Down syndrome cell adhesion molecule 2: Biological Overview | References
Gene name - Down syndrome cell adhesion molecule 2
Synonyms - CG42256, Dscam2
Cytological map position - 65E6-65E7
Function - receptor
Symbol - Dscam2
FlyBase ID: FBgn0265296
Genetic map position - 3L:7,155,528..7,183,017 [-]
Classification - immunoglobulin superfamily member
Cellular location - surface transmembrane
|Recent literature||Bosch, D. S., van Swinderen, B. and Millard, S. S. (2015). Dscam2 affects visual perception in Drosophila melanogaster. Front Behav Neurosci 9: 149. PubMed ID: 26106310
Dscam2, a cell surface protein that mediates cellular repulsion, plays a crucial role in the development of the Drosophila melanogaster visual system. Dscam2 generates boundaries between neighboring modules in the fly optic lobe; in Dscam2 mutants this visual system modularity is compromised. Although developmental wiring defects have been well described in the Dscam2 mutant, behavioral consequences have not been investigated. To address this, the visual behavior of Dscam2 mutant flies was examined. Using a phototaxis assay, it was ascertained that these flies are not blind, but have a reduced phototaxic response. Through population-based and single fly optomotor assays, it was found that Dscam2 mutant flies can track motion but that their response is opposite to control flies under defined experimental conditions. In a fixation paradigm, which allows tethered flies to control the angular position of a visual stimulus, mutant flies' responses were diametrically opposed to those seen in control flies. These data suggest that modest changes in the modularity of the fly visual system in the Dscam2 mutant can dramatically change the perception of specific visual cues and modify behavior (Bosch, 2015).
|Tadros, W., Xu, S., Akin, O., Yi, C. H., Shin, G. J., Millard, S. S. and Zipursky, S. L. (2016). Dscam proteins direct dendritic targeting through adhesion. Neuron 89: 480-493. PubMed ID: 26844831
Cell recognition molecules are key regulators of neural circuit assembly. The Dscam family of recognition molecules in Drosophila has been shown to regulate interactions between neurons through homophilic repulsion. This is exemplified by Dscam1 and Dscam2, which together repel dendrites of lamina neurons, L1 and L2, in the visual system. By contrast, this study shows that Dscam2 directs dendritic targeting of another lamina neuron, L4, through homophilic adhesion. Through live imaging and genetic mosaics to dissect interactions between specific cells, Dscam2 was shown to be required in L4 and its target cells for correct dendritic targeting. In a genetic screen, Dscam4 was identified as another regulator of L4 targeting which acts with Dscam2 in the same pathway to regulate this process. This ensures tiling of the lamina neuropil through heterotypic interactions. Thus, different combinations of Dscam proteins act through distinct mechanisms in closely related neurons to pattern neural circuits.
|Kerwin, S. K., Li, J. S. S., Noakes, P. G., Shin, G. J. and Millard, S. S. (2018). Regulated alternative splicing of Drosophila Dscam2 is necessary for attaining the appropriate number of photoreceptor synapses. Genetics 208(2): 717-728. PubMed ID: 29208630
How the brain makes trillions of synaptic connections using a genome of only 20,000 genes is a major question in modern neuroscience. Alternative splicing is one mechanism that can increase the number of proteins produced by each gene, but its role in regulating synapse formation is poorly understood. In Drosophila, photoreceptors form a synapse with multiple postsynaptic elements including lamina neurons L1 and L2. L1 and L2 express distinct isoforms of the homophilic repulsive protein Dscam2, and since these isoforms cannot bind to each other, cell-specific expression has been proposed to be necessary for preventing repulsive interactions that could disrupt the synapse. This study shows that the number of synapses are reduced in flies that express only one isoform, and L1 and L2 dendritic morphology is perturbed. It is proposed that these defects result from inappropriate interactions between L1 and L2 dendrites. It is concluded that regulated Dscam2 alternative splicing is necessary for the proper assembly of photoreceptor synapses.
