InteractiveFly: GeneBrief

Downs syndrome cell adhesion molecule 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | | References

BIOLOGICAL OVERVIEW

Dscam, a Drosophila homolog of human Down syndrome cell adhesion molecule (DSCAM), an immunoglobulin superfamily member, was isolated by its affinity to Dreadlocks (Dock), an SH3/SH2 adaptor protein required for axon guidance. Dscam, Dock and Pak, a serine/threonine kinase, act together to direct pathfinding of Bolwig's nerve, which contains a subclass of sensory axons, to an intermediate target in the embryo. Dscam also is required for the formation of axon pathways in the embryonic central nervous system. cDNA and genomic analyses reveal the existence of multiple forms of Dscam with a conserved architecture containing variable immunoglobulin (Ig) and transmembrane (TM) domains. Alternative splicing can potentially generate more than 38,000 Dscam isoforms. This molecular diversity is likely to contribute to the specificity of neuronal connectivity (Schmucker, 2000).

To gain insight into the mechanisms by which growth cones integrate guidance cues, a combined biochemical and genetic analysis of the Dock signal transduction pathway has been pursued. Dock is an adaptor protein containing 3 SH3 domains and a single SH2 domain, and is closely related to mammalian Nck. dock mutants show defects in axon guidance in the adult fly visual system and in the embryonic nervous system. Based on the role of the adaptor protein Grb-2 in linking receptor tyrosine kinases to Ras, it is proposed that Dock links guidance receptors to downstream regulators of the actin cytoskeleton. Pak, a p21-activated serine/threonine kinase, acts downstream of Dock in adult photoreceptor neurons. Dock binds through its second SH3 domain to Pak and Pak binds directly to Rho family GTPases, evolutionarily conserved regulators of the actin-based cytoskeleton. Genetic studies reveal that both Pak's kinase activity and its interaction with Rho family GTPases are essential for axon guidance (Schmucker, 2000 and references therein).

Dscam binds directly to multiple domains of Dock and is widely expressed on axons in the embryonic nervous system. Dscam is required for recognition of an intermediate targeting determinant for Bolwig's nerve: Dock and Pak are required for this step, and Dscam shows dosage-sensitive interactions with both dock and Pak. Based on these studies, it is proposed that Dscam recognizes a guidance signal(s) and translates it into changes in the actin-based cytoskeleton through Dock and Pak (Schmucker, 2000).

Since Dscam mutants are early larval lethal and Dscam is expressed on many axons in the developing embryonic CNS, assessment was made of whether mutations in Dscam disrupt axon pathways. Axon tracts in stage 16 embryos were visualized with monoclonal antibody BP102 and monoclonal antibody 1D4 an antibody to Fasciclin II. In wild-type embryos, mAbBP102 stains in a ladder-like pattern within the ventral nerve cord, highlighting the connectives and commissures. Dscam mutant embryos exhibit mild disorganization of these tracts. mAb1D4 stains three continuous fascicles on both sides of the midline. In Dscam, breaks in the connectives, predominantly of the outer two fascicles, are observed. Defects exhibit variable expressivity. In addition, axon bundles aberrantly cross the midline. These defects are seen in all embryos of two different heteroallelic combinations (Schmucker, 2000).

To gain insight into Dscam's role in guidance at a defined choice point for which both Dock and Pak also are required, focus was placed on Dscam's role in Bolwig's nerve axon guidance. The projection of Bolwig's nerve elaborates a simple and well-studied pathway. Bolwig's organ, a larval photosensitive structure, contains 12 photoreceptor neurons that extend a single bundle of axons during embryogenesis to their targets in the brain. Bolwig's nerve extends posteriorly along the embryonic optic stalk to the surface of the optic lobe. It then grows across the surface of the optic lobe to a presumptive intermediate target neuron, P2. At P2, the growth cones pause before projecting to their synaptic targets in the brain. Phenotypes can be readily assessed in embryos double-stained for Bolwig's nerve and P2, using mAb22C10 and anti-Bsh (Brain-specific homeobox protein), respectively. In stage 16 embryos carrying Dscam^E1 or Dscam^P over a deficiency, some 53%-58% of Bolwig's nerve projections were defective. In half of the 'abnormal projections' the entire nerve mistargets, whereas in the remainder only a subset of axons does. Mistargeting axons either project past P2 or stop prematurely. Many of the projections scored as wild type, however, show abnormal expansion of Bolwig's nerve terminus at P2. These guidance defects are unlikely to reflect an indirect consequence of defects in Bolwig's organ differentiation, because organ and cellular morphology in Dscam mutants are indistinguishable from wild type. Bolwig's nerve guidance phenotypes in Dscam mutants are similar to those seen in dock and Pak mutants. The penetrance of dock and Pak are higher than in Dscam. The incomplete penetrance of Dscam may reflect residual function due to maternal contribution. Alternatively, this may reflect redundancy: two additional genes encoding Dscam-related proteins have been identified in the fly genome (Schmucker, 2000).

Dscam RNA is expressed in Bolwig's organ as well as more generally within the CNS and PNS. The protein product is exclusively expressed on axon processes. To assess whether selective expression of Dscam in Bolwig's nerve is sufficient to rescue the mutant, a transgene encoding full-length Dscam driven by the GMR promoter (a strong transcriptional driver providing Bolwig's organ-specific expression) was constructed and it was introduced into the germline by P element DNA transformation. Two independent insertions were characterized. In a wild-type (or a Dscam mutant) background, 100% of the embryos carrying one or two copies of GMR-Dscam exhibit strong axon guidance phenotypes. Individual axons project in abnormal directions over the surface of the optic lobe and rarely contact P2. It is unclear whether this reflects the sensitivity of Bolwig's nerve guidance to increased levels of Dscam or misexpression in Bolwig's organ of an inappropriate isoform, or both. Due to the large size of the Dscam locus (61 kb), whether or not the wild-type gene rescues the mutant phenotype could not be assessed. In any case, the dominant phenotype precludes assessing transgene rescue of the mutant phenotype. In contrast, GMR-dock rescues 85% of dock mutant embryos (Schmucker, 2000).

Whether dock and Pak are functional components of a Dscam guidance pathway was assessed through genetic analysis. Mistargeting defects of Bolwig's nerve were observed in some 44% of the embryos heterozygous for both Dscam and dock. In contrast, only 4%-6% and 10%-13% of embryos heterozygous for either dock or Dscam, respectively, show defects. Similarly, whereas some 38% of embryos heterozygous for both Dscam and Pak have an abnormal Bolwig's nerve, only 5% were defective in embryos heterozygous for Pak. The synergistic interactions between Dscam, dock, and Pak, the similarity of complete loss-of-function phenotypes, and the physical interactions between these proteins are consistent with their acting together to mediate recognition between the Bolwig's nerve growth cones and P2 (Schmucker, 2000).

It is envisioned that during Bolwig's nerve guidance a signal from P2, an intermediate target, attracts Bolwig's nerve growth cones. In dock, Pak, and Dscam mutants, Bolwig's nerve frequently comes in close proximity to P2 and projects past it or deflects away from it. Hence, Dscam is likely to play a prominent role in recognizing a local attractive signal at P2. Since the Dscam phenotype in the CNS is more severe than that of dock, it is likely that Dscam also functions through other downstream components in other neurons (Schmucker, 2000).

How might binding of Dscam on a filopodial process to a ligand at P2 promote attraction? Two alternative models are proposed. (1) Dock and Dscam may form a complex in the absence of ligand. This interaction would be mediated by the SH3-1 and SH3-3 domains in Dock and two closely spaced PXXP sites in Dscam's cytoplasmic tail. Dock's SH3-2 domain may bind to the polyproline stretch in the C-terminal tail; this may prevent recruitment of Pak to the membrane in the absence of ligand. Upon encountering an attractive ligand at P2, however, it is envisioned that Dscam becomes tyrosine phosphorylated. This may promote rearrangement of the complex with Dock binding through its SH2 domain to phosphotyrosine. This conformational change cannot strictly depend upon the SH2 domain, as the SH3-1 and SH3-3 domains can functionally compensate for the SH2 domain. This conformational change would unmask the SH3-2 domain, thus facilitating recruitment of Pak to the complex. Pak, in turn, would promote actin reorganization and subsequent movement of the growth cone to P2. Alternatively, in the absence of ligand the cytoplasmic tail of Dscam may be in a conformation not accessible to Dock binding. In this model, ligand binding would promote both a conformational change in Dscam's cytoplasmic domain and tyrosine phosphorylation. Dock may then associate with Dscam and recruit Pak to the membrane. Candidates for additional components in this signaling pathway have been identified through biochemical and genetic studies and include a nonreceptor tyrosine kinase, an adaptor protein linked to actin, and a nonreceptor tyrosine phosphatase. A precise mechanistic understanding of the relationship between Dscam, Dock, and Pak and these additional components will require identification of the Dscam ligand and detailed biochemical studies (Schmucker, 2000).

Does human DSCAM signal through Nck and Pak? While the extracellular region of human and Drosophila Dscam share identical domain structures and highly related amino acid sequences, the intracellular domains appear unrelated. This may indicate that the human and fly proteins signal downstream via different pathways or that the interactions with Nck are mediated by other proline-rich sequences, phosphotyrosine residues or other associated proteins. Alternatively, there may be multiple mammalian DSCAM genes, one of which shares sequence homology with the cytoplasmic domain of the fly protein. While a partial sequence of a second DSCAM-related gene has been identified on human chromosome 11 (accession no. BAA86446), its cytoplasmic domain is conserved with human DSCAM and is unrelated to Drosophila Dscam (Schmucker, 2000).

Multiple forms of Dscam have been identified. These different forms encode proteins with the same architecture but with sequence variations in three different Ig domains and the transmembrane domain. Genomic analysis reveals that exons encoding these alternative domains are tandemly arranged. There are 12 alternative forms of exon 4 (encoding the N-terminal half of Ig2); 48 alternative forms of exon 6 (encoding the N-terminal half of Ig3); 33 alternative forms of exon 9 (encoding Ig7), and 2 alternative forms of exon 17 (encoding transmembrane domains). DNA sequence analysis supports the view that multiple forms of Dscam are generated by alternative splicing. If all combinations are permissible, 38,016 forms of Dscam may be generated. This level of diversity is comparable to that achieved in individual antibody protein subunits through rearrangement prior to further diversification via TdT-mediated nucleotide insertion and somatic mutation. For instance, it is estimated that, in humans, recombination generates some 9,180 variable heavy chains (51 VH segments, 30 D segments and 6 J segments). An extraordinary diversity of Dscam isoforms is expressed during development. As the mechanisms utilized to generate splicing of one of two alternative exons in many other well-characterized genes are poorly understood, it seems premature to entertain models for the molecular mechanisms regulating the 'either-or' splicing patterns for the 12, 48, and 33 densely packed alternative exons in Dscam. Alternative forms of human DSCAM have not been found in searches of available genomic sequences and ESTs. Hence, either alternatively spliced forms of DSCAM are not generated in mammals or diversity exists among DSCAM-related genes (Schmucker, 2000).

Several other cell surface proteins involved in neuronal connectivity or implicated in the process show considerable molecular diversity. The vertebrate olfactory receptors, encoded by a large gene family containing in excess of 1000 different sequences, play a key role in determining glomerular specificity in the olfactory bulb, although they do not appear to be the sole specificity determinants. Multiple forms of neurexins also have been proposed to play a role in synapse specificity. Alternative splicing of neurexins encoded by three different genes may give rise to more than a 1000 different isoforms differing in size and amino acid sequence. In contrast to neurexins, all Dscam isoforms share a common domain architecture. While loss-of-function studies have not yet critically addressed the role of neurexins in forming neuronal connections, their localization to synapses makes them intriguing candidates for contributing to connection specificity (Schmucker, 2000 and references therein).

Two cadherin cell adhesion molecule subfamilies containing multiple forms also have been implicated in specificity. There are some 20 different classic cadherins expressed in mammals. They are localized to synapses and, in some cases, show restricted patterns of expression correlating with specific functional pathways. Recently, a second family of cadherins, cadherin-related neuronal receptors (CNRs), have been identified in mice through a protein interaction screen and are expressed at synapses (Kohmura, 1998). Fifty-two different forms of CNRs have been identified in three different clusters in the human genome (Wu, 1999). The mRNAs encoding the mouse CNRs are expressed in a diffuse and rather general pattern within the nervous system. In the olfactory bulb, two different CNRs are coexpressed in a single neuron while a smaller fraction of neurons express only one or the other CNR. This raises the intriguing possibility that different combinations of CNRs in different neurons could impart connection specificity. Given the conservation between guidance receptors in flies and mammals, it is surprising that CNRs were not identified in the recently published DNA sequence of the Drosophila genome. While it is still unclear whether DSCAM-related genes in mammals may be alternatively spliced, different organisms may have evolved different strategies to generate diversity among related guidance receptors, as a means of specifying complex patterns of neuronal connectivity (Schmucker, 2000 and references therein).

The massive use of alternative exons in a combinatorial fashion to generate thousands of different protein isoforms all sharing a common architecture with differences in amino acid sequence is unprecedented within the nervous system. The physiological importance of these different isoforms remains entirely unclear. Diverse forms of Dscam may recognize distinct isoforms of specific ligands. That such alterations could lead to changes in ligand–receptor interaction are supported by the findings that alternative forms of Ig repeats in murine FGF receptor 1, generated by alternative splicing, exhibit marked differences in affinity for bFGF. Alternatively, Dscam may form dimers with other forms of Dscam or act as a coreceptor to modulate the activities of other guidance receptors. In this context, it has been proposed that accurate targeting of motoneurons to their specific muscle targets requires input from multiple receptor pathways. Future studies will identify proteins binding to the extracellular domain of Dscam, address whether different forms of Dscam are expressed in specific neurons, and test the functional importance of diversity (Schmucker, 2000).

A double S shape provides the structural basis for the extraordinary binding specificity of Dscam isoforms

Drosophila Dscam encodes a vast family of immunoglobulin (Ig)-containing proteins that exhibit isoform-specific homophilic binding. This diversity is essential for cell recognition events required for wiring the brain. Each isoform binds to itself but rarely to other isoforms. Specificity is determined by 'matching' of three variable Ig domains within an approximately 220 kD ectodomain. This study presents the structure of the homophilic binding region of Dscam, comprising the eight N-terminal Ig domains (Dscam(1-8)). Dscam(1-8) forms a symmetric homodimer of S-shaped molecules). This conformation, comprising two reverse turns, allows each pair of the three variable domains to "match" in an antiparallel fashion. Structural, genetic, and biochemical studies demonstrate that, in addition to variable domain "matching," intramolecular interactions between constant domains promote homophilic binding. These studies provide insight into how "matching" at all three pairs of variable domains in Dscam mediates isoform-specific recognition (Sawaya, 2008).

Probabilistic splicing of Dscam1 establishes identity at the level of single neurons

The Drosophila Dscam1 gene encodes a vast number of cell recognition molecules through alternative splicing. These exhibit isoform-specific homophilic binding and regulate self-avoidance, the tendency of neurites from the same cell to repel one another. Genetic experiments indicate that different cells must express different isoforms. How this is achieved is unknown, as expression of alternative exons in vivo has not been shown. This study modified the endogenous Dscam1 locus to generate splicing reporters for all variants of exon 4. Splicing was shown not to occur in a cell-type-specific fashion, that cells sharing the same anatomical location in different individuals express different exon 4 variants, and that the splicing pattern in a given neuron can change over time. It is concluded that splicing is probabilistic. This is compatible with a widespread role in neural circuit assembly through self-avoidance and is incompatible with models in which specific isoforms of Dscam1 mediate homophilic recognition between processes of different cells (Miura, 2013).

This study took a genetic approach to visualize Dscam1 isoform expression. By monitoring exon 4 splicing as a surrogate for isoform expression in neurons, splicing was assessed in vivo broadly throughout the developing nervous system and in specific cell types, assessed splicing in single identified cells between different animals, and followed splicing in the same cell at different times during development. It was demonstrated that splicing is probabilistic in class IV da neurons where it is required for dendritic self-avoidance. The patterns of splicing in both the MB and L1/L2 neurons, where Dscam1 is required for axon branch self-avoidance and appropriate pairing at multiple contact synapses, respectively, are also consistent with a probabilistic mode of splicing (Miura, 2013).

The analysis of exon 4 splicing in class IV da neurons revealed that, on average each neuron expresses multiple exon 4 variants. The sum of the average expression probability of all the alternative variants of exon 4 in the class IV da neurons is 393 ± 38%, arguing the splicing of about four variants in a single neuron. If the splicing mechanism were probabilistic, not only at the level of single neurons but also at the level of each round of mRNA processing, it might be expected that the most abundantly spliced variant (i.e., exon 4.2) would be expressed in all neurons, albeit at varying levels, given enough rounds of transcription. Thus, the scattered splicing pattern within a neuronal population may reflect a splicing mechanism in which the same variant is included in multiple mRNAs whereas others are excluded (e.g., through the assembly of stable splicing complex associated with chromatin). Alternatively, this pattern of expression may result from low copy numbers of total Dscam1 mRNAs in each class IV da neuron (Miura, 2013).

The expression of multiple isoforms in each neuron is a key to robust self-avoidance. Previous studies using RT-PCR analysis on single MB neurons also indicated that each neuron expresses multiple variants of exon 9. Monte Carlo simulations and mathematical modeling suggest that expression of multiple isoforms in a neuron through probabilistic splicing can provide a robust mechanism to endow each neuron with a unique cell surface identity. Indeed, this robustness is supported by the observation that the differential Dscam1 expression in L1 and L2 neurons arises from probabilistic splicing in these neurons. The large number of isoforms encoded by Dscam1 is likely to be sufficient to offset the reduced diversity caused by biased exon usage. In addition, dynamic splicing further minimizes the risk that neighboring neurons share the same Dscam1 isoforms for an extended period (Miura, 2013).

Recent studies suggest that a similar mechanism for self-avoidance has evolved in vertebrates. In the mouse retina and cerebellum, self-avoidance is mediated by a large family of isoform- specific homophilic binding proteins encoded by the clustered protocadherin g locus (Lefebvre, 2012). RT-PCR analyses showed that Purkinje cells express different isoforms, and this is consistent with probabilistic expression of multiple protocadherin g isoforms in each neuron. In this case, regulation appears to be at the level of alternative promoter choice rather than alternative splicing. Thus, probabilistic expression may have evolved as a common strategy, albeit via different molecular mechanisms, by which neurons acquire unique self-identities (Miura, 2013).

Slit and Receptor tyrosine phosphatase 69D confer spatial specificity to axon branching via Dscam1

Axonal branching contributes substantially to neuronal circuit complexity. Studies in Drosophila have shown that loss of Dscam1 receptor diversity can fully block axon branching in mechanosensory neurons. This paper reports that cell-autonomous loss of the receptor tyrosine phosphatase 69D (RPTP69D) and loss of midline-localized Slit inhibit formation of specific axon collaterals through modulation of Dscam1 activity. Genetic and biochemical data support a model in which direct binding of Slit to Dscam1 enhances the interaction of Dscam1 with RPTP69D, stimulating Dscam1 dephosphorylation. Single-growth-cone imaging reveals that Slit/RPTP69D are not required for general branch initiation but instead promote the extension of specific axon collaterals. Hence, although regulation of intrinsic Dscam1-Dscam1 isoform interactions is essential for formation of all mechanosensory-axon branches, the local ligand-induced alterations of Dscam1 phosphorylation in distinct growth-cone compartments enable the spatial specificity of axon collateral formation (Dascenco, 2015).

This study reports on a molecular mechanism regulating Dscam1 activity in growth cones and provides insight in the regulation and spatial specificity of axon collateral formation. Biochemical and genetic results are consistent with the molecular model that the specificity of mechanosensory (ms)-axon branching arises from a spatially restricted change of Dscam1 phosphorylation in growth cone (Dascenco, 2015).

Previous studies on the function of Dscam1 have established the model that isoform-specific homophilic Dscam1-Dscam1 interactions trigger repulsion between sister dendrites. This controls for regular spacing of sister dendrites in a process termed neurite self-avoidance. In addition, cell-intrinsic and isoform-specific interactions have also been shown to be important in sensory axons for growth-cone sprouting and branching. Importantly, for both of these functions, it is thought that Dscam1 signaling is primarily dependent on and initiated by homophilic binding of matching isoforms present on sister neurites. The results reported in this study provide evidence that Dscam1-Dscam1 interactions in axonal growth cones are subject to branch-specific modulation by extrinsic cues. Binding of the ligand Slit to Dscam1 can locally enhance cis-interactions with the receptor tyrosine phosphatase RPTP69D as well as the dephosphorylation of Dscam1. Although homophilic Dscam1 interactions can be considered to play an initial permissive role in all neurite-neurite interactions in a sprouting growth cone, the spatial restriction of an extrinsic Dscam1 ligand likely initiates functional disparity of Dscam1 signaling across different growth-cone compartments (Dascenco, 2015).

The biochemical data support the notion that RPTP69D directly dephosphorylates Dscam1 at specific cytoplasmic tyrosines. Three candidate tyrosines were identified for the regulation of Dscam1 phosphorylation: Y1857, Y1890, and Y1981. Two of the tyrosine residues, Y1857 and Y1890, are part of consensus SH2-binding sites and therefore are likely involved in regulating recruitment of SH2-domain-containing adaptor molecules. Given that these mutations diminish the Dscam1 GOF effects, it seems reasonable to speculate that they are required for downstream signaling and/or receptor turn-over or trafficking. Surprisingly, the single Y1981F mutation causes strong dominant interference with axon branching where the phenotypic effects are qualitatively indistinguishable from a loss of Dscam1 isoform diversity, which is thought to increase the probability of matching isoform interactions (i.e., GOF activity). The primary amino acid sequence surrounding Y1981 does not reveal any distinct signaling motif. However, in silico 3D protein modeling based on structural predictions suggests that phosphorylation of Y1981 could directly result in structural changes of the Dscam1 cytoplasmic domain and thereby influence Dscam1 activity (Dascenco, 2015).

Biochemical results suggest that Slit can enhance Dscam1-RPTP69D complex formation and Dscam1 dephosphorylation. Furthermore, Slit-N can directly bind to the N-terminal Ig domains of Dscam1 (Ig1-4) with an affinity comparable to that of other guidance cue/receptor interactions, suggesting that Slit-N can function as a bona fide Dscam1 ligand. Numerous studies have shown that the repellent as well as the branch-promoting function of vertebrate Slit require the function of Robo receptors. The current results show that for the formation of specific axon collaterals of Drosophila ms-neurons, Slit functions via Dscam1 in a Robo1-3-independent pathway (Dascenco, 2015).

Slit is one of the best characterized 'axon-repellent' cues and also contributes to axon branching. Imaging single ms-axons and growth-cone branching, this study found that in Slit mutant animals, only filopodia or micropodia with a midline-directed growth direction are reduced, consistent with a positive role of Slit in promoting the extension of specific branches. In contrast, branch-point initiation in ms-neurons is likely independent of Slit or RPTP69D (Dascenco, 2015).

Given that high Slit protein concentrations are likely only encountered by filopodia- or micropodia-like extensions that reach the midline proximity, the Slit-Dscam1-RPTP69D interactions are likely only occurring in a spatially restricted sub-compartment of the branching growth cone. It is envisioned that the Dscam1-RPTP69D interactions in ms-axons constitute a molecular selection process, which depends on Dscam1-RPTP69D complex formation in a subset of axonal processes that encounter sufficient Slit protein. As a result, Dscam1 dephosphorylation by RPTP69D is increased locally and triggers a response by either promoting axon-branch extension or blocking repulsion (Dascenco, 2015).

The loss of only a subset of axon branches in RPTP69D/Slit mutants suggests that there are multiple molecular control pathways accounting for the selection of different axon collaterals or the extension of the main axon shaft. Although this study has focused on RPTP69D and Slit, it is most likely that other co-receptors and extracellular cues control the activity of Dscam1 in growth cones (Dascenco, 2015).

Dendrite self-avoidance is controlled by Dscam

Dendrites distinguish between sister branches and those of other cells. Self-recognition can often lead to repulsion, a process termed 'self-avoidance.' Dendrite self-avoidance in Drosophila da sensory neurons requires cell-recognition molecules encoded by the Dscam locus. By alternative splicing, Dscam encodes a vast number of cell-surface proteins of the immunoglobulin superfamily. Interactions between identical Dscam isoforms on the cell surface underlie self-recognition, while the cytoplasmic tail converts this recognition to dendrite repulsion. Sister dendrites expressing the same isoforms engage in homophilic repulsion. By contrast, Dscam diversity ensures that inappropriate repulsive interactions between dendrites sharing the same receptive field do not occur. The selectivity of Dscam-mediated cell interactions is likely to be widely important in the developing fly nervous system, where processes of cells must distinguish between self and nonself during the construction of neural circuits (Matthews, 2007).

These data demonstrate a cell-autonomous role for Dscam function in self-avoidance in all classes of Drosophila da neurons. da neurons associate closely with the epidermis as they extend across the body wall; thus, their dendrites create a two-dimensional meshwork in which developing branches frequently encounter other dendrites. This is in contrast to the layout of the CNS, in which axons and dendrites usually elaborate in three dimensions. By examining da neurons, it was possible to analyze the behavior of individual branches within a single dendritic arbor at high resolution. This allowed critical quantitative examination of the mechanisms underlying selective recognition between dendrites. The data show that when deficient in Dscam function, individual dendrites do not recognize sister branches and fail to initiate repulsion, leading to a breakdown in self-avoidance. Individual branches of Dscam mutant cells often failed to evenly disperse across their territory. Additionally, processes from specific da neurons gathered at nonrandom, discrete target sites within their territory (see below) (Matthews, 2007).

Dscam is likely to play a similar role in the CNS based on axonal- and dendritic-arborization phenotypes. In the olfactory system, for example, the terminal processes of single mutant olfactory receptor neurons, projection neurons, and interneurons form clumps. While this might reflect a phenotype of self-avoidance, the resolution of these studies was not sufficient to distinguish between self-avoidance and other mechanisms such as branch extension and synapse formation. By contrast, single-branch resolution has been achieved for Dscam defects in the axonal projections of MB neurons. Dscam is required for proper segregation of sister axon branches, and analogous to the self-avoidance control in da neurons, specific isoforms do not appear to provide instructive cues for this segregation event. It has been argued that this reflects a role for Dscam in mediating self-recognition and repulsion between these axons. In da neurons, dendrite self-avoidance defects were separable from growth, branching, and targeting errors and were fully penetrant. Thus, these data directly implicate Dscam in self-avoidance and demonstrate this role at the level of interactions between individual branches (Matthews, 2007).

