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
|Odierna, G. L., Kerwin, S. K., Harris, L. E., Shin, G. J., Lavidis, N. A., Noakes, P. G. and Millard, S. S. (2020). Dscam2 suppresses synaptic strength through a PI3K-dependent endosomal pathway. J Cell Biol 219(6). PubMed ID: 32259198
Dscam2 is a cell surface protein required for neuronal development in Drosophila; it can promote neural wiring through homophilic recognition that leads to either adhesion or repulsion between neurites. This study reports that Dscam2 also plays a post-developmental role in suppressing synaptic strength. This function is dependent on one of two distinct extracellular isoforms of the protein and is autonomous to motor neurons. The PI3K enhancer, Centaurin gamma 1A, was linked to the Dscam2-dependent regulation of synaptic strength, and it was shown that changes in phosphoinositide levels correlate with changes in endosomal compartments that have previously been associated with synaptic strength. Using transmission electron microscopy, an increase was found in synaptic vesicles at Dscam2 mutant active zones, providing a rationale for the increase in synaptic strength. This study provides the first evidence that Dscam2 can regulate synaptic physiology and highlights how diverse roles of alternative protein isoforms can contribute to unique aspects of brain development and function.
|Galindo, S. E., Shin, G. J., Millard, S. S. and Grueber, W. B. (2023). Regulated alternative splicing of Dscam2 is required for somatosensory circuit wiring. bioRxiv. PubMed ID: 36909552
Axon and dendrite placement and connectivity is guided by a wide range of secreted and surface molecules in the developing nervous system. Nevertheless, the extraordinary complexity of connections in the brain requires that this repertoire be further diversified to precisely and uniquely regulate cell-cell interactions. One important mechanism for molecular diversification is alternative splicing. Drosophila Down syndrome cell adhesion molecule (Dscam2) undergoes cell type-specific alternative splicing to produce two isoform-specific homophilic binding proteins. Regulated alternative splicing of Dscam2 is important for dendrite and axon patterning, but how this translates to circuit wiring and animal behavior is not well understood. This study examined the role of cell-type specific expression of Dscam2 isoforms in regulating synaptic partner selection in the larval somatosensory system. Synaptic partners in the nociceptive circuit express different Dscam2 isoforms. Forcing synaptic partners to express a common isoform resulted in nociceptive axon patterning defects and attenuated nocifensive behaviors, indicating that a role for Dscam2 alternative splicing is to ensure that synaptic partners do not express matching isoforms. These results point to a model in which regulated alternative splicing of Dscam2 across populations of neurons restricts connectivity to specific partners and prevents inappropriate synaptic connections.
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 (Millard, 2007).
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 (Tadros, 2016).
This paper shows that Dscam2 and Dscam4 act together to regulate dendritic targeting. The genetic data presented in this study, coupled with previous biochemical studies, are consistent with Dscam2 using homophilic interactions to adhere L4 dendrites to multiple lamina neurons. In all other contexts examined previously, Dscam2 promotes repulsion. To further understand the adhesive role of Dscam2 in circuit assembly, a genetic screen was carried out to identify other genes with a similar phenotype. This led to the identification of Dscam4 mutants with an L4 targeting phenotype indistinguishable from that of Dscam2. Dscam2 and Dscam4 promote adhesion between L4 dendritic filopodia and their target fascicles, as revealed through live imaging. In this way, these proteins regulate the tiling of L4 dendritic processes across the lamina neuropil. Thus, Dscam family members act in different combinations through either adhesion or repulsion to pattern neural circuits (Tadros, 2016).
Previously, it was reported that Dscam2 regulates tiling of the axon terminals of L1 neurons in the medulla. It was proposed that this arrangement of processes is a consequence of homotypic interactions (i.e., interactions between cells of the same type) between L1 axon terminals in adjacent columns. This model was based on three observations. First, L1 axon terminals initially expand to contact those of their immediate neighbors and then retract. Second, the axon terminals of Dscam2 mutant L1 neurons remain expanded, invading the territory of their neighbors, in genetically mosaic animals. And third, wild-type L1s also exhibit the same defect when confronted with Dscam2 mutant lamina neurons in adjacent columns (Tadros, 2016).
