDN/dgo-/+, stbm-/+ was comparable with the enhancement of sev>DE-cadDN by heterozygosity of a shg null allele). The localization of PCP protein complexes at the level of adherens junctions is consistent with the idea that PCP factors can influence DE-cad function. The mechanism of this regulation remains unclear. The RhoA-RNAi transgene, which was expressed only in R3/R4 precursors during the initiation of ommatidial rotation, enhanced sev>DE-cadDN associated under-rotation defects. Although a RhoA requirement in multiple cellular processes makes it difficult to dissect its specific role in rotation, the specificity of the phenotype (enhanced under-rotation in sev>DEcad/RhoAIR) suggests a role for RhoA in the regulation of cadherin-mediated cell movement (Mirkovic, 2006).

Although Egfr signaling appears to be required for the precise 90° rotation, its role in the process - promoting motility or antagonizing it - has remained unclear. The genetic data suggest that Egfr signaling acts positively to promote rotation, since a reduction in Egfr signaling enhances the sev>DE-cadDN under-rotation phenotype. This may reflect a positive role for Egfr signaling in the regulation of DE-cad activity or turnover at the membrane, as suggested from human tumor cell lines. Affecting the function of endocytic pathway components can also have an effect on ommatidial rotation. This might be mediated by Egfr signaling, as is thought to be the case in human cancer cells, leading to recycling and redistribution of E-cad at the plasma membrane (Mirkovic, 2006).

Drosophila DN-cadherins, which are encoded by the adjacent cadN and cadN2 genes, are the main cadherins expressed in the nervous system. In developing photoreceptors they participate in axon guidance, and in pupal eye discs they mediate terminal patterning of the retina [through specific expression in cone cells. During PCP establishment, DN-cad1 is concentrated at the border between R3/R4 precursors, in a pattern largely complementary to DE-cad. This suggested a possible combinatorial role for DE-cad and DN-cad in rotation, with DN-cad either providing a structural role in rotating clusters, or participating in signaling cascades that regulate cell movement. Analysis of DN-cad mutant clones in discs during rotation demonstrated a specific function; many mutant clusters have completed rotation well before wild-type clusters of the same stage. These data indicate that DN-cadherins function to slow down rotation, serving an opposing function to DE-cad (Mirkovic, 2006).

The balance and complementary distribution of DE-cad and DN-cad appear crucial for correct rotation to occur. Mild overexpression of DN-cad1 in R3/R4 (sev>DN-cad) is sufficient to interfere with the process, possibly by affecting DE-cad levels. Consistently, DN-cad1 overexpression enhances sev>DE-cadDN induced under-rotation and overexpression clones of DN-cad1 cause a decrease in endogenous DE-cad levels. Alternatively, the negative effect of DN-cad on DE-cad might be through competition for ß-catenin, since sev>DE-cadDN is partially rescued by UAS-ArmS2, although since sev>DN-cad is not enhanced by arm dose reduction this appears less likely. Interestingly, sev>DN-cad is enhanced by co-expression of full-length DE-cad and full-length Arm. These phenotypes resemble those of a strong sev>DN-cad line, suggesting that DN-cad is stabilized by increased levels of available Arm, and also that co-overexpression of two cadherins may interfere with optimal turnover rate at the membrane (Mirkovic, 2006).

Effects of Mutation or Deletion

Cadherin-N is encoded by l(2)36Da, a gene contained within an interval subject to deletion mapping by Steward and Nusslein-Volhard. All previously isolated six l(2)36Da alleles exhibit abnormal staining patterns for Cadherin-N. Mutant surviving adults exhibit strongly uncoordinated or reduced locomotion (Iwai, 1997 and references).

Loss-of-function mutations of the gene resulted in either embryonic lethality or uncoordinated locomotion of adults. In the central nervous system of null mutant embryos, subsets of ipsilateral axons display a variety of aberrant trajectories including failure of position shifts, defective bundling, and errors in directional migration of growth cones. These results suggest that processes of axon patterning critically depend on DN-cadherin-mediated axon-axon interactions (Iwai, 1997).

Using visual behavioral screens in Drosophila, multiple alleles of N-cadherin have been identified. Removal of N-cadherin selectively from photoreceptor neurons (R cells) causes deficits in specific visual behaviors that correlate with disruptions in R cell connectivity. These defects include disruptions in the pattern of neuronal connections made by all three classes of R cells (R1-R6, R7, and R8). N-cadherin is expressed in both R cell axons and their targets. By inducing mitotic recombination in a subclass of eye progenitors, mutant R7 axons surrounded by largely wild-type R cell axons and a wild-type target were generated. R7 axons lacking N-cadherin mistarget to the R8 recipient layer. N-cadherin may be required for R7 to recognize processes within the R7 recipient layer. This may represent the initial contact involved in the formation of specific synaptic connections. As the relationship between layer selection and the formation of specific synapses has not been explored, this view remains speculative (Lee, 2001).

The distribution of N-cadherin protein was assessed at multiple stages during late larval and pupal development using antibodies specific to either its extracellular or intracellular domain. Similar results were obtained with both antibodies. N-cadherin is expressed on all R cells as they differentiate, and can be observed on the R8 axon as soon as it extends into the optic lobe. Strong expression was visible within the lamina plexus, where R1-R6 axons terminate, and within the medulla, including the region containing R7 and R8 termini. The expression pattern at the third larval stage is thus consistent with N-cadherin acting within R cell axons to control local topographic map formation in both the lamina and the medulla. N-cadherin immunoreactivity is observed also in the medulla neuropil, the developing lamina neuron L5 and the subretinal glial cells. N-cadherin may also function within these cells to contribute to patterns of R cell connectivity (Lee, 2001).