The simplest model for a direct role for Dscam in self-recognition is one in which identical Dscam ectodomains on the surfaces of isoneuronal dendrites recognize each other and induce a subsequent repulsive signal that is mediated by domains in the cytoplasmic tail. This model is supported by both in vitro and in vivo data presented in this paper. First, identical Dscam isoforms expressed in two cell populations in vitro induced their aggregation in an isoform-specific manner, showing that Dscam provides cells with the ability to distinguish between different cell surfaces. Second, ectopic expression of identical Dscam isoforms on the dendrites of different cells, which normally overlap, promoted growth away from each other (Matthews, 2007).

How can in vitro adhesion be reconciled with in vivo repulsion? The data suggest that the dendrites of da neurons convert an initial Dscam-dependent cell-surface interaction into a repulsive response, which leads to dendrite separation and receptive field elaboration. da dendrites expressing a form of Dscam in which the cytoplasmic domain was replaced with GFP formed stable bridges. These data are reminiscent of studies demonstrating that complexes of ephrin-A2 and EphA3 are intermediates in heterophilic repulsive interactions in cell culture. Ephrin-A2 is normally cleaved by a metalloprotease, and cleavage-resistant mutations lead to more stable interactions between growth cones and target cells (Matthews, 2007).

The signal transduction mechanism promoting repulsion is poorly understood. At least some self-avoidance activity derives from sequences encoded by exon 18 in the Dscam cytoplasmic tail, which includes a polyproline motif. In previous studies, the Dock adaptor protein was shown to bind to this region as well as to other sites on the cytoplasmic domain and to act downstream of Dscam in axon guidance. While Dock has been implicated in the repulsive signaling downstream from the slit receptor, Robo, loss-of-function Dock mutations caused no obvious self-avoidance defects in da neurons. Dock may not function in self-avoidance or, alternatively, it may be redundant with other signaling pathways. The Tricornered (Trc) signaling pathway was previously shown to regulate tiling and self-avoidance in class IV neurons; however, examination of animals carrying transheterozygous mutant combinations did not uncover a genetic interaction between trc and Dscam (Matthews, 2007).

Alternative splicing of Dscam pre-mRNA can generate an enormous number of distinct cell-surface receptors. Is Dscam diversity, or any specific Dscam isoform, necessary for self-avoidance in individual da neurons? The results argue that while diversity is not strictly required for self-recognition and repulsion, it is crucial to prevent inappropriate repulsive interactions from occurring between the dendrites of different cells. This may be a central function for Dscam diversity both in different functional groups of sensory neurons in the PNS, which must sample input from overlapping regions of the body wall, and in regions of the CNS with much more highly intermingled dendritic and axonal processes (Matthews, 2007).

Previous data suggest an analogous function for Dscam diversity in mediating the sorting of axons in the developing MB. MB axon phenotypes were partially rescued by expression of single isoforms, whereas ectopic expression across multiple cells gave dominant effects, in which axons were guided to improper targets. These data together with studies described in this paper indicate that axons that project along a common fascicle or dendrites with overlapping fields must express sufficiently different isoform repertoires. Supporting this scenario, expression of Dscam isoforms in MB neurons, as well as photoreceptor subtypes, appears to be specified through a stochastic mechanism whereby each neuron expresses a biased, yet largely nonspecific set of isoforms. Given the complexity of the Dscam locus, it is reasonable to expect that different roles for diversity will be observed in different cell populations or even different processes of a cell. For example, these results are not incompatible with Dscam diversity also contributing to wiring in a more deterministic fashion wherein specific isoforms are required for elaborating different aspects of neural circuits (Matthews, 2007).

Dendritic arbors respond to numerous intrinsic and extrinsic cues during morphogenesis. How might self-avoidance mechanisms operate in the context of these other patterning events during the assembly of neural circuits? Dscam mutant phenotypes in da neurons provide insight into this problem. Mutant isoneuronal dendrites freely overlapped along their length, and the dendrites of some cells collected into tight bundles at stereotyped locations along the body wall. Mutant dendrites rarely grew beyond these specific sites of termination. Interestingly, wild-type dendrites normally projected to these same foci but provided a more diffuse coverage of the surrounding area, very likely because self-avoidance prohibited their overlap. These observations together suggest that Dscam mutant phenotypes reveal coordinates on the body wall that are attractive to dendrites and that there is an important interplay in da neurons between self-avoidance signaling and dendrite guidance mechanisms (Matthews, 2007).

One implication of these observations for circuit assembly is that self-avoidance is likely crucial for the spreading of highly branched dendritic processes that might otherwise tend to fasciculate or respond in unison to localized extrinsic guidance signals. In this way, self-avoidance might act throughout the nervous system to establish properly targeted and fully sampled territories. The analogies between Dscam mutant phenotypes in the brain and those of da neurons described in this study support this notion. Based on these findings, it is proposed that Dscam-mediated self-avoidance plays a widespread role in patterning the fly nervous system. Since Dscam diversity is not seen in vertebrate neurons, it is speculated that analogous mechanisms might exist in which stochastic expression of other families of cell-surface recognition molecules provide the capacity for self-avoidance in the vertebrate brain (Matthews, 2007).

REGULATION

Alternative Splicing of Dscam

Drosophila Dscam gene encodes an axon guidance receptor that can express 38,016 different mRNAs by virtue of alternative splicing. The Dscam gene contains 95 alternative exons that are organized into four clusters of 12, 48, 33, and 2 exons each. Although numerous Dscam mRNA isoforms can be synthesized, it remains to be determined whether different Dscam isoforms are synthesized at different times in development or in different tissues. The alternative splicing of the Dscam exon 4 cluster, which contains 12 mutually exclusive alternative exons, has been investigated and found to be developmentally regulated. The most highly regulated exon, 4.2, is infrequently used in early embryos but is the predominant exon 4 variant used in adults. Moreover, the developmental regulation of exon 4.2 alternative splicing is conserved in D. yakuba. In addition, different adult tissues express distinct collections of Dscam mRNA isoforms. Given the role of Dscam in neural development, these results suggest that the regulation of alternative splicing plays an important role in determining the specificity of neuronal wiring. In addition, this work provides a framework to determine the mechanisms by which complex alternative splicing events are regulated (Celotto, 2001).

All of the exon 4 variants are very similar in size, ranging from 159 to 171 nucleotides (nt; 4.11 = 159 nt; 4.1, 4.2, 4.3, 4.5, 4.6, 4.7 = 162 nt; 4.9 = 168 nt; and 4.4, 4.8, 4.10, 4.12 = 171 nt). Likewise, the 12 possible RT-PCR products obtained using primers in the constant exons 3 and 5 differ from one another by only 12 nt. As a result, traditional methods such as agarose gel electrophoresis cannot be used to analyze the alternative splicing of exon 4. By separating the RT-PCR products on a SSCP gel, which separates molecules based on conformational differences, the majority of the 12 RT-PCR products could be distinguished from one another. The identity of each band was assigned in two ways: (1) standards were generated using PCR products generated from 12 cDNA clones containing each exon 4 variant spliced to exons 3 and 5 (lanes 2-13); (2) each band from a reaction was excised from the SSCP gel, cloned, and sequenced (Celotto, 2001)

Although the majority of the RT-PCR products migrate at a distinct position in the gel, the RT-PCR products obtained from some mRNAs comigrate. Specifically, RT-PCR products containing exons 4.3 and 4.12 migrate as a single band as do the RT-PCR products synthesized from mRNAs containing exons 4.5, 4.7, and 4.9. Using this method, the relative frequency with which the majority of the exon 4 variants are utilized within each RNA sample can be determined (Celotto, 2001).

To determine if Dscam alternative splicing is regulated, the frequency at which each Dscam exon 4 variant is utilized throughout development was measured. Total RNA harvested from flies at various stages of development was used as a template for RT-PCR reactions with primers in exons 3 and 5 and the RT-PCR products were resolved on SSCP gels. The frequency at which most of the exon 4 variants are utilized does not change significantly throughout development. However, the splicing of two exons, 4.2 and 4.8, appears to be highly regulated (Celotto, 2001).

Exon 4.2 displays the most striking developmental changes. Only ~1% of the Dscam transcripts in 0- to 12-hr embryos contain exon 4.2. However, in first instar larvae (L1), exon 4.2-containing transcripts make up ~20% of the total Dscam mRNAs. An analysis of RNA isolated hourly from embryos raised at 22° reveals that Dscam transcripts containing exon 4.2 first appear at hour 12, which corresponds to embryonic stage 15. The relative abundance of Dscam transcripts containing exon 4.2 remains high throughout the remainder of development and, in adults, ~44% of the total Dscam mRNAs contain exon 4.2. This represents a 40- to 50-fold increase in exon 4.2 utilization between embryos and adults (Celotto, 2001).

The expression of Dscam mRNAs containing exon 4.8 is the opposite of the expression pattern of exon 4.2-containing transcripts. Approximately 20% of all Dscam mRNAs in embryos contain exon 4.8. The abundance of exon 4.8-containing transcripts decreases throughout the remainder of development and in adults only ~1% of the total Dscam mRNAs contain exon 4.8. It is concluded that alternative splicing of some of the Dscam exon 4 variants is dramatically regulated throughout development (Celotto, 2001).

The diversity of Dscam proteins generated by alternative splicing is thought to play an important role in determining the specificity of neuronal wiring. One prediction of this model is that neurons in different tissues would express different Dscam isoforms to direct their axons to specific addresses. To begin testing this model, the relative abundance was examined of each exon 4-containing Dscam mRNA isoform in different adult tissues (Celotto, 2001).

RNA was harvested from antennae, heads, wings, and legs dissected from adult flies. These RNA samples were subjected to RT-PCR and the products were separated on SSCP gels. These results show that the collection of Dscam transcripts is significantly different in each body part examined. For example, whereas ~45% of the total Dscam transcripts in legs and ~42% of the transcripts in wings contain exon 4.2, only ~16% of Dscam transcripts isolated from heads contain exon 4.2 (lanes 2-4). Similar differences are observed for many of the other exon 4 variants. It is concluded that the alternative splicing of the Dscam exon 4 variants is regulated in a tissue-specific manner (Celotto, 2001).

The most striking change observed is the developmental regulation of exon 4.2, which is not utilized in early embryos. To determine whether the alternative splicing of exon 4.2 has been conserved in other Drosophila species, the genomic DNA encompassing the exon 3-5 region of the Dscam gene was cloned from D. yakuba, which is estimated to have diverged from D. melanogaster 7-15 million years ago. The sequence of the exon 4 region of the D. yakuba Dscam gene is similar to the D. melanogaster gene throughout its length. Like D. melanogaster, the D. yakuba Dscam gene contains 12 variants of exon 4. The exon 4 variants are on average 95% identical between D. melanogaster and D. yakuba at the nucleotide level. Most of the exonic nucleotide changes are silent third-position changes. As a result, the protein sequences encoded by these exons are nearly identical between the two species (Celotto, 2001).

As expected, the sequences of the intron are more divergent than the exons. The nucleotide sequence of the introns separating the exon 4 variants is an average of 82% identical between D. melanogaster and D. yakuba. The introns between exons 3 and 4.1 and between exons 4.12 and 5 are 78% and 77% identical between the two species (Celotto, 2001).

A comparison of the splice site sequences flanking each of the exon 4 variants revealsthat the 5' splice sites are more conserved between the two species than the 3' splice sites. For example, between the two species there are only 2 nucleotide changes among the 12 exon 4 5' splice sites, whereas there are 25 nucleotide changes among the 12 exon 4 3' splice sites (Celotto, 2001).

Whether the developmental pattern of exon 4.2 alternative splicing observed in D. melanogaster is conserved in D. yakuba was tested. As with D. melanogaster, it was found that D. yakuba Dscam transcripts containing exon 4.2 are not expressed in embryos but are expressed in both larvae and adults. The relative abundance of the exon 4.8-containing transcripts decreases throughout D. yakuba development as in D. melanogaster. However, in both cases, the magnitude of the changes is lower in D. yakuba than in D. melanogaster. It is concluded that a similar developmental pattern of Dscam exon 4-regulated alternative splicing occurs in both D. melanogaster and D. yakuba (Celotto, 2001).

Stochastic yet biased expression of multiple Dscam splice variants by individual cells

Drosophila Dscam is essential for axon guidance and has 38,016 possible alternative splice forms. This diversity can potentially be used to distinguish cells. The Dscam mRNA isoforms expressed by different cell types and individual cells were analyzed. The choice of splice variants expressed is regulated both spatially and temporally. Different subtypes of photoreceptors express broad yet distinctive spectra of Dscam isoforms. Single-cell RT-PCR has documented that individual cells express several different Dscam isoforms and allows an estimation of the diversity that is present. For example, it is estimated that each R3/R4 photoreceptor cell expresses 14-50 distinct mRNAs chosen from the spectrum of thousands of splice variants distinctive of the R3/R4 cell type. Thus, the Dscam repertoire of each cell is different from those of its neighbors, providing a potential mechanism for generating unique cell identity in the nervous system and elsewhere (Neves, 2004).

Synonymous codon usage is typically biased towards translationally superior codons in many organisms. In Drosophila, genomic data indicates that translationally optimal codons and splice optimal codons are mostly mutually exclusive, and adaptation to translational efficiency is reduced in the intron-exon boundary regions where potential exonic splicing enhancers (ESEs) reside. In contrast to genomic scale analyses on large datasets, a refined study on a well-controlled set of samples can be effective in demonstrating the effects of particular splice-related factors. Down syndrome cell adhesion molecule (Dscam) has the largest number of alternatively spliced exons (ASEs) known to date, and the splicing frequency of each ASE is accessible from the relative abundance of the transcript. Thus, these ASEs comprise a unique model system for studying the effect of splicing regulation on synonymous codon usage. Codon Bias Indices (CBI) in the 3' boundary regions were reduced compared to the rest of the exonic regions among 48 and 33 ASEs of exon 6 and 9 clusters, respectively. These regional differences in CBI were affected by splicing frequency and distance from adjacent exons. Synonymous divergence levels between the 3' boundary region and the remaining exonic region of exon 6 ASEs were similar. Additionally, another sensitive comparison of paralogous exonic regions in recently retrotransposed processed genes and their parental genes revealed that, in the former, the differences in CBI between what were formerly the central regions and the boundary regions gradually became smaller over time. Thus, analysis of the multiple ASEs of Dscam allowed direct tests of the effect of splice-related factors on synonymous codon usage and provided clear evidence that synonymous codon usage bias is restricted by exonic splicing signals near the intron-exon boundary. A similar synonymous divergence level between the different exonic regions suggests that the intensity of splice-related selection is generally weak and comparable to that of translational selection. Finally, the leveling off of differences in codon bias over time in retrotransposed genes meets the direct prediction of the tradeoff model that invokes conflict between translational superiority and splicing regulation, and strengthens the conclusions obtained from Dscam (Takahashi, 2009).

Hidden RNA pairings counteract the "first-come, first-served" splicing principle to drive stochastic choice in Dscam1 splice variants

Drosophila melanogaster Dscam1 encodes 38,016 isoforms via mutually exclusive splicing; however, the regulatory mechanism behind this is not fully understood. This study found a set of hidden RNA secondary structures that balance the stochastic choice of Dscam1 splice variants (designated balancer RNA secondary structures). In vivo mutational analyses revealed the dual function of these balancer interactions in driving the stochastic choice of splice variants, through enhancement of the inclusion of distal exon 6s by cooperating with docking site-selector pairing to form a stronger multidomain pre-mRNA structure and through simultaneous repression of the inclusion of proximal exon 6s by antagonizing their docking site-selector pairings. Thus, this study provides an elegant molecular model based on competition and cooperation between two sets of docking site-selector and balancer pairings, which counteracts the "first-come, first-served" principle. These findings provide conceptual and mechanistic insight into the dynamics and functions of long-range RNA secondary structures (Dong, 2022).

Pre-mRNA alternative splicing is a major source of proteomic and functional diversity. In humans, ~95% of multi-exon genes are subjected to alternative splicing, and splicing defects are associated with a variety of genetic diseases. Common alternative splicing mechanisms include exon skipping, intron retention, alternative 5' or 3' splice site usage, and mutually exclusive splicing. Mutually exclusive splicing occurs when only one exon from a cluster of variable exons is spliced into a specific mRNA product. The most astonishing example of this can be found in the Drosophila melanogaster Down's syndrome cell adhesion molecule 1 (Dscam1) gene, which potentially generates 38,016 different isoforms through mutually exclusive splicing of exon clusters 4, 6, 9, and 17. Growing evidence has revealed that the notable diversity of Dscam1 isoforms is required for both neuronal wiring and immune defense (Dong, 2022).

In an attempt to explain how only one exon variant of Dscam1 is selected at a time from a cluster of exons, several models based on competing RNA secondary structures have been proposed. This mechanism was initially found in the exon 6 cluster of Dscam1, where the intronic docking site downstream of exon 5 can pair competitively with selector sequences upstream of each exon 6 variant. Similar structural arrangements have been identified in the exon 4 and exon 9 clusters of Dscam1; however, in contrast to the docking site located upstream of the exon 6 cluster, the docking sites for the exon 4 and exon 9 clusters are located within their respective downstream introns. The heterogeneous nuclear ribonucleoprotein 36 (hrp36) also ensures splicing fidelity in the exon 6 cluster but not in the exon 4 or exon 9 cluster. Moreover, a locus control region immediately upstream of the docking site is involved in the inclusion of the nearest exon when the docking site is paired with its upstream selector sequence (Dong, 2022).

A long-standing question regarding mutually exclusive splicing is how docking site-selector pairing is regulated. Proximal selector sequences are only a few hundred nucleotides away from the docking site, whereas distal selector sequences are located over 10,000 nucleotides away from the docking site. If docking site-selector pairing were dictated on a cotranscriptional 'first-come, first-served' basis, there would be a bias toward docking site-proximal exon 6 variants; however, this is not observable in all tissues. These splicing outcomes in terms of exon 6 inclusion cannot be explained by compensation from splice site strength, because distal exon 6s do not display a preference for stronger splice sites compared to proximal exon 6s. The predicted thermodynamic stability of docking site-selector pairing does not correlate with the selection frequency of the associated exons. The frequency of some exon 6 variants actually increased following deletion of the docking site in a previous study examining D. melanogaster /Drosophila virilis (Dme/Dvi) Dscam1 mutant constructs. It is therefore likely that unknown mechanisms regulate selection of the Dscam1 exon 6 cluster (Dong, 2022).

The current study found a previously unknown RNA secondary structure hidden within the exon 6 cluster that balances the stochastic selection of Dscam1 splice variants (designated balancer RNA secondary structures). Targeted mutational analyses using a CRISPR-Cas9 system revealed that hidden RNA pairings drive the stochastic choice of splice variants by simultaneously enhancing the inclusion of distal exon 6s by facilitating docking site-selector pairing and by repressing the inclusion of proximal exon 6s. The balancers and docking site-selector base pairings cooperated to form a strong multidomain structure that enhanced distal exon inclusion. From this, a molecular model was developed for the regulation of the stochastic, mutually exclusive splicing of Dscam1 exon 6 based on competition and cooperation between two sets of docking site-selector and balancer RNA secondary structures. This work suggests that Dscam1 has evolved compensatory mechanisms that balance the distance and strength of docking site-selector base pairing to drive the stochastic selection of Dscam1 splice variants and provides an explanation for the lack of obvious 5' to 3' preference during exon 6 variant selection. Moreover, genetic analysis indicated that disruption of docking site pairing or balancer base pairing led to growth and neuronal anomalies, suggestive of their physiological significance. An additional framework for the regulation of complex, mutually exclusive splicing and new insight into the role of long-range RNA secondary structures in gene regulatory networks (Dong, 2022).

At first glance, these previously unknown balancer RNA secondary structures appeared to be similar to the docking site and selector sequence base pairing structures. Both structures were composed of two types of conserved elements: an upstream element, which was located in the intron downstream of the constitutive exon 5, and multiple downstream elements, which were located in the exon 6 cluster body. The upstream element could pair competitively with various downstream elements. However, the two types of competing RNA secondary structures were fundamentally different in both their function and mechanism. In contrast to the base pairing between the docking site and various exon 6 upstream selectors, the balancer RNA secondary structures tended to be located in the central and 3' regions of the exon 6 cluster. Moreover, the balancer RNA secondary structures themselves did not promote the inclusion of exon 6 variants but rather enhanced the inclusion of distal exon 6s by strengthening docking site-selector base pairing or inhibited the inclusion of proximal exon 6 s by antagonizing docking site-selector base pairing. These data suggest that the regulatory function of balancer RNA secondary structures was dependent on the base pairing between the docking site and selector sequence. Furthermore, the formation of balancer RNA secondary structures enhanced the inclusion of multiple exon 6s in a proximity-dependent manner. For example, the Ubs6.1-Dbs6.40 RNA secondary structure may act as a fulcrum that guides the docking site to pair with the selector upstream of exon 6.41 or 6.42. Thus, base pairing between the docking site and a selector sequence may activate selection of the most proximal exon variant 6, whereas the balancer sites are thought to be effective over a long range. It is concluded that while docking site-selector base pairing interactions determined which exon 6 variant was selected, the balancer base pairings drove the stochastic choice of exon 6 variants (Dong, 2022).

This study provides a reasonable explanation for the long-standing puzzle in the choice of exon 6 variants of Dscam1. By combining various techniques including informatics, evolutionary analyses, and in vivo mutagenesis experiments, a new model was generated depicting the regulation of exon 6 variant inclusion. According to a proposed model (see Model for balancer RNA secondary structures in balancing the stochastic choice of Dscam1 splice variants), for a 5'-distal exon 6 (i.e., exon 6.z) to be included in mature Dscam1 mRNA, the docking site-selector interaction may cooperate with multiple balancer base pairings to form a stronger multidomain RNA secondary structure; balancer base pairings stabilize the docking site-selector interaction, thereby increasing the frequency of exon 6.z inclusion. For a 5'-proximal exon 6 (i.e., exon 6.x) to be included in mature mRNA, the docking site may pair with the selector sequence to form short RNA secondary structures containing few domains. Therefore, 5'-proximal exon 6 inclusion relies upon docking site-selector base pairing strength. Simultaneously, balancer secondary structures also inhibit the inclusion of 5'-proximal exon 6s by antagonizing their docking site-selector base pairing interactions. For a middle exon 6 to be included in the mature mRNA, strong intermediate RNA secondary structures may be formed. Although docking site-selector pairings did not exhibit an obvious preference for docking site-distal exon 6s, the combined overall base pairings exhibited a low-to-high strength gradient from docking site-proximal to docking site-distal exon 6s. In this study, it was found that docking site deletion led to a bias toward the inclusion of docking site-distal exon 6s (except for exon 6.1), whereas deleting upstream balancers led to increased docking site-proximal exon 6 inclusion. Therefore, it is speculated that the gradient of pairing strength compensates for the docking site-proximal to docking site-distal preference of the exon 6 variants caused by the first-come, first-served principle. Thus, the final splicing outcome did not exhibit an obvious docking site-proximal preference in exon 6 variants. This finding suggests that flies have evolved an intricate compensatory pairing mechanism that drives the stochastic choice of Dscam1 splice variants (Dong, 2022).

The conservation of balancer RNA secondary structures and genetic analyses highlight their regulatory benefits and physiological roles. First, it would be advantageous to increase balancer base pairing interactions alongside the duplication of exon clusters. Moreover, balancer pairing compensation could provide specific advantages to large exon clusters (i.e., Dscam1) through a variety of mechanisms. An intriguing possibility is that the balancer base pairing interaction induces the juxtaposition of the docking site and its downstream selector sequences, thereby enhancing their pairing interaction. For example, Ubs6.1-Dbs6.40 base pairing acts as a fulcrum that guides the docking site to pair with multiple selector sequences. Balancer base pairing interactions not only reduce the base pairing distance across large RNA molecules but also help with avoiding the formation of nonspecific secondary structures and heterogeneous nuclear ribonucleoprotein complexes that might affect the accuracy or efficiency of specific docking site-selector base pairings. Thus, balancer base pairing strategies may have been evolutionarily favorable in complex alternative splicing units such as Dscam1, in which long-distance base pairing is required. Coordination and competition between diverse balancer and docking site-selector base pairing interactions have increased the flexibility and efficiency of folding-mediated splicing. Phenotypic analyses revealed obvious defects in these mutants bearing disruption of two types of RNA secondary structures, suggesting that two types of RNA secondary structures coordinate to ensure the proper frequency of exon 6 variants, which is required for normal development (Dong, 2022).

Is this type of functional RNA secondary structures common in vivo? Competing RNA secondary structures were initially identified in the exon 6 cluster of Dscam1, which at that time was believed to be unique. Similar structural codes have recently been revealed in several exon clusters, such as Drosophila 14-3-3Ξ, exon 4 and 9 clusters in Drosophila Dscam1, srp, RIC-3, Branchiostoma MRP, human dynamin 1, and CD55. In addition, this docking site-mediated model governs the regulation of 3'-end alternative splicing in Drosophila PGRP-LC, CG42235, and Pip genes. Therefore, competing RNA secondary structures may be a broadly applicable mechanism to regulate mutually exclusive splicing. In addition to mutually exclusive exons, competing RNA secondary structures may regulate variable exon skipping of human SF1 and alternative backsplicing of human POLR2A. Moreover, this study observed similar balancer RNA secondary structures in the Dscam1 exon 9 cluster and MRP1. Analogous nested RNA secondary structures have been found in the catalog of pairs of conserved complementary regions (PCCRs) in human protein-coding genes. Likewise, two groups of RNA structural modules have been shown to operate together to dynamically regulate alternative splicing in the human Ate1 gene. It is therefore likely that the framework of balancer RNA secondary structures developed in this study is widely applicable to the regulation of mutually exclusive splicing and other RNA processing events (Dong, 2022).

Moreover, some questions related to the functional regulation of RNA structures should be further addressed. First, why are these exons in the stem-loop not spliced compared to flanking exons? Previous studies have shown that RNA pairing can repress the splicing of exons within a loop. Moreover, Dscam1 exon 6 is kept silent through weak splice sites in combination with splicing repressors, such as the heterogeneous nuclear ribonucleoprotein hrp36. When the docking site pairs with certain selector sequence, the distant enhancers specifically activate the proximal alternative exon by promoting recognition of the splicing site and/or dissociation of the repressors, while other exons in a stem-loop and downstream exons away from the RNA loop are repressed. Second, can the balancer and docking site-selector base pairings form a long-range pseudoknot? It is likely that two sets of RNA secondary structures form a long-range pseudoknot. However, the mutational evidence in the present study supports the notion that balancer secondary structures act to antagonize docking site-selector base pairing of proximal exon 6s. Therefore, it is proposed that these elements function by forming RNA secondary structures rather than pseudoknot structures (Dong, 2022).

