- Email: [email protected]
Role of self-avoidance in neuronal wiring
Available online at www.sciencedirect.com
ScienceDirect Role of self-avoidance in neuronal wiring Yoshiaki Kise1,2 and Dietmar Schmucker1,2 Addresses 1 Vesalius Research Center, VIB, Leuven, Belgium 2 Department of Oncology, University of Leuven, School of Medicine, Leuven, Belgium Corresponding author: Schmucker, Dietmar (dietmar.schmucker@ vib-kuleuven.be)
Current Opinion in Neurobiology 2013, 23:983–989 This review comes from a themed issue on Development of neurons and glia Edited by Samuel Pfaff and Shai Shaham For a complete overview see the Issue and the Editorial Available online 22nd October 2013 0959-4388/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.conb.2013.09.011
Introduction Recent progress in studying neuronal wiring has made it clear that a cellular mechanism termed self-avoidance is a key process underlying the patterning of highly branched neurites in both vertebrates and invertebrates (Figure 1). Self-avoidance refers to the developmental mechanism by which neurites from the same neuron rarely overlap each other, while neurites from other neurons are allowed to overlap extensively. Often, sister branches of a branching neuron grow within a spatially confined tissue area (or even across a small 2-dimensional (2D) tissue surface) yet they strictly avoid crisscrossing. As a result the neurons are forming non-overlapping dendritic or axonal arborization patterns, which cover an evenly spaced sensory territory or forming multiple arbors connecting with different synaptic targets. The evidence that neurons utilize a cellular mechanism of self-avoidance was already shown by Kramer and colleagues in leech mechanosensory neurons about 30 years ago [1,2]. However, the molecular basis of self-avoidance had just begun to be revealed during the last decade by the studies of the cell surface receptors Drosophila Dscam1, vertebrate DSCAM, and clustered Protocadherins. Loss of Dscam1 function in Drosophila causes dendrite self-crossing or fasciculation in dendritic arborization (da) neurons [3–5] or olfactory projection neurons [6], and also causes defects in axonal branch segregation in mushroom body neurons [7]. Similarly, loss of DSCAM or clustered Protocadherin-gamma function causes abnormal dendrite fasciculation or clumping in cells of the mouse retina [8,9,10]. Here, we review how these receptors function in neurite self-avoidance and summarize recent studies illustrating www.sciencedirect.com
novel molecular and cellular mechanisms of self-avoidance.
Self-avoidance and self-recognition The developmental mechanism of establishing highly branched neurite patterns does not need to be deterministic (i.e. guided along a specific path) but can be highly variable as long as few growth parameters are obeyed. Such parameters are repulsion among sister neurites (i.e. self-avoidance), initial growth direction of the primary neurite and (intrinsic or extrinsic) growth stop signals, which often includes inhibitory interactions with neighboring ‘same-type’ neurons. Clear indications of self-avoidance have first been described for mechanosensory neurons that innervate the epidermis of leech animals [1,2] and provided an experimental system for elucidating the cellular mechanisms that regulate sister branch spacing. The observations by Kramer and colleagues led them to suggest that axon branches directly compete for territory (Figure 1b). Such competition among sister branches has now been described for numerous different neuronal cell types, most prominently in neurons with highly branched dendritic compartments. A significant amount of evidence suggests that contact mediated self-repulsion plays an important role in selfavoidance. This is well supported by both cellular and molecular studies. Although other strategies, such as presence of limiting trophic factor or an activity dependent selection, may also play roles in certain cell types [11]. Self-avoidance implies that the underlying molecular mechanism depends on a selective recognition discriminating between neurites of other neurons (non-self) and sister branches of the same neuron (self). However, it is important to emphasize that the process of self-recognition is conceptually separate from the process of self-avoidance. Molecularly these processes may or may not depend on the same regulators. For example in Drosophila multi-dendritic neurons the Dscam1 receptors are important for neurite self-recognition as well as for controlling self-avoidance (Figures 1c and 2a). In contrast, dendrite self-avoidance in C. elegans PVD neurons is regulated by DCC, Netrin, and UNC-5 interactions, molecules that are not involved in selfrecognition (Figure 1e). We would therefore like to discuss first two molecular systems that underlie the process of self-recognition where direct links to self-avoidance can be made. In a Current Opinion in Neurobiology 2013, 23:983–989
984 Development of neurons and glia
Figure 1
(b)
(a) wild type
HmLAR2 RNAi
(c)
Self-avoidance wild type
Dscam1 null
(d)
Loss of self-avoidance
magnification
(e)
2° 1° 3° 4° wild type wild type
UNC-6 (Netrin) null
Pcdh-γ null Current Opinion in Neurobiology
Self-avoidance is important for neuronal wiring. (a) Self-avoidance in leech comb cell. Comb cell extends processes in parallel without overlapping (left). However, processes often cross over each other by HmLAR2 RNAi (right) [42]. (b) Self-avoidance in leech mechanosensory neuron. Mechanosensory Pv neuron extends axons in major field (blue) and minor field (pink). Sister branches do not overlap in both fields. In addition, major field branches do not invade minor field, and vice versa (left). Ablation of an axon of a minor field allows axons from the major field axon to spread into minor field territory (right) [2]. (c) Self-avoidance in Drosophila dendritic arborization neurons (da neurons). Wild type dendrites show self avoidance and do not cross each other. However, Dscam1 null dendrites show self-crossing. See arrows in magnified image [3,4,5]. (d) Self-avoidance in mouse retinal SACs. Wild type SAC denrdrites exhibit self-avoidance (left). Pcdh-g deficient SAC dendrites show self-crossing and form bundles (right) [10]. (e) Self-avoidance in C. elegans PVD neuron. PVD dendrites form a non-overlapping tree pattern (left). Arrowheads indicate self-avoidance between tertiary (38) dendrites. UNC-6 (Netrin) mutant shows overlap (arrows) of the adjacent 38 dendrites (right) [41].