N-cadherin remains expressed on R1-R6 cell axons as they select lamina targets during midpupal development. Strong expression is also observed within several lamina target neurons in each column at this stage. Conversely, significant N-cadherin staining on any lamina glial cells could not be detected, although the possibility that N-cadherin might be expressed on these cells at a low level cannot be excluded. These observations are consistent with a direct role for N-cadherin during target selection within the lamina (Lee, 2001).

To assess the expression of N-cadherin at the developmental stage when the layer-specific targeting of R7 is taking place, N-cadherin expression was followed during the early phases of pupal development. At this stage of development, there is a gradient of developmental stages distributed across the medial/lateral axis of the medulla with the youngest R cell axons arriving at the lateral edge. The R7 and R8 terminals lie in two distinct layers in early pupal development with strong mAb24B10 and N-cadherin colocalization observed in the presumptive R8 layer and with weaker staining within the future R7 layer. Because R7 axons arrive later than R8s, there is a clear gradient of innervation within the R7 layer. In addition to expression in R7 and R8, N-cadherin is expressed in the region of the medulla neuropil between them but is not expressed at high levels in the region immediately above R8 or below R7 (Lee, 2001).

To confirm that the R8 and R7 terminals are indeed separate at this early stage, expression of an R7-specific axonal marker, PM181-Gal4, driving a membrane-tethered GFP reporter (UAS-mCD8-GFP), was examined. Expression of this marker in R7 commences prior to axonogenesis and, hence, can be used to label R7 growth cones early in their development. In contrast, mAb24B10 recognizes an antigen expressed in the R7 cell approximately 12 hr later. Using this R7-specific marker, it was observed that the R7 growth cone arrives in the medulla and immediately extends past the R8 layer. Moreover, as R8 but not R7 expresses mAb24B10 at this early stage, it was also confirmed that the R8 axon does not extend into the R7 target layer (Lee, 2001).

N-cadherin has multiple guidance functions in different neuronal cell types of the visual system. In R8 cells, N-cadherin is required for the formation of the normal topographic map. The defects found in null mutants likely reflect a role for N-cadherin in mediating interactions between R8 axons. N-cadherin is also essential for R1-R6 axons to choose correct synaptic partners in the lamina. In N-cadherin mutants, R1-R6 axons do not defasciculate from the ommatidial bundle and fail to reach their targets. This defect likely reflects a loss of N-cadherin-mediated interactions between R cell axons or between R cell growth cones and lamina neurons. N-cadherin is also required for R7 target specificity (Lee, 2001).

By analyzing individual N-cadherin mutant R7s in a mostly wild-type background, a function of N-cadherin in target specificity was uncovered. In particular, individual N-cadherin mutant R7 axons fail to terminate in the R7 target layer, M6, and instead terminated in the R8 target layer, M3. One simple model to explain the role of N-cadherin in R7 target selection is that it mediates homophilic adhesion between R7 growth cones and processes in the medulla neuropil. This adhesive interaction may promote R7 axon extension into the M6 layer. Indeed, the expression pattern of N-cadherin within the medulla neuropil is consistent with this view: N-cadherin is expressed throughout the region of R7 axon extension. Alternatively, N-cadherin could stabilize contact between the R7 terminus and processes in its target layer. Consistent with this notion, N-cadherin expression is observed later in pupal development in both the R7 terminals and surrounding medulla neuropil (Lee, 2001).

This analysis suggests that, just as there is a choice point at which R cell axons decide whether to terminate in the lamina or continue through to the medulla, there is a choice point for R7 and R8 axons in the medulla. Here R8 axons remain in the presumptive M3 layer while R7 axons extend further and terminate within presumptive M6. R7 layer selection occurs immediately upon entry of R7 terminals into the medulla region. Remarkably, at this early stage the termination sites differ by only 2-3 µm in distance. Further experiments examining mutant R7 growth cones as they extend into a normal target will resolve whether N-cadherin is required in R7 for the initial selection of the appropriate layer or stabilization of the R7-target interaction (Lee, 2001).

Since N-cadherin is expressed by both R7 and R8, differential expression of N-cadherin cannot account for the different choices made by these two growth cones. Additional regulatory mechanisms are envisioned to account for the differences in R7 and R8 target selection. Interestingly, mutations in a receptor tyrosine phosphatase, PTP69D, exhibit R7 targeting defects similar to those observed in N-cadherin mutants, although it is not known whether PTP69D functions in R7 cells or other R cell axons. Regulation of N-cadherin activity by receptor tyrosine phosphatases has been reported: the vertebrate receptor tyrosine phosphatase µ (PTPµ) can physically associate with N-cadherin and modulate its activity. In particular, disrupting PTPµ function in vitro slows outgrowth of retinal ganglion axons on an N-cadherin substrate (Burden-Gulley, 1999). This result suggests that PTPµ can positively regulate N-cadherin-mediated interactions that are required for axon outgrowth. It will be interesting to assess in future experiments whether differential regulation of N-cadherin activity by receptor tyrosine phosphatases at the R7/R8 choice point accounts for the differences in target selection made by these two axons (Lee, 2001).

Diverse functions of N-Cadherin in dendritic and axonal terminal arborization of olfactory projection neurons

The cadherin superfamily of cell adhesion molecules have been proposed to play important roles in determining synaptic specificity in developing nervous systems. Function was studied of N-cadherin in Drosophila second order olfactory projection neurons (PNs), each of which must selectively target their dendrites to one of 50 glomeruli. The results do not support an instructive role for N-cadherin in selecting dendritic targets; rather, N-cadherin is essential for PNs to restrict their dendrites to single glomeruli. Mosaic analyses suggest that N-cadherin mediates dendro-dendritic interactions between PNs and thus contributes to refinement of PN dendrites to single glomeruli. N-cadherin is also essential for the development of PN axon terminal arbors in two distinct central targets: regulating branch stability in the lateral horn and restricting high-order branching in the mushroom body. Although the N-cadherin locus potentially encodes eight alternatively spliced isoforms, transgenic expression of one isoform is sufficient to rescue all phenotypes (Zhu, 2004).