This study has provided sufficient evidence for the splicing regulation of docking site-selector and balancer RNA secondary structures in the Dscam1 gene. Future challenges include directly demonstrating the existence of the purported and/or unknown base pairs and mapping RNA-protein interactions and the effects on the choice of different exons. It will also be important to test whether these variable exons are spliced cotranscriptionally and, if so, how far the RNA polymerase progresses before splicing is detected (Dong, 2022).

Transmembrane/juxtamembrane domain-dependent Dscam distribution and function during mushroom body neuronal morphogenesis

Besides 19,008 possible ectodomains, Drosophila Dscam contains two alternative transmembrane/juxtamembrane segments, respectively, derived from exon 17.1 and exon 17.2. Would specific Dscam isoforms mediate formation and segregation of axonal branches in the Drosophila mushroom bodies (MBs)? Removal of various subsets of the 12 different exon 4 variants does not affect MB neuronal morphogenesis, while expression of a Dscam transgene only partially rescues Dscam mutant phenotypes. Interestingly, differential rescuing effects are observed between two Dscam transgenes that each possess one of the two possible versions of exon 17. Axon bifurcation/segregation abnormalities are better rescued by the exon 17.2-containing transgene, but coexpression of both transgenes is required for rescuing mutant viability. Meanwhile, exon 17.1 targets ectopically expressed Dscam-GFP to dendrites while Dscam[exon 17.2]-GFP is enriched in axons; only Dscam[exon 17.2] affects MB axons. These results suggest that exon 17.1 is minimally involved in axonal morphogenesis and that morphogenesis of MB axons probably involves multiple distinct exon 17.2-containing Dscam isoforms (Wang, 2004).

MB alpha/ß neurons homozygous for the C22-1 deficiency undergo normal morphogenesis despite loss of three-quarters of Dscam isoforms. It was therefore wondered whether a single Dscam isoform was sufficient for supporting MB morphogenesis. This possibility was assessed by supplementing Dscam null mutant MB neurons with specific Dscam isoforms. One challenge in such rescuing experiments is to drive expression of Dscam transgenes in a physiologically relevant manner (Wang, 2004).

Fortunately, a 4.5 kb genomic fragment that lies immediately 5′ to the Dscam start codon appears to be sufficient for driving transgene expression in the endogenous Dscam expression pattern. Fusing the 4.5 kb Dscam genomic fragment with GAL4, it was observed that this GAL4 driver can selectively and efficiently induce expression of UAS-controlled transgenes in both the peripheral and central nervous systems through different developmental stages. To examine transgene expression in more detail and to compare its pattern directly with endogenous Dscam's protein distribution pattern, the 4.5 kb Dscam genomic fragment was then fused with a Dscam cDNA that had been modified to encode a chimeric protein with GFP at Dscam's carboxyl terminal. In the wandering larval CNS, Dscam-GFP is broadly enriched in neuropil-like structures. Interestingly, endogenous Dscam is distributed in a similar pattern, as revealed by immunostaining with an anti-Dscam peptide antibody, encouraging use of the isolated 4.5 kb genomic fragment in driving the expression of various engineered Dscam transgenes in the rescuing experiments (Wang, 2004).

Dscam's ectodomain contains three variable regions that are encoded by exon 4, exon 6, and exon 9, respectively. Analysis of expressed Dscam sequences has revealed differential expression of distinct exon 4 alternatives in different tissues and at different developmental stages. For instance, the most highly regulated exon, 4.2, rarely exists in early embryos but is the predominant exon 4 variant present in adult. However, no requirement was detected for any specific Dscam exon 4 variant during MB morphogenesis. Given that the usage of exon alternatives is most regulated in the exon 9 cluster, it will be interesting to determine whether, in contrast with exon 4, specific exon 9 alternatives are required for normal MB morphogenesis. Nevertheless, consistent with the notion that the identities of individual Dscams' ectodomains might not be critical for MB neuronal morphogenesis, it was found that Dscam isoforms with a fixed ectodomain can mediate divergent segregation of axonal branches in most Dscam mutant MB neurons. It is possible that Dscam isoforms with another ectodomain may not rescue Dscam mutant MB neurons' morphogenetic defects at all, but this possibility can be largely ignored since similar rescuing results have been obtained when they supplemented Dscam null mutant MB neurons with various single-isoform Dscam transgenes (Zhan, 2004). Based on these results, it is likely that Dscam proteins with different ectodomains are equally potent in governing MB neuronal morphogenesis. But multiple isoforms with distinct ectodomains are still needed to fully support normal MB morphogenesis, given that Dscam isoforms with one fixed ectodomain significantly but partially rescue Dscam mutant phenotypes. Similar arguments could explain why Dscam isoforms with one fixed ectodomain are sufficient for rescuing organism lethality but fail to mediate normal brain development in rescued Dscam⁻ flies. However, partial rescue can be alternatively explained by other possibilities. For instance, driven by an arbitrary Dscam promoter, Dscam cDNA-genomic hybrid transgenes might not be expressed in identical spatiotemporal patterns as endogenous Dscam. Nevertheless, the involvement of multiple distinct Dscam isoforms is further suggested by the demonstration (Zhan, 2004) that multiple distinct Dscam ectodomains are expressed in any given MB neuron examined via single-cell RT-PCR (Wang, 2004).

Dscam's transmembrane/juxtamembrane domain is encoded by either exon 17.1 or exon 17.2. Interestingly, three independent lines of experiments have all demonstrated the possible involvement of Dscam proteins with different transmembrane/juxtamembrane segments in the morphogenesis of dendrites versus axons. (1) Only one of the two Dscam cDNA-genomic hybrid transgenes, which specifically vary in exon 17, significantly rescues Dscam mutant axonal morphogenetic defects. (2) Ectopically expressed Dscam is either localized to dendrites or enriched in axons, depending on the exon 17-encoding juxtamembrane/transmembrane variable segment. (3) Ectopic expression of Dscam isoforms with different exon 17 alternatives disrupts different developmental processes. All of these results are consistent with the notion that exon 17.1-containing Dscam isoforms are selectively targeted to dendrites and, thus, have minimal effects on axonal morphogenesis. In vivo, it is possible that basic morphogenesis of MB axons exclusively involves exon 17.2-containing Dscam isoforms and that Dscams with exon 17.1, which are specifically targeted to dendrites, might regulate morphogenesis and/or functions of dendrites. Axonal morphogenesis normally takes place before complex dendritic elaboration followed by synapse formation. Interestingly, exon 17.2-containing Dscam isoforms, but not exon 17.1 Dscams, can rescue early larval lethality in Dscam mutant organisms. These results imply little involvement of exon 17.1-containing Dscams in early neuronal morphogenetic processes and indirectly suggest possible roles for dendritic-targeted Dscams in the maturation of dendrites and/or synapse formation and modulation. These notions are further supported by the fact that Dscams with exon 17.1 are required for helping exon 17.2-containing Dscam isoforms to rescue Dscam mutants into the adult stage. However, it remains to be shown that endogenous Dscam proteins with exon 17.1 are selectively localized in dendrites, and Dscam's roles in dendritic morphogenesis and/or functions remain to be elucidated. In addition, although several cell surface proteins are known to exhibit polarized distribution in neurons and their sorting signals are being gradually identified, the exon 17.1-encoding juxtamembrane/transmembrane domain likely carries a novel dendrite-targeting motif based on its amino acid composition. A possible axon-targeting signal is likewise present in the juxtamembrane/transmembrane segment encoded by the Dscam exon 17.2 (Wang, 2004).

In summary, this study shows that specific Dscam isoforms are either targeted to dendrites or enriched in axons, raising the possibility that every single neuron might have distinct sets of Dscam molecules located in dendrites versus axons. Thus, simply by coupling different Dscam ectodomains with exon 17.1 versus exon 17.2, individual neurons could simultaneously send different messages to and/or respond differentially to their upstream and downstream neurons. Formation and modulation of neuronal connections might be fine tuned through the regulation of the compositions of Dscam proteins across synapses. In addition, it is suggested that only exon 17.2-containing Dscam isoforms are involved in governing axonal morphogenesis during early development of the nervous system. Finally, although no single ectodomain appears to be indispensable and various ectodomains might be functionally exchangeable, normal development of the Drosophila brain probably needs multiple distinct Dscam ectodomains (Wang, 2004).

Alternative splicing of Drosophila Dscam generates axon guidance receptors that exhibit isoform-specific homophilic binding

Dscam is an immunoglobulin (Ig) superfamily protein required for the formation of neuronal connections in Drosophila. Through alternative splicing, Dscam potentially gives rise to 19,008 different extracellular domains linked to one of two alternative transmembrane segments, resulting in 38,016 isoforms. All isoforms share the same domain structure but contain variable amino acid sequences within three Ig domains in the extracellular region. Different isoforms exhibit different binding specificity. Each isoform binds to itself but does not bind or binds poorly to other isoforms. The amino acid sequences of all three variable Ig domains determine binding specificity. Even closely related isoforms sharing nearly identical amino acid sequences exhibit isoform-specific binding. It is proposed that this preferential homophilic binding specificity regulates interactions between cells and contributes to the formation of complex patterns of neuronal connections (Wojtowicz, 2004).

This study demonstrates that a set of 11 different Dscam isoforms show surprising homophilic binding specificity. Each isoform preferentially binds to itself over different isoforms. Should this binding property extend to the entire spectrum of Dscam isoforms, this would provide enormous potential for regulating interactions between neurites during the establishment of neuronal connections (Wojtowicz, 2004).

Several lines of evidence support the view that Dscam proteins on opposing cell surfaces bind to each other. Mammalian Dscams have been shown to promote cell aggregation when transfected into cultured mouse cells. Dscam mediates interactions between cells in vivo. The trajectory of interneurons overexpressing a single isoform of Dscam is disrupted upon encountering midline cells that also overexpress the same Dscam isoform. That this reflects direct interactions between Dscam proteins on opposing cell surfaces is supported by the biochemical experiments presented in this paper. Dscam binding has been localized to the N-terminal eight Ig domains. Since this region contains the three variable Ig domains, it raised the possibility that differences within these domains could modulate interactions between isoforms (Wojtowicz, 2004).

All Dscam isoforms tested exhibited preferential binding to self over other isoforms. Isoforms differing in any one of the three variable Ig domains do not bind to each other, or show marked differences in binding. While binding between different isoforms was undetectable in the pull-down assay from S2 cell extracts, the possibility remains that weak interactions between different isoforms exist that are below the limit of detection of this assay. Quantification of the sensitivity of the pull-down assay demonstrated that 10-fold less protein on the Western blots would not have been reliably detected. Therefore, if heterophilic binding occurs between the isoforms tested, it is significantly weaker than the isoform-specific homophilic interaction. That interactions between different isoforms do occur under milder conditions (e.g., no detergent) is underscored by the binding of two isoforms of Dscam differing in seven amino acids in the bead binding-to-cells assay. Hence, it is speculated that, while each isoform preferentially binds to itself, isoforms also exhibit a range of weaker binding interactions with other isoforms (Wojtowicz, 2004).

All three variable Ig domains played a crucial role in binding specificity; swapping any one resulted in a marked reduction or a complete loss of binding. It is anticipated that future biochemical and structural studies will provide insights into the molecular basis of isoform-specific recognition. In the meantime, the simplest model for the 'matching' of alternative Ig domains is that each variable Ig domain interacts with the same variable Ig domain in an opposing molecule. The binding of all three Ig domains is likely required to stabilize otherwise weak interactions between individual variable domains (Wojtowicz, 2004).

How might isoform-specific binding contribute to wiring the fly brain? While the notion that each neuron may express one or only a few isoforms that specify interactions with other neurons is attractive, recent studies argue that single neurons express multiple isoforms and that even neurons of the same class express different and largely nonoverlapping sets of them. Hence, it is highly unlikely that any two neurons will express an identical set of isoforms. This ensures that the only neurites that express an identical set of isoforms are those from the same neuron (Wojtowicz, 2004).

Studies on developing mushroom body (MB) neurons provide support for the view that interactions between identical isoforms play a crucial role in mediating interactions between two neurites of the same cell. MB neurons extend axons that bifurcate at a common branch point, and the resulting sister branches segregate to different pathways. A prominent feature of the loss-of-function phenotype in these neurons is a failure of sister branches to segregate. A simple model to account for this is that identical isoforms of Dscam on sister branches bind to each other and induce a contact-dependent repulsive interaction perhaps analogous to repulsive interactions between Eph receptors and ephrin ligands. Prior to bifurcation, MB axons project together within a fascicle, and, following bifurcation, each sister branch also extends within a fascicle with other MB axon branches. To allow fasciculation, it is likely important that the array of Dscam isoforms expressed on each MB axon is different from its neighbors. Indeed, expression analysis reveals that, as with other neuronal subclasses, individual MB neurons express multiple isoforms and largely nonoverlapping arrays of them. Hence, while the specific isoforms of Dscam expressed in MB neurons may be unimportant, it may be crucial that neighboring MB axons express different isoforms. In support of this view, expression of a single Dscam isoform in multiple MB neurons induces a dominant phenotype characterized by defasciculation of MB axons, whereas expression of a single isoform in a single mutant neuron rescues the defect in the segregation of sister branches. The notion that interactions between identical isoforms induces a repellent response is consistent with other loss- and gain-of-function studies (Wojtowicz, 2004 and references therein).

It seems unlikely that Dscam acts only in a cell-autonomous fashion to mediate interactions between processes of the same neuron. In the absence of Dscam, defasciculation of axons has been observed both in the developing mushroom body and in Bolwig's nerve. Perhaps weaker signals resulting from interactions between different isoforms or between a small fraction of identical isoforms expressed on different neurons may promote adhesive interactions leading to fasciculation. Interestingly, recent studies have argued that different levels of Eph/ephrin signaling result in qualitatively different responses; high levels induce contact-dependent repulsion, while lower levels promote contact-dependent attraction (Wojtowicz, 2004 and references therein).

In summary, it is proposed that the nature of the interactions between Dscam isoforms on the surface of neurites produces qualitatively or quantitatively different intracellular signals influencing the development of neurites. Signaling may be modulated by the number of identical isoforms shared by two neurites, the level of expression of each isoform, and the binding affinity or avidity of different isoforms. For instance, high signaling levels produced by interactions between neurites of the same neuron expressing an identical array of Dscam isoforms would induce repulsion. Conversely, lower signals produced by weaker interactions between neurites of different cells expressing few or no identical Dscam isoforms would promote growth along one another, thereby allowing fasciculation. Since other Ig superfamily proteins have been shown to interact with multiple proteins, it remains possible that other Dscam phenotypes may reflect interactions with additional cell surface or soluble ligands that may or may not exhibit isoform-specific interactions (Wojtowicz, 2004).

Alternative splicing of Dscam has been highly conserved over some 250 million years separating the fly, the mosquito, and the bee. This observation, combined with the biochemistry reported in this study and genetic data establishing a role for Dscam in neuronal connectivity, supports the hypothesis that Dscam isoforms function as molecular tags contributing to the formation of precise patterns of neuronal connections (Wojtowicz, 2004 and references therein).

While Dscam diversity has been highly conserved during insect evolution, the mouse and human Dscam genes do not undergo extensive alternative splicing. In addition to Dscam, there are a number of genes in the fly genome with arrays of three or more alternatives for a given exon that encode related amino acid sequences. It is striking, however, that no mammalian genes appear to share a similar arrangement, although genes containing only two alternatives for a given exon are common in the mammalian genome. This suggests that diversification of gene function in the mammalian genome has not occurred through the massive cassette-like strategy utilized to generate biochemically distinct isoforms of Drosophila Dscam. Other mechanisms may have evolved in mammals to generate comparable diversity in neuronal cell surface proteins. These may include the use of large families of related proteins encoded by separate genes (e.g., odorant receptors), smaller families of proteins used in a combinatorial fashion (e.g., CNRs, MHC class II, classical cadherins), gradients of receptors and ligands (e.g., Ephrins and Eph receptors), or a combination of multiple genes, alternative transcription start sites, and alternative splicing, as in the case of neurexins (Wojtowicz, 2004 and references therein).

It is concluded that Dscam plays a widespread role in regulating the formation of neuronal connections in Drosophila. Recent expression studies revealed that different neurons express different combinations of Dscam isoforms endowing each neuron with a discrete molecular identity. The biochemical studies described in this study demonstrate that different Dscam isoforms have striking differences in binding specificity. It is proposed that a general function of Dscam diversity is to promote repellent interactions between neurites from the same cell expressing the same array of Dscam isoforms in a cell-autonomous fashion. Differences in the arrays of isoforms expressed in different neurons may also contribute to the patterning of neuronal connections. Whether Dscam diversity is indeed crucial to patterning neuronal connections in flies awaits additional analyses in which the number and type of Dscam isoforms expressed in different neurons are systematically manipulated (Wojtowicz, 2004).

Extensive diversity of Ig-superfamily proteins in the immune system of insects

The extensive somatic diversification of immune receptors is a hallmark of higher vertebrates. However, whether molecular diversity contributes to immune protection in invertebrates is unknown. Evidence is presented that Drosophila immune-competent cells have the potential to express more than 18,000 isoforms of the Ig-superfamily receptor Down syndrome cell adhesion molecule (Dscam). Secreted protein isoforms of Dscam were detected in the hemolymph and hemocyte-specific loss of Dscam impaired the efficiency of phagocytic uptake of bacteria, possibly due to reduced bacterial binding. Importantly, the molecular diversity of Dscam transcripts generated through a mechanism of alternative splicing is highly conserved across major insect orders, suggesting an unsuspected molecular complexity of the innate immune system of insects (Watson, 2005).

Immunoglobulin-domain-containing proteins constitute the largest repertoire of surface receptors in animals and serve many functions in molecular recognition, cell adhesion and signaling. Most striking is the exceptional diversity of antigen-specific receptors of the adaptive immune system in higher vertebrates, which depends on somatic gene rearrangement and clonal selection. However, somatic rearrangement of highly diverse immune receptors has been considered to exist in a relatively small number of animal species restricted to the jawed vertebrates (Watson, 2005).

A single Drosophila Dscam gene has been identified as a member of the Ig-superfamily and its essential function in neuronal wiring has been characterized. Gene organization of Dscam comprises clusters of variable exons flanked by constant exons. Although mechanistically entirely different from somatic rearrangements, alternative splicing of the Dscam gene combines constant and variable exons by mutually exclusive splicing, and potentially generates as many as 19,008 different extracellular domains. Therefore, it is conceivable that a large protein-isoform repertoire with the potential for recognizing diverse ligands and epitopes could be generated. To explore this, a comparative and functional analysis of Dscam expression in immunecompetent cells of flies and other insects was undertaken (Watson, 2005).

Fat body cells and hemocytes (i.e., insect blood cells) constitute important cells of the insect immune system. Most proteins in insect hemolymph, the insect equivalent of blood serum, are produced in fat body cells which also secrete anti-microbial peptides that constitute an important component of the humoral immune defense. In contrast, hemocytes are involved in cellular defense strategies such as phagocytosis and wound repair (Watson, 2005 and references therein).

In situ hybridization of tissue from 3rd instar Drosophila larvae with a Dscam-specific probe revealed Dscam expression in fat body cells. For a comparison of Dscam expression in immune and neural tissue mRNA was isolated from larval hemocytes, fat body and brain tissue. Hemocyte-specific GFP expression allowed for the purification of hemocytes by Fluorescence Activated Cell Sorting (FACS). RT-PCR analysis and sequencing of ~50 cDNAs revealed that the majority of Dscam mRNAs in hemocytes, fat body and brain contain unique exon 4-6 combinations (Watson, 2005).

For a global assessment of alternative splicing in different cell types custom made oligo-arrays were used. Microarrays contained specific 50-mer oligo-probes for all alternatively spliced exons. Dscam mRNA sequences were amplified by RT-PCR and cDNAs were fluorescently labeled and hybridized to the microarrays. 59 of the 60 alternative exon 4 and exon 6 sequences were found to be expressed in all 3 cell types. In brain tissue 32 exon 9 sequences were expressed. However, only a subset (total of 14) was expressed in fat body and a slightly different subset (total of 15) was expressed in hemocytes. Based on relative expression levels, it is estimated that 80%-90% of all Dscam mRNAs in hemocytes and fat body contain either exon: 9.6, 9.9, 9.13, 9.30, or 9.31, demonstrating that exon 9 splice variants in fat body and hemocytes are distinct from those found in brain. Considering all of the alternative exons detected (12 exon 4, 47 exon 6, 16 exon 9, and 2 exon 17), it is calculated that this potentially allows for the generation of more than 18,000 diverse receptor isoforms in fat body cells and hemocytes (Watson, 2005).

Antibodies were raised against extracellular (D-ex1, D-ex2) and intracellular (D-cy) domains of Dscam. All antibodies recognized an ~210 kDa endogenous form of Dscam in extracts from cultured S2 cells, a cell line thought to be derived from embryonic hemocytes and shown to share many characteristics with hemocytes. A 210 kDa form of Dscam was also confirmed in purified larval hemocytes, fat body tissue, and at comparatively high levels in brain. Immunoprecipitations from fat body extracts revealed three Dscam forms possibly representing truncated forms generated by proteolytic cleavage. Unexpectedly, it was found that S2 cell conditioned medium contains a soluble Dscam protein of ~160 kDa and secreted Dscam protein of the same molecular weight is also present in hemolymph serum (Watson, 2005).

Liquid chromatography and tandem mass spectrometry (LC-MS/MS) directly confirmed that S2 cells secrete Dscam isoforms. Coverage of the secreted forms by the identified peptides amounts to more than 50% of the entire extracellular part of Dscam. Importantly, some of the identified peptides confirmed the presence of alternatively spliced sequences including five Ig- 2 sequences and at least twelve Ig-3 sequences. In agreement with the expression profiling of exon 9 (Ig 7) sequences, three distinct Ig7 domains (i.e., Ig76, Ig79 and Ig713) were identified, that correspond to most abundantly expressed exon 9 sequences. Considering the protein sequencing results, the presence of secreted Dscam in hemolymph, and the large pool of diverse Dscam mRNAs in fat body or hemocytes, it is possible that thousands of Dscam isoforms circulate in the hemolymph of Drosophila (Watson, 2005).

Attempts were made to determine if Dscam proteins are functionally required in immune-competent cells. However, since animals with homozygous amorphic mutations in Dscam die as embryos, it was not possible to directly test this in null mutant animals. Nevertheless, it was possible to purify GFP-labeled hemocytes from Dscam mutant larvae that carry a trans-allelic combination of hypomorphic (Dscam39) and amorphic (Dscam20) mutations. Immunoblotting showed that Dscam20/Dscam39 animals have a strong overall reduction in protein level. One important function of hemocytes is the ingestion of bacterial pathogens by phagocytosis. Wild type and Dscam deficient hemocytes were challenged with heat-killed fluorescently labeled E. coli and the number of hemocytes containing fluorescent bacteria was determined ('Phagocytic Index'). Normal hemocytes exhibited highly efficient phagocytosis and 85%-90% had taken up bacteria after 10 minutes. In contrast, only 55% of Dscam mutant cells had taken up bacteria (Watson, 2005).

To investigate more directly the possible role of Dscam in immune defenses, three questions were addressed. (1) Is Dscam cell autonomously required for phagocytosis in hemocytes? (2) Can antibodies that specifically bind extracellular Ig-domains of Dscam acutely interfere with phagocytosis? (3) Can Dscam isoforms directly bind to pathogens (Watson, 2005)?

Expression of double-stranded RNA (i.e., RNAi) was used to suppress Dscam expression in transgenic flies. A hemolectin promoter region-GAL4 fusion, termed Hml-GAL4, was used for activating expression exclusively in embryonic and larval hemocytes. Hemocytes with Dscam-specific knock-down showed a significantly reduced rate of phagocytosis, with less than 60% of the cells containing bacteria. This partial inhibition may reflect RNAi-mediated knock-down in only a subset of the highly heterogeneous cell population of larval hemocytes. Therefore S2 cells, which represent a less heterogeneous cell population also capable of phagocytosis were examined, and anti-Dscam antibodies were used to block Dscam function. It was reasoned that the short application of anti-Dscam antibodies, in contrast to continuous RNAi, may be less likely to influence general hemocyte characteristics or development. Treatment of S2 cells with polyclonal anti-Dscam serum D-ex1 result in a 30% decrease in the phagocytic index. It is possible that the anti-Dscam antibody may not directly block Dscam-bacteria interactions, or may have additional indirect influences on the process of phagocytosis. However, the reduction of phagocytosis is consistent with the loss-of-function in vivo analysis and in vitro binding studies. Taken together, partial but significant reduction in phagocytosis could be achieved by genetic inhibition of expression in hemocytes and by blocking Dscam protein interactions (Watson, 2005).

Flow cytometry was used to measure whether different Dscam isoforms are capable of binding directly to bacteria. Validity of a standard binding assay was tested using a polyclonal antibody that specifically recognizes E. coli epitopes, and the same assay was used to test binding of different recombinant Dscam isoforms. All isoforms contained C-terminal Fc tags, which were used for detection using fluorescently labeled protein A. Isoforms are designated by the combination of alternative variable Ig domains. Dscam-1.30.30-Fc and Dscam- 7.27.25-Fc contain all of the extracellular domains, whereas Dscam-7.27.13-Fc contains only the N-terminal 9 Ig plus the first FNIII domain. Dscam-7.27.25-Fc and Dscam-7.27.13-Fc could bind to live DH5alpha E. coli bacteria. Binding of Dscam-7.27.13-Fc to E. coli suggests that the 10 N-terminal domains containing all three variable Ig domains are sufficient for binding. In contrast, binding of isoform Dscam-1.30.30-Fc to E. coli is barely detectable, and therefore similar to Fc-peptides alone or control Ig-domains containing anti-heavy chain (mouse) antibodies. It is possible that lack of binding of Dscam-1.30.30-Fc is unique to just this isoform and it may not generally reflect the presence of distinct pools of binding and non-binding isoforms (Watson, 2005).

Therefore, it remains an important task to examine in future studies binding properties of other isoforms. Importantly, the molecular basis of Dscam binding to bacteria is presently unknown and an assessment of binding specificity will crucially depend on the identification of potentially distinct epitopes on bacteria (Watson, 2005).