second part we discuss several cellular and molecular systems, which are primarily dedicated to self-avoidance.
Molecular systems that control selfrecognition Drosophila Dscam1
In Drosophila Dscam1 is required for both, self-recognition and self-avoidance. Dscam1 is a member of the immunoglobulin super family (IgSF) class of surface receptors. The large extracellular domain contains three Ig domains (Ig2, Ig3, Ig7), which are hypervariable due to the existence of many tandemly arrayed alternative exons coding for Ig2, Ig3, Ig7, respectively [12]. Combinatorial Current Opinion in Neurobiology 2013, 23:983–989
but mutually exclusive use of the alternative exons allows the generation of maximally 18 612 protein isoforms. Several studies have examined the significance of this diversity and found that the remarkably high preference of homophilic binding [13–16] provides the molecular specificity for discriminating between cell-surfaces displaying matching (identical or highly similar) or nonmatching Dscam1 isoforms. As such, the molecular diversity of Dscam1 provides the molecular basis for selfrecognition [3–5,17–19]. Genetic studies were instrumental in showing that Dscam1 specificity and function provide the molecular www.sciencedirect.com
Role of self-avoidance in neuronal wiring Kise and Schmucker 985
Figure 2
(a) Repulsion response
Neurite 1
Ig7 Ig2, Ig3 and Ig7
Ig3 Ig2
Ig2 Ig3 Ig7
Repulsion response
(b) Repulsion response
Neurite 2
Neurite 1
EC2 and EC3 EC1 EC2 EC3
Repulsion response
Neurite 2 Current Opinion in Neurobiology
Isoform specific homophilic binding can provide a basis for self–non-self recognition: (a) Homophilic interaction of Drosophila melanogaster Dscam1. Trans interactions of Dscam1 isoforms with identical Ig 2, 3 and 7 domains support stable homophilic binding, whereas Dscam1 molecules with nonmatching domains fail to interact. Only the first eight Ig domains were shown [26]. (b) Homophilic interaction of clustered protocadherins in vertebrates. Protocadherin gamma isoforms are capable of forming tetrameric cis-clusters, which are thought to interact homophilically in trans. The specificity of the trans-interactions are likely mediated by the second and third extracellular domains. However, as a cautionary note, currently no structural data are available confirming these proposed interactions. Similarly, the importance of tetramer formation for the in vivo function of protocadherins has not yet been tested [10,27].