The olfactory neural circuit presents a fascinating wiring problem. From flies to mammals, olfactory receptor neurons (ORNs) expressing a common olfactory receptor have widely distributed cell body positions in sensory epithelia, yet their axons converge onto the same glomeruli in the antennal lobe/olfactory bulb, thereby forming the first spatial odor map in the brain. In Drosophila, second order olfactory projection neurons (PNs), the main postsynaptic targets of ORNs, send dendrites into specific glomeruli according to their lineage and birth order. Furthermore, PNs of a particular glomerular class exhibit stereotypical axon branching patterns and terminal fields in the lateral horn, one of the higher order olfactory centers, allowing stereotypical transfer and transformation of olfactory information farther into the brain. Thus, during the construction of the fly olfactory system, a given ORN must choose one of 50 glomeruli to target its axons, while a given PN must also choose one of 50 glomeruli to target its dendrites, and furthermore the PN must coordinate its dendritic target choice with its axon terminal arborization pattern in higher olfactory centers. Evidence suggests that at least the initial development of the olfactory circuit is independent of sensory input; for instance, the antennal lobe glomerular assembly, as well as the axon terminal arborization pattern, are largely complete before the first signs of olfactory receptor expression. Thus, wiring seems to be controlled predominantly by genetic programs. The molecular logic that underlies this striking wiring specificity is largely unknown (Zhu, 2004).

The wiring specificity of the fly olfactory system has been studied from the perspective of the second order PNs. Because lineage and birth order provide important information to determine where a PN should send its dendrites, it is likely that PNs of different glomerular classes express different combinations of transcription factors. A specific set of transcription factors might then direct the expression of a unique combination of cell surface receptors within PNs of a particular glomerular class, allowing their dendrites to choose a specific location in the antennal lobe and their axons to exhibit a specific terminal arborization pattern in higher olfactory centers. Indeed, a pair of POU domain transcription factors, Acj6 and Drifter, are differentially expressed in the two main PN lineages and regulate wiring specificity of PNs of these two lineages (Zhu, 2004).

To study cell-autonomous functions of candidate genes for their roles in PN wiring specificity, the MARCM strategy was used to generate positively labeled homozygous mutant clones in an otherwise unlabeled and largely heterozygous genetic background. Analysis focused on projection neurons that express GH146-GAL4, which accounts for 90 of the estimated 150-200 PNs. The majority of GH146-positive PNs (from here on referred to as PNs) are of the anterodorsal (adPNs) and lateral (lPNs) neuroblast lineages that innervate stereotypical, intercalated, yet nonoverlapping sets of glomeruli. These PNs can be visualized as neuroblast clones or single-cell clones using MARCM. Strong loss-of-function mutants in a number of cadherin superfamily cell adhesion molecules including N-cadherin, E-cadherin, Flamingo, Fat, and Dachsous. The only mutant that shows a detectable phenotype in PN dendrite or axon patterning is N-cadherin. The allele N-cadherinM19, which appears to be a complete loss-of-function allele, was used: it contains a stop codon in the extracellular domain, is nearly protein null when assayed with an antibody against an extracellular epitope before the stop codon, and it behaves genetically as a null in embryonic nervous system patterning (Zhu, 2004).

Developmental studies reveal that the dendrites of N-cadherin mutant PNs occupy the same positions as their wild-type counterparts during early pupal development, arguing against a function for N-cadherin in initial dendritic targeting. Furthermore, N-cadherin-/- dorsal and lateral neuroblast clones largely preserves their dendritic innervation pattern. It remains possible that N-cadherin may function in determining individual PN glomerular choices within either the dorsal or lateral neuroblast lineage. However, analysis of single-cell clones of DL1 and DA1 class (of which independent criteria are available to verify their identities) argues against this possibility. In contrast, the dendritic overspill phenotype is observed in every N-cadherin-/- single-cell clone, supporting a permissive role of N-cadherin in determining connection specificity in the antennal lobe. Analysis of the role of N-cadherin in ORNs also does not support a major instructive role in target selection of ORN axons (Zhu, 2004).

The fact that the N-cadherin locus encodes eight alternatively spliced isoforms makes it attractive to suppose that this molecular diversity is used for selective synaptic adhesion, thereby contributing to synaptic specificity. Yet it has been shown that UAS-GAL4-mediated transgene expression of a single isoform is sufficient to rescue dendritic overspill phenotypes of all classes of PNs examined. This observation suggests that differential expression of N-cadherin at the level of transcription or alternative splicing is not required for PN dendritic targeting specificity (Zhu, 2004).

At present the possibility exists that N-cadherin plays an instructive role in determining targeting specificity of a subset of PNs that were not analyzed (for instance GH146-negative PNs). It is also possible that N-cadherin may play a role in connection specificity in higher olfactory centers, especially in the lateral horn (Zhu, 2004).

This study nevertheless has uncovered an important and novel function for N-cadherin in restricting dendritic targeting to single glomeruli. Every PN class examined exhibited a remarkably similar phenotype: instead of dendrites of a single PN targeting to a single glomerulus, dendrites from N-cadherin mutant PNs spread to neighboring glomeruli. Developmental studies revealed a major function of N-cadherin in dendritic refinement. In wild-type animals, dendrites from a single PN initially occupy a proportionally larger area of the antennal lobe, presumably overlapping with dendrites of other PN classes. They then refine their dendritic domain to single glomeruli by 50 hr APF, concomitant with glomerular maturation. N-cadherin mutant dendrites initially occupy dendritic domains of roughly similar size, but they then fail to restrict their domains to a single glomerulus (Zhu, 2004).