Although the detailed molecular basis of Dscam function in immune-competent cells is not known, the results are consistent with the possibility that Dscam acts as a signaling receptor or co-receptor during phagocytosis. In addition, binding of Dscam isoforms to bacteria may reflect the possibility that diverse secreted Dscam isoforms are involved in opsonizing invading pathogens in the hemolymph. Comparative genomic analysis of Dscam-like sequences show high conservation of orthologous Dscam genes in Diptera and Hymenoptera orders. To explore Dscam expression and alternative splicing in other insect orders, Dscam gene structure and expression in the flour beetle Tribolium castaneum (Coleoptera) and the silk moth Bombyx mori (Lepidoptera) were examined. Orthologous genes were identified in both species and all Dscam-like domains were found to be highly conserved. The expression of alternative Dscam isoforms was confirmed by cloning and characterizing 32 cDNAs from Tribolium RNA (Tr-Dscam). Alternatively spliced mRNA segments of Tr-Dscam matched corresponding Ig-2, Ig-3 and Ig-7 segments of Drosophila Dscam. RT-PCR and sequencing of Dscam mRNA extracted from fat body tissue of Tribolium larvae revealed nine different isoform sequences (out of 16 cDNAs). These results suggest that expression of diverse Dscam isoforms in immunecompetent fat body cells is conserved among highly diverged insect species (Watson, 2005).

This study provides evidence for a potentially extensive repertoire of thousands of Ig-domain-containing proteins in immune-competent cells of insects, which represent an estimated 60% of metazoan species. Recently, novel and diverse receptor sequences have been identified in jawless vertebrates (lamprey), in protochordates (amphioxus), and in mollusks (freshwater snail). It has also been reported that a large class of scavenger receptors (with an estimated 1,200 scavenger receptor cystein-rich (SRCR) domains) are expressed in putative immune effector cells (coelomocytes) of echinoderms. Similarly, immune responses of crustaceans apparently utilize an extensive set of diverse antimicrobial peptides. Although most animals have not acquired adaptive immunity, this apparently broad conservation of receptor diversity strongly suggests important functions and future studies will have to further address whether the presence of diverse immune receptors in invertebrates increases the effectiveness of immune responses of individual animals. Alternatively, given the relative short life span of many invertebrates, it may be that immune receptor diversity is less important ontogenetically but rather enhances the adaptive potential of animal populations to changing environmental and pathogenic threats (Watson, 2005).

Mutually exclusive splicing of the insect Dscam pre-mRNA directed by competing intronic RNA secondary structures

Drosophila Dscam encodes 38,016 distinct axon guidance receptors through the mutually exclusive alternative splicing of 95 variable exons. Importantly, known mechanisms that ensure the mutually exclusive splicing of pairs of exons cannot explain this phenomenon in Dscam. Two classes of conserved elements have been identified in the Dscam exon 6 cluster, which contains 48 alternative exons -- the docking site, located in the intron downstream of constitutive exon 5, and the selector sequences, which are located upstream of each exon 6 variant. Strikingly, each selector sequence is complementary to a portion of the docking site, and this pairing juxtaposes one, and only one, alternative exon to the upstream constitutive exon. The mutually exclusive nature of the docking site:selector sequence interactions suggests that the formation of these competing RNA structures is a central component of the mechanism guaranteeing that only one exon 6 variant is included in each Dscam mRNA (Graveley, 2005).

Comparative sequence analysis was used to identify RNA sequence elements that could potentially be involved in the regulation of Dscam alternative splicing. The sequences of the Dscam genes of 16 different insects were extracted from GenBank as either preassembled genes or as individual sequence reads from the trace archives that were subsequently assembled into a contig covering the gene. The organisms analyzed consisted of 13 Dipteran species, including 11 Drosophila species (D. melanogaster, D. simulans, D. yakuba, D. erecta, D. ananassae, D. pseudoobscura, D. persimilis, D. willistoni, D. mojavensis, D. virilis, and D. grimshawi) and two mosquito species (Anopheles gambiae [malaria mosquito] and Aedes aegypti [yellow fever mosquito]), the Lepidopteran Bombyx mori (silk worm), the Hymenopteran Apis mellifera (honeybee), and the Coleopteran Tribolium castaneum (red flour beetle). Together these organisms encompass four major taxanomic groups of insects that last shared a common ancestor at least 300 million years ago. The Dscam genes of each organism can each potentially generate tens of thousands of isoforms by alternative splicing, though the exact number of alternative exons differs in most species (Graveley, 2005).

Multiple sequence alignment of the Dscam genes from these 16 species revealed a number of conserved intronic elements, the majority of which were located in the exon 6 cluster. The most highly conserved element in the entire Dscam gene, which is greater than 60,000 bp in D. melanogaster, is located in the intron between the constitutive exon 5 and exon 6.1 and will be referred to as the docking site. The docking site is a 66 nt sequence element in D. melanogaster that is 90%-100% identical in the 10 other Drosophila species examined. Moreover, the central 24 nt of the docking site is nearly invariant in all 16 species. The only exceptions are D. willistoni, which contains a 2 nt insertion at position 34 of the docking site, and A. mellifera, which contains two T to C transitions at positions 22 and 28. A docking site consensus sequence can be derived from the first 37 nt of the alignment (Graveley, 2005).

The second class of conserved elements that were identified will be referred to as selector sequences. The initial selector sequences were identified as relatively conserved sequences in the introns upstream of some of the exon 6 variants. Some of these elements are related to one another. For instance, the selector sequences upstream of exons 6.5, 6.19, and 6.43 all contain the sequence CAGGCAG, while the selector sequences upstream of exons 6.28, 6.36, and 6.44 contain sequences that deviate from CAGGCAG by only one nucleotide. However, this is not universally true since the exon 6.12 selector sequence does not contain this motif. By searching the remaining exon 6 cluster for sequences that are similar to but not identical to the initially identified selector sequences, a potential selector sequence was identified upstream of each exon 6 variant that was also similar in other Drosophila species. An alignment of the selector sequences located upstream of all 48 D. melanogaster exon 6 variants, together with some flanking sequence, revealed that all of the selector sequences overlap with one another to a certain extent. This alignment was used to generate a consensus selector sequence. Importantly, this consensus sequence does not resemble any known splicing regulatory elements or splice site sequences (Graveley, 2005).

Strikingly, the central 28 nt of the consensus selector sequence is complementary to the docking site consensus sequence. Moreover, all 48 predicted D. melanogaster selector sequences are complementary to the docking site. Because the selector sequences all overlap with one another to some extent, the docking site is predicted to interact with only one selector sequence at a time. Thus, the docking site:selector sequence interactions would simultaneously juxtapose exon 5 with the exon 6 variant that is to be included and could explain how the alternative splicing of these 48 exons is mutually exclusive (Graveley, 2005).

The interactions between the selector sequences and the docking site are supported by a number of observations. The docking site is nearly invariant, consistent with the notion that it engages in multiple mutually exclusive interactions. Any mutation in the docking site would affect the interaction with most, if not all, of the selector sequences and therefore interfere with the splicing of the entire exon 6 cluster. In contrast, mutations within a selector sequence would only affect the splicing of the downstream exon 6 variant. Consistent with this, the selector sequences are much less conserved than the docking site. Nonetheless, orthologous selector sequences that contain nucleotide differences can still form similar interactions with the docking site (Graveley, 2005).

Although the docking sites of the non- Drosophila species have diverged from the Drosophila docking site to some extent, potential selector sequences exist upstream of each exon 6 variant in these other species. This is particularly striking given the apparent high rate of recombination within the exon 6 region. The docking site in the honey bee A. mellifera is the most divergent from the docking site in D. melanogaster and contains two U to C changes in the most highly conserved portion. However, putative selector sequences exist upstream of each A. mellifera exon 6 variant and are predicted to interact with the A. mellifera docking sequence with a thermodynamic stability similar to those in D. melanogaster. Most importantly, the two nucleotides that are different in the A. mellifera docking site engage in base-pairing interactions in the majority of the predicted docking site:selector sequence secondary structures. Many of the docking site:selector sequence structures in all species other than the honeybee contain U-A base pairs at these positions, while C-G base pairs exist at these positions in the honeybee structures. This provides several independent examples of compensatory double mutations that maintain the structural integrity of the docking site:selector sequence interactions. Together, these observations strongly support a model in which the selector sequences interact with the docking site in a mutually exclusive manner (Graveley, 2005).

Several mechanisms have been identified that serve to guarantee that pairs of alternative exons are spliced in a mutually exclusive manner. However, none of the known mechanisms can explain how the alternative splicing of genes containing more than two mutually exclusive exons occurs such that only one exon is included. The Dscam gene is an extreme example of this since the exon 4, 6, and 9 clusters contain 12, 48, and 33 exons, respectively. This study describes the docking site and the selector sequences -- two classes of conserved sequence elements within the Dscam exon 6 cluster that have the potential to engage in base-pairing interactions. The mutually exclusive nature of the interactions of the selector sequences with the docking site suggests that the formation of these structures is a central component of the mechanism ensuring that only one of the exon 6 variants is included (Graveley, 2005).

It is quite intriguing that each of the Dscam mRNAs isolated from the fly contains only one of the 48 exon 6 variants despite the fact that each exon is flanked by what appear to be functional splice sites. Thus, the mechanism that exists to prevent multiple exon 6 variants from being included must operate with a high degree of fidelity. A protein has been identified in an RNAi screen that appears to function to prevent all of the exon 6 variants from being spliced together -- when depleted by RNAi, multiple, even adjacent, exon 6 variants are included in the mRNA and they are accurately spliced together (Y. Savva, J. Park, and B.R.G., unpublished data reported in Graveley, 2005). This finding demonstrates that the exon 6 variants are in fact capable of being spliced together but that protein factors exist that function to repress this reaction (Graveley, 2005).

Based on these two sets of observations, a model can be proposed to explain how the alternative splicing of the exon 6 cluster is mutually exclusive. A key component of this model is that a protein(s) acts to both repress the splicing of each exon 6 variant and to prevent the exon 6 variants from being spliced together. It is proposed that the selector sequence upstream of the exon 6 variant that is to be included interacts with the docking site and that this interaction somehow relieves the repression on the downstream exon 6 variant, and as a result, it can be spliced to exon 5. Finally, the exon 6 variant that is then spliced to exon 5 could only be spliced to exon 7 because the exon 6 variants downstream of the included exon would still be repressed. As a result, only one exon 6 variant would be included in the mRNA (Graveley, 2005).

Although the docking site:selector sequence interactions are strongly supported by their evolutionary conservation and some compensatory mutations in the honeybee A. mellifera, this model will obviously need to be experimentally tested with mutations and compensatory mutations that disrupt and restore the docking site:selector sequence interactions. Due to the size (14,000 bp in D. melanogaster) and complexity (48 exons) of the exon 6 cluster, several attempts have been made to generate minigene constructs that lack several of the alternative exons. However, none of the constructs made to date are accurately spliced in tissue culture cells. Thus, these experiments may need to be conducted in the fly using the entire exon 6 cluster or perhaps even the entire Dscam gene. Nonetheless, once a system is in place, it will be interesting to test whether the strength of the docking site:selector sequence interactions contribute to the frequency at which each exon 6 variant is used. At first glance, however, it does not appear that the predicted thermodynamic stability of each docking site:selector sequence interaction correlates with the frequency with which each exon 6 variant is used in flies. Moreover, contrary to what one would expect if splicing occurs cotranscriptionally and the docking site:selector sequence interactions are the driving force of exon 6 selection, the exon 6 variants closest to exon 5 are not chosen more frequently than other exons. Thus, the mechanism involved in selecting a specific exon 6 variant may be distinct from the interaction between the selector sequence and the docking site (Graveley, 2005).

It will also be interesting to determine precisely what the docking site:selector sequence interaction does. The structures of the docking site:selector sequence interactions are somewhat reminiscent of those that direct site-specific RNA editing. Though it is formally possible that some components of the RNA editing machinery could play a role in Dscam alternative splicing, RNAi depletion of ADAR does not affect alternative splicing of Dscam. An alternate possibility is that the docking site:selector sequence structures serve as binding sites for a protein that somehow inactivates the repression of the downstream exon 6 variant. An intriguing possibility is that the interaction juxtaposes the exon 6 variant to a splicing regulatory element upstream of the docking site. Interestingly, an additionally highly conserved sequence element is located immediately adjacent to the docking site that is predicted to form a 20 bp stem-loop structure that is supported by multiple compensatory mutations. However, the function and relevance of this stem-loop structure is not immediately obvious (Graveley, 2005).

Are competing base-pairing interactions a common mechanism that evolved to negotiate the splicing of genes containing multiple mutually exclusive exons? At first glance, it does not appear so. Conserved elements similar to the docking site and selector sequences are not readily apparent in either the exon 4 or exon 9 clusters of Dscam, nor in other genes containing multiple mutually exclusive exons (C. elegans unc-32 and D. melanogaster Myosin heavy chain, ATPα, GluClα, slowpoke, hephaestus, and Thiolester containing protein II). Thus, the use of competing base-pairing interactions may be unique to the Dscam exon 6 cluster. Moreover, additional experimental and comparative genomic work suggests that the mechanisms of mutually exclusive splicing of each cluster in Dscam are quite possibly different. This suggests that multiple distinct and independent mechanisms to ensure the mutually exclusive splicing of clusters of three or more exons may have evolved multiple times. This is not entirely surprising, however, since multiple, distinct mechanisms are known to exist to guarantee that only one exon is included when only two alternative exons need to be chosen from (Graveley, 2005).

Curiously, vertebrate genes that contain a region with more than two mutually exclusive exons have not been identified. This suggests that the vertebrate spliceosome may have lost the ability to negotiate pre-mRNAs containing more than two mutually exclusive exons. Alternatively, insects and worms (and perhaps other metazoans) may have evolved the ability to cope with the challenge of including only one alternative exon among a multitude of possible choices after they last shared a common ancestor with higher eukaryotes. Due to the fact that multiple mutually exclusive exons can be successfully used to generate such a tremendous diversity of proteins from a single gene, it is striking that genes with this organization are not more common in general and appear to be all together absent from vertebrates (Graveley, 2005).

The iStem, a long-range RNA secondary structure element required for efficient exon inclusion in the Drosophila Dscam pre-mRNA

The Drosophila Dscam gene encodes 38,016 different proteins, due to alternative splicing of 95 of its 115 exons, that function in axon guidance and innate immunity. The alternative exons are organized into four clusters, and the exons within each cluster are spliced in a mutually exclusive manner. This study describes an evolutionarily conserved RNA secondary structure that is called the Inclusion Stem (iStem) that is required for efficient inclusion of all 12 variable exons in the exon 4 cluster. Although the iStem governs inclusion or exclusion of the entire exon 4 cluster, it does not play a significant role in determining which variable exon is selected. Thus, the iStem is a novel type of regulatory element that simultaneously controls the splicing of multiple alternative exons (Kreahling, 2005).

To begin characterizing the RNA sequence elements involved in Dscam exon 4 alternative splicing, an exon 4 minigene was generated. A portion of the Dscam gene beginning in exon 3 and ending in the intron downstream of exon 5 was cloned into a Drosophila expression vector containing the inducible metallothionein promoter to generate pDscamWT. This vector was transiently transfected into Drosophila S2 cells, and transcription was induced by the addition of CuSO₄. Splicing was then analyzed by RT-PCR using a primer in exon 5 and a minigene-specific primer that anneals upstream of exon 3. Analysis of these PCR products on denaturing polyacrylamide gels revealed that the majority of the transcripts contain one of the exon 4 variants. However, approximately 15% of the transcripts lacked an exon 4 variant and instead contained exon 3 spliced directly to exon 5. The profile of exon 4 variants that are utilized was analyzed both by sequencing cloned RT-PCR products and by resolving the RT-PCR products on a single-strand conformational polymorphism gel, which separates the molecules based on conformation rather than size. The majority of the transcripts from pDscamWT contain exons 4.12, 4.1, and 4.11, although exons 4.8, 4.10, 4.6, and 4.4 were also detectibly utilized. Thus, in S2 cells, transcripts derived from the minigene are spliced in a mutually exclusive manner and multiple exon 4 variants are selected. This system is therefore well suited for identifying and analyzing cis-acting sequences involved in Dscam exon 4 splicing (Kreahling, 2005).

RNA sequences required for various aspects of exon 4 splicing were identified by generating deletions throughout the entire minigene and testing their effects on splicing in transfection experiments. Several of these deletions had profound effects on exon 4 splicing. This study focuses on an interesting set of deletions that removed portions of the 1,412-nucleotide (nt) intron between exons 3 and 4.1. Deletion of a 670-nt fragment encompassing nt 422 to 1090 of the intron (pDscamDelta1) had no effect on splicing of the minigene. However, extending the 5' boundary of this deletion to position 224 of the intron (pDscamDelta2) resulted in a dramatic increase in exon 4 skipping, as compared to pDscamWT results. Additional deletions that progressively decrease the 3' boundary of the deletion defined a 105-nt region (pDscamDelta5) that, when deleted, also resulted in a significant increase in exon 4 skipping. In contrast, a slightly smaller 58-nt deletion encompassing nucleotides 224 to 280 (pDscamDelta6) displayed no more exon 4 skipping than the wild-type construct. Thus, an element located between nt 280 and 422 of this intron (defined by the deletion boundaries in pDscamDelta1 and pDscamDelta6) is required for efficient exon 4 inclusion (Kreahling, 2005).

The sequence of the 105-nt segment deleted in pDscamDelta5 contains a pyrimidine-rich region and resembles a 3' splice site. Although both U2 snRNP and U2AF can bind to this element in nuclear extracts, mutations that disrupt the binding of these splicing factors have little if any effect on exon 4 skipping. This led to a hypothesis that this element may not function as a protein binding site in vivo. Detailed sequence analysis revealed that a 27-nt segment of this element could potentially base pair with a sequence located 18 nt downstream of exon 3, forming a structure consisting of a 27-bp stem containing a 2-nt internal bulge and a 275-nt loop. It was hypothesized that if the sequence element initially identified functions to promote exon 4 inclusion by forming this stem-loop structure, disrupting this structure should affect the efficiency of exon 4 inclusion. This was tested by disrupting the stem with deletions that removed either the 5' or 3' half of the stem. Indeed, a dramatic increase was observed in exon 4 skipping—deleting the 27 nt that make up either the 5' or 3' half of the stem resulted in ~55-fold or ~60-fold increases in the ratio of exon 4 exclusion/inclusion, respectively. This suggests that disrupting the formation of this RNA secondary structure has a significant effect on exon 4 splicing and that this structure is important for efficiently including an exon 4 variant. Hereafter, this RNA structural element will be referred to as the iStem (Kreahling, 2005).

Due to the evolutionarily conserved proximity of the iStem to exon 3 and the fact that the iStem affects the inclusion of all 12 exon 4 variants equally, it seems most likely that the iStem acts on the 5' splice site of exon 3. One possibility is that the iStem promotes the assembly of a specific protein complex at the 5' splice site of exon 3 that confers upon exon 3 the ability to splice to one of the exon 4 variants. The iStem could do this by serving as a binding site for a splicing regulator or splicing regulatory complex (Kreahling, 2005).

What types of regulators could recognize the iStem? If a protein or complex interacts with the iStem, it would need to do so in a sequence-independent manner. An RNA interference screen was recently conducted to identify proteins that regulate Dscam alternative splicing. Although none of the Drosophila double-stranded RNA binding proteins tested had an impact on the splicing of exon 4, depletion of several DExH/D-box proteins resulted in an increase in exon 4 skipping (Park, 2004). One of these DExH/D-box proteins identified in the screen is Rm62, the Drosophila homolog of the human p68 helicase. Interestingly, p68 helicase has been shown to modulate the binding of U1 snRNP to 5' splice sites and functions as an alternative splicing regulator. Thus, it is possible that the iStem serves as a binding site for a DExH/D-box protein (such as Rm62) that interacts with U1 snRNP bound to the 5' splice site of exon 3, resulting in a complex that is competent to splice to one of the exon 4 variants. In the absence of the iStem, or the DExH/D-box protein, the complex would not assemble at the 5' splice site of exon 3 and, as a result, the exon 4 variants would be skipped. Testing this model will require the development of an in vitro splicing system for Dscam; such a system is currently unavailable (Kreahling, 2005).

Tracking the evolution of alternatively spliced exons within the Dscam family

The Dscam gene in the fruit fly Drosophila melanogaster contains twenty-four exons, four of which are composed of tandem arrays that each undergo mutually exclusive alternative splicing, potentially generating 38,016 protein isoforms. This degree of transcript diversity has not been found in mammalian homologs of Dscam. This study examines the molecular evolution of exons within this gene family to locate the point of divergence for this alternative splicing pattern. Using the fruit fly Dscam exons 4, 6, 9 and 17 as seed sequences, sixteen genomes were iteratively searched for homologs, and then phylogenetic analyses of the resulting sequences were performed to examine their evolutionary history. Homologs were found in the nematode, arthropod and vertebrate genomes, including homologs in several vertebrates where Dscam had not been previously annotated. Among these, only the arthropods contain homologs arranged in tandem arrays indicative of mutually exclusive splicing. No homologs to these exons were found within the Arabidopsis, yeast, tunicate or sea urchin genomes but homologs to several constitutive exons from fly Dscam were present within tunicate and sea urchin. Comparing the rate of turnover within the tandem arrays of the insect taxa (fruit fly, mosquito and honeybee), it was found the variants within exons 4 and 17 are well conserved in number and spatial arrangement despite 248-283 million years of divergence. In contrast, the variants within exons 6 and 9 have undergone considerable turnover since these taxa diverged, as indicated by deeply branching taxon-specific lineages. These results suggest that at least one Dscam exon array may be an ancient duplication that predates the divergence of deuterostomes from protostomes but that there is no evidence for the presence of arrays in the common ancestor of vertebrates. The different patterns of conservation and turnover among the Dscam exon arrays provide a striking example of how a gene can evolve in a modular fashion rather than as a single unit (Crayton, 2006).

Structural basis of Dscam isoform specificity

The Dscam gene gives rise to thousands of diverse cell surface receptors thought to provide homophilic and heterophilic recognition specificity for neuronal wiring and immune responses. Mutually exclusive splicing allows for the generation of sequence variability in three immunoglobulin ecto-domains, D2, D3 and D7. This study reports X-ray structures of the amino-terminal four immunoglobulin domains (D1-D4) of two distinct Dscam isoforms. The structures reveal a horseshoe configuration, with variable residues of D2 and D3 constituting two independent surface epitopes on either side of the receptor. Both isoforms engage in homo-dimerization coupling variable domain D2 with D2, and D3 with D3. These interactions involve symmetric, antiparallel pairing of identical peptide segments from epitope I that are unique to each isoform. Structure-guided mutagenesis and swapping of peptide segments confirm that epitope I, but not epitope II, confers homophilic binding specificity of full-length Dscam receptors. Phylogenetic analysis shows strong selection of matching peptide sequences only for epitope I. It is proposed that peptide complementarity of variable residues in epitope I of Dscam is essential for homophilic binding specificity (Meijers, 2007).

This study has provided a structural analysis of the recognition specificity of two variable immunoglobulin domains of Drosophila Dscam. Although the D1-D4 structures reported here contain only two variable domains, and it remains to be determined how D7 contributes to binding, biochemical analysis in the context of the full-length Dscam receptor is consistent with an essential contribution of the variable peptide segments of epitope I to the homophilic-binding specificity of Dscam. Swapping the peptide segment containing epitope I but not epitope II resulted in a full switch in binding specificity between two isoforms. This strongly suggests that in a Dscam dimer the matching epitope I peptides enable binding, and non-matching ones inhibit homophilic binding, thereby functioning as a specificity module. The strong sequence conservation of epitope I residues is consistent with a high evolutionary selection pressure preserving a limited set of homophilic-binding interfaces. Although an involvement of epitope II in binding of non-Dscam ligands has not been tested experimentally, the apparently faster-evolving sequence variability in epitope II would be consistent with immune receptor adaptations to dynamic alterations in host-pathogen interactions. It is therefore hypothesized that this structural separation of homophilic and heterophillic binding (that is potentially self and non-self recognition) in Dscam may have enabled the parsimonious use of the same gene in creating a large receptor diversity in both the nervous system and immune system (Meijers, 2007).

Quantitative profiling of Drosophila melanogaster Dscam1 isoforms reveals no changes in splicing after bacterial exposure

The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, a long read RNA sequencing method was used to directly investigate the Dscam1 splicing pattern after exposing adult Drosophila melanogaster and a S2 cell line to live Escherichia coli. After bacterial exposure both models showed increased expression of immune-related genes, indicating that the immune system had been activated. However there were no changes in total Dscam1 mRNA expression. RNA sequencing further showed that there were no significant changes in individual exon expression and no changes in isoform splicing patterns in response to bacterial exposure. Therefore these studies do not support a change of D. melanogaster Dscam1 isoform diversity in response to live E. coli. Nevertheless, in future this approach could be used to identify potentially immune-related Dscam1 splicing regulation in other host species or in response to other pathogens (Armitage, 2014 PubMed).

Specification of dendritogenesis site in Drosophila aCC motoneuron by membrane enrichment of Pak1 through Dscam1

Precise positioning of dendritic branches is a critical step in the establishment of neuronal circuitry. However, there is limited knowledge on how environmental cues translate into dendrite initiation or branching at a specific position. Through a combination of mutation, RNAi, and imaging experiments, this study found that a Dscam-Dock-Pak1 hierarchical interaction defines the stereotypical dendrite growth site in the Drosophila aCC motoneuron. This interaction localizes the Cdc42 effector Pak1 to the plasma membrane at the dendrite initiation site before the activation of Cdc42. Ectopic expression of membrane-anchored Pak1 overrides this spatial specification of dendritogenesis, confirming its function in guiding Cdc42 signaling. It was further discovered that Dscam1 localization in aCC occurs through an inter-neuronal contact that involves Dscam1 in the partner MP1 neuron. These findings elucidate a mechanism by which Dscam1 controls neuronal morphogenesis through spatial regulation of Cdc42 signaling and, subsequently, cytoskeletal remodeling (Kamiyama, 2015).