basis for self-avoidance in Drosophila [3–7,18–20]. Given the numerous examples of developmental defects in axonal as well as dendritic branch patterning, it seems possible that Dscam1 is required for self-avoidance in all Drosophila neurons with branched neurite compartments. www.sciencedirect.com
The central model for Dscam1 function in self-avoidance has been most thoroughly established in the studies of the four classes of multidendritic sensory neurons termed dendritic arbor (da) neurons [3–5]. These studies showed that cell autonomous loss of Dscam1 results in aberrant Current Opinion in Neurobiology 2013, 23:983–989
986 Development of neurons and glia
self-crossing of dendrites or even dendrite fasciculation (Figure 1c). Within the distinct area of a dendritic field of da neurons the growing sister dendrites occasionally encounter each other. As these neurites emanate from the same cell body they express identical sets of Dscam1 isoforms. Upon contact of neurite membranes the matching Dscam1 isoforms engage in homophilic binding and trigger signaling events, which result in repulsion (Figure 2a). In contrast, contact with neurites from other cells does rarely juxtapose identical isoforms and therefore does not trigger repulsion. Indeed, expression profiling of several neuronal cell types are consistent with each cell expressing a unique combination of Dscam1 isoforms, thereby endowing each neuron with a distinct cell-surface identity [20,21]. Moreover, analysis of mutants encoding reduced numbers of isoforms established that thousands of isoforms are required to discriminate self and of non-self [18,19]. These Drosophila studies provide an example in which the neurite repulsion of sister neurites is executed by molecularly coupling self-recognition to repulsion. Important evidence that direct binding between Dscam1 isoforms is indeed the molecular basis for self-avoidance in vivo has recently been provided by use of de novo engineered chimeric isoforms [22]. In these experiments pairs of chimeric isoforms that bind each other but not to themselves were tested in transgenic animals. It was found that isoforms that lack homophilic binding did support self-avoidance only weakly, yet coexpression of complementary chimeric isoforms within the same neuron was sufficient to mediate self-avoidance. Depending on the neuron number and the extent of neurite-neurite contacts in a developing nervous system, the specificity of self-recognition depends on mechanisms excluding expression of matching isoform sets between different neurons. Theoretical modeling suggested that in the case of Dscam1 a minimum of some 5000 different isoforms might be required for excluding the likelihood of expression of matching isoforms between cells and thereby limiting Dscam mediated repulsion to sister neurites [19]. However, it should be noted that many questions related to this model are currently open: How much isoform matching between cells is required for triggering repulsion, how is repulsion triggered upon homophilic binding, what controls the number of isoforms per cell, is there a reason for the high cell-type specific bias in isoform expression? [21,23]. Clustered protocadherins in vertebrates
Given the profound requirement of Dscam1 in selfrecognition and self-avoidance in flies it seems puzzling that DSCAM genes in vertebrates did not reveal any sign of a large molecular diversity. This left it unclear how neurite self-recognition might be achieved in vertebrate neurons [24]. It was even more surprising that the recent Current Opinion in Neurobiology 2013, 23:983–989
loss-of-function studies in mice revealed that the vertebrate DSCAM and DSCAML1 genes have a role in self-avoidance of dendrites in the retina [8,9]. This raised the question which molecular system is responsible for self and non-self recognition in vertebrates [25], and suggested the possibility that — unlike Dscam1 in flies — molecularly self-recognition in vertebrates may not be directly coupled to self-avoidance [26]. Much clarification of these issues and questions were provided by two recent studies on clustered protocadherin genes in vertebrates. In a nutshell these studies provided firstly, evidence for a previously underappreciated molecular diversity of protocadherins [27]; secondly, evidence for homophilic binding specificity of diverse protocadherin clusters [27]; thirdly, evidence for a role of Pcdh-g in self-avoidance [10]. Therefore, these studies strongly support the hypothesis first proposed by Sanes and Zipursky that in vertebrates the clustered Protocadherins (Pcdhs) have an analogous role to Dscam1 in invertebrates and are important for both, self-recognition and self-avoidance [25]. In the mouse genome, there are three Pcdh gene clusters called Pcdh-a, Pcdh-b, and Pcdh-g which generate 14, 22, and 22 cadherin-like receptors, which are widely expressed in the nervous system. The first exon of each Pcdh-a and Pcdh-g mRNA encodes the entire extracellular domain and very short part of the cytoplasmic domain. This ‘variable’ part of the receptor is cis-spliced to constant exons encoding a common cytoplasmic domain for the alpha and gamma cluster. Each Pcdh-b gene consists of a single exon, which encodes a transmembrane receptor