Dendritic refinement of second order olfactory neurons appears to be a common feature in both flies and mammals. Mammalian mitral/tufted cells, equivalent to PNs, also send their apical dendrites into several glomerular structures during early development, subsequently refining to a single glomerulus. The mechanism of this refinement is unclear. Interestingly, loss of odor-evoked neuronal activity of ORNs does not affect mitral cell dendritic refinement. Likewise, Drosophila PN dendritic refinement occurs before any OR receptor expression and before morphologically mature synapses are detected in the antennal lobe, suggesting an activity-independent process (Zhu, 2004).

What kind of cell-cell interactions does N-cadherin mediate during PN dendritic refinement? Previously, N-cadherin has been proposed to mediate axonal target selection in the visual system of Drosophila. During development, PN dendritic refinement coincides with ORN axonal invasion into the antennal lobe; N-cadherin is expressed in both ORNs and PNs at this time. Based on these observations, it is attractive to hypothesize that N-cadherin mediates homophilic interactions between ORN axons and PN dendrites to refine neural processes. However, no positive evidence was found for N-cadherin-mediated interaction between ORN axons and PN dendrites in PN dendritic refinement. Rather, the reverse MARCM analysis demonstrates that N-cadherin in other PNs of the same lineage, including PNs that occupy the same glomerulus, is required for the uniglomerular targeting of wild-type PNs (Zhu, 2004).

What cellular mechanisms could explain both the autonomous and nonautonomous effects of N-cadherin in uniglomerular targeting? The simplest model is that N-cadherin expressed on the surface of PN dendrites confers proper adhesiveness to the dendrite during and after the initial targeting event. Loss of N-cadherin results in reduced cell adhesion, allowing dendrites to more easily invade the neighboring glomeruli or shooting out of the confines of the antennal lobe. The non-cell-autonomous effect of N-cadherin revealed by the reverse MARCM analysis suggests that their interaction partners are dendrites of other PNs, most likely of the same class and thereby occupying the same general domain. In this scenario, direct PN dendrite-dendrite interaction mediated by N-cadherin contributes to confining dendrites to individual glomeruli (Zhu, 2004).

Other alternative scenarios are less likely. For instance, the reverse MARCM phenotype could be explained if altered PN patterning as a result of the unlabeled mutant neuroblast clones leads to altered ORN axons, which then indirectly affect the labeled wild-type PN. However, at least at the VA1lm glomerulus, it has been shown that altered PN dendritic patterning does not result in a corresponding alteration of ORN axon arborization (Zhu, 2004).

Since N-cadherin is expressed in all PN classes, it must work together with other cell surface proteins eventually to refine dendrites to their class-specific glomeruli. Some of these cell surface proteins may allow dendrites to be targeted to their appropriate spatial locations; others may mediate repulsive interactions among dendrites of different PN classes (Zhu, 2004).

Based primarily on the developmental timing of PN dendritic patterning and ORN axon arrival, as well as studies of cellular constituents of the developing antennal lobe, it has been proposed that PN-PN interactions are utilized in creating a prototypic dendritic map before the arrival of ORN axons. Furthermore, since functional studies from mammals to moths suggest an autonomous role for ORNs in organizing glomerular pattern, it is proposed that ORN axons and PN dendrites may be capable of generating independent maps; ORN-PN interaction would then enable the registration of the two maps with one another. Analysis of the N-cadherin mutant phenotype and the interaction between mutant ORNs or PNs lends support to these hypotheses. (1) It is shown that PN-PN interactions do occur and are essential at least for the uniglomerular targeting of dendrites. (2) The fact that dendritic overspill of PNs does not result in a corresponding ORN axon overspill supports the view that ORNs deploy targeting strategies independent of their future postsynaptic partners. At the same time, disruption of N-cadherin in ORNs does not affect the initial dendritic refinement of those neurons. It is possible that further ORN-PN interaction during the synapse maturation period could play a stabilizing role in the maintenance of synaptic connections, possibly explaining why disruption of N-cadherin in ORNs results in a more diffuse dendritic projection in the adult (Zhu, 2004).

This analysis has revealed that N-cadherin has diverse functions in a single type of neuron, olfactory projection neurons. In addition to its important roles in restricting the dendrites of each PN to a single glomerulus in the antennal lobe, N-cadherin is required for PN axon terminal arborization in both the mushroom body and the lateral horn. Do these diverse functions of N-cadherin reflect local actions of N-cadherin in these three sites, or could N-cadherin directly act in one site, with the phenotypes in other sites being secondary consequences due to intraneuronal communication? Several lines of evidence support the notion that N-cadherin acts locally and independently in PN dendrites and axons: (1) N-cadherin is highly expressed in all three sites; (2) based on developmental analysis of the mutant phenotype, N-cadherin is required in overlapping time windows in these three sites. For instance, the antennal lobe phenotype suggests a continuous role of N-cadherin from before 18 hr APF to close to 50 hr APF, whereas its function in the lateral horn for DL1 PNs appears to be at 36-42 hr APF. The lack of clear-cut sequential actions makes the intraneuronal communication model less likely. (3) N-cadherin affects the dendrites of all classes of PNs but the axons of only a subset of PNs. For instance, although N-cadherin mutant DA1 single-cell clones exhibited dendritic phenotypes, the highly specific targeting of lateral horn terminal arborization to the anterior corner for the DA1 PN was unaffected in the N-cadherin mutant. Such uncoupling argues against the intraneuronal signaling model (Zhu, 2004).