Protein Interactions

It has been proposed, based on mutational analyses of domain requirements for Dreadlocks (Dock) in axon guidance, that Dock interacts with upstream guidance signals in a redundant fashion through both SH3 and SH2 domains. The Dock SH2 domain interacts directly with Dscam. Binding is disrupted by pretreatment with alkaline phosphatase. The SH3 domains of Dock also directly interact with Dscam. Interactions between different SH3 domains and Dscam were assessed in a yeast two-hybrid assay and in GST pulldown experiments. In yeast, full-length Dock interacts strongly with the cytoplasmic domain of Dscam. Each of the three SH3 domains tested individually in yeast show a comparatively reduced level of interaction. This suggests Dock interacts through multiple SH3 domains with Dscam (Schmucker, 2000).

The interaction sites between different SH3 domains and Dscam were mapped. Two putative SH3 binding sites (PXXP1 and PXXP2) separated by 40 amino acids are found in the N-terminal portion of the Dscam cytoplasmic domain; a C-terminal polyproline sequence (PEPPP) is also present. Site-directed mutagenesis of the PXXP sites revealed that the first SH3 domain (SH3-1) binds preferentially to PXXP1 and the third SH3 domain (SH3-3) binds to PXXP2. GST-SH3-1 and GST-SH3-3 interact with the N-terminal half of the cytoplasmic tail of Dscam containing the PXXP sites, but only weakly to the C-terminal half encompassing the polyproline sequence. Conversely, the second SH3 domain (SH3-2) binds preferentially to the C-terminal polyproline motif. That PXXP1 and PXXP2 sequences are the primary interaction sites between Dock and Dscam is strongly supported by the marked reduction in interaction between Dock and the cytoplasmic domain of Dscam carrying point mutations in both these sites. Residual binding may be due to interaction between SH3-2 and the C-terminal polyproline sequence. In summary, these data indicate that Dscam binds directly to Dock through both SH3 and SH2 domains, consistent with genetic studies arguing for redundancy between these domains (Schmucker, 2000).

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, the SH2 domain of Dock was used to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which has been shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system. The smallest protein in this complex p63) has now been identified. p63 has been named DSH3PX1 because it appears to be the Drosophila ortholog of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. DSH3PX1 interacts with the Drosophila ortholog of Wasp, a protein component of actin polymerization machinery, and DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking (Worby, 2001).

Complementary chimeric isoforms reveal Dscam1 binding specificity in vivo

Dscam1 potentially encodes 19,008 ectodomains of a cell recognition molecule of the immunoglobulin (Ig) superfamily through alternative splicing. Each ectodomain, comprising a unique combination of three variable (Ig) domains, exhibits isoform-specific homophilic binding in vitro. Although it has been proposed that the ability of Dscam1 isoforms to distinguish between one another is crucial for neural circuit assembly, via a process called self-avoidance, whether recognition specificity is essential in vivo has not been addressed. This issue was tackled by assessing the function of Dscam1 isoforms with altered binding specificities. Pairs of chimeric isoforms were generated that bind to each other (heterophilic) but not to themselves (homophilic). These isoforms failed to support self-avoidance or did so poorly. By contrast, coexpression of complementary isoforms within the same neuron restored self-avoidance. These data establish that recognition between Dscam1 isoforms on neurites of the same cell provides the molecular basis for self-avoidance (Wu, 2012).

Linking cell surface receptors to microtubules: Tubulin Folding Cofactor D mediates Dscam functions during neuronal morphogenesis

Formation of functional neural networks requires the coordination of cell surface receptors and downstream signaling cascades, which eventually leads to dynamic remodeling of the cytoskeleton. Although a number of guidance receptors affecting actin cytoskeleton remodeling have been identified, it is relatively unknown how microtubule dynamics are regulated by guidance receptors. This study used Drosophila olfactory projection neurons to study the molecular mechanisms of neuronal morphogenesis. Dendrites of each projection neuron target a single glomerulus of approximately 50 glomeruli in the antennal lobe, and the axons show stereotypical pattern of terminal arborization. In the course of genetic analysis of the dachsous mutant allele (ds^UAO71), this study identified a mutation in the tubulin folding cofactor D gene (TBCD) as a background mutation. TBCD is one of five tubulin-folding cofactors required for the formation of alpha- and beta-tubulin heterodimers. Single-cell clones of projection neurons homozygous for the TBCD mutation displayed disruption of microtubules, resulting in ectopic arborization of dendrites, and axon degeneration. Interestingly, overexpression of TBCD also resulted in microtubule disruption and ectopic dendrite arborization, suggesting that an optimum level of TBCD is crucial for in vivo neuronal morphogenesis. It was further found that TBCD physically interacts with the intracellular domain of Down syndrome cell adhesion molecule (Dscam), which is important for neural development and has been implicated in Down syndrome. Genetic analyses revealed that TBCD cooperates with Dscam in vivo. This study may offer new insights into the molecular mechanism underlying the altered neural networks in cognitive disabilities of Down syndrome (Okumura, 2015).

Structural basis of Dscam1 homodimerization: Insights into context constraint for protein recognition

The Drosophila neural receptor Dscam1 (Down syndrome cell adhesion molecule 1) plays an essential role in neuronal wiring and self-avoidance. Dscam1 potentially encodes 19,008 ectodomains through alternative RNA splicing and exhibits exquisite isoform-specific homophilic binding, which makes it an exceptional example for studying protein binding specificity. However, structural information on Dscam1 is limited, which hinders illumination of the mechanism of Dscam1 isoform-specific recognition. Whether different Dscam1 isoforms adopt the same dimerization mode remains a subject of debate. This study presents 12 Dscam1 crystal structures, provide direct evidence indicating that all isoforms adopt a conserved homodimer geometry in a modular fashion, identifies two mechanisms for the Ig2 binding domain to dispel electrostatic repulsion during dimerization, decodes Ig2 binding specificity by a central motif at its symmetry center, uncovers the role of glycosylation in Dscam1 homodimerization, and finds electrostatic potential complementarity to help define the binding region and the antiparallel binding mode. A concept is proposed that the context of a protein may set restrictions to regulate its binding specificity, which provides a better understanding of protein recognition (Li, 2016).

Dscam1 forms a complex with Robo1 and the N-terminal fragment of Slit to promote the growth of longitudinal axons

The Slit protein is a major midline repellent for central nervous system (CNS) axons. In vivo, Slit is proteolytically cleaved into N- and C-terminal fragments, but the biological significance of this is unknown. Analysis in the Drosophila ventral nerve cord of a slit allele (slit-UC) that cannot be cleaved revealed that midline repulsion is still present but longitudinal axon guidance is disrupted, particularly across segment boundaries. Double mutants for the Slit receptors Dscam1 and robo1 strongly resemble the slit-UC phenotype, suggesting they cooperate in longitudinal axon guidance, and through biochemical approaches, it was found that Dscam1 and Robo1 form a complex dependent on Slit-N. In contrast, Robo1 binding alone shows a preference for full-length Slit, whereas Dscam1 only binds Slit-N. Using a variety of transgenes, it was demonstrated that Dscam1 appears to modify the output of Robo/Slit complexes so that signaling is no longer repulsive. These data suggest that the complex is promoting longitudinal axon growth across the segment boundary. The ability of Dscam1 to modify the output of other receptors in a ligand-dependent fashion may be a general principle for Dscam proteins (Alavi, 2016).

DEVELOPMENTAL BIOLOGY

Dscam is expressed on axons in the embryonic CNS. Dscam RNA is expressed in Bolwig's organ as well as more generally within the CNS and PNS. The protein product is exclusively expressed on axon processes (Schmucker, 2000).

Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies

Dscam is an immunoglobulin (Ig) superfamily member that regulates axon guidance and targeting in Drosophila. Alternative splicing potentially generates 38,016 isoforms differing in their extracellular Ig and transmembrane domains. Dscam mediates the sorting of axons in the developing mushroom body (MB). This correlates with the precise spatiotemporal pattern of Dscam protein expression. MB neurons express different arrays of Dscam isoforms and single MB neurons express multiple isoforms. Two different Dscam isoforms differing in their extracellular domains introduced as transgenes into single mutant cells partially rescued the mutant phenotype. Expression of one isoform of Dscam in a cohort of MB neurons induces dominant phenotypes, while expression of a single isoform in a single cell does not. It is proposed that different extracellular domains of Dscam share a common function and that differences in isoforms expressed on the surface of neighboring axons influence interactions between them (Zhan, 2004).

Dscam is expressed selectively in young MB axons and is required to form a single highly organized nerve bundle and for the segregation of MB axon branches. Using DNA microchip analysis, it was demonstrated that MB neurons express many different Dscam isoforms, that different isoforms combinations are expressed in single cells, and that they differ from the array of isoforms expressed in photoreceptor neurons. Single isoforms containing different extracellular domains substantially rescue the null mutant phenotype in single mutant cells. Rescue activity was restricted to TM2-containing isoforms. This likely reflects differences in the localization of TM1 and TM2 isoforms in MB neurons. Expression of single Dscam isoforms in cohorts of MB neurons induced strong dominant phenotypes. Conversely, expression of a single isoform in isolated neurons did not. It is concluded that different Dscam isoforms have a common function largely accounting for the ability of single isoforms to provide partial rescuing activity. It is proposed that appropriate patterning in the MB requires that each MB axon express Dscam isoforms differently from its neighbors (Zhan, 2004).

Previous studies have demonstrated that Dscam is required for MB formation. Using genetically mosaic animals, it was discovered that the removal of Dscam in large clones results in massive defects in adult MB structure, including collapse and fusion of lobes. In single-cell clones, while Dscam mutant MB neurons invariably bifurcated at the base of the peduncle, branches frequently fail to segregate to different lobes. The developmental analysis described here demonstrates an earlier additional requirement for Dscam function in regulating MB development (Zhan, 2004).

A striking feature of MB organization is the precise topographic relationship between dendrites, axons, and synapses of MB neurons. These neurons fall into morphologically and molecularly (e.g., neurotransmitter expression) distinct classes. The axons in the peduncle are arranged in a concentric manner with a smooth gradient of developmental age in which the oldest fibers are located peripherally. Dendrites are arranged in a zonal fashion that shares a simple topographic relationship to axons not only in the peduncle but also in their layered organization in the lobes. Although MBs in different insects vary considerably in their size and complexity, they share a common topographic organization. This conservation of structure argues that the spatial relationship between different cellular elements in the MB is of functional significance (Zhan, 2004).

Developmental studies have revealed a common mechanism for promoting fiber organization in the MB. In Drosophila, the honey bee, cricket, and cockroach, different MB neuronal classes (e.g., gamma, alpha'ß', and alphaß) are generated in a sequential fashion from a common neuroblast. For instance, gamma neurons are generated prior to alpha'ß' and are followed by alphaß. Cells generated from each neuroblast form a separate cluster from which a single fascicle projects into the peduncle. At the entrance to the peduncle, axons from different fascicles sort such that young axons project into the center of the developing peduncle where they displace progressively older axons peripherally. The relative position of axons established in the peduncle is conserved in the dendritic calyces and lobes. Since MB neurons are generated in a parallel sequential fashion from multiple neuroblasts, mechanisms must exist to promote the selective association of axons of the same age (Zhan, 2004 and references therein).

Based on the Dscam expression pattern and mutant phenotypes as well as biochemical studies, it is proposed that Dscam promotes the association between young axons. Dscam protein is first observed on MB axons as they converge in the peduncle, and it remains expressed on these axons as they branch into the lobes. Expression is dynamic, and Dscam protein is downregulated as fibers mature. In the absence of Dscam, a single core fiber in the peduncle and lobes may be replaced by multiple phalloidin-positive fascicles (typically two or three). These ectopic core fibers are surrounded by a thin layer of axons that stain with neither phalloidin nor Fas II in a manner reminiscent of the organization of the single fiber bundle that forms in the wild-type peduncle. Indeed, it is proposed that in the absence of Dscam young fibers emerging from neurons generated from the same neuroblasts frequently do not converge in the peduncle but rather remain separate. Hence, Dscam ensures that MB neurons generated by different neuroblasts at the same time selectively fasciculate to form a single integrated neuropil (Zhan, 2004).

Defects in the lobe structure may be secondary consequences of earlier defects in the peduncle, or Dscam also may be required in an independent step for different MB branches to select the appropriate pathways in the developing lobes. The branch segregation defects may reflect a role for Dscam in repelling sister branches, thus promoting extension into different lobes. The notion that Dscam could mediate repulsive response is consistent with the phenotypes associated with the overexpression of a single isoform in the MB, as described in this study, as well as in Bolwig's nerve and in olfactory neurons, as well as loss-of-function phenotypes in the olfactory system (Zhan, 2004 and references therein).

The primary motivation for these studies on Dscam function in the MB was to assess whether the diversity of Dscam proteins produced by alternative splicing is functionally important. Microarray data revealed that MB neuronal populations express many different isoforms of Dscam and that single neurons express multiple isoforms. The array of isoforms made in MB neurons is distinct from other neurons, such as photoreceptors isolated from the developing eye disc. While incorporation of alternative exon 4s and 6s into transcripts appeared largely random in these different cell populations, preferential use of alternative exon 9s was observed for other neurons in the developing brain. These findings are consistent with genetic studies showing that the deletion of various alternative exon 4s does not lead to MB defects, arguing for redundancy between them (Zhan, 2004).

Despite the complex pattern of Dscam isoforms expressed in MB neurons, single isoforms of Dscam encoded by cDNA transgenes, including an isoform containing an alternative exon 9 rarely expressed in these neurons, substantially rescues the adult branch segregation phenotype observed in single mutant cells. Whether rescue reflects restoration of the core fiber phenotype in the peduncle or appropriate segregation of axons at the base of the peduncle upon bifurcation is not known. Regardless, these studies argue that all isoforms of Dscam containing TM2 share a common underlying molecular function (Zhan, 2004).

While Dscam proteins containing different extracellular domains provide similar rescuing activity, this is restricted to TM2-containing isoforms. This correlates with differences in protein localization. TM2-containing isoforms are distributed along the entire cell surface, while TM1-containing isoforms were largely excluded from axons. These observations suggest that sequences within TM1, TM2, or both play important roles in regulating the subcellular distribution of Dscam (Zhan, 2004).

In summary, these studies argue that TM1- and TM2-containing isoforms are sorted to different neuronal compartments. It is important to emphasize, however, that the protein localization data are based solely on protein expressed in excess of endogenous levels. Indeed, while transgenes expressing TM1-containing proteins are not sufficient to provide substantial rescue activity, a missense mutation within TM1 leads to defects in MB development similar to Dscam nulls. Hence, it is likely that mechanisms exist to deliver TM1-containing isoforms to axons. Recent biochemical experiments suggest that this may occur through cis interactions with TM2-containing isoforms (Zhan, 2004).

Several lines of evidence suggest that Dscam diversity may be required for normal MB development: (1) expression of a single isoform in a single mutant neuron provides only partial rescue; (2) expression of single isoforms in multiple MB neurons induces a strong dominant phenotype; (3) single MB neurons express multiple isoforms, and different neurons express different combinations of them. While different isoforms share a common feature of homophilic binding, single isoforms show a striking specificity for identical isoforms. They bind strongly to the same isoform and weakly, if at all, to different isoforms. It remains possible that the dominant phenotypes induced when many neurons express a single isoform may, in part, reflect differences in expression levels intrinsic to the method of producing large and small clones using the MARCM system. However, given the biochemical data, it is highly likely that interactions between neuronal cell surfaces expressing the same isoform of Dscam will be different from neurons expressing different isoforms (Zhan, 2004).

It is proposed that Dscam plays two distinct functions in MB neurons: one based on interactions between isoforms expressed on the surface of different neurons and the other based on interactions between isoforms on branches of the same neuron. It is envisioned that these interactions promote different levels of signal, with low levels promoting contact-dependent attraction, leading to fasciculation, and high levels promoting contact-dependent repulsion. In both the peduncle and lobes, Dscam on different MB growth cones promotes core fiber formation. As different MB neurons express different combinations of Dscam isoforms, this fasciculation function may be a consequence of low signaling levels. This may result from weak interactions between different isoforms or interactions between the small fraction of identical isoforms that may be shared by different MB neurons. At the base of the peduncle, MB growth cones bifurcate and then segregate to different lobes. In the absence of Dscam, axons bifurcate, as in wild-type, but frequently fail to segregate. Here, interactions between identical isoforms of Dscam expressed on sister branches of the same neurons may produce a strong signal activating a repellent response, thereby preventing axons from extending into the same lobe. Indeed, the diversity of Dscam expression on MB axons ensures that only sister branches express an identical set of Dscam isoforms. Upon segregating to the two lobes, weaker interactions between Dscam on axons of different neurons promote fasciculation. Since the biochemical properties of Dscam proteins and the gain- and loss-of-function phenotypes of Dscam throughout the nervous system are complex, Dscam diversity may contribute to wiring specificity in different ways in different developmental contexts (Zhan, 2004).

Dynein-dynactin complex is essential for dendritic restriction of TM1-containing Drosophila Dscam

Many membrane proteins, including Drosophila Dscam, are enriched in dendrites or axons within neurons. However, little is known about how the differential distribution is established and maintained. Dscam isoforms carrying exon 17.1 (Dscam[TM1]) are largely restricted to dendrites, while Dscam isoforms with exon 17.2 (Dscam[TM2]) are enriched in axons. This study investigated the mechanisms underlying the dendritic targeting of Dscam[TM1]. Through forward genetic mosaic screens and by silencing specific genes via targeted RNAi, it was found that several genes, encoding various components of the dynein-dynactin complex, are required for restricting Dscam[TM1] to the mushroom body dendrites. In contrast, compromising dynein/dynactin function did not affect dendritic targeting of two other dendritic markers, Nod and Rdl. Tracing newly synthesized Dscam[TM1] further revealed that compromising dynein/dynactin function did not affect the initial dendritic targeting of Dscam[TM1], but disrupted the maintenance of its restriction to dendrites. The results of this study suggest multiple mechanisms of dendritic protein targeting. Notably, dynein-dynactin plays a role in excluding dendritic Dscam, but not Rdl, from axons by retrograde transport (Yang, 2008).

Multiple lines of evidence indicate that the dynein/dynactin complex has an important function in maintaining proper distribution of dendritic Dscam in MB neurons. First, mutations in three components (Lis1, Dmn and p24) of the dynein/dynactin complex were recovered based on mislocalization of dendritic Dscam through a MARCM-based genetic mosaic screen. Second, silencing other components of the complex with RNAi also resulted in mistargeting of dendritic Dscam to axons. Third, disrupting dynein/dynactin function with dominant-negative Glued reproduced the mislocalization phenotype. Further, newly synthesized Dscam[TM1] was preferentially targeted to dendrites. Interestingly, compromising dynein/dynactin function did not affect the targeting from cell bodies to dendrites but disrupted the continuous exclusion of dendritic Dscam from axons. Altogether, these findings show that dynein/dynactin normally acts to prevent Dscam[TM1] from entering axons by retrograde axonal transport (Yang, 2008).

Acute induction by TARGET, in which GAL4-dependent expression of UAS-transgene is acutely controlled by a temperature-sensitive GAL4 repressor, GAL80ts, revealed two mechanisms underlying the dendritic distribution of Dscam[TM1]. Newly synthesized Dscam[TM1] was largely excluded from axons, suggesting directed dendritic targeting and the involvement of selective transport in the dendritic distribution of Dscam[TM1]. Though dynein/dynactin is essential for restricting Dscam[TM1] to dendrites, knocking down dynein/dynactin function did not disrupt the directed dendritic targeting. This leads to the belief that dynein/dynactin is required for preventing dendritic Dscam from misdistributing into axons. When dynein/dynaction function was compromised, newly synthesized Dscam[TM1] remained consistently targeted to dendrites but later leaked into axons. Dendritic Dscam gradually filled the axons; and it took about six hours for Dscam[TM1] to reach the axon termini. This protracted process of mislocalization suggests that dendritic Dscam passively leaks into the axons, and that dynein/dynactin-mediated retrograde axonal transport normally acts to rapidly move leaked Dscam[TM1]-containing vesicles out of the axons. In summary, these phenomena not only demonstrate a dynein-dynactin-independent mechanism of selective transport that preferentially targets Dscam[TM1]-containing vesicles to dendrites, but also implicate the involvement of retrograde axonal transport in preventing accumulation of Dscam[TM1] in axons. These two independent mechanisms act together to ensure restriction of dendritic Dscam to the dendrites (Yang, 2008).

Although the dynein/dynactin complex is essential for maintaining dendritic distribution of Dscam[TM1], the results do not reveal whether mislocalized Dscam[TM1] is on the plasma membrane or in vesicles inside the cytoplasm. It is possible that dendritic Dscam passively leaks into axons either through membrane diffusion or mistargeting of vesicles. Since blocking endocytosis with temperature-sensitive shibire mutant showed no obvious effect on Dscam dendritic distribution, the model is favored that dynein/dynactin acts to prevent axonal accumulation of Dscam[TM1] by actively moving mistargeted Dscam[TM1]-containing vesicles out of axons by retrograde axonal transport (Yang, 2008).

Dscam[TM1]-containing cargos are primarily targeted to dendrites via a dynein/dynactin-independent process. In addition, they are effectively excluded from the axons by dynein/dynactin-mediated retrograde axonal transport. However, dynein/dynactin is not routinely needed for excluding dendritic proteins from the axons. Since no biological process can be carried out with absolute fidelity, it is conceivable that dendritic molecules of most kinds may accidentally leak into the axons. Some salvage mechanism(s) should exist for actively clearing mislocalized molecules to prevent any significant accumulation in the wrong places. One of the possibilities is that dynein/dynactin mediates retrograde axonal transport and can serve as a general mechanism for removing dendritic molecules out of axons. This hypothesis remains to be tested thoroughly. Nonetheless, blocking dynein/dynactin function did not affect the distribution of two other dendritic markers checked. Nod-β-gal is a reliable minus-end reporter of microtubules, and misdistribution of Nod-β-gal in MB axons has been shown in short stop mutant clones, in which microtubule polarity is perturbed. Absence of Nod-β-gal from the axons of dynein/dynactin mutant neurons demonstrates that the microtubules in axons remained uniformly polarized with minus ends pointing toward cell bodies, and rules out the possibility that dendritic Dscam became mislocalized due to abnormal microtubule organization. As to Rdl-HA, which, like Dscam[TM1], is a membrane protein, a lack of effect on its somatodendritic distribution indicates that dynein/dynactin is selectively involved in preventing dendritic Dscam from leaking into the axons. Diverse mechanisms may be utilized to efficiently clear different dendritic proteins in axons (Yang, 2008).

Regarding the mechanism(s) of selective transport, directed dendritic targeting apparently requires motor proteins that selectively move cargos toward the dendrites. Since dendrites, but not axons, carry microtubules with minus ends pointing away from cell bodies, potential candidates that underlie directed dendritic targeting include all minus-end-directed microtubule motors. Notably, dynein/dynactin is dispensable to the initial dendritic targeting of Dscam[TM1] or the continuous dendritic restriction of Rdl, arguing against any critical role for minus-end-directed dynein/dynactin in transporting cargos into the dendrites. Other microtubule motors that might support such directional movement include dendrite-specific plus-end-directed motors (e.g. KIF17 and KIF21B), though it remains mysterious how a plus-end-directed motor can be well restricted to dendrites. In theory, forward genetic mosaic screens will ultimately allow uncovering of the diverse mechanisms of dendritic protein targeting. Encouragingly, mutants have been obtained that exhibit different mislocalization phenotypes, further characterization of which should shed additional light on neuron polarity and its underlying cellular/molecular mechanisms. Notably, in DC-B9 mutant clones, mistargeted Dscam[TM1]::GFP existed abundantly in the MB peduncle, preferentially accumulated at the end of the peduncle, but never extended into the axon lobes. This intriguing phenotype suggests presence of distribution barriers not only in the beginning of axons but also at the junction between the proximal axon domain (peduncle) and the distal axon segment (lobe), and implies another possible mechanism for restricting Dscam[TM1] to the dendritic membrane (Yang, 2008).

Furthermore, the functional roles of each subunit of the dynein/dynactin complex have not been fully determined. Although several studies of the dynein light chains in mammalian cells indicate that dynein subunits can be functionally specialized, studies in Drosophila show that strong loss-of-function mutations in different dynein/dynactin subunits show extensive overlap in the resulting mutant phenotypes. The current data indicate that Lis1, Dmn, Glued, p24, p25, Dhc64C, Dhc62B, and Dlc90F all participate in the complete function of dynein/dynactin complex in maintaining dendritic distribution of Dscam. This result supports the idea that all the dynein/dynactin subunits work together to fulfill its diverse functions, and loss of any subunits may result in different degrees of similar dynein/dynactin-dysfunctional phenotypes (Yang, 2008).

With respect to Dscam targeting motifs, the cytoplasmic juxtamembrane domain of Dscam may dictate its TM-dependent subcellular localization. However, further structure-distribution analysis only allowed location of an axonal targeting motif to the cytoplasmic juxtamembrane region of TM2, leaving its dendritic targeting motif(s) still undetermined. In addition, using the same system it could not be determined whether any of the mutants recovered here also affects the axonal targeting of Dscam[TM2], since transgenic Dscam[TM2] becomes uniformly distributed upon overexpression following an analogous induction. The involvement of multiple mechanisms in targeting specific Dscams to specific neuronal domains further supports the notion that Dscam isoform compositions in the dendrites versus axons of the same neurons need to be independently regulated, elucidation of the physiological significance of which promises to shed new light on how the brain develops and operates (Yang, 2008).

In summary, this study has uncovered a scavenger mechanism for maintaining dendritic distribution of Dscam[TM1] and provide an in vivo model to study neuron polarity and differential protein targeting. On top of the many known functions of dynein/dynactin (including mitosis, vesicular transport, retrograde signaling, neuronal migration), dynein/dynactin helps restrict certain dendritic proteins to the somatodendritic domain of neurons by preventing them from spreading into the axons. Notably, multiple independent mechanisms act together to locate Dscam[TM1] to dendrites; and diverse mechanisms are utilized to target different dendritic proteins to the dendrites (Yang, 2008).

EFFECTS OF MUTATION

To critically assess whether Dscam is required for axon guidance, loss-of-function mutations were identified. Dscam is localized to region 43B1-B3. Four lethal complementation groups have been mapped to this region. Sequences abutting a P element lethal insertion into one of these complementation groups, l(2)43Bc, were identical to sequences in a P1 genomic clone containing Dscam sequences. This insertion maps 1200 nucleotides downstream from exon 4.1; exon 4 encodes part of the second Ig domain. Two inversion alleles of l(2)43Bc also break within the Dscam locus. In(LR)43b71kIA breaks in the 8.8 kb intron separating exons 2 and 3. A second inversion, In(2R)DX8, also breaks within the Dscam locus, although the precise breakpoint was not identified. A single EMS allele, l(2)43Bc¹, fails to complement the inversions and the P allele. All alleles were early larval lethal. They have been renamed as follows: l(2)43Bc¹ = Dscam^E1; In(LR)43b71kIA = Dscam^X1; In(2R)Dx8 = Dscam^X2; and the P allele = Dscam^p (Schmucker, 2000).