|Li, J. S. S. and Sean Millard, S. (2019). Deterministic splicing of Dscam2 is regulated by Muscleblind. Sci Adv 5(1): eaav1678. PubMed ID: 30746474
Alternative splicing increases the proteome diversity crucial for establishing the complex circuitry between trillions of neurons. To provide individual cells with different repertoires of protein isoforms, however, this process must be regulated. Previously, it was found that the mutually exclusive alternative splicing of Drosophila Dscam2 produces two isoforms (A and B) with unique binding properties. This splicing event is cell type specific, and the transmembrane proteins that it generates are crucial for the development of axons, dendrites, and synapses. This study shows that Muscleblind (Mbl) controls Dscam2 alternative splicing. Mbl represses isoform A and promotes the selection of isoform B. Mbl mutants exhibit phenotypes also observed in flies engineered to express a single Dscam2 isoform. Consistent with this, mbl expression is cell type specific and correlates with the splicing of isoform B. This study demonstrates how the regulated expression of a splicing factor is sufficient to provide neurons with unique protein isoforms crucial for development.
Sensory processing centers in both the vertebrate and the invertebrate brain are often organized into reiterated columns, thus facilitating an internal topographic representation of the external world. Cells within each column are arranged in a stereotyped fashion and form precise patterns of synaptic connections within discrete layers. These connections are largely confined to a single column, thereby preserving the spatial information from the periphery. Other neurons integrate this information by connecting to multiple columns. Restricting axons to columns is conceptually similar to tiling. Axons and dendrites of neighbouring neurons of the same class use tiling to form complete, yet non-overlapping, receptive fields. It is thought that, at the molecular level, cell-surface proteins mediate tiling through contact-dependent repulsive interactions, but proteins serving this function have not yet been identified. This study shows that the immunoglobulin superfamily member Dscam2 restricts the connections formed by L1 lamina neurons to columns in the Drosophila visual system. The data support a model in which Dscam2 homophilic interactions mediate repulsion between neurites of L1 cells in neighbouring columns. It is proposed that Dscam2 is a tiling receptor for L1 neurons (Millard, 2007).
The Drosophila visual system is a modular structure. The retina contains 750 simple eyes, each containing eight photoreceptor neurons or R cells (R1-R8). R cells project into the brain, where they make connections within two neuropils, the lamina and medulla. R1-R6 neurons target to the lamina, where they form synapses with lamina neurons (L1-L5). R7, R8 and L1-L5 form connections in single columns within layers in the medulla, and each column contains one axon of each of these cell types. As a consequence of this wiring pattern, each column processes motion (lamina neurons) and colour (R7 and R8) from a single point in space. Although some progress has been made in understanding how neurons select different layers within each of the 750 columns (Clandinin, 2002), the molecular mechanisms that restrict synaptic connections to a single column are not known (Millard, 2007).
Dscam2 belongs to a conserved family of cell-surface proteins expressed in the nervous systems of many different organisms. Down syndrome cell adhesion molecule (DSCAM) was originally identified as an open reading frame in a region of human chromosome 21 critical for Down's syndrome. There are four Dscam genes in the fly genome (Dscam, and Dscam2-4). They encode type I transmembrane proteins that share about 30% sequence identity and have a common extracellular domain comprising ten immunoglobulin and six fibronectin type III repeats. These proteins have divergent cytoplasmic tails. The genomic organization of each fly Dscam family member differs considerably. Dscam encodes four cassettes of alternative exons that can potentially generate 38,016 different proteins through mutually exclusive alternative splicing. Dscam has a function in forming neural circuits throughout the fly brain. Dscam isoforms bind homophilically, and in vivo studies indicate that these interactions promote repulsion. Dscam2-4 do not show extensive isoform diversity, and in this way these family members are more similar to mammalian DSCAMs. Dscam2 has two alternative immunoglobulin 7 domains that share about 50% sequence identity and are referred to as Dscam2A and Dscam2B. Given the structural similarities between Dscam and Dscam2 and the prominent expression of Dscam2 on neurites in the developing brain, it is proposed that interactions between Dscam2 proteins are required for patterning neuronal connections (Millard, 2007).