Interestingly, axonal phenotypes in the two higher order olfactory centers appear qualitatively different. In the lateral horn, where PN axon terminal arborization is highly stereotyped according to glomerular class, a strikingly specific perturbation of selective terminal branches is observed in two classes of PNs. Developmental studies clearly demonstrate that N-cadherin affects lateral horn axon terminal branch stability, rather than its initial growth. This represents the first demonstration of selective stabilization of terminal branches mediated by a cadherin, which may well be important in establishing the specificity of synaptic connection of PN axons with their postsynaptic targets. Because of the lack of knowledge about the postsynaptic neurons and their dendritic fields in the lateral horn, the significance of loss of particular branches in synaptic specificity shall await future investigations. Finally, it was previously shown that the DL1 dorsal axon branch was also specifically disrupted in PNs deprived of the POU transcription factor Acj6. Interestingly, these ultimately similar phenotypes are mechanistically distinct: Acj6 affects dorsal branch formation, whereas N-cadherin affects its stabilization (Zhu, 2004).

The axonal branching pattern of PNs in the mushroom body is considerably less stereotyped than that in the lateral horn in wild-type animals. Here, at least for one class of PNs (DL1), N-cadherin functions to restrict exuberant higher-order branches, in contrast to its role in stabilizing selected branches in the lateral horn for the same class of PNs (Zhu, 2004).

It is possible that N-cadherin-mediated adhesion is differentially interpreted in different cellular contexts for specific purposes. Mechanistically, the diverse functions of N-cadherin could be accomplished by N-cadherin working together with different partners at the cell surface or utilizing different intracellular signaling pathways. At the cell surface, a Robo-mediated inhibition of N-cadherin function has recently been proposed. Intracellularly, the cytoplasmic domains of classic cadherins interact with β-, γ-, and δ-catenins. Components of Rho GTPase pathways have also been implicated in regulating cadherin-mediated cell-cell adhesions. The differential distribution of these cell surface partners and intracellular signaling components in different cellular compartments may explain the different actions of N-cadherin. While the molecular mechanisms await further investigations, this study underscores the pleiotropic functions of this important class of cell adhesion proteins in multiple aspects of olfactory circuit assembly (Zhu, 2004).

Afferent induction of olfactory glomeruli requires N-Cadherin

Drosophila olfactory receptor neurons (ORNs) elaborate a precise internal representation of the external olfactory world in the antennal lobe (AL), a structure analagous to the vertebrate olfactory bulb. ORNs expressing the same odorant receptor innervate common targets in a highly organized neuropilar structure inside the AL, the glomerulus. During normal development, ORNs target to specific regions of the AL and segregate into subclass-specific aggregates called protoglomeruli prior to extensive intermingling with target dendrites to form mature glomeruli. Using a panel of ORN subclass-specific markers, it has been demonstrated that in the adult AL, N-cadherin (N-cad) mutant ORN terminals remain segregated from dendrites of target neurons. N-cad plays a crucial role in protoglomerulus formation but is largely dispensible for targeting to the appropriate region of the AL. It is proposed that N-cad, a homophilic cell adhesion molecule, acts in a permissive fashion to promote subclass-specific sorting of ORN axon terminals into protoglomeruli (Hummel, 2004).

A striking feature of olfactory system organization is the evolutionarily conserved arrangement of ORN terminals into an odortopic map. Here, ORNs expressing the same odorant receptors innervate common targets in a highly organized neuropilar structure, the glomerulus. Mosaic animals in which N-cad was selectively removed from ORNs are largely devoid of glomeruli. ORN targeting to the appropriate region of the AL was not dependent upon N-cad. Developmental analysis reveals that N-cad is essential for protoglomerulus formation at an early stage of AL development. The analysis of N-cad thus supports the notion that targeting and glomerulus formation are distinct steps in constructing an olfactory sensory map (Hummel, 2004).

A series of studies in both vertebrate and invertebrates has underscored the key role played by ORNs in regulating glomerulus formation. Surgical ablation of the antennal primordium from the moth Manduca sexta results in the loss of glomerular structures, as does the genetic disruption of ORN development in Drosophila. Mouse ORNs form glomerular structures when forced to innervate ectopic sites in response to surgical or genetic ablation of the olfactory bulb. These interactions may be instructive, since the pattern of sexually dimorphic glomeruli in gynandromorphs in the moth reflects the sex of the ORNs, not the sex of AL cells. The molecular mechanisms that mediate the intrinsic capacity of ORNs to sort out in a subclass-specific manner are unknown. The finding that genetically mosaic animals in which ORNs deficient in N-cad lack glomeruli provides further evidence that ORNs are essential for glomerular development and simultaneously identify a molecular component required in this process (Hummel, 2004).

Developmental studies revealed a stereotyped pattern of glomerulus development in flies. ORNs send axons into the AL from early (18 hr) through late (100 hr) pupal development. Axons extend into the nerve fiber layer surrounding the incipient AL. Most ORNs project through the commissure to the contralateral AL. Studies with a panneuronal marker reveal that ORNs extend processes from the nerve fiber layer into the developing dendritic layer immediately after they reach the AL, on their way to the commissure. Using the 72OK marker (which labels axons that converge onto two ventral-medial glomeruli, VM1 and VM4), it has been demonstrated that axons of the same ORN subclass extend thin processes into a restricted area of the AL corresponding to the approximate position of the future glomerulus. These processes quickly grow and rapidly condense into a protoglomerulus (Hummel, 2004).