Axon bifurcation results in the formation of sister branches, and divergent segregation of the sister branches is essential for efficient innervation of multiple targets. Axon bifurcation occurs when one growth cone is split into two and the two growth cones diverge. The directional segregation of the growth cones and thus the sister branches derived from a single axon are essential for propagating neural signals in divergent directions. If axon bifurcation leads to formation of 'twin' growth cones, how can these growth cones faithfully project away from each other and toward different targets? This simultaneous extension and divergence of twin growth cones might be achieved simply by mutual repulsion mechanisms. Alternatively, during or soon after bifurcation, individual growth cones might further differentiate and acquire distinct sets of guidance receptors through interactions among one another or with different glial cells. From a genetic mosaic screen, it has been found that a lethal mutation in Dscam specifically perturbs segregation of axonal branches in the mushroom bodies. Single axon analysis further reveals that Dscam mutant axons generate additional branches, which randomly segregate among the available targets. Moreover, when only one target remains, branching is suppressed in wild-type axons while Dscam mutant axons still form multiple branches at the original bifurcation point. It is concluded that Dscam controls axon branching and guidance such that a neuron can innervate multiple targets with minimal branching (Wang, 2002).

To facilitate mosaic analysis in the complex CNS, a novel genetic mosaic system, called MARCM has been developed, in which only the homozygous cells lacking GAL80 are uniquely labeled in mosaic tissues. Using the MARCM genetic mosaic system, the wild-type development of the Drosophila MB has been described with unprecedented single-cell resolution. Moreover, by creating clones of MB neurons homozygous for various mutations in mosaic organisms, it is possible to elucidate the molecular mechanisms controlling different aspects of MB development. Interestingly, from a genetic mosaic screen designed for identifying novel mutations that cause specific defects in the development of MB neurons, a lethal mutation was found that specifically hinders the formation of bifurcated axon bundles. If the mutant clone is created before the birth of neurons that normally project two perpendicular fascicles, these neurons will instead extend their axons in only one of the two original directions. One mutant neuroblast clone in the whole MB can alter the projections of the other three wild-type clonal units. Phenotypic analysis of single neurons further reveals that mutant axons give rise to additional branches at the bifurcation point and that the branches are distributed randomly among the accessible pathways. When only one path exists, wild-type neurons project a single process along the path while mutant neurons generate multiple branches. In addition, defects in axon extension are observed in certain mutant MB neurons. Mapping by recombination and complementation reveals this interesting lethal mutation is an allele of Dscam, suggesting a novel molecular mechanism for mediating formation and guidance of axonal branches (Wang, 2002).

The MBs are paired structures, one in each brain lobe; and each MB is derived from four neuroblasts (Nbs), each of which generates a similar set of three distinct types of neurons. Gamma neurons, which are generated prior to the mid-3^rd instar stage, project axons into the gamma lobe at the adult stage; alpha'/ß' neurons, which are generated in late 3^rd instar, have bifurcated axons that form the alpha' and ß' lobes; and alpha/ß neurons, which are generated after puparium formation (PF), project their bifurcated axonal branches into the alpha and ß lobes. In a genetic mosaic screen, a novel lethal mutation, l(2R)MB99, was identified that causes various defects in the guidance of bifurcated alpha/ß axons (Wang, 2002). When subsets of cells within wild-type MB Nb clones are selectively marked using GAL4-201Y, all labeled axons extend either into the gamma lobe or the alpha and ß lobes; the alpha lobe extends dorsally while the gamma and ß lobes extend medially toward the midline. Only a small number of the late pupal-born alpha/ß neurons are marked, so the alpha and ß lobes appear very thin and faint as compared with the gamma lobe. In addition, these labeled axons occupy the center of the alpha and ß lobes. These GAL4-201Y-marked alpha and ß lobes are hereafter referred to as the core alpha and ß lobes. Notably, the core alpha and ß lobes are morphologically indistinguishable, consistent with the fact that individual alpha/ß axons bifurcate into two branches that project away from each other into the alpha and ß lobes, respectively. A total of 50 l(2R)MB99 mutant Nb clones were collected for detailed phenotypic analysis. In contrast with normal looking gamma lobes, abnormal alpha/ß lobes are observed in every Nb clone homozygous for the l(2R)MB99 mutation. Being much thicker and denser than in wild-type clones, the core alpha/ß lobes seem to be composed of many more axons in the l(2R)MB99 mutant clones. But, unlike wild-type alpha/ß axons, most mutant core alpha/ß processes appear to fail to reach the ends of the alpha/ß lobes. Because no change in the number of cell bodies can be detected (about 30 GAL4-201Y-positive alpha/ß neurons in each Nb clone), the morphological changes observed in mutant core alpha/ß bundles imply that individual alpha/ß axons acquire supernumerary but short-ending branches in mutant clones. In addition, dramatic changes in the configuration of the alpha/ß lobes are observed in 38% of mutant Nb clones. Instead of bifurcating axons at a right angle, these mutant clones project all of their alpha/ß axons in only one direction, either dorsally or medially. Interestingly, these uni-directionally extending mutant processes can exist as two distinct bundles running side by side, or can be fasciculated into a single bundle. Another phenotype that suggests defects in the divergent segregation of alpha/ß axonal branches is detected in about 20% of mutant Nb clones, where differences in the thickness of the axon bundles exist between the dorsal and medial fascicles. These complicated, wide-ranging abnormalities underscore the importance of the phenotypic analysis of individual axons (Wang, 2002).

Thus Dscam is involved in regulating the bifurcation of MB axons. Instead of having two branches that project away from each other, Dscam mutant axons give rise to supernumerary branches, through repeated bifurcation, that fail to extend in divergent directions. It is likely that homophilic interactions between identical Dscam molecules mediate novel mechanisms that coordinate induction of axon bifurcation with segregation of sister branches. In addition, Dscam mutant axons can alter the projections of wild-type axons within the same MB. Further investigations into the cellular basis of such non-cell-autonomous effects will shed new light on how the insect olfactory learning and memory center acquires its normal projection patterns during development (Wang, 2002). Several different scenarios can account for the observed correlation of ectopic bifurcation with abnormal segregation in Dscam mutant axons. One possibility is that Dscam activity might play a direct role in both divergent segregation of sister branches and suppression of ectopic bifurcation. These two activities could occur via a common mechanism or through two independent signaling events. Alternatively, the role of Dscam in divergent segregation of sister branches could be a secondary consequence of its suppression of axon bifurcation, or vice versa. The idea is favored that Dscam directly controls both formation and segregation of axonal branches because in single mutant cells, abnormal segregation is not always coupled with the generation of additional branches and vice versa. Most likely, Dscam normally prevents sister branches from extending along the same path and consequently suppresses additional bifurcation after the sister branches have occupied all available paths (Wang, 2002).

One mechanism that can explain coordination of axon bifurcation with divergent segregation is Dscam-dependent growth cone collapse. When sister growth cones contact each other, homophilic interactions between Dscam molecules may lead to growth cone collapse or fusion. Thus, when axons bifurcate, only divergently split growth cones could survive, initiating separation of sister branches. Moreover, new growth cones continue to bifurcate until all target sites receive branches; but only one growth cone derived from any individual neuron could traverse a given path because the Dscam-mediated homophilic interactions would immediately collapse adjacent sister growth cones into one. This mechanism would couple formation of axonal branches to divergent guidance of sister branches. The final result would be that individual axons would innervate multiple targets through minimal bifurcation. In contrast, sister branches may extend along the same path after loss of Dscam activity. Consequently, when bifurcation is induced in mutant axons, they randomly distribute their branches and often generate additional branches through repeated bifurcation (Wang, 2002).

This model also suggests that induction of axon bifurcation may not always lead to formation of sister branches. Growth cones split in response to bifurcation-inducing signals. However, if twin growth cones cannot migrate divergently, then no axon bifurcation would be expected after futile cycles of splitting followed by collapse. This mechanism can explain why bifurcation is suppressed in wild-type axons when only one trajectory is left. Despite the lack of bifurcated bundles, induction of axon bifurcation persists in mosaic MBs since Dscam mutant axons still generate multiple branches at the original bifurcation point. Taken together, these observations further support the proposal that divergent guidance is essential for the survival of twin growth cones in the presence of normal Dscam activity (Wang, 2002).

Remarkably, Drosophila Dscam may exist in numerous isoforms through alternative splicing, and Dscam is widely required for axon arborization in distinct CNS neurons. Given that various Dscam isoforms exhibit distinct features in their extracellular domains while sharing common intracellular structures, it is likely that activation of a common Dscam-dependent signaling pathway is dynamically and differentially regulated in distinct growth cones expressing distinct sets of Dscam's. Although immunohistochemistry using an anti-Dscam Ab has revealed general expression of Dscam in most pupal brain structures, it remains unclear whether distinct Dscam isoforms are expressed in different neurons. However, delicate cell type-specific controls of Dscam signaling could be achieved if homophilic interactions were restricted to identical or certain pairs of Dscam isoforms. In addition, activation of Dscam might lead to different developmental changes, depending on the types of cellular structures that are involved. For instance, Dscam-Dscam interactions between split growth cones may result in reunification of growth cones. In contrast, homophilic Dscam binding could mediate contact-dependent attraction of growth cones when the interactions occur between growth cones and their guiding substrates. Such Dscam-mediated growth cone guidance might be crucial to normal extension of GAL4-201Y-positive alpha/ß neurons as well as correct pathfinding of the Bolwig's nerve. If the Dscam pathway is widely used to mediate various growth cone behaviors, it is understandable that numerous Dscam isoforms would be needed to confer diverse activation patterns in different types of growth cones (Wang, 2002).

Analysis of the non-cell-autonomous effects of Dscam mutant clones provides convincing evidence that the first-born alpha'/ß' neurons play a crucial role in shaping the projection patterns of all later-born MB neurons. If Nb clones are induced after the initiation of alpha'/ß' neuron production, both dorsal and medial MB lobes are always observe. In contrast, if early alpha'/ß' neurons are made homozygous for a Dscam mutation, absence of either the dorsal or medial lobe is observed in about one-third of mosaic MBs. Interestingly, consistent patterns exist between alpha'/ß' lobes and alpha/ß lobes. Given that alpha'/ß' neurons, unlike gamma neurons, maintain their projection patterns through metamorphosis, the effects of early alpha'/ß' neurons on the final organization of MB lobes support roles of alpha'/ß' axons as pioneer axons that guide the projections of adult-specific MB neurons. Because alpha'/ß' axons and alpha/ß axons form distinct bundles, the effects of alpha'/ß' axons on the projections of alpha/ß axons may be due to pioneer axon-mediated changes in the arrangement of local glial cells. Since one MB is composed of four indistinguishable clonal units, how several cells within one unit can dominate the entire MB projection patterns remains to be elucidated (Wang, 2002).

The demonstration of Drosophila Dscam's essential roles in the bifurcation of MB axons immediately suggests many lines of research for future investigation of the molecular mechanisms underlying growth cone splitting. For instance, it remains to be determined whether the Dock/Pak signaling pathway is involved in bifurcation of various axons. In addition, it is uncertain whether the vertebrate Dscam plays similar roles during development of the CNS. Identification of additional molecules that control bifurcation of axons in Drosophila may generate new insights into how similar neuronal morphogenetic processes are regulated in vertebrates (Wang, 2002).

Different classes of olfactory receptor neurons (ORNs) in Drosophila innervate distinct targets, or glomeruli, in the antennal lobe of the brain. Specific ORN classes require the cell surface protein Dscam (Down Syndrome Cell Adhesion Molecule) to synapse in the correct glomeruli. Dscam mutant ORNs frequently terminate in ectopic sites both within and outside the antennal lobe. The morphology of Dscam mutant axon terminals in either ectopic or cognate targets is abnormal. Target specificity for other ORNs is not altered in Dscam mutants, suggesting that different ORNs use different strategies to regulate wiring. Multiple forms of Dscam RNA are detected in the developing antenna, and Dscam protein is localized to developing ORN axons. A role is proposed for Dscam protein diversity in regulating ORN target specificity (Hummel, 2003).

Dscam expression was assessed in the developing olfactory system using both in situ hybridization and immunohistology. Between 30% to 40% of pupal development, Dscam RNA is seen in the third antennal segment that contains differentiating ORNs. At this stage of development, many ORNs extend axons into the antennal lobe. Within the target region, Dscam RNA is observed in most, and perhaps all, neuronal cell bodies surrounding the antennal lobe; this region includes the cell bodies of both projection neurons and local interneurons. Dscam protein is highly enriched in ORN axons in early to mid pupal development, with little immunoreactivity apparent on cell bodies, as assessed using an antibody directed to a domain shared by all Dscam isoforms. Similarly, little Dscam immunoreactivity was observed on cell bodies of interneurons and projection neurons. At 40% pupal development, Dscam protein is seen on ORN axons in the peripheral nerve fiber layer surrounding the developing antennal lobe. Dscam is also detected on the centrally located dendritic processes of antennal lobe neurons. At later stages, Dscam immunoreactivity is seen uniformly distributed within developing glomeruli and is markedly downregulated in the antennal lobe of adult flies. In contrast to the vertebrate olfactory system, ORNs in flies are not generated throughout the life of the animal, and hence, it would not be surprising that genes that regulate targeting in the fly olfactory system would be downregulated in the adult (Hummel, 2003).

To assess the diversity of Dscam isoforms expressed in the developing ORNs, third antennal segments at 30% pupal development were isolated and the RNA isoforms expressed were analyzed using RT-PCR. Seventy-two different clones from six different antennae were sequenced, and sixty-eight of these comprised different combinations of alternative exons 4, 6, and 9 (encoding amino acids in Ig2, Ig3, and Ig7, respectively). No striking preferential patterns of alternative exon utilization or combinations of exons were apparent. While Dscam protein is largely expressed in ORNs at this stage, the possibility cannot be ruled out that Dscam RNA but not protein is expressed in other cells. Since markers for different subclasses of ORNs are expressed after glomeruli are formed, it has not yet been possible to assess the forms of Dscam expressed in different subclasses of ORNs as they target to specific glomeruli. Nevertheless, these data are consistent with many different forms of Dscam protein being produced by ORNs, since they project into the target region and that individual ORNs express multiple Dscam isoforms (Hummel, 2003).

Dscam plays a complex role in regulating the formation of specific connections in the fly olfactory system. In the absence of Dscam, two classes of antennal ORNs, Or47a and GH298, project into the antennal lobe, as they do in wild-type, but terminate at ectopic sites they encounter as they project to their cognate glomeruli. That Dscam prevents inappropriate targeting is underscored by the robust mistargeting of three classes of maxillary palp ORNs to neuropil outside the antennal lobe. In addition, ORN axons that reach the correct glomerulus do not elaborate a normal terminal region. They fail to extend thin processes throughout the cognate glomerulus as in wild-type and remain tightly segregated in local domains within the glomerulus. All ORNs studied that branch in wild-type to the contralateral glomerulus frequently fail to do so in Dscam mutants. It seems likely that defects in branching result from abnormalities in the interactions between ORNs and their cognate ipsilateral glomeruli. For some ORNs, branches target accurately to their cognate glomeruli on the contralateral antennal lobe, whereas in others they terminate in inappropriate locations. While there are differences in Dscam phenotypes in different ORNs, as well as their penetrance and expressivity, these observations support a critical role for Dscam in mediating interactions between growth cones and neuropil within potential target regions (Hummel, 2003).

Are alternative forms of Dscam required for targeting? In recent studies, attempts have been made to assess whether expression of a single form of Dscam would rescue the targeting defects in specific olfactory receptor neurons. Since expression in ORNs leads to a severe dominant phenotype with loss of glomerular structure, it has not been possible to critically address the importance of multiple isoforms for targeting using this experimental approach. In future studies, the importance of alternative splicing in target selection will be assessed by analyzing loss-of-function mutants in which the number of alternative exons is reduced at the endogenous locus and by expressing additional isoforms of Dscam in ORNs (Hummel, 2003).

It is thought that ORNs and projection neurons express molecular labels that allow them to match-up in distinct glomeruli Could the alternative forms of Dscam provide such labels? This is indeed a possibility, since Dscam is expressed on both ORNs and projection neurons during glomerular formation. That Dscam could act as a short-range attractant is consistent with the observation that mammalian Dscam proteins can bind to each other when expressed in transfected cells. In Drosophila, forms expressed on specific ORNs and the appropriate target projection neurons would selectively adhere to each other. Alternatively, Dscam could act as a short-range repellent. Projection neurons that extend dendrites selectively into only a single glomerulus may express a diverse set of Dscam isoforms, and different ORN subclasses express forms of Dscam that interact with isoforms expressed in dendrites of all projection neurons, except those in their cognate glomeruli. As a consequence, ORNs connect to their appropriate targets by being excluded from all other potential targets. Repulsion may be mediated by interactions between the same Dscam isoforms or between different isoforms; different isoforms may modulate the extent of repulsion between ORNs and target glomeruli, and hence, competitive interactions between ORN classes may contribute to specificity (Hummel, 2003).

While Dscam is necessary for ORN targeting, it is clear that it is not the only ORN determinant regulating this process. Indeed, some ORN targeting is independent of Dscam (i.e., Or22a and Or23a), and in many cases, subclasses affected by Dscam mutations exhibit only partially penetrant phenotypes. This may reflect the overlapping function of other Dscams (i.e., Dscams 2-4; these Dscams do not come in multiple forms) that are expressed within the olfactory system or the activity of redundant pathways utilizing other cell surface recognition molecules. Indeed, other targeting proteins, such as N-cadherin and Flamingo, contribute to targeting of some but not all classes of ORNs. These studies allude to a complex combinatorial mechanism regulating olfactory receptor neuron targeting in Drosophila. The availability of markers for different neurons exhibiting different targeting specificities within a common ganglion provides a unique opportunity to explore the mechanisms regulating the formation of diverse patterns of synaptic connectivity (Hummel, 2003).

Dendritic patterning by Dscam and synaptic partner matching in the Drosophila antennal lobe

In the olfactory system of Drosophila melanogaster, axons of olfactory receptor neurons (ORNs) and dendrites of second-order projection neurons typically target 1 of ~50 glomeruli. Dscam, an immunoglobulin superfamily protein, acts in ORNs to regulate axon targeting. Dscam acts in projection neurons and local interneurons to control the elaboration of dendritic fields. The removal of Dscam selectively from projection neurons or local interneurons leads to clumped dendrites and marked reduction in their dendritic field size. Overexpression of Dscam in projection neurons causes dendrites to be more diffuse during development and shifts their relative position in adulthood. Notably, the positional shift of projection neuron dendrites causes a corresponding shift of its partner ORN axons, thus maintaining the connection specificity. This observation provides evidence for a pre- and post-synaptic matching mechanism independent of precise glomerular positioning (Zhu, 2006).

The shift of position of the projection neurons of the VA1d glomerulus, caused by the overexpression of Dscam in VA1d neurons, offers a unique opportunity to investigate the logic of olfactory circuit assembly. Because projection neuron dendrites form a coarse spatial map before ORN axons invade, ORN axons could use the projection neuron dendritic map as a cue to determine their spatial position in the antennal lobe. To date, however, there is no experimental support for this hypothesis. Alternative proposals are that ORN axons recognize non-projection neuron cues within or surrounding the antennal lobe and/or that they self-organize through axon-axon interaction to reach their spatially invariant targets (Zhu, 2006).

The overexpression of Dscam in projection neurons alters the initial spatial map of the projection neuron dendrites before ORN invasion; therefore, it was asked whether the change in the spatial map of projection neuron dendrites affects the targeting of the corresponding ORN axons. If projection neuron dendritic maps are used as cues for ORN axon targeting, one would predict a corresponding shift of ORN axon position. Alternatively, if the spatial positions of ORN axons are solely determined by interactions among ORN axons or with non-projection neuron cues, changing the spatial map of projection neuron dendrites would lead to a mismatch of projection neuron dendrites and ORN axons and would result in a change in connection specificity (Zhu, 2006).

To distinguish between these two possibilities, ORN axon innervation patterns in the antennal lobe were examined in response to the dendritic map shift induced by Dscam overexpression in projection neuron dendrites. The ORNs innervating the VA1d glomerulus express Or88a. To label these ORN axons, the transgene Or88a-CD2 was used in which the Or88a promotor drives the expression of the membrane marker CD2. In the same brain, Mz19⁺ projection neurons were labeled by the membrane marker mCD8-GFP using the Gal4/UAS system. In the wild type, Or88a axons synapse with Mz19⁺ projection neuron dendrites innervating VA1d, which is adjacent to the DA1 glomerulus. In 13 brain hemispheres where Mz19⁺ projection neurons also overexpressed Dscam, the spatial position of the VA1d projection neuron dendrites shifted ventrally, away from the DA1 glomerulus. Notably, Or88a axons shifted to a ventral position accordingly in each case, maintaining the correct connections with the VA1d projection neuron dendrites. In the seven hemispheres in which Dscam overexpression did not induce a dendritic shift, Or88a axon targeting was normal. These results demonstrated that Or88a axons recognized VA1d projection neuron dendrites, thereby maintaining their connection specificity even when these dendrites were shifted to a different position (Zhu, 2006).

Overexpressing Dscam in Mz19⁺ projection neurons shifted the VA1d dendrites to a ventral position equivalent to VA1lm, which is normally innervated by ORN axons expressing Or47b. Would Or47b axons still innervate the same position in the antennal lobe and therefore connect incorrectly with the ventrally shifted VA1d projection neuron dendrites? Or would they avoid the ventrally shifted VA1d dendrites and target a new position? To address this question, the Or47b axons were labelled using the transgene Or47b-CD2. It was found that, in each hemisphere, when Dscam overexpression caused a ventral shift of VA1d dendrites, Or47b axons shifted to a dorsal position and innervated the glomerulus situated ectopically between DA1 and VA1d, avoiding the VA1d dendrites completely. In hemispheres where no projection neuron dendritic position shift occurred, Or47b axons targeted normal positions. Note that for this VA1lm glomerulus, neither the pre- nor the postsynaptic partners had been subjected to any genetic modifications. Their positional shifts were the sole result of the expression of Dscam in projection neurons in glomeruli neighboring VA1lm. Together, these data supported the notion that glomerular targeting, at least in the two ORN classes examined, requires specific recognition between ORN afferents and projection neuron dendrites (Zhu, 2006).

Individual neurons express different arrays of isoforms in a largely stochastic fashion. It is proposed that such expression provides each neuron with a unique identity. Biochemical studies demonstrate that Dscam promotes isoform-specific binding: isoforms sharing the same extracellular domain bind to each other, whereas isoforms that are different do not. Furthermore, interactions between identical Dscam proteins seem to promote contact-dependent repulsion. One phenotype in the Dscam mutant axon is the failure to segregate sister branches, which is also the case for projection neuron axons (Zhu, 2006).

How might Dscam contribute to dendrite branching? To efficiently innervate the target areas, some neurons possess an intraneuronal tiling mechanism such that dendritic branches of the same neuron do not overlap. On the basis of expression studies in other neurons, it is proposed that the array of Dscam isoforms expressed on a dendrite's surface provides each projection neuron and local interneuron with a mechanism by which to distinguish its own dendrites from those of neighboring cells. Thus, Dscam isoforms expressed on sister branches will be the same; however, they will share little, if any, overlap with the isoforms expressed on dendrites of other projection neurons or local interneurons innervating the same glomurulus. Thus, Dscam diversity provides a mechanism by which dendrites from the same neuron can avoid each other as they elaborate their receptive fields, while overlapping with dendritic processes of other cells within the neuropil (Zhu, 2006).

Developmental studies have led to a model in which ORNs and projection neurons initially develop spatial maps that are independent of each other. When the two spatial maps converge, the connection specificity between a given ORN-projection neuron pair can be determined solely by matching the positional coordinates of the respective spatial maps for ORN axons and projection neuron dendrites. Such an extreme model would predict that if a change is made to the positions of projection neuron dendrites before and independent of the arrival of ORN axons, the positional maps of ORN axons and projection neuron dendrites would be misaligned. Under these circumstances, ORN axons should innervate projection neuron dendrites positioned as they would be in the wild type, rather than as they are in the reordered state; thus the ORN axons would select the incorrect target. Alternatively, these two maps may be rather coarse and specific recognition between appropriate ORN axons and their dendritic targets may be required to refine the map (Zhu, 2006).

The findings that changing the spatial map of the projection neuron dendrites also leads to corresponding changes of the ORN axon map strongly argues against a strict spatially regulated matching of the afferent axonal and dendritic maps. The data indicate that ORN axons are influenced by their corresponding postsynaptic projection neuron dendrites in determining their spatial position in the antennal lobe, at least at the local level for the two classes of ORNs analyzed. This finding by no means contradicts the contribution of projection neuron-independent mechanisms in ORN axon targeting. Such mechanisms may be used to set up a coarse map for the ORN axons, limiting the approximate spatial position that a given ORN axon can target. ORN axons would then be faced with only a few projection neuron dendritic targets in the neighborhood, which they can sample through ORN-projection neuron recognition in order to solidify the final synaptic partners. Sensory input and synaptic activity are unlikely to play a role in matching ORN axons and projection neuron dendrites because this process is completed before olfactory receptor expression and synaptic maturation. It is more likely that these processes recognize each other through yet to be identified 'chemoaffinity tags' (Zhu, 2006).

Homophilic Dscam interactions control complex dendrite morphogenesis

Alternative splicing of Dscam results in up to 38,016 different receptor isoforms proposed to interact by isoform-specific homophilic binding. Dscam controls cell-intrinsic aspects of dendrite guidance in all four classes of dendrite arborization (da) neurons. Loss of Dscam in single neurons causes a strong increase in self-crossing. Restriction of dendritic fields of neighboring class III neurons appeared intact in mutant neurons, suggesting that dendritic self-avoidance, but not heteroneuronal tiling, may depend on Dscam. Overexpression of the same Dscam isoforms in two da neurons with overlapping dendritic fields forced a spatial segregation of the two fields, supporting the model that dendritic branches of da neurons use isoform-specific homophilic interactions to ensure minimal overlap. Homophilic binding of the highly diverse extracellular domains of Dscam may therefore limit the use of the same 'core' repulsion mechanism to cell-intrinsic interactions without interfering with heteroneuronal interactions (Hughes, 2007).