This paper has uncovered a role for Dscam2 in regulating L4 dendritic tiling that occurs in a mechanistically different fashion. Developmental and reverse MARCM experiments suggested that Dscam2 mediates L4 tiling in a manner analogous to its role in L1 axons, that is via Dscam2-mediated homotypic interactions between L4 dendrites innervating the same cartridge. Additional genetic mosaic studies, however, ruled this out. By modifying MARCM (i.e., DL-MARCM), it was possible to assess the targeting of wild-type L4 dendrites encountering dendrites from neighboring L4 mutant neurons. In all cases, these wild-type dendrites targeted normally. Thus, while L4 tiling relies upon Dscam2-mediated homophilic recognition, it does not result solely from homotypic interactions between neighboring L4 dendrites (Tadros, 2016).
Developmental analyses raised the alternative possibility that Dscam2 is used by L4 dendrites to promote interactions with the target fascicle. Indeed, targeting defects were observed in L4 dendrites innervating cartridges with mutant L1 or L2 neurons using DL-MARCM. Thus, in this context, tiling arises from heterotypic interactions. As the penetrance of this cell non-autonomous phenotype was weaker than removing Dscam2 from L4, it is proposed that L1 and L2 act redundantly, perhaps with other lamina neurons (e.g. L3 and L5 which were not tested), to promote Dscam2-dependent recognition between L4 dendrites and the target fascicle. This is consistent with the expression of Dscam2B and Dscam2A in L1 and L2 neurons, respectively, and the redundant role for these isoforms in L4 uncovered through the mosaic analysis of isoform-specific knockin alleles. Thus, L4 tiling arises through heterotypic interactions via Dscam2-mediated homophilic recognition. This is consistent with studies in both vertebrates and invertebrates in which cell ablation experiments indicate that, in some contexts, tiling may arise through heterotypic interactions (Tadros, 2016).
As previously reported, removal of Dscam2 from either L1 or L2 results in ectopic branching of these neurons. This observation raised the possibility that wild-type L4s follow these branches in a Dscam2-independent fashion and that this underlies the Dscam2 non-autonomous effect on wild-type L4s. Although this cannot be out, the interpretation is preferred that these data reflect a Dscam2-dependent effect independent of the ectopic L1 or L2 phenotype for two reasons. First, of the two instances in which a wild-type L4 produced an extra branch upon encountering a Dscam2 mutant L1, one L1 did not form an ectopic branch. And second, in most cases where either L1 (2/3) or L2 (18/23) displayed an extra branch phenotype, the associated L4 did not (Tadros, 2016).
The functional significance of this tiling and its disruption in Dscam2 mutants remain open questions. A recent behavioral study has suggested that L4 functions in responding to progressive, i.e., front-to-back, as opposed to regressive motion. This response would be consistent with L4's asymmetric dendritic arrangement, having one dendrite projecting anteriorly and two posteriorly. While this is an attractive model, another study using slightly different methodology suggests no role for L4 in directional selectivity. Critically assessing the importance of the spatial arrangement of L4 dendrites on behavior will require selective removal of Dscam2 from all L4 neurons in an otherwise wild-type background (Tadros, 2016).
Dscam paralogs promote cell adhesion in vitro. However, in all cases examined previously, genetic and phenotypic analyses of Dscam1 and Dscam2 mutations are consistent with a repulsive function for these proteins upon homophilic binding in vivo. In addition, gain-of-function studies in Drosophila support this view. Targeted expression of single Dscam1 isoforms promotes dendritic repulsion between class I and class III dendritic arborization (da) sensory neurons. This repulsive interaction is converted into adhesion by removing the cytoplasmic domain or selectively removing specific motifs, underscoring the role of cytoplasmic signaling in converting Dscam1-mediated homophilic binding to repulsion. And finally, forced expression of either Dscam2A or Dscam2B isoforms in closely apposed neurons in the visual system promotes repulsion between them (Tadros, 2016).