Protoglomeruli are largely peripheral to the mass of dendrites of AL projection neurons. Glomerulus formation ensues as ORN processes and PN dendrites intermingle. The sequence of events leading to glomerulus formation is in many ways phylogenetically conserved. It is important to note, however, that the dynamics of glomerulus formation differ between different subclasses of mouse ORNs. Nevertheless, in both vertebrates and invertebrates, the formation of protoglomeruli largely comprising the processes of ORNs occurs prior to extensive intercalation with dendrites of target neurons (Hummel, 2004).

N-cad mutant ORNs do not form protoglomeruli. 72OK ORNs lacking N-cad extend processes into the appropriate region of the lobe. In striking contrast to wild-type, however, rather than condensing into protoglomeruli at 35% pupal development (PD), ORN axons extend farther throughout the ventral medial region of the AL before retracting back to the surface of the neuropil. They remain segregated from the dendrites of target neurons. While markers for other subsets of ORNs early in development are not available, it is likely that the process visualized with 72OK will extend to other ORNs, as developmental analysis with a panneuronal marker reveals a widespread failure of protoglomerulus formation. Furthermore, adult N-cad mutant ORN processes terminate on the surface of the lobe in close proximity to the position of the glomerulus in a wild-type animal. These studies establish N-cad as a key regulator of the cellular interactions underlying the formation of protoglomeruli (Hummel, 2004).

The subclass-specific convergence of ORN axons reflects specific interactions between ORN terminals within the target region. In both the mouse and fly, the cell bodies of different ORN subclasses are scattered throughout the nasal epithelium and the antenna, respectively. ORN axons do not segregate into subclass-specific fascicles as they project to the target. Association between axon terminals takes place within the target region itself. That different ORNs show affinity for fibers of the same subclass is supported by studies in both organisms. In the mouse extratoes mutant, the olfactory bulb fails to form. ORNs terminate instead in a fibrocellular mass lacking PNs and only a small number of LNs. Despite the lack of target neurons, P2 neurons sort out into a glomerular-like structure. ORNs expressing the same odorant receptors show selective affinity. Mice expressing odorant receptor transgenes in ORNs that lie in inappropriate epithelial zones form ectopic glomeruli in regions of the olfactory bulb distinct from those formed by ORNs expressing the endogenous receptor. In some cases, however, axons from ORNs expressing the endogenous receptors are recruited into these ectopic glomeruli. A similar conclusion has been reached in Drosophila, exploring targeting defects in ORNs mutant for Dscam. ORNs lacking Dscam target to inappropriate regions of the AL; indeed, several subclasses of ORNs terminate in the wrong ganglion in these mutants. Nevertheless, ORNs of the same subclass sort out from fibers of other ORN classes to form separate, often adjacent, ectopic glomeruli (Hummel, 2004 and references therein).

The homophilic cell adhesion activity of N-cad supports a simple model for its function in the developing fly olfactory system. Here N-cad acts to promote interactions between ORN terminals of the same subclass. It is likely that this interaction is largely permissive in nature because N-cad is widely expressed, and removal of N-cad from ORNs leads to a similar axonal phenotype for all 10 ORN subclasses tested. In contrast to the permissive function for N-cad in protoglomerulus formation in flies, odorant receptors appear to play an instructive role in this process in vertebrates. Genetic and expression studies indicate that odorant receptors do not play a role in ORN convergence in flies, although it remains possible that a subset of these receptors not yet tested will, as in vertebrates, serve this function (Hummel, 2004).

Could N-cad play an instructive role in sorting? As ORNs target to the appropriate region of the lobe, the sorting problem is reduced to sorting among a small number of different ORN subclasses within a local region. Modulation of N-cad activity in different classes may provide sufficient specificity for sorting. Indeed, cells in culture expressing different levels of cadherins sort out from one another. As panneuronal expression of a single form of N-cad in ORNs rescues the N-cad mutant targeting phenotypes, it is unlikely that different levels of N-cad protein specified at the level of transcription or different isoforms produced by alternative splicing underlie specific sorting. Presumably, other cell surface components in fly ORNs mediate subclass-specific sorting to protoglomeruli modulating the core homophilic cell adhesion activity of N-cad (Hummel, 2004).

N-cad may not mediate adhesion between ORNs, but rather may promote interactions between ORNs and target dendrites. In the fly visual system, for instance, N-cad is required in R7 photoreceptor neurons to select the appropriate target region. Genetic mosaic studies support the view that this reflects interactions between R7 growth cones and their targets rather than between R7 neurons and other photoreceptor neurons. Since N-cad is expressed on both ORNs and target neurons as ORNs enter the target region, it is expressed at the right place and time to mediate interactions between them. However, recent studies (Zhu, 2004) indicate that glomerulus formation does not require N-cad in PN dendrites. N-cad may also play a subsequent role in mediating interactions between ORNs and targets, but in order to critically address this issue, it will be necessary to assess glomerulus development in animals in which N-cad is removed after formation of protoglomeruli (Hummel, 2004).

While adhesion between ORN terminals has emerged as a common theme from work in both vertebrate and invertebrate systems, specific interactions between axons within the target region may be a more widespread phenomenon. Indeed, in the Drosophila visual system, the exquisite target specificity of different subclasses of R1-R6 photoreceptor axons relies upon interactions between them. In wild-type animals, R1-R6 axons from a single ommatidium project away from each other to a distinct set of targets. This requires N-cad and it has been proposed that N-cad mediates adhesive interactions between R cells and their targets. While biochemical studies support an adhesive function for classical cadherins, the N-cad phenotypes in R1-R6 and ORNs are also consistent with a repellent function. N-cad on R cell axons may promote repulsive interactions between them rather than mediating adhesion between R cell axons and target cells. In the olfactory system, N-cad may promote repulsive interactions between different classes of ORNs, thereby leading to their segregation rather than promoting interactions between axon terminals of the same class. That N-cad may mediate repellent interactions between neurites is supported by genetic analysis by Zhu (2004) of a requirement for N-cad in mediating interactions between PN dendrites. It has been proposed that repellent interactions between R8 axons play a crucial role in elaborating a precise topographic map in the fly visual system and that the protocadherin Flamingo serves this function. Whether interactions between afferents are adhesive or repulsive, or indeed whether they interact in a more complex and instructive fashion, these studies raise the important issue that afferent interactions may play crucial and hitherto poorly appreciated roles in the elaboration of specific patterns of synaptic connectivity (Hummel, 2004).