To assess the function of Dscam2, protein-null mutations in the gene were generated by homologous recombination. The Dscam2 mutants were viable but had marked defects in R-cell projections into the medulla. Using a panel of cell-type specific markers in the medulla, widespread defects were observed in axonal and dendritic organization. As wiring defects in one class of neurons may indirectly affect other classes, it was not possible to accurately assess the function of Dscam2 in homozygous mutant animals (Millard, 2007).
To identify a specific cell type that requires Dscam2, it was removed from subsets of neurons by using genetic mosaic techniques. Four cell types (R7, R8, L1 and L2) were targeted that connect to specific layers within each medulla column. To assess whether Dscam2 was required in R7 and R8, genetically mosaic animals were generated in which mutant R7 and R8 cells projected into an otherwise wild-type brain. R7 and R8 neurons lacking Dscam2 formed patterns of projections that were indistinguishable from their wild-type counterparts (Millard, 2007).
The analysis was extended to a subset of lamina neurons, L1 and L2. L1 axons arborize in two medulla layers, m1 and m5. In contrast, L2 axons form a single terminal arborization at the m2 layer. To assess whether Dscam2 is required in L1 and L2 neurons, single mutant cells were generated in an otherwise wild-type background, using the MARCM technique. To do this, FLP recombinase was expressed under the control of a Dachshund (Dac) enhancer to induce recombination selectively in lamina precursor cells just before their final cell division. In wild-type controls, fewer than ten lamina neurons were labelled per optic lobe. Of these, 90% were L1 neurons and 10% were L2. Wild-type L1 and L2 cells arborized in the correct layers and were restricted to a single column. Other lamina neurons were not labelled by this procedure (Millard, 2007).
Dscam2 mutant L1 neurons arborized in the correct layers. These arbors, however, were no longer restricted to a single column and often extended over several columnar units. These neurons formed terminal structures within the appropriate layers in adjacent columns. Phenotypes were observed in m1, in m5 or in both of these layers. In some cases (less than 10%) L1 axons bifurcated between m1 and m5 and each branch targeted to the appropriate layer in adjacent columns. In marked contrast to mutant L1 neurons, the terminal arbors of mutant L2 neurons were indistinguishable from the wild type. In summary, Dscam2 is required within L1 neurons to restrict arbors to a single column. Conversely, R7, R8 and L2 axons are restricted to a single column by Dscam2-independent mechanisms (Millard, 2007).
How might Dscam2 restrict L1 processes to a single column? Columnar restriction in the medulla is reminiscent of dendritic tiling. Here dendrites of neighbouring cells of the same class do not overlap. Although the molecular mechanisms underlying tiling are not known, it has been proposed that they involve homotypic repulsion between cells of the same type. If Dscam2 restricts L1 processes in this manner then it would be predicted, first, that Dscam2 would exhibit homophilic binding; second, that L1 processes expressing Dscam2 would contact each other during development and then retract to a single column; and third, that wild-type L1 axonal processes would extend into adjacent columns in which L1 neurons were Dscam2 mutant (Millard, 2007).
To assess whether Dscam2 exhibits homophilic binding, cell aggregation assays and pull-down experiments were used. Two S2 cell populations expressing different Dscam2 isoforms (Dscam2A and Dscam2B) segregated into isoform-specific clusters. Similar results were obtained from mixing experiments between Dscam2 and either Dscam or Dscam3. Confirming this binding specificity, Dscam2 ectodomains fused to human Fc bound only to the full-length Dscam2 proteins with the identical ectodomain. In summary, Dscam2 interacts with itself in an isoform-specific manner and does not bind to other Dscam family members (Millard, 2007).