This study shows that Dscam has an important cell-intrinsic function in dendrite development of da neurons. Dscam is required for steering the growth of sister branches to ensure correct dendrite morphogenesis but is not required for other mechanisms of dendrite patterning. Dscam loss-of-function mutations result in strong disruption of dendrite morphogenesis in different classes of da sensory neurons. The phenotypic defects included uneven spacing of dendritic branches, a strong increase in dendritic self-crossing, and highly abnormal dendritic fascicles or tangles. All the observed phenotypes are consistent with the possibility that loss of Dscam results in a lack of self-avoidance of sister dendrites. Consistent with a role of Dscam in dendrite-dendrite repulsion, it was found that Dscam overexpression in da neurons, which normally have overlapping dendritic fields, forced the respective dendrites to segregate from each other. In addition, gain-of-function phenotypes resulting from overexpression of single Dscam isoforms or a Dscam isoform lacking the cytoplasmic domain are also consistent with the possibility that repulsion between sister branches is controlled by Dscam signaling. It is therefore suggested that in da neurons, direct isoform-specific homophilic Dscam-Dscam interactions result in signal transduction events that lead to repulsion of dendrites expressing identical Dscam isoforms. This model is consistent with previous biochemical studies, Dscam's role in bifurcating MB axons, and Dscam's function in projection neurons of the olfactory system (Hughes, 2007).

Previous expression studies have shown that single photoreceptor neurons of the same type express different Dscam isoforms. Similarly, expression of a large diversity of Dscam isoforms has also been found in olfactory neurons and MB neurons. Based on these findings and the observation that experimentally forced expression of identical isoforms in da neurons causes dominant phenotypes, it is highly likely that different da neurons also express diverse Dscam isoforms. Considering the large diversity of Dscam isoforms, the possibility that dendrites from different neurons present identical Dscam isoforms seems minimal. In contrast, dendritic sister branches of the same cell, even though they are likely expressing multiple isoforms, will at significant frequency encounter homophilic Dscam-Dscam interactions. This model is consistent with the finding that the expression of the same Dscam isoform causes the segregation of normally overlapping dendritic fields. Based on this one might expect that a functionally critical threshold of Dscam diversity must exist. However, no obvious morphogenesis defects of class I neurons, such as changes in self-crossing, number of dendritic termini, or dendritic area, were detected in homozygous animals bearing the reduced diversity allele Dscam^C22-1. Also no obvious defects were detected in dendrite morphogenesis of class IV neurons in Dscam^C22-1 animals. Although it is possible that some aspects of dendrite morphogenesis are altered in Dscam^C22-1 and were not identified in these experiments, the possibility is favored that a few thousands or even significantly fewer Dscam isoforms are sufficient to still ensure nonoverlapping expression of identical isoforms in neighboring da neurons. However, it is proposed that reducing the diversity of Dscam isoforms below a certain threshold would lead to scenarios where different neighboring da neurons express the same isoforms and it would not be possible to limit Dscam-mediated repulsion of dendrites to cell-intrinsic sister dendrite interactions. This would likely lead to strong morphogenesis and functional defects throughout the Drosophila PNS (Hughes, 2007).

Dendrite development of da neurons requires at least four distinguishable patterning mechanisms: (1) growth of dendrites and dendritic branches emanating from the same cell has to be controlled such that relatively even spacing between dendrites with minimal overlap is achieved (self-avoidance); (2) for any given class or type of neuron, the dendritic growth has to obey a characteristic polarity and likely limits the extension of the primary dendritic branches (dendrite architecture); (3) the degree of branching has to be adapted to the type of sensory neuron (stereotyped branching); (4) inhibitory interactions with nearby neurons are needed to control the size of dendritic fields such that a complete but nonredundant innervation of a receptive area by functionally uniform groups of neurons is achieved (heteroneuronal tiling) (Hughes, 2007).

It has been speculated that self-avoidance and tiling might depend on the same molecular mechanism and may not require distinct signals. In such a scenario, isoneuronal dendrites could be developmentally identical to 'like' heteroneuronal dendrites. In this study it was suggested that this may not be the case. Dscam function is required for correct spacing of dendrites due to self-avoidance of sister branches but is unlikely required for other mechanisms of dendrite patterning. These results suggest that the repulsive mechanism or mechanisms underlying hetero-neuronal tiling are molecularly different from the mechanism controlling repulsive interactions underlying self-avoidance. It seems likely that homophilic Dscam-Dscam interactions represent the major molecular system controlling isoneuronal dendrite-dendrite repulsion in Drosophila. In this specific context of dendrite morphogenesis, the diversity of Dscam ensures that this repulsive function is restricted to cell-intrinsic interactions, as only dendrites of the same cell are likely to express identical isoforms. As such, the molecular diversity of Dscam is less likely to provide each neuron with a unique 'identity' but rather provides a molecular buffer for enabling 'tolerance' between neurons (Hughes, 2007).

Several studies have revealed examples consistent with the notion that Dscam signaling can lead to neurite repulsion. It has been proposed that this repulsive function can be mediated by direct homophilic Dscam-Dscam interactions. For example, it has been shown that the trajectory of interneurons overexpressing a single isoform of Dscam is disrupted upon encountering midline cells that overexpress the identical isoform. The strongest support for a direct Dscam-Dscam interaction has been provided by a series of impressive biochemical experiments, in which it was shown that from a randomly chosen set of 11 Dscam isoforms, each one binds to itself but not to others. All three variable Ig domains of Dscam are required for homophilic binding specificity. In addition, recent studies described that overexpression of Dscam in a subset of projection neurons connecting with specific glomeruli (termed DA1 and DC3) resulted in a strong gain-of-function phenotype, again consistent with a repulsive interaction due to homophilic Dscam interactions. This gain-of-function phenotype was found to be dependent on Dscam signaling, as a deletion of the cytoplasmic domain in a Dscam isoform (Dscam1.30.30.1ΔC) blocked this dominant phenotype. Similarly, it was found that overexpression of Dscam1.30.30.1ΔC in ddaE neurons blocked the repulsion of sister dendrites and instead lead to abnormal fasciculation. Although the endogenous physiological function revealed by these experiments is unclear, they nevertheless are consistent with the hypothesis that Dscam can function as a cell-surface receptor mediating neurite repulsion (Hughes, 2007).

How is the homophilic Dscam-Dscam interaction transformed into a repulsive action rather than a stable adhesion? Dscam has been initially identified as a tyrosine phosphorylated receptor functioning upstream of the adaptor molecule Dock. Dock binds to Dscam via SH2 as well as SH3 domains and serves to recruit the effector kinase Pak to the plasma membrane where it can be activated by Rac or Cdc42. Pak has been implicated in several signaling pathways that control cytoskeletal rearrangement, including pathways underlying neurite repulsion. By examining the effect of a constitutively membrane-bound form of Pak in da neurons, it was found that Pak signaling can influence dendrite morphogenesis. However, loss-of-function analysis provided no evidence for a direct role of Pak in dendrite morphogenesis or self-avoidance of class I neurons. Therefore, at least in class I neurons, Dscam signaling likely bypasses Pak and utilizes alternative downstream components that have yet to be identified. Although a signaling pathway controlling heteroneuronal tiling and branching in da neurons has been described, signaling pathways that control self-avoidance are currently unknown (Hughes, 2007).

It is important to note that Dscam function is not only required for controlling neurite repulsion. For example, it has been proposed that Dscam controls axon guidance of Bolwig's nerve by signaling through Dock and Pak in response to an as of yet unknown guidance cue present at an intermediate target. In early developing MB fibers, Dscam is required for axon bundling and fasciculation, thereby mediating adhesive interactions. In addition, one distinct function of Dscam in mechanoreceptor neurons appears to mediate a growth-promoting role rather than repulsion. Importantly, the role of Dscam diversity in the development of mechanosensory neuron projections suggests the possibility that Dscam is not only involved in homophilic interactions of neurites emanating from the same cell. In fact, it has been proposed that in the somatosensory system Dscam isoforms have instructive roles controlling targeting decisions of axonal branches. Future studies will have to address the molecular differences that allow for such a versatile use of Dscam receptors (Hughes, 2007).

Drosophila sensory neurons require Dscam for dendritic self-avoidance and proper dendritic field organization

A neuron's dendrites typically do not cross one another. This intrinsic self-avoidance mechanism ensures unambiguous processing of sensory or synaptic inputs. Moreover, some neurons respect the territory of others of the same type, a phenomenon known as tiling. Different types of neurons, however, often have overlapping dendritic fields. Dscam is required for dendritic self-avoidance of all four classes of Drosophila dendritic arborization (da) neurons. However, neighboring mutant class IV da neurons still exhibit tiling, suggesting that self-avoidance and tiling differ in their recognition and repulsion mechanisms. Introducing 1 of the 38,016 Dscam isoforms to da neurons in Dscam mutants is sufficient to significantly restore self-avoidance. Remarkably, expression of a common Dscam isoform in da neurons of different classes prevented their dendrites from sharing the same territory, suggesting that coexistence of dendritic fields of different neuronal classes requires divergent expression of Dscam isoforms (Soba, 2007).

Given the necessity for different classes of da neurons to express at least one isoform of Dscam to ensure self-avoidance, it is important to explore the possible consequences of having multiple da neurons in the same region express the same Dscam isoform. Different classes of da neurons presumably respond to different sensory inputs, and hence their coverage of overlapping regions of the body wall will allow the animal to detect different types of sensory stimuli at the same physical locations. Expression of a common Dscam isoform made it almost impossible for different classes of da neurons to occupy the same territory, which is likely associated with deprivation of all but one type of sensory input at any one location. These considerations provide a plausible explanation for the existence of a vast number of Dscam splice variants. For Dscam to mediate self-recognition manifested as dendritic self-avoidance in da neurons, it may be crucial to limit the expression of each of the Dscam isoforms to one or a small subset of neurons in any region of the nervous system. This allows neighboring neurons to interact in ways distinct from the interactions that occur between isoneuronal processes of an individual neuron. Indeed, microarray analyses of individual Drosophila photoreceptor cells and mushroom body neurons have found distinct Dscam isoforms expressed in neighboring cells. This supports the idea that an intricate splicing mechanism ensures that individual neurons can distinguish between self and nonself. Given that divergent Dscam isoform expression is required for coexistence of da neurons in the same territory, it is likely that other mechanisms are involved in recognition of neighboring class IV da neurons for the purpose of tiling (Soba, 2007).

Intriguingly, even duplicated class I neurons most likely express distinct Dscam isoforms, since they occupy the same territory, but repel each other when expressing a common Dscam isoform. While the specific isoforms expressed by individual da neurons are currently unknown, the general principle of enabling dendrites of a single neuron to avoid one another without imposing recognition and avoidance of neighboring neurons underscores the importance of Dscam diversity. Since Dscam has been shown to primarily interact in an isoform-specific manner, Dscam-based self-recognition and self-avoidance depends on expression of the same isoforms. This is illustrated by the repulsive function of Dscam in vivo, where it induces branch retraction and avoidance of dendrites expressing identical isoforms. The self-avoidance phenomenon seems to be conserved in axonal development as well: in mushroom body neurons Dscam ensures the proper segregation and targeting of the two axonal processes to different lobes (Soba, 2007).

This study reveals significant differences between self-avoidance, which encompasses isoneuronal crossing and bundling of dendrites, and tiling. Dscam is essential for dendritic self-avoidance (dendritic crossing and bundling) of all classes of da neurons, but not for tiling of class IV da neurons. Thus, different recognition mechanisms are used for self-avoidance and tiling. The phenomenon of tiling, in which the dendrites of different neurons of the same class avoid one another, relies on components of the Hpo/Trc/Fry pathway. Whereas this signaling pathway also contributes to the avoidance of isoneuronal dendritic crossing of class IV da neurons, it is not required to prevent bundling of class IV da neuron dendrites, nor is it important for self-avoidance of the other three classes of da neurons. It is an interesting open question as to what intracellular machineries are employed to prevent the dendrites of each da neuron in class I-III from crossing one another. The involvement of the Hpo/Trc/Fry pathway in preventing crossing, but not bundling, of dendrites from the same class IV da neuron further suggests the possibility of multiple mechanisms for self-avoidance. While fasciculation of multiple processes, e.g., those in axon guidance, is a widely used mechanism to ensure accurate projection to common target areas, bundling of isoneuronal dendrites defeats the purpose of dendritic field coverage and nonredundant signal processing. Given the ample opportunities for neighboring dendritic branches of the same neuron to bundle, there is likely a specialized mechanism that repels branching dendrites from each other as they respond to cues for their extension (Soba, 2007).

Robust discrimination between self and non-self neurites requires thousands of Dscam1 isoforms

Dscam genes encode neuronal cell recognition proteins of the immunoglobulin superfamily. In Drosophila, Dscam1 generates 19,008 different ectodomains by alternative splicing of three exon clusters, each encoding half or a complete variable immunoglobulin domain. Identical isoforms bind to each other, but rarely to isoforms differing at any one of the variable immunoglobulin domains. Binding between isoforms on opposing membranes promotes repulsion. Isoform diversity provides the molecular basis for neurite self-avoidance. Self-avoidance refers to the tendency of branches from the same neuron (self-branches) to selectively avoid one another. To ensure that repulsion is restricted to self-branches, different neurons express different sets of isoforms in a biased stochastic fashion. Genetic studies demonstrated that Dscam1 diversity has a profound role in wiring the fly brain. This study shows how many isoforms are required to provide an identification system that prevents non-self branches from inappropriately recognizing each other. Using homologous recombination, mutant animals encoding 12, 24, 576 and 1,152 potential isoforms were generated. Mutant animals with deletions encoding 4,752 and 14,256 isoforms were also analysed. Branching phenotypes were assessed in three classes of neurons. Branching patterns improved as the potential number of isoforms increased, and this was independent of the identity of the isoforms. Although branching defects in animals with 1,152 potential isoforms remained substantial, animals with 4,752 isoforms were indistinguishable from wild-type controls. Mathematical modelling studies were consistent with the experimental results that thousands of isoforms are necessary to ensure acquisition of unique Dscam1 identities in many neurons. It is concluded that thousands of isoforms are essential to provide neurons with a robust discrimination mechanism to distinguish between self and non-self during self-avoidance (Hattori, 2009).

A STRIPAK component Strip regulates neuronal morphogenesis by affecting microtubule stability

During neural development, regulation of microtubule stability is essential for proper morphogenesis of neurons. Recently, the striatin-interacting phosphatase and kinase (STRIPAK) complex was revealed to be involved in diverse cellular processes. However, there is little evidence that STRIPAK components regulate microtubule dynamics, especially in vivo. This study shows that one of the core STRIPAK components, Strip, is required for microtubule organization during neuronal morphogenesis. Knockdown of Strip causes a decrease in the level of acetylated alpha-tubulin in Drosophila S2 cells, suggesting that Strip influences the stability of microtubules. Strip physically and genetically interacts with tubulin folding cofactor D (TBCD), an essential regulator of alpha- and beta-tubulin heterodimers. Furthermore, the genetic interaction is demonstrated between strip and Down syndrome cell adhesion molecule (Dscam), a cell surface molecule that is known to work with TBCD. Thus, it is proposed that Strip regulates neuronal morphogenesis by affecting microtubule stability (Sakuma, 2015).

EVOLUTIONARY HOMOLOGS

Insect DSCAMs

Two additional genes encoding Dscam-related proteins, CG12536 and CG8619, have been identified in the fly genome (Schmucker, 2000).

The Drosophila Dscam gene encodes an axon guidance receptor and can generate 38,016 different isoforms via the alternative splicing of 95 variable exons. Dscam contains 10 immunoglobulin (Ig), six Fibronectin type III, a transmembrane (TM), and cytoplasmic domains. The different Dscam isoforms vary in the amino acid sequence of three of the Ig domains and the TM domain. The organization of the Dscam gene from three members of the Drosophila subgenus (D. melanogaster, D. pseudoobscura, and D. virilis), the mosquito Anopheles gambiae, and the honeybee Apis mellifera have been compared. Each of these organisms contains numerous alternative exons and can potentially synthesize tens of thousands of isoforms. Interestingly, most of the alternative exons in one species are more similar to one another than to the corresponding alternative exons in the other species. These observations provide strong evidence that many of the alternative exons have arisen by reiterative exon duplication and deletion events. In addition, these findings suggest that the expression of a large Dscam repertoire is more important for the development and function of the insect nervous system than the actual sequence of each isoform (Graveley, 2004).

More than one way to produce protein diversity: duplication and limited alternative splicing of an adhesion molecule gene in basal arthropods

Exon duplication and alternative splicing evolved multiple times in metazoa and are of overall importance in shaping genomes and allowing organisms to produce many fold more proteins than there are genes in the genome. No other example is as striking as the one of the Down syndrome cell adhesion molecule (Dscam) of insects and crustaceans (pancrustaceans) involved in the nervous system differentiation and in the immune system. To elucidate the evolutionary history of this extraordinary gene, Dscam homologs were investigated in two basal arthropods, the myriapod Strigamia maritima and the chelicerate Ixodes scapularis. In both, Dscam diversified extensively by whole gene duplications resulting in multigene expansions. Within some of the S. maritima genes, exons coding for one of the immunoglobulin domains (Ig7) duplicated and are mutually exclusively alternatively spliced. The results suggest that Dscam diversification was selected independently in chelicerates, myriapods, and pancrustaceans and that the usage of Dscam diversity by immune cells evolved for the first time in basal arthropods. It is proposed an evolutionary scenario for the appearance of the highly variable Dscam gene of pancrustaceans, adding to the understanding of how alternative splicing, exon, and gene duplication contribute to create molecular diversity associated with potentially new cellular functions (Brites, 2013).

Improvement of Dscam homophilic binding affinity throughout Drosophila evolution

Drosophila Dscam1 is a cell-surface protein that plays important roles in neural development and axon tiling of neurons. It is known that thousands of isoforms bind themselves through specific homophilic interactions, a process which provides the basis for cellular self-recognition. Detailed biochemical studies of specific isoforms strongly suggest that homophilic binding, i.e. the formation of homodimers by identical Dscam1 isomers, is of great importance for the self-avoidance of neurons. Due to experimental limitations, it is currently impossible to measure the homophilic binding affinities for all 19,000 potential isoforms. This study reconstructed the DNA sequences of an ancestral Dscam form (which likely existed approximately 40 ~ 50 million years ago) using a comparative genomic approach. On the basis of this sequence, a working model was established to predict the self-binding affinities of all isoforms in both the current and the ancestral genome, using machine-learning methods. Detailed computational analysis was performed to compare the self-binding affinities of all isoforms present in these two genomes. The results revealed that 1) isoforms containing newly derived variable domains exhibit higher self-binding affinities than those with conserved domains, and 2) current isoforms display higher self-binding affinities than their counterparts in the ancient genome. As thousands of Dscam isoforms are needed for the self-avoidance of the neuron, it is proposed that an increase in self-binding affinity provides the basis for the successful evolution of the arthropod brain. These data provide an excellent model for future experimental studies of the binding behavior of Dscam isoforms. The results of this analysis indicate that evolution favored the rise of novel variable domains thanks to their higher self-binding affinities, rather than selection merely on the basis of simple expansion of isoform diversity; this particular selection process would established a powerful mechanism required for neuronal self-avoidance. Thus, this study reveals a new molecular mechanism for the successful evolution of arthropod brains (Wang, 2014; PubMed).

Zebrafish DSCAM

The Dscam is a protein overexpressed in the brains of Down syndrome patients and implicated in mental retardation. Dscam is involved in axon guidance and branching in Drosophila, but cellular roles in vertebrates have yet to be elucidated. To understand its role in vertebrate development, the zebrafish homolog of Dscam was cloned and it was shown to share high amino acid identity and structure with the mammalian homologs. Zebrafish dscam is highly expressed in developing neurons, similar to what has been described in Drosophila and mouse. When dscam expression is diminished by morpholino injection, embryos display few neurons and their axons do not enter stereotyped pathways. Zebrafish dscam is also present at early embryonic stages including blastulation and gastrulation. Its loss results in early morphogenetic defects. dscam knockdown results in impaired cell movement during epiboly as well as in subsequent stages. It is proposed that migrating cells utilize dscam to remodel the developing embryo (Yimlamai, 2005).

Mammalian DSCAM genes

Down syndrome (DS), a major cause of mental retardation, is characterized by subtle abnormalities of cortical neuroanatomy, neurochemistry and function. Recent work has shown that chromosome band 21q22 is critical for many of the neurological phenotypes of DS. A gene, DSCAM (Down syndrome cell adhesion molecule), has now been isolated from chromosome band 21q22.2-22.3. Homology searches indicate that the putative DSCAM protein is a novel member of the immunoglobulin (Ig) superfamily that represents a new class of neural cell adhesion molecules. The sequence of cDNAs indicates alternative splicing and predicts two protein isoforms, both containing 10 Ig-C2 domains, with nine at the N-terminus and the tenth located between domains 4 and 5 of the following array of six fibronectin III domains, with or without the following transmembrane and intracellular domains. Northern analyses reveals the transcripts of 9.7, 8.5 and 7.6 kb primarily in brain. These transcripts are differentially expressed in substructures of the adult brain. Tissue in situ hybridization analyses of a mouse homolog of the DSCAM gene has revealed broad expression within the nervous system at the time of neuronal differentiation in the neural tube, cortex, hippocampus, medulla, spinal cord and most neural crest-derived tissues. Given its location on chromosome 21, its specific expression in the central nervous system and neural crest, and the homologies to molecules involved in neural migration, differentiation, and synaptic function, it is proposed that DSCAM is involved in neural differentiation and contributes to the central and peripheral nervous system defects in DS (Yamakawa, 1998).

Down Syndrome (DS) caused by trisomy 21 is the most common birth defect associated with mental retardation. To understand the cellular function of DSCAM protein, human DSCAM cDNA was transfected into mouse fibroblast L cells and its expression was analysed. On Western blot analysis, antibodies raised against recombinant DSCAM-Ig3 recognize a 198 kDa protein band in the membrane fraction of DSCAM transfected L cells. Stable transformants expressing DSCAM show uniform surface expression. DSCAM-expressing transfectants exhibit enhanced adhesive properties, aggregating with faster kinetics and forming aggregates in a homophilic manner. Divalent cations are not required for this cell aggregation. These results demonstrate that DSCAM is a cell adhesion molecule that can mediate cation-independent homophilic binding activity between DSCAM expressing cells (Agarwala, 2000).

Down Syndrome Cell Adhesion molecule (DSCAM) is a member of the immunoglobulin superfamily, and represents a novel class of neuronal cell adhesion molecules. In order to understand the cellular functions of DSCAM, full-length mouse and human cDNA clones have been isolated, and its expression analysed during mouse development and differentiation. Sequence analysis of the human DSCAM cDNA predicts at least 33 exons that are distributed over 840 kb. When compared to human DSCAM, the mouse homolog shows 90% and 98% identity at the nucleotide and amino acid levels, respectively. In mouse, DSCAM is located on 16C, the syntenic region for human chromosome band 21q22 and also the region duplicated in mouse DS models. DSCAM gene is predicted to encode an approximately 220-kDa protein, and its expression shows dynamic changes that correlate with neuronal differentiation during mouse development. These results suggest that DSCAM may play critical roles in the formation and maintenance of specific neuronal networks in brain (Agarwala, 2001a).

DSCAM, a conserved gene involved in neuronal differentiation, is a member of the Ig superfamily of cell adhesion molecules. A human DSCAM (Down syndrome cell adhesion molecule) paralog, DSCAML1, is located on chromosome 11q23. The deduced DSCAML1 protein contains 10 Ig domains, six fibronectin-III domains, and an intracellular domain, all of which are structurally identical to DSCAM. When compared to DSCAM, DSCAML1 protein shows 64% identity to the extracellular domain and 45% identity to the cytoplasmic domain. In the mouse brain, DSCAML1 is predominantly expressed in Purkinje cells of the cerebellum, granule cells of the dentate gyrus, and in neurons of the cerebral cortex and olfactory bulb. Biochemical and immunofluorescence analyses indicate that DSCAML1 is a cell surface molecule that targets axonal features in differentiated PC12 cells. DSCAML1 exhibits homophilic binding activity that does not require divalent cations. Based on its structural and functional properties and similarities to DSCAM, it is suggested that DSCAML1 may be involved in formation and maintenance of neural networks. The chromosomal locus for DSCAML1 makes it an ideal candidate for neuronal disorders (such as Gilles de la Tourette and Jacobsen syndromes) that have been mapped on 11q23 (Agarwala, 2001b).

Dscam, a novel cell-adhesion molecule belonging to the Ig-superfamily mediates homophilic intercellular adhesion and is expressed abundantly in the nervous system during development. To gain better understanding of the role of Dscam in neuronal differentiation, an antibody was raised and its protein product was characterized. Anti-Dscam antibody detects an approximately 200-kDa protein band in human and mouse brain lysates. Immunohistochemical studies show that during embryonic development of mice, mouse Dscam is expressed throughout the neuronal tissues and also in nonneuronal tissues such as lung, liver, and limb buds. In adult brain Dscam expression is predominant in the cerebellum, hippocampus, and olfactory bulb. Immunofluorescence double labeling of hippocampal and cerebellar primary cultures reveals that Dscam is associated with axonal and dendritic processes. In view of its cellular localization and spatiotemporal expression pattern, it is suggested that Dscam is involved in cell-cell interactions during axonal-dendritic development, and maintenance of functional neuronal networks (Agarwala, 2001c).

CNS development involves neural patterning, neuronal and axonal migrations, and synapse formation. DSCAM, a chromosome 21 axon guidance molecule, is expressed by CNS neurons during development and throughout adult life. DSCAM and its chromosome 11 paralog DSCAML1 exhibit inverse ventral-dorsal expression patterns in the developing spinal cord and distinct, partly inverse, expression patterns in the developing cortex, beginning in the Cajal-Retzius cells. In the adult cortex, DSCAM predominates in layer 3/5 pyramidal cells and DSCAML1 predominates in layer 2 granule cells. In the cerebellum, DSCAM is stronger in the Purkinje cells and DSCAML1 in the granule cells. The predicted DSCAML1 protein contains 60 additional N-terminal amino acids which may contribute to its distinct expression pattern and putative function. It is proposed that the DSCAMs comprise novel elements of the pathways mediating dorsal-ventral patterning and cell-fate specification in the developing CNS (Barlow, 2002).