that lacks a cytoplasmic domain. Each variable Pcdh exon has its own promoter. Expression of different Pcdh receptors appears to be stochastic and different express different Pcdh combinations neurons [28,29,30,31–38]. A recent breakthrough finding by Schreiner and Weiner [27], showed that the recognition specificity of Pcdhs might be much higher than expected considering the relatively simple exon arrangement at the Pcdh locus. Specifically, they used biochemistry and binding assays to show that Pcdhs can form tetrameric cis-complexes when overexpressed (Figure 2b). Cell-binding assays further showed that tetramers can serve as ‘binding units’ for homophilic trans-interactions and that the specificity of those homophilic interactions depends on the subunit composition (i.e. type of monomeric Pcdh). As a result, the number of possible diverse Pcdh ‘tetramer-units’ amounts to thousands of different homophilic binding units even for just the Pcdh-g cluster alone. Whether these proposed tetrameric binding units are functionally important in vivo remains to be tested rigorously. Recent findings using immuno-precipitations from zebrafish www.sciencedirect.com
Role of self-avoidance in neuronal wiring Kise and Schmucker 987
tissue provide the first encouraging support of potential multimeric receptor units in showing that Pcdh-g and Pcdh-a can be co-immunoprecipitated [39]. One very recent genetic study of the role of Pcdh-g revealed a striking requirement of Pcdh-g for self-recognition as well as self-avoidance in the mouse retina [10]. Pcdh-g is expressed in retinal starburst amacrine cells (SACs) whose dendrites exhibit self-avoidance, but overlap between neighboring SACs. In a conditional Pcdh-g mutant retina where the common cytoplasmic domain for all Pcdh-g is deleted, SAC dendrites show self-crossing and fasciculation defects (Figure 1d). The same phenotype is observed even when the Pcdh-g gene is deleted in single SACs, indicating that Pcdh-g is cell-autonomously required for dendrite self-avoidance. Expression of a single isoform in mutant cells can rescue the Pcdh-g self-avoidance defects. However, if a single isoform is uniformly expressed, overlap of dendrites between neighboring SACs is significantly decreased, suggesting ectopic heteroneuronal repulsion. It is important to note that this phenotype is weak, presumably because Pcdh-a and Pcdh-b are also expressed in these cells and counteract the ectopic repulsion. Nevertheless, these results indicate that the diversity of Pcdh-g is required for self and nonself recognition. Therefore, invertebrates and vertebrates use a highly similar strategy for self-recognition and selfavoidance during neuronal wiring.
Specialized systems that control selfavoidance Although fly Dscam and vertebrate clustered Pcdhs have a role in both self-recognition and self-avoidance, selfavoidance does not strictly depend on self–non-self discrimination. In principle specific expression of repellent molecules within a neuron, but not the surrounding cells may suffice to avoid crisscrossing of self-neurites. However, the demands on the specificity of such an exclusive system are high and therefore may only be found in systems with sparse neuronal populations and little neurite overlap. One example of ‘pre-specified’ cell-autonomous repulsive interactions are the transmembrane Sema6A and PlexA4 interactions, which have been shown to be required for horizontal cell dendritic self-avoidance in mouse retina [40]. Horizontal cells in PlexA4 or Sema6A knockout retina exhibit abnormal dendrite elaboration with reduced overall coverage of neurites in the outer plexiform layer (OPL). The analysis at single cell level revealed that PlexA4 -/- horizontal cells exhibited reduced dendritic self-avoidance. However, in contrast to the phenotype observed in DSCAM mutant retina, horizontal cell mosaic spacing is normal. Another ligand/receptor pair has been found to mediate self-avoidance in PVD nociceptive neurons in C. elegans www.sciencedirect.com
[41]. In this case UNC-6 (Netrin) and its receptors UNC-40 (DCC) and UNC-5 are required for dendritic self-avoidance. UNC-6 is secreted from ventral cells and UNC-40 captures UNC-6 at the surface of PVD dendrites. Sequestered UNC-6 interacts with UNC-5 in trans to mediate repulsive responses. It is likely that UNC-40 also mediates self-avoidance independent of UNC-6 and UNC-5 because expression of an UNC-40 mutant form, which lacks an intracellular domain cannot rescue the selfavoidance defect in UNC-40 mutants, suggesting that there is an additional UNC-40 ligand for self-avoidance. The comb cell in leech is one of the classical models for the study of neurite self-avoidance. Comb cells are found in the peripheral body wall of leech embryo and extend approximately 70 parallel processes bilaterally without overlapping with one another ([42], Figure 1a). The LARlike receptor tyrosine phosphatase, HmLAR2 is highly expressed in comb cells and the inhibition of HmLAR2 results in crossing-over of sibling processes [42]. In addition, HmLAR2 ectodomain can bind to itself, suggesting that HmLAR2 may have a role in homophilic binding mediated self-avoidance in leech. Moreover, the example of da neurons in Drosophila larvae illustrates that