The molecular mechanisms underlying the formation of an odortopic map presents a complex problem in cellular recognition. A major advance in this field was the discovery that odorant receptors in the mouse play an instructive role in ORN targeting. Given the striking conservation in the cellular organization in the olfactory system of the mouse and fly, it is surprising that fly odorant receptors do not serve this key targeting function. Target specificity in this system must rely on other cellular recognition mechanisms. The Ig superfamily protein Dscam plays a crucial role in targeting some, but not all, ORNs. In the absence of Dscam, some subclasses of ORNs target to inappropriate regions of the AL, where they form ectopic glomeruli. Hence, Dscam contributes to target specificity, whereas N-cad acts at a later step to promote the formation of protoglomeruli. It is anticipated that further studies on N-cad and Dscam, as well as other cell surface and signaling proteins identified in ongoing genetic screens for targeting mutants in the olfactory system, will lead to a detailed molecular understanding of the cellular interactions underlying the exquisite specificity of neuronal connections in the fly olfactory system (Hummel, 2004).

An isoform-specific allele of Drosophila N-cadherin disrupts a late step of R7 targeting

Drosophila N-cadherin is required for the formation of precise patterns of connections in the fly brain. Alternative splicing is predicted to give rise to 12 N-cadherin isoforms. An N-cadherin allele, N-cad18Astop, was identified that eliminates the six isoforms containing alternative exon 18A. This allele strongly disrupts the connections of R7 photoreceptor neurons. During the first half of pupal development, N-cadherin is required for R7 growth cones to terminate within a temporary target layer in the medulla. N-cadherin isoforms containing exon 18B are sufficient for this initial targeting. By contrast, 18A isoforms are preferentially expressed in R7 during the second half of pupal development and are necessary for R7 to terminate in the appropriate synaptic layer in the medulla neuropil. Transgene rescue experiments suggest that differences in isoform expression, rather than biochemical differences between isoforms, underlie the 18A isoform requirement in R7 neurons (Nern, 2005).

Multiple interactions control synaptic layer specificity in the Drosophila visual system

How neurons form synapses within specific layers remains poorly understood. In the Drosophila medulla, neurons target to discrete layers in a precise fashion. This study demonstrates that the targeting of L3 neurons to a specific layer occurs in two steps. Initially, L3 growth cones project to a common domain in the outer medulla, overlapping with the growth cones of other neurons destined for a different layer through the redundant functions of N-Cadherin (CadN) and Semaphorin-1a (Sema-1a). CadN mediates adhesion within the domain and Sema-1a mediates repulsion through Plexin A (PlexA) expressed in an adjacent region. Subsequently, L3 growth cones segregate from the domain into their target layer in part through Sema-1a/PlexA-dependent remodeling. Together, these results and recent studies argue that the early medulla is organized into common domains, comprising processes bound for different layers, and that discrete layers later emerge through successive interactions between processes within domains and developing layers (Pecot, 2013).

Although the growth cones of L1, L3, and L5 neurons target to different layers, they initially overlap within a common domain in the outer medulla. Based on biochemical interactions and the mistargeting phenotypes and protein expression patterns described in this paper, it is envisioned that CadN-dependent adhesive interactions restrict processes to the outer medulla and that PlexA-expressing tangential neurons prevent Sema-1a expressing growth cones from projecting into the inner medulla. L2 and L4 growth cones also appear to initially target to a common domain within the distal outer medulla, but do not require Sema-1a and CadN for this targeting step and thus utilize an alternative mechanism. Interestingly, the morphology of L2 and L4 neurons does rely on Sema-1a and CadN function, indicating that within lamina neurons, these molecules regulate different aspects of targeting. This is supported by the expression of Sema-1a and CadN in all lamina neuron subclasses during development (Pecot, 2013).

In mice separate channels encoding light increments (ON) and decrements (OFF) are spawned in the outer retina and relayed to different sublaminas of the inner plexiform layer (IPL). The current findings are reminiscent of recent studies in the mouse IPL (Matsuoka, 2011) in which Kolodkin and colleagues demonstrated that the processes of different subclasses of PlexA4-expressing amacrine cells are segregated to different OFF layers and that this requires both PlexA4 and Sema6A. Although these proteins act in a more traditional fashion as a receptor and ligand, respectively, they are expressed in a complementary fashion early in development when the developing neuropil is very thin, with PlexA expressed in the nascent OFF layer and Sema6A in the developing ON layers. This raises the intriguing possibility that, as in the medulla, different cells initially target to common domains, from which they then segregate into discrete layers. As Cadherin proteins are differentially expressed in a layered fashion in the developing IPL and defects in targeting are incomplete in both Sema6A and PlexA4 mutants (Matsuoka, 2011), it is possible that, as in the medulla, Semaphorin/Plexin repulsion acts in parallel with cadherin-based adhesion to control layer-specific patterning within the developing IPL (Pecot, 2013).