By contrast, several observations argue that Dscam2 and Dscam4 promote adhesion between L4 dendrites and their target fascicles. In fixed samples, wild-type L4 dendrites target to lamina axon fascicles, while Dscam2, Dscam4, or Dscam2/4 double mutant dendrites extend laterally along the lattice of R cell growth cones surrounding the target fascicles. Indeed, using multicolor flip out (MCFO), extensive intermingling of L4 dendrites and L1 processes was observed in the target fascicle. In live imaging, L4 dendritic processes become progressively anchored to their target fascicles. By contrast, in mutants, the interactions between L4 dendrites and the target fascicles were transient, while interactions with R cell growth cones were sustained. This tight association in mutants could reflect a loss of repulsive interactions between L4 dendrites and R cells. Genetic mosaic analysis, however, indicate that this is highly unlikely. Together, these data support a model in which Dscam2 and Dscam4 act together to promote adhesion between L4 dendritic growth cones and their lamina targets. In the absence of this Dscam2/Dscam4-mediated adhesion, an adhesive interaction between L4 dendrites and the surrounding R cell growth cones is suggested to be unmasked. Indeed, transcript profiling of R cells and L4 neurons during development suggest that they both express several recognition molecules that are candidates for mediating this interaction (Tadros, 2016).
Although a model is favored in which Dscam2 and Dscam4 mediate selective adhesive interactions with the lamina fascicle, the data do not rule out a model in which Dscam2 and Dscam4 regulate L4 targeting in a permissive fashion. For instance, Dscam2/4 may restrict the exploratory phase to fascicles in the immediate vicinity through adhesive interactions with both lamina axons and R cell growth cones. Indeed, in live imaging, mutant L4 dendrites extend to additional fascicles on either side of their normal targets at a very early stage of filopodial exploration. In this model, another cell recognition molecule, acting in an instructive way, would promote association of L4 dendrites with the lamina axon fascicle. Characterization of other genes acting in this process and higher resolution live imaging may allow critical distinguishion between an instructive and a permissive role for Dscam2 and Dscam4 in L4 dendritic targeting (Tadros, 2016).
It is becoming increasingly clear that Drosophila Dscams regulate dendritic patterning in different ways. Dscam1 is alternatively spliced to give rise to greater than 19,000 ectodomain isoforms, with greater than 18,000 exhibiting homophilic binding specificity. This property, coupled with probabilistic expression, endows each neuron with a unique identity and allows for discrimination between self and non-self (i.e., self-avoidance). Dscam2, on the other hand, is alternatively spliced to give rise to only two isoforms. These act in combination with Dscam1 isoforms to regulate the appropriate association of L1 and L2 dendrites at multiple contact synapses. Here, L1 and L2 express different Dscam2 isoforms in a mutually exclusive fashion. It is thought that the unique identity acquired by the dendrites of these cells, through alternative splicing of both Dscam1 and Dscam2, allows them to appropriately discriminate between self and non-self at multiple contact synapses. In these contexts, Dscam proteins promote repulsion. As this study has shown, Dscam2 in combination with Dscam4 promotes adhesive interactions during dendritic targeting. This may reflect a unique property of a Dscam2/Dscam4 protein complex, differences in output of different spliced variants of the cytoplasmic domains, or differences in levels of expression. Precedents for each of these potential mechanisms have been reported in other intercellular signaling proteins. Additional experiments will be needed to distinguish between these different possibilities (Tadros, 2016).
In summary, this study has shown that two different combinations of Dscam proteins regulate the patterning of different dendritic elements in the developing lamina in diverse ways. As all four Dscam proteins are broadly expressed within the developing lamina, distinct paralogs, and isoforms of them, either alone or in combination may pattern dendrites in different ways. Furthermore, as Dscam proteins also interact with other proteins in both cis and trans, they have the potential to pattern circuits in many different ways (Tadros, 2016).
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
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
date revised: 28 December 2023
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