neurites often grow restricted to a 2D space. The Drosophila da neurons establish their dendritic field between an epithelial cell layer and the extracellular matrix (ECM) at the larval body wall. High-resolution confocal imaging, electron microscopy, and markers for enclosed dendrites revealed that loss of integrin a (mew) or b (mys) subunit increases the dendrite enclosure leading to apparent dendrite crossing in class IV da neurons [43,44]. However, these aberrant dendrite crossings are not the result of defective self-avoidance. Moreover, dendrite crossing phenotypes previously observed in mutants of fry, trc, and the Torc2 complex, which were suggested to be signaling components responsible for hetero-neuronal tiling, may not be caused by a lack of homotypic dendrite-dendrite repulsion, but rather due to a lack of growth restriction to 2D space (i.e. lack of direct dendrite-dendrite contact). Because integrin overexpression in these mutant backgrounds rescues dendrite-crossing phenotypes, it is likely that there exists a contact-mediated homotypic repulsion (i.e. tiling) pathway, which has not yet been identified. As evident from these examples, cell surface receptors play a key role in self-avoidance in general. Control of surface receptor expression by transcription factors may in some cases be an important regulatory step in modulating aspects of self-recognition and/or self-avoidance. The transcriptional factor SatB2 is expressed in the layer II/ III pyramidal neurons of the mouse cerebral cortex at early postnatal stages during which their specific dendritic arborization occurs. While SatB2 loss-of-function causes Current Opinion in Neurobiology 2013, 23:983–989
988 Development of neurons and glia
cell-fate specification defects, a precisely timed SatB2 shRNA knock-down in layer II/III neurons by in utero electroporation results in dendrite self-crossing, dendrite fasciculation, and soma clumping between neighboring neurons [45]. As these defects have striking similarity with phenotypes observed in retinal cells of DSCAM/ DSCAML1 mutant mice, the expression of surface receptors such as DSCAM/DSCAML1, clustered Pcdhs or other related receptors in cortical neurons may be regulated by SatB2, revealing a potentially direct link of self-avoidance components to cell type specific differentiation programs of cortical neurons.
Concluding remarks A substantial body of work has firmly established the importance of self-recognition and self-avoidance as an essential mechanism contributing to the wiring of the nervous system. It demonstrated the central role of surface receptors such as Dscam1 in flies and clustered Pcdhs in mammals. However, important questions relating to the molecular principles of receptor expression, receptorreceptor interactions, and receptor signal transduction pathways remain challenges for future work. It is currently entirely unclear how each neuron avoids expressing all isoforms of Dscam1 or clustered Pcdhs and why there is a strong splicing bias even though the canonical model of Dscam1 or Pcdh function implies sufficiency of stochastic expression. What is the molecular mechanism of converting homophilic binding into repulsion signaling? Or what drives and controls oligomeric assembly of Pcdh receptors? Are tetrameric receptor units indeed the main functional unit in vivo? Furthermore, we anticipate that future studies will reveal molecular cross-talk between self-recognition mechanisms and other neuron-specific pathways patterning dendritic or axonal branching.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Kramer AP, Kuwada JY: Formation of the receptive fields of leech mechanosensory neurons during embryonic development. J Neurosci 1983, 3:2474-2486.
2.
Kramer AP, Stent GS: Developmental arborization of sensory neurons in the leech Haementeria ghilianii. II. Experimentally induced variations in the branching pattern. J Neurosci 1985, 5:768-775.
3.
Hughes ME, Bortnick R, Tsubouchi A, Ba¨umer P, Kondo M, Uemura T, Schmucker D: Homophilic Dscam interactions control complex dendrite morphogenesis. Neuron 2007, 54:417-427.
4.
Matthews BJ, Kim ME, Flanagan JJ, Hattori D, Clemens JC, Zipursky SL, Grueber WB: Dendrite self-avoidance is controlled by Dscam. Cell 2007, 129:593-604.
5.
Soba P, Zhu S, Emoto K, Younger S, Yang SJ, Yu HH, Lee T, Jan LY, Jan YN: Drosophila sensory neurons require Dscam for dendritic self-avoidance and proper dendritic field organization. Neuron 2007, 54:403-416.
Current Opinion in Neurobiology 2013, 23:983–989
6.
Zhu H, Hummel T, Clemens JC, Berdnik D, Zipursky SL, Luo L: Dendritic patterning by Dscam and synaptic partner matching in the Drosophila antennal lobe. Nat Neurosci 2006, 9:349-355.
7.
Wang J, Zugates CT, Liang IH, Lee CH, Lee T: Drosophila Dscam is required for divergent segregation of sister branches and suppresses ectopic bifurcation of axons. Neuron 2002, 33:559-571.
8.
Fuerst PG, Koizumi A, Masland RH, Burgess RW: Neurite arborization and mosaic spacing in the mouse retina require DSCAM. Nature 2008, 451:470-474.
9.
Fuerst PG, Bruce F, Tian M, Wei W, Elstrott J, Feller MB, Erskine L, Singer JH, Burgess RW: DSCAM and DSCAML1 function in selfavoidance in multiple cell types in the developing mouse retina. Neuron 2009, 64:484-497.