To assess whether L1 processes contact each other during development and whether Dscam2 is expressed in these layers, wild-type L1 arborization patterns and Dscam2 antibody staining were examined during pupal development. Using MARCM to label L1 cells, growth cone expansions and immature interstitial branches were observed at 30 h after puparium formation (APF). About 10 h later, m1 and m5 arbors were exuberant, not restricted to columns, and neurites from neighbouring labelled cells contacted each other. During subsequent development these processes retracted and were restricted to a single column by 70 h APF. Dscam2 was expressed within these layers throughout this time course. Expression peaked at 40 h APF and was markedly reduced by 70 h APF, by which time L1 arbors were restricted to a single column. It is not possible to determine which cells within these two layers account for the Dscam2 immunoreactivity; however, the results of genetic studies make it likely that minimally, L1 processes are Dscam2 positive. Dscam2 is also found in other layers, but at only low levels or not at all in R7 and R8 growth cones (Millard, 2007).
If L1 axons are restricted to a single column by Dscam2 homophilic interactions, then wild-type L1 arbors should display a phenotype when they contact mutant axons lacking Dscam2. To address this, reverse MARCM was used. As with MARCM, both wild-type and mutant lamina neurons are generated, but in reverse MARCM only the wild-type cells are labelled. As the frequency of generating labelled cells is low, the likelihood that a labelled wild-type L1 axon and a mutant lamina axon will be present in the same or an adjacent column is correspondingly low. In control experiments, labelled wild-type cells were restricted to columns in a wild-type genetic background. In contrast, of 466 wild-type L1 neurons examined using reverse MARCM, 15 neurons were observed extending processes into adjacent columns. Thus, Dscam2 homophilic interactions are required for restricting L1 arbors to columns (Millard, 2007).
Since both L1 and L2 mutant neurons are generated by Dac-FLP induced MARCM, Dscam2 could restrict L1 arbors either through repulsive interactions between L1 axons in adjacent columns or through adhesive interactions between L1 and L2 axons in the same column. Interactions with L2 axons are unlikely for two reasons: first, although L2 axons extend through the m1 layer, and thus could mediate interactions with L1 processes in this layer, they do not extend to the m5 layer, and second, the reverse MARCM phenotype is exclusively asymmetric, suggesting that the mutant axon resides in an adjacent column. In MARCM experiments, 61% of the mutant arbors extended in both directions, but under reverse MARCM conditions none of the phenotypes were bidirectional. These data argue that Dscam2 mediates axonal tiling between L1 processes in neighbouring columns (Millard, 2007).
Columnar restriction is a common organizing principle used by many sensory systems that relay spatial information from the periphery to processing centres in the brain. As a result of the reiterative nature of these circuits, multiple targets are available in close proximity to each other within the same layer. Local repulsion between axonal processes of identical neurons in adjacent columns, which make connections with these targets, provides a developmental strategy for preserving the spatial information in each circuit. This study shows that Dscam2 is a homophilic tiling receptor for L1 neurons. Axonal tiling ensures that synaptic connections are made exclusively with targets in a single column (Millard, 2007).
The functions of Dscam and Dscam2 have intriguing similarities and differences. Although both promote homophilic repulsion between neurites, they do so in different cellular contexts. Since each neuron expresses a unique set of Dscam isoforms, neurites from the same cell selectively recognize and repel each other. This process, called 'self avoidance', facilitates the uniform coverage of synaptic fields in the nervous system. By contrast, Dscam2 mediates repulsive interactions between neurites of the same cell type. This process, called tiling, limits connections to a local area. Tiling and self avoidance therefore act in concert to pattern dendritic and axonal fields in the nervous system.
Search PubMed for articles about Drosophila Dscam2
Clandinin, T. R. and Zipursky, S. L. (2002). Making connections in the fly visual system. Neuron 35: 827-841. PubMed ID: 12372279
Millard, S. S., Flanagan, J. J., Pappu, K. S., Wu, W. and Zipursky, S. L. (2007). Dscam2 mediates axonal tiling in the Drosophila visual system. Nature 447(7145): 720-4. PubMed ID: 17554308
date revised: 7 June 2008
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