Taken together, these studies suggest that the restriction of processes to a common domain prior to their segregation into distinct layers may be a developmental strategy used in both the medulla and the vertebrate IPL. This step-wise process may represent a more general strategy for reducing the molecular diversity required to establish synaptic connections by limiting the potential synaptic partners that growth cones and nascent dendritic arbors encounter within the developing neuropil (Pecot, 2013).

After targeting to a common domain within the outer medulla, L3 growth cones undergo stereotyped changes in shape and position that lead to segregation into the M3 layer. Initially, L3 growth cones are spear-like, spanning much of the depth of the incipient outer medulla. They then expand and elaborate a myriad of filopodia before resolving into flattened synaptic terminals within the M3 layer. This transformation is marked by two prominent steps: extension of processes from one side of the lateral region of the growth cone into the incipient M3 layer and retraction of the leading edge of the growth cone from the incipient M5 layer (part of the domain shared by L1 and L5 growth cones) (Pecot, 2013).

It has been suggested that CadN may regulate the extension within M3, as this step is partially perturbed in CadN mutant growth cones. However, as CadN mutations affect the initial position of L3 growth cones within the outer medulla, the extension defect within the M3 layer may be indirect. By contrast, in sema-1a mutant growth cones, initial targeting is indistinguishable from wild-type, so defects in retraction away from the incipient M5 layer are likely to reflect a direct role for Sema-1a in this later step in growth cone reorganization. PlexA RNAi phenocopies a sema-1a null mutation and, thus, PlexA is also required for retraction and is likely to function on medulla tangential fibers, where it is most strongly expressed. In support of this, the tip of the L3 growth cone that retracts is in close proximity to these PlexA-expressing fibers (Pecot, 2013).

The function of Sema-1a/PlexA signaling in sculpting L3 growth cones appears to be distinct mechanistically from the earlier role it plays in confining the growth cones to a common domain. During initial targeting, PlexA acts as a barrier to L3 growth cones and prevents them from projecting beyond the outer medulla. Thus, at this early step, Sema-1a/PlexA interaction provides a stop signal for the leading edge of L3 (uncovered in double mutants with CadN). In the second step, however, Sema-1a/PlexA signaling promotes retraction into the M3 layer. How these diverse outputs of Sema-1a/PlexA signaling arise is unclear. Sema-1a may be coupled to different downstream effectors at each step, modified by association with other receptor subunits, or may be modulated by other extracellular signaling pathways (Pecot, 2013).

CadN may also play a role in the retraction of L3 growth cones away from the domain shared with L1 and L5 growth cones. In early pupal stages, disrupting CadN function, while leaving growth cone morphology largely spear-like, causes L3 axons to project deeper within the medulla. Under these conditions, Sema-1a function is sufficient to prevent the growth cones from extending beyond the outer medulla. Subsequently, CadN mutant L3 growth cones fail to move away from the outer medulla's proximal edge into the developing M3 layer and thus remain within the most proximal layer, M6. This suggests that CadN, while acting in parallel with Sema-1a to restrict L3 growth cones to the outer medulla initially, may also be required at later stages for movement of the L3 leading edge into the M3 layer. As CadN has been shown previously to regulate neurite outgrowth over cultured astrocytes, it may be required for L3 growth cones to move along adjacent processes. However, the initial projection of L3 axons into the medulla is not affected by CadN mutations, indicating that other components control this process. It also remains possible that the defect in growth cone retraction results indirectly from CadN's earlier role in targeting; this earlier role may account for the defects in growth cone extension within M3 (Pecot, 2013).

Disrupting CadN function in different neurons affects targeting in unique ways. For example, L5 axons lacking CadN target to the proper layer, but extend inappropriately within the layer into neighboring columns (Nern, 2008). In addition, CadN mutant R7 growth cones display abnormal morphology and, in contrast to mutant L3 growth cones, initially target correctly, but retract to a more superficial medulla region. Collectively, these findings demonstrate that CadN regulates divergent features of growth cone targeting in different contexts. This likely reflects molecular diversity between different growth cones and illustrates the importance of understanding how molecules act in combination to generate target specificity (Pecot, 2013).

These studies add to previous findings suggesting that column assembly relies on a precisely orchestrated sequence of interactions between different neuronal cell types (Nern, 2008; Timofeev, 2012). This study shows that, as L1, L3, and L5 growth cones expressing Sema-1a enter the medulla, they meet the processes of newly arriving tangential fibers expressing PlexA, which acting in parallel with CadN, prevents extension of these growth cones into the inner medulla. This timing may permit other Sema-1a-expressing growth cones to extend into the inner medulla at earlier stages; these growth cones may then use Sema-1a/PlexA signaling for patterning connections in the inner medulla or deeper neuropils of the lobula complex. Subsequent sculpting of the L3 growth cone, mediated by Sema-1a/PlexA and perhaps CadN, leads to its reorganization into an expanded terminal within M3. As L3 growth cones become restricted to the M3 layer, Netrin, secreted from L3 growth cones, becomes concentrated within the M3 layer, and this, in turn, attracts R8 growth cones to the M3 layer, as recently described by Salecker and colleagues (Timofeev, 2012; Pecot, 2013 and references therein).

Given the extraordinary cellular complexity of the medulla neuropil, with over 100 different neurons forming connections in different medulla layers, and the few mechanistic clues to layer specific targeting that have emerged so far, a complex interplay between different sets of neurons is envisioned to be required to assemble the medulla circuit. The availability of specific markers for many of these neurons, techniques to follow the expression of even widely expressed proteins at the single cell level as is described in this study, and the ability to genetically manipulate single cells during development provide a robust system for uncovering the molecular logic regulating the layered assembly of axon terminals, dendritic arbors, and synaptic connectivity (Pecot, 2013).


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Cadherin-N: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 March 2017

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