10. Lefebvre JL, Kostadinov D, Chen WV, Maniatis T, Sanes JR: Protocadherins mediate dendritic self-avoidance in the mammalian nervous system. Nature 2012, 488:517-521. This study shows for the first time that Pcdh-g is required for dendritic self-avoidance in retinal starburst amacrine cells. It also provides evidence that its isoform diversity is important to allow for heteroneuronal interactions. The data strongly support the notion that Dscam in arthropods and Protocadherin in vertebrates serve analogous roles during dendrite patterning. 11. Grueber WB, Sagasti A: Self-avoidance and tiling: mechanisms of dendrite and axon spacing. Cold Spring Harb Perspect Biol 2010, 2:a001750. 12. Schmucker D, Clemens JC, Shu H, Worby CA, Xiao J, Muda M, Dixon JE, Zipursky SL: Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 2000, 101:671-684. 13. Wojtowicz WM, Flanagan JJ, Millard SS, Zipursky SL, Clemens JC: Alternative splicing of Drosophila Dscam generates axon guidance receptors that exhibit isoform-specific homophilic binding. Cell 2004, 118:619-633. 14. Wojtowicz WM, Wu W, Andre I, Qian B, Baker D, Zipursky SL: A vast repertoire of Dscam binding specificities arises from modular interactions of variable Ig domains. Cell 2007, 130:1134-1145. 15. Meijers R, Puettmann-Holgado R, Skiniotis G, Liu JH, Walz T, Wang JH, Schmucker D: Structural basis of Dscam isoform specificity. Nature 2007, 449:487-491. 16. Sawaya MR, Wojtowicz WM, Andre I, Qian B, Wu W, Baker D, Eisenberg D, Zipursky SL: A double S shape provides the structural basis for the extraordinary binding specificity of Dscam isoforms. Cell 2008, 134:1007-1018. 17. Chen BE, Kondo M, Garnier A, Watson FL, Pu¨ettmann-Holgado R, Lamar DR, Schmucker D: The molecular diversity of Dscam is functionally required for neuronal wiring specificity in Drosophila. Cell 2006, 125:607-620. 18. Hattori D, Demir E, Kim HW, Viragh E, Zipursky SL, Dickson BJ: Dscam diversity is essential for neuronal wiring and selfrecognition. Nature 2007, 449:223-227. 19. Hattori D, Chen Y, Matthews BJ, Salwinski L, Sabatti C, Grueber WB, Zipursky SL: Robust discrimination between self and non-self neurites requires thousands of Dscam1 isoforms. Nature 2009, 461:644-648. 20. Zhan XL, Clemens JC, Neves G, Hattori D, Flanagan JJ, Hummel T, Vasconcelos ML, Chess A, Zipursky SL: Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies. Neuron 2004, 43:673-686. 21. Neves G, Zucker J, Daly M, Chess A: Stochastic yet biased expression of multiple Dscam splice variants by individual cells. Nat Genet 2004, 36:240-246. 22. Wu W, Ahlsen G, Baker D, Shapiro L, Zipursky SL: Complementary chimeric isoforms reveal Dscam1 binding specificity in vivo. Neuron 2012, 74:261-268. This paper provides direct evidence for the importance of Dscam1 homophilic binding for self-avoidance in vivo by using complementary pairs of chimeric Dscam isoform that bind to each other but not homophilically. Pairwise but not single expression within the same neuron supports self-avoidance. www.sciencedirect.com
Role of self-avoidance in neuronal wiring Kise and Schmucker 989
23. Sun W, You X, Gogol-Do¨ring A, He H, Kise Y, Sohn M, Chen T, Klebes A, Schmucker D, Chen W: Ultra-deep profiling of alternatively spliced Drosophila Dscam isoforms by circularization-assisted multi-segment sequencing. EMBO J 2013, 32:2029-2038. This paper provides the first quantitative expression analysis of Dscam1 isoform and shows that most of possible isoforms are expressed in vivo. It also demonstrates that there is a strong tissue- and developmental stagespecific selectivity in isoform expression. The ‘how’ and ‘why’ of such a significant splicing bias is currently unknown. 24. Schmucker D, Flanagan JG: Generation of recognition diversity in the nervous system. Neuron 2004, 44:219-222. 25. Zipursky SL, Sanes JR: Chemoaffinity revisited: dscams, protocadherins, and neural circuit assembly. Cell 2010, 143:343-353. 26. Schmucker D, Chen B: Dscam and DSCAM: complex genes in simple animals, complex animals yet simple genes. Genes Dev 2009, 23:147-156. 