DSCAM and DSCAML1 function in self-avoidance in multiple cell types in the developing mouse retina

DSCAM and DSCAM-LIKE1 (DSCAML1) serve diverse neurodevelopmental functions, including axon guidance, synaptic adhesion, and self-avoidance, depending on the species, cell type, and gene family member studied. The function of DSCAM and DSCAML1 was examined in the developing mouse retina. In addition to a subset of amacrine cells, Dscam was expressed in most retinal ganglion cells (RGCs). RGCs had fasciculated dendrites and clumped cell bodies in Dscam(-/-) mice, suggesting a role in self-avoidance. Dscaml1 was expressed in the rod circuit, and mice lacking Dscaml1 had fasciculated rod bipolar cell dendrites and clumped AII amacrine cell bodies, also indicating a role in self-avoidance. Neurons in Dscam or Dscaml1 mutant retinas stratified their processes appropriately in synaptic laminae in the inner plexiform layer, and functional synapses formed in the rod circuit in mice lacking Dscaml1. Therefore, DSCAM and DSCAML1 function similarly in self-avoidance, and are not essential for synaptic specificity in the mouse retina (Fuerst, 2009).

Novel axon projection after stress and degeneration in the Dscam mutant retina

The Down syndrome cell adhesion molecule gene (Dscam) is required for normal dendrite patterning and promotes developmental cell death in the mouse retina. Loss-of-function studies indicate that Dscam is required for refinement of retinal ganglion cell (RGC) axons in the lateral geniculate nucleus, and this study reports and describes a requirement for Dscam in the maintenance of RGC axon projections within the retina. Mouse Dscam loss of function phenotypes related to retinal ganglion cell axon outgrowth and targeting have not been previously reported, despite the abundance of axon phenotypes reported in Drosophila Dscam1 loss and gain of function models. Analysis of the Dscam deficient retina was performed by immunohistochemistry and Western blot analysis during postnatal development of the retina. Conditional targeting of Dscam and Jun was performed to identify factors underlying axon-remodeling phenotypes. A subset of RGC axons were observed to project and branch extensively within the Dscam mutant retina after eye opening. Axon remodeling was preceded by histological signs of RGC stress. These included neurofilament accumulation, axon swelling, axon blebbing and activation of JUN, JNK and AKT. Novel and extensive projection of RGC axons within the retina was observed after upregulation of these markers, and novel axon projections were maintained to at least one year of age. Further analysis of retinas in which Dscam was conditionally targeted with Brn3b or Pax6alpha Cre indicated that axon stress and remodeling could occur in the absence of hydrocephalus, which frequently occurs in Dscam mutant mice. Analysis of mice mutant for the cell death gene Bax, which executes much of Dscam dependent cell death, identified a similar axon misprojection phenotype. Deleting Jun and Dscam resulted in increased axon remodeling compared to Dscam or Bax mutants. Retinal ganglion cells have a very limited capacity to regenerate after damage in the adult retina, compared to the extensive projections made in the embryo. This study found that DSCAM and JUN limit ectopic growth of RGC axons, thereby identifying these proteins as targets for promoting axon regeneration and reconnection (Fernandes, 2015).

Chelicerata sDscam isoforms combine homophilic specificities to define unique cell recognition

Thousands of Down syndrome cell adhesion molecule (Dscam1) isoforms and ∼60 clustered protocadhrein (cPcdh) proteins are required for establishing neural circuits in insects and vertebrates, respectively. The strict homophilic specificity exhibited by these proteins has been extensively studied and is thought to be critical for their function in neuronal self-avoidance. In contrast, significantly less is known about the Dscam1-related family of ∼100 shortened Dscam (sDscam) proteins in Chelicerata. Chelicerata sDscamα and some sDscamβ protein trans interactions are strictly homophilic, and the trans interaction is meditated via the first Ig domain through an antiparallel interface. Additionally, different sDscam isoforms interact promiscuously in cis via membrane proximate fibronectin-type III domains. Cell-cell interactions depend on the combined identity of all sDscam isoforms expressed. A single mismatched sDscam isoform can interfere with the interactions of cells that otherwise express an identical set of isoforms. Thus, these data support a model by which sDscam association in cis and trans generates a vast repertoire of combinatorial homophilic recognition specificities. It is proposed that in Chelicerata, sDscam combinatorial specificity is sufficient to provide each neuron with a unique identity for self-nonself discrimination. Surprisingly, while sDscams are related to Drosophila Dscam1, the results mirror the findings reported for the structurally unrelated vertebrate cPcdh. Thus, these findings suggest a remarkable example of convergent evolution for the process of neuronal self-avoidance and provide insight into the basic principles and evolution of metazoan self-avoidance and self-nonself discrimination (Zhou, 2020).

DSCAM protein interactions

DSCAM is a member of the immunoglobulin superfamily that maps to a Down syndrome region of chromosome 21q22.2-22.3. Genetic and biochemical studies have shown that in Drosophila, Dscam activates Pak1 via the Dock adaptor molecule. The extracellular domain of human DSCAM is highly homologous to the Drosophila protein; however, the intracellular domains of both human and Drosophila DSCAM share no obvious sequence identity. To study the signaling mechanisms of human DSCAM, the interaction between DSCAM and potential downstream molecules was investigated. DSCAM was shown to directly bind to Pak1 and stimulates Pak1 phosphorylation and activity, unlike Drosophila, where an adaptor protein Dock mediates the interaction between Dscam and Pak1. DSCAM activates both JNK and p38 MAP kinases. Furthermore, expression of the cytoplasmic domain of DSCAM induces a morphological change in cultured cells that is JNK-dependent. These observations suggest that human DSCAM also signals through Pak1 and may function in axon guidance similar to the Drosophila Dscam (Li, 2004).

DSCAM is a netrin receptor that collaborates with DCC in mediating turning responses to netrin-1

During nervous system development, spinal commissural axons project toward and across the ventral midline. They are guided in part by netrin-1, made by midline cells, which attracts the axons by activating the netrin receptor DCC. However, previous studies suggest that additional receptor components are required. This study reports that the Down's syndrome Cell Adhesion Molecule (DSCAM), a candidate gene implicated in the mental retardation phenotype of Down's syndrome, is expressed on spinal commissural axons, binds netrin-1, and is necessary for commissural axons to grow toward and across the midline. DSCAM and DCC can each mediate a turning response of these neurons to netrin-1. Similarly, Xenopus spinal neurons exogenously expressing DSCAM can be attracted by netrin-1 independently of DCC. These results show that DSCAM is a receptor that can mediate turning responses to netrin-1 and support a key role for netrin/DSCAM signaling in commissural axon guidance in vertebrates (Ly, 2008).

Regulation of mammalian DSCAM expression

The development of CNS neuronal networks involves processes including neuroblast migration, axonal pathfinding, and synaptogenesis. To evaluate the role of the axonal guidance molecule DSCAM in CNS connectivity, a lacZ reporter construct, Pr1.8-betagal, was generated containing a 1.8kb fragment of the human DSCAM promoter region, and its expression in four E12.5 transgenic mouse embryos was analyzed. Pr1.8-betagal drives lacZ expression in the choroid plexus and roof of the fourth ventricle, the floor plate of the fourth ventricle, pons and medulla oblongata, and the eye, limb buds, and dorsal root ganglion. This recapitulates a subset of DSCAM expression as demonstrated by in situ hybridization, supporting this 1.8kb fragment as a component of the endogenous DSCAM promoter. The Pr1.8-betagal expression pattern supports a role for DSCAM in CNS development, providing an endogenous promoter to investigate the contribution of DSCAM to Down syndrome neural defects (Barlow, 2002).

Neurite arborization and mosaic spacing in the mouse retina require DSCAM

To establish functional circuitry, retinal neurons occupy spatial domains by arborizing their processes, which requires the self-avoidance of neurites from an individual cell, and by spacing their cell bodies, which requires positioning the soma and establishing a zone within which other cells of the same type are excluded. The mosaic patterns of distinct cell types form independently and overlap. The cues that direct these processes in the vertebrate retina are not known. This study shows that some types of retinal amacrine cells from mice with a spontaneous mutation in Down syndrome cell adhesion molecule (Dscam), a gene encoding an immunoglobulin-superfamily member adhesion molecule, have defects in the arborization of processes and in the spacing of cell bodies. In the mutant retina, cells that would normally express Dscam have hyperfasciculated processes, preventing them from creating an orderly arbor. Also, their cell bodies are randomly distributed or pulled into clumps rather than being regularly spaced mosaics. These results indicate that mouse DSCAM mediates isoneuronal self-avoidance for arborization and heteroneuronal self-avoidance within specific cell types to prevent fasciculation and to preserve mosaic spacing. These functions are analogous to those of Drosophila DSCAM and DSCAM2. DSCAM may function similarly in other regions of the mammalian nervous system, and this role may extend to other members of the mammalian Dscam gene family (Fuerst, 2008).

DSCAM and DSCAML1 regulate the radial migration and callosal projection in developing cerebral cortex

Down syndrome cell adhesion molecule (Dscam) is essential for self-avoidance and tiling of dendritic development in sensory neurons in Drosophila. Recent studies also show that DSCAM together with its closely related protein DSCAML1 functions in dendritic self-avoidance of a certain types of interneuron in mammalian retina. However, the functions of these DSCAMs in developing mammalian cerebral cortex are not well understood. This study reduced the expression of DSCAM or DSCAML1 in mouse cortical neurons by RNA interference both in vitro and in vivo. Knockdown of DSCAM or DSCAML1 was found to increase the complexity of proximal dendritic branching, and impedes the axon growth in cultured neurons. In vivo knockdown experiments showed that both DSCAM and DSCAML1 contribute to normal radial migration and callosal projection during the postnatal development. This results indicate an important role of DSCAM and DSCAML1 in the development of cortical neural network (Zhang, 2014).

DSCAM promotes refinement in the mouse retina through cell death and restriction of exploring dendrites

A gain-of-function mouse allele of the Down syndrome cell adhesion molecule (Dscam) was developed and used to complement loss-of-function models. The role of Dscam in promoting cell death, spacing, and laminar targeting of neurons was assayed in the developing mouse retina. It was found that ectopic or overexpression of Dscam is sufficient to drive cell death. Gain-of-function studies indicate that Dscam is not sufficient to increase spatial organization, prevent cell-to-cell pairing, or promote active avoidance in the mouse retina, despite the similarity of the Dscam loss-of-function phenotype in the mouse retina to phenotypes observed in Drosophila Dscam1 mutants. Both gain- and loss-of-function studies support a role for Dscam in targeting neurites; DSCAM is necessary for precise dendrite lamination, and is sufficient to retarget neurites of outer retinal cells after ectopic expression. It was further demonstrated that DSCAM guides dendrite targeting in type 2 dopaminergic amacrine cells, by restricting the stratum in which exploring retinal dendrites stabilize, in a Dscam dosage-dependent manner. Based on these results a single model is proposed to account for the numerous Dscam gain- and loss-of-function phenotypes reported in the mouse retina whereby DSCAM eliminates inappropriately placed cells and connections (Li, 2015).

Replacing the PDZ-interacting C-termini of DSCAM and DSCAML1 with epitope tags causes different phenotypic severity in different cell populations

Different types of neurons in the retina are organized vertically into layers and horizontally in a mosaic pattern that helps ensure proper neural network formation and information processing throughout the visual field. The vertebrate Dscams (DSCAM and DSCAML1) (see Drosophila Dscam4 and Dscam1, respectively) are cell adhesion molecules that support the development of this organization by promoting self-avoidance at the level of cell types, promoting normal developmental cell death, and directing vertical neurite stratification. To understand the molecular interactions required for these activities, this study tested the functional significance of the interaction between the C-terminus of the Dscams and multi-PDZ domain-containing scaffolding proteins in mouse. It was hypothesized that this PDZ-interacting domain would mediate a subset of the Dscams' functions. Instead, it was found that in the absence of these interactions, some cell types develop almost normally, while others resemble complete loss of function. Thus, there is a differential dependence on this domain for Dscams' functions in different cell types (Garrett, 2016).

DSCAM gene triplication causes excessive GABAergic synapses in the neocortex in Down syndrome mouse models

Down syndrome (DS) is caused by the trisomy of human chromosome 21 (HSA21). A major challenge in DS research is to identify the HSA21 genes that cause specific symptoms. Down syndrome cell adhesion molecule (DSCAM) is encoded by a HSA21 gene. Previous studies have shown that the protein level of the Drosophila homolog of DSCAM determines the size of presynaptic terminals. However, whether the triplication of DSCAM contributes to presynaptic development in DS remains unknown. This study shows that DSCAM levels regulate GABAergic synapses formed on neocortical pyramidal neurons (PyNs). In the Ts65Dn mouse model for DS, where DSCAM is overexpressed due to DSCAM triplication, GABAergic innervation of PyNs by basket and chandelier interneurons is increased. Genetic normalization of DSCAM expression rescues the excessive GABAergic innervations and the increased inhibition of PyNs. Conversely, loss of DSCAM impairs GABAergic synapse development and function. These findings demonstrate excessive GABAergic innervation and synaptic transmission in the neocortex of DS mouse models and identify DSCAM overexpression as the cause. They also implicate dysregulated DSCAM levels as a potential pathogenic driver in related neurological disorders (Liu, 2023).

REFERENCES

Search PubMed for articles about Drosophila Downs syndrome cell adhesion molecule 1

Agarwala, K. L., et al. (2000). Down syndrome cell adhesion molecule DSCAM mediates homophilic intercellular adhesion. Brain Res. Mol. Brain Res. 79: 118-126. 10925149

Agarwala, K. L., et al. (2001a). DSCAM, a highly conserved gene in mammals, expressed in differentiating mouse brain. Biochem. Biophys. Res. Commun. 281(3): 697-705. 11237714

Agarwala, K. L., et al. (2001b). Cloning and functional characterization of DSCAML1, a novel DSCAM-like cell adhesion molecule that mediates homophilic intercellular adhesion. Biochem. Biophys. Res. Commun. 285(3): 760-72. 11453658

Agarwala, K. L., et al. (2001c). Dscam is associated with axonal and dendritic features of neuronal cells. J. Neurosci. Res. 66(3): 337-46. 11746351

Alavi, M., Song, M., King, G.L., Gillis, T., Propst, R., Lamanuzzi, M., Bousum, A., Miller, A., Allen, R. and Kidd, T. (2016). Dscam1 forms a complex with Robo1 and the N-terminal fragment of Slit to promote the growth of longitudinal axons. PLoS Biol 14: e1002560. PubMed ID: 27654876

Armitage, S. A., Sun, W., You, X., Kurtz, J., Schmucker, D. and Chen, W. (2014). Quantitative profiling of Drosophila melanogaster Dscam1 isoforms reveals no changes in splicing after bacterial exposure. PLoS One 9: e108660. PubMed ID: 25310676

Barlow, G. M., et al. (2002). Mammalian DSCAMs: roles in the development of the spinal cord, cortex, and cerebellum? Biochem. Biophys. Res. Commun. 293(3): 881-91. 12051741

Barlow, G. M., et al. (2002). DSCAM: an endogenous promoter drives expression in the developing CNS and neural crest. Biochem. Biophys. Res. Commun. 299(1): 1-6. 12435380

Brites, D., Brena, C., Ebert, D. and Du Pasquier, L. (2013). More than one way to produce protein diversity: duplication and limited alternative splicing of an adhesion molecule gene in basal arthropods. Evolution 67: 2999-3011. PubMed ID: 24094349

Celotto, A. M. and Graveley, B. R. (2001). Alternative splicing of the Drosophila Dscam pre-mrna is both temporally and spatially regulated. Genetics 159: 599-608. 11606537

Crayton, M. E., Powell, B. C., Vision, T. J. and Giddings, M. C. (2006). Tracking the evolution of alternatively spliced exons within the Dscam family. BMC Evol. Biol. 6: 16. 16483367

Dascenco, D., Erfurth, M. L., Izadifar, A., Song, M., Sachse, S., Bortnick, R., Urwyler, O., Petrovic, M., Ayaz, D., He, H., Kise, Y., Thomas, F., Kidd, T. and Schmucker, D. (2015). Slit and Receptor tyrosine phosphatase 69D confer spatial specificity to axon branching via Dscam1. Cell 162: 1140-1154. PubMed ID: 26317474

Dong, H., Xu, B., Guo, P., Zhang, J., Yang, X., Li, L., Fu, Y., Shi, J., Zhang, S., Zhu, Y., Shi, Y., Zhou, F., Bian, L., You, W., Shi, F., Yang, X., Huang, J., He, H. and Jin, Y. (2022). Hidden RNA pairings counteract the "first-come, first-served" splicing principle to drive stochastic choice in Dscam1 splice variants. Sci Adv 8(4): eabm1763. PubMed ID: 35080968

Fernandes, K. A., Bloomsburg, S. J., Miller, C. J., Billingslea, S. A., Merrill, M. M., Burgess, R. W., Libby, R. T. and Fuerst, P. G. (2015). Novel axon projection after stress and degeneration in the Dscam mutant retina. Mol Cell Neurosci 71: 1-12. PubMed ID: 26691152

Fuerst, P. G., Koizumi, A., Masland, R. H. and Burgess, R. W. (2008). Neurite arborization and mosaic spacing in the mouse retina require DSCAM. Nature 451(7177): 470-4. PubMed citation: 18216855

Fuerst, P. G., et al. (2009). DSCAM and DSCAML1 function in self-avoidance in multiple cell types in the developing mouse retina. Neuron 64(4):484-97. PubMed Citation: 19945391

Garrett, A.M., Tadenev, A.L., Hammond, Y.T., Fuerst, P.G. and Burgess, R.W. (2016). Replacing the PDZ-interacting C-termini of DSCAM and DSCAML1 with epitope tags causes different phenotypic severity in different cell populations. Elife 5. PubMed ID: 27637097

Graveley, B. R., et al. (2004). The organization and evolution of the dipteran and hymenopteran Down syndrome cell adhesion molecule (Dscam) genes. RNA 10(10): 1499-506. 15383675

Graveley, B. R. (2005). Mutually exclusive splicing of the insect Dscam pre-mRNA directed by competing intronic RNA secondary structures. Cell 123(1): 65-73. 16213213

Hattori, D., et al. (2009). Robust discrimination between self and non-self neurites requires thousands of Dscam1 isoforms. Nature 461: 644-648. PubMed Citation: 19794492

Hughes, M. E., et al. (2007). Homophilic Dscam interactions control complex dendrite morphogenesis. Neuron 54(3): 417-27. Medline abstract: 17481395

Hummel, T., et al. (2003). Axonal targeting of olfactory receptor neurons in Drosophila is controlled by Dscam. Neuron 37: 221-231. 12546818

Kamiyama, D., McGorty, R., Kamiyama, R., Kim, M. D., Chiba, A. and Huang, B. (2015). Specification of dendritogenesis site in Drosophila aCC motoneuron by membrane enrichment of Pak1 through Dscam1. Dev Cell 35: 93-106. PubMed ID: 26460947

Kohmura, N., Senzaki, K., Hamada, S., Kai, N., Yasuda, R., Watanabe, M., Ishii, H., Yasuda, M., Mishina, M. and Yagi, T. (1998). Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex. Neuron 20: 1137-1151. PubMed Citation: 9655502

Kreahling, J. M. and Graveley, B. R. (2005). The iStem, a long-range RNA secondary structure element required for efficient exon inclusion in the Drosophila Dscam pre-mRNA. Mol. Cell. Biol. (23): 10251-60. 16287842

Li, S., Sukeena, J. M., Simmons, A. B., Hansen, E. J., Nuhn, R. E., Samuels, I. S. and Fuerst, P. G. (2015). DSCAM promotes refinement in the mouse retina through cell death and restriction of exploring dendrites. J Neurosci 35: 5640-5654. PubMed ID: 25855178

Li, S. A., Cheng, L., Yu, Y. and Chen, Q. (2016). Structural basis of Dscam1 homodimerization: Insights into context constraint for protein recognition. Sci Adv 2: e1501118. PubMed ID: 27386517

Li, W. and Guan, K. L. (2004). The Down syndrome cell adhesion molecule (DSCAM) interacts with and activates Pak. J. Biol. Chem. 279(31): 32824-31. 15169762

Liu, H., Caballero-Floran, R. N., Hergenreder, T., Yang, T., Hull, J. M., Pan, G., Li, R., Veling, M. W., Isom, L. L., Kwan, K. Y., Huang, Z. J., Fuerst, P. G., Jenkins, P. M. and Ye, B. (2023). DSCAM gene triplication causes excessive GABAergic synapses in the neocortex in Down syndrome mouse models. PLoS Biol 21(4): e3002078. PubMed ID: 37079499

Ly, A., et al. (2008). DSCAM is a netrin receptor that collaborates with DCC in mediating turning responses to netrin-1. Cell 133(7): 1241-54. PubMed Citation: 18585357

Matthews, B. J., Kim, M. E., Flanagan, J. J., Hattori, D., Clemens, J. C., Zipursky, S. L. and Grueber, W. B. (2007). Dendrite self-avoidance is controlled by Dscam. Cell 129(3): 593-604. Medline abstract: 17482551

Meijers, R., et al. (2007). Structural basis of Dscam isoform specificity. Nature 449: 487-491. Medline abstract: 17721508

Miura, S. K., Martins, A., Zhang, K. X., Graveley, B. R. and Zipursky, S. L. (2013). Probabilistic splicing of Dscam1 establishes identity at the level of single neurons. Graphical Abstract

Lefebvre, J. L., Kostadinov, D., Chen, W. V., Maniatis, T. and Sanes, J. R. (2012). Protocadherins mediate dendritic self-avoidance in the mammalian nervous system. Nature 488: 517-521. PubMed ID: 22842903

Okumura, M., Sakuma, C., Miura, M. and Chihara, T. (2015). Linking cell surface receptors to microtubules: Tubulin Folding Cofactor D mediates Dscam functions during neuronal morphogenesis. J Neurosci 35: 1979-1990. PubMed ID: 25653356

Park, J. W., et al (2004). Identification of alternative splicing regulators by RNA interference in Drosophila. Proc. Natl. Acad. Sci. USA 101: 15974-15979. 15492211

Sakuma, C., Okumura, M., Umehara, T., Miura, M. and Chihara, T. (2015). A STRIPAK component Strip regulates neuronal morphogenesis by affecting microtubule stability. Sci Rep 5: 17769. PubMed ID: 26644129

Sawaya, M. R., Wojtowicz, W. M., Andre, I., Qian, B., Wu, W., Baker, D., Eisenberg, D. and Zipursky, S. L. (2008). A double S shape provides the structural basis for the extraordinary binding specificity of Dscam isoforms. Cell 134(6): 1007-1018. PubMed ID: 18805093

Schmucker, D., et al. (2000). Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 101: 671-684. PubMed Citation: 10892653

Soba, P., et al. (2007). Drosophila sensory neurons require Dscam for dendritic self-avoidance and proper dendritic field organization. Neuron 54(3): 403-16. Medline abstract: 17481394

Takahashi, A. (2009). Effect of exonic splicing regulation on synonymous codon usage in alternatively spliced exons of Dscam. BMC Evol. Biol. 9: 214. PubMed Citation: 19709440

Wang, G. Z., Marini, S., Ma, X., Yang, Q., Zhang, X. and Zhu, Y. (2014). Improvement of Dscam homophilic binding affinity throughout Drosophila evolution. BMC Evol Biol 14: 186. PubMed ID: 25158691

Wang, J., et al. (2002). Drosophila Dscam is required for divergent segregation of sister branches and suppresses ectopic bifurcation of axons. Neuron 33: 559-571. 11856530

Wang, J., et al. (2004). Transmembrane/juxtamembrane domain-dependent Dscam distribution and function during mushroom body neuronal morphogenesis. Neuron 43: 663-672. 15339648

Watson, F. L., et al. (2005). Extensive diversity of Ig-superfamily proteins in the immune system of insects. Science 309(5742): 1874-8. 16109846

Wojtowicz, W. M., Flanagan, J. J., Millard, S. S., Zipursky, S. L. and Clemens, J. C. (2004). Alternative splicing of Drosophila Dscam generates axon guidance receptors that exhibit isoform-specific homophilic binding. Cell 118(5): 619-33. 15339666

Worby, C. A., et al. (2001). The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton. J. Biol. Chem. 276(45): 41782-9. 11546816

Wu, Q. and Maniatis, T. (1999). A striking organization of a large family of human neural cadherin-like cell adhesion genes. Cell 97: 779-790. PubMed Citation: 10380929

Wu, W., Ahlsen, G., Baker, D., Shapiro, L. and Zipursky, S. L. (2012). Complementary chimeric isoforms reveal Dscam1 binding specificity in vivo. Neuron 74(2): 261-8. PubMed Citation: 22542180

Yimlamai, D., Konnikova, L., Moss, L. G. and Jay, D. G. (2005). The zebrafish down syndrome cell adhesion molecule is involved in cell movement during embryogenesis. Dev. Biol. 279(1): 44-57. 15708557

Yamakawa, K., et al. (1998). DSCAM: a novel member of the immunoglobulin superfamily maps in a Down syndrome region and is involved in the development of the nervous system. Hum. Mol. Genet. 7: 227-237. PubMed Citation: 9426258

Yang, J. S., Bai, J. M. and Lee, T. (2008). Dynein-dynactin complex is essential for dendritic restriction of TM1-containing Drosophila Dscam. PLoS One (10): e3504. PubMed Citation: 18946501

Zhan, X. L., Clemens, J. C., Neves, G., Hattori, D., Flanagan, J. J., Hummel, T., Vasconcelos, M. L., Chess, A. and Zipursky, S. L. (2004). Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies. Neuron 43(5): 673-86. 15339649

Zhang, L., Huang, Y., Chen, J. Y., Ding, Y. Q. and Song, N. N. (2015).DSCAM and DSCAML1 regulate the radial migration and callosal projection in developing cerebral cortex. Brain Res 1594: 61-70. PubMed ID: 25451118

Zhou, F., Cao, G., Dai, S., Li, G., Li, H., Ding, Z., Hou, S., Xu, B., You, W., Wiseglass, G., Shi, F., Yang, X., Rubinstein, R. and Jin, Y. (2020). Chelicerata sDscam isoforms combine homophilic specificities to define unique cell recognition. Proc Natl Acad Sci U S A. PubMed ID: 32963097

Zhu, H., et al. (2006). Dendritic patterning by Dscam and synaptic partner matching in the Drosophila antennal lobe. Nature Neuroscience 9: 349-355. 16474389

date revised: 5 November 2023

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.