27. Schreiner D, Weiner JA: Combinatorial homophilic interaction between gamma-protocadherin multimers greatly expands the molecular diversity of cell adhesion. Proc Natl Acad Sci U S A 2010, 107:14893-14898. This breakthrough publication provides for the first time evidence that the recogniton specificity of Pcdhs might be much higher than previously thought. It describes that Pcdhs can form tetramers, which can bind homophilically. Specificity of homophilic binding was found to be controlled by Pcdh isoform composition of the respective tetrameric complexes (see Figure 2b). 28. Weiner JA, Jontes JD: Protocadherins, not prototypical: a complex tale of their interactions, expression, and functions. Front Mol Neurosci 2013, 6:4. 29. Yagi T: Molecular codes for neuronal individuality and cell assembly in the brain. Front Mol Neurosci 2012, 5:45. 30. Hirayama T, Tarusawa E, Yoshimura Y, Galjart N, Yagi T: CTCF is required for neural development and stochastic expression of clustered Pcdh genes in neurons. Cell Rep 2012, 2:345-357. This paper characterizes the stochastic expression of Pcdh genes and identifies CTCF as an important activator of clustered Pcdh expression. 31. Golan-Mashiach M, Grunspan M, Emmanuel R, Gibbs-Bar L, Dikstein R, Shapiro E: Identification of CTCF as a master regulator of the clustered protocadherin genes. Nucleic Acids Res 2012, 40:3378-3391. 32. Monahan K, Rudnick ND, Kehayova PD, Pauli F, Newberry KM, Myers RM, Maniatis T: Role of CCCTC binding factor (CTCF) and cohesin in the generation of single-cell diversity of protocadherin-a gene expression. Proc Natl Acad Sci U S A 2012, 109:9125-9130. 33. Guo Y, Monahan K, Wu H, Gertz J, Varley KE, Li W, Myers RM, Maniatis T, Wu Q: CTCF/cohesin-mediated DNA looping is required for protocadherin a promoter choice. Proc Natl Acad Sci U S A 2012, 109:21081-21086.
www.sciencedirect.com
34. Ribich S, Tasic B, Maniatis T: Identification of long-range regulatory elements in the protocadherin-alpha gene cluster. Proc Natl Acad Sci U S A 2006, 103:19719-19724. 35. Kehayova P, Monahan K, Chen W, Maniatis T: Regulatory elements required for the activation and repression of the protocadherin-alpha gene cluster. Proc Natl Acad Sci U S A 2011, 108:17195-17200. 36. Yokota S, Hirayama T, Hirano K, Kaneko R, Toyoda S, Kawamura Y, Hirabayashi M, Hirabayashi T, Yagi T: Identification of the cluster control region for the protocadherin-beta genes located beyond the protocadherin-gamma cluster. J Biol Chem 2011, 286:31885-31895. 37. Hirano K, Kaneko R, Izawa T, Kawaguchi M, Kitsukawa T, Yagi T: Single-neuron diversity generated by Protocadherin-b cluster in mouse central and peripheral nervous systems. Front Mol Neurosci 2012, 5:90. 38. Kawaguchi M, Toyama T, Kaneko R, Hirayama T, Kawamura Y, Yagi T: Relationship between DNA methylation states and transcription of individual isoforms encoded by the protocadherin-alpha gene cluster. J Biol Chem 2008, 283:12064-12075. 39. Biswas S, Emond MR, Jontes JD: The clustered protocadherins Pcdha and Pcdhg form a heteromeric complex in zebrafish. Neuroscience 2012, 219:280-289. 40. Matsuoka RL, Jiang Z, Samuels IS, Nguyen-Ba-Charvet KT, Sun LO, Peachey NS, Che´dotal A, Yau KW, Kolodkin AL: Guidance-cue control of horizontal cell morphology, lamination, and synapse formation in the mammalian outer retina. J Neurosci 2012, 32:6859-6868. 41. Smith CJ, Watson JD, VanHoven MK, Colo´n-Ramos DA, Miller DM 3rd: Netrin (UNC-6) mediates dendritic self-avoidance. Nat Neurosci 2012, 15:731-737. This study reveals a novel mechanism controlling self-avoidance in C. elegans nociceptice neurons. It demonstrates that the diffusible guidance cue Netrin (Unc-6) and its receptors interact in trans on sister dendrites and thereby triggering repulsion (i.e. self-avoidance) in C. elegans. 42. Baker MW, Macagno ER: The role of a LAR-like receptor tyrosine phosphatase in growth cone collapse and mutualavoidance by sibling processes. J Neurobiol 2000, 44:194-203. 43. Kim ME, Shrestha BR, Blazeski R, Mason CA, Grueber WB: Integrins establish dendrite–substrate relationships that promote dendritic self-avoidance and patterning in Drosophila sensory neurons. Neuron 2012, 73:79-91. 44. Han C, Wang D, Soba P, Zhu S, Lin X, Jan LY, Jan YN: Integrins regulate repulsion-mediated dendritic patterning of Drosophila sensory neurons by restricting dendrites in a 2D space. Neuron 2012, 73:64-78. 45. Zhang L, Song NN, Chen JY, Huang Y, Li H, Ding YQ: Satb2 is required for dendritic arborization and soma spacing in mouse cerebral cortex. Cereb Cortex 2012, 22:1510-1519.
Current Opinion in Neurobiology 2013, 23:983–989
ScienceDirect Role of self-avoidance in neuronal wiring Yoshiaki Kise1,2 and Dietmar Schmucker1,2 Addresses 1 Vesalius Research Center, VIB, Leuven, Belgium 2 Department of Oncology, University of Leuven, School of Medicine, Leuven, Belgium Corresponding author: Schmucker, Dietmar (dietmar.schmucker@ vib-kuleuven.be)
Current Opinion in Neurobiology 2013, 23:983–989 This review comes from a themed issue on Development of neurons and glia Edited by Samuel Pfaff and Shai Shaham For a complete overview see the Issue and the Editorial Available online 22nd October 2013 0959-4388/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.conb.2013.09.011
Introduction Recent progress in studying neuronal wiring has made it clear that a cellular mechanism termed self-avoidance is a key process underlying the patterning of highly branched neurites in both vertebrates and invertebrates (Figure 1). Self-avoidance refers to the developmental mechanism by which neurites from the same neuron rarely overlap each other, while neurites from other neurons are allowed to overlap extensively. Often, sister branches of a branching neuron grow within a spatially confined tissue area (or even across a small 2-dimensional (2D) tissue surface) yet they strictly avoid crisscrossing. As a result the neurons are forming non-overlapping dendritic or axonal arborization patterns, which cover an evenly spaced sensory territory or forming multiple arbors connecting with different synaptic targets. The evidence that neurons utilize a cellular mechanism of self-avoidance was already shown by Kramer and colleagues in leech mechanosensory neurons about 30 years ago [1,2]. However, the molecular basis of self-avoidance had just begun to be revealed during the last decade by the studies of the cell surface receptors Drosophila Dscam1, vertebrate DSCAM, and clustered Protocadherins. Loss of Dscam1 function in Drosophila causes dendrite self-crossing or fasciculation in dendritic arborization (da) neurons [3–5] or olfactory projection neurons [6], and also causes defects in axonal branch segregation in mushroom body neurons [7]. Similarly, loss of DSCAM or clustered Protocadherin-gamma function causes abnormal dendrite fasciculation or clumping in cells of the mouse retina [8,9,10]. Here, we review how these receptors function in neurite self-avoidance and summarize recent studies illustrating www.sciencedirect.com
novel molecular and cellular mechanisms of self-avoidance.
Self-avoidance and self-recognition The developmental mechanism of establishing highly branched neurite patterns does not need to be deterministic (i.e. guided along a specific path) but can be highly variable as long as few growth parameters are obeyed. Such parameters are repulsion among sister neurites (i.e. self-avoidance), initial growth direction of the primary neurite and (intrinsic or extrinsic) growth stop signals, which often includes inhibitory interactions with neighboring ‘same-type’ neurons. Clear indications of self-avoidance have first been described for mechanosensory neurons that innervate the epidermis of leech animals [1,2] and provided an experimental system for elucidating the cellular mechanisms that regulate sister branch spacing. The observations by Kramer and colleagues led them to suggest that axon branches directly compete for territory (Figure 1b). Such competition among sister branches has now been described for numerous different neuronal cell types, most prominently in neurons with highly branched dendritic compartments. A significant amount of evidence suggests that contact mediated self-repulsion plays an important role in selfavoidance. This is well supported by both cellular and molecular studies. Although other strategies, such as presence of limiting trophic factor or an activity dependent selection, may also play roles in certain cell types [11]. Self-avoidance implies that the underlying molecular mechanism depends on a selective recognition discriminating between neurites of other neurons (non-self) and sister branches of the same neuron (self). However, it is important to emphasize that the process of self-recognition is conceptually separate from the process of self-avoidance. Molecularly these processes may or may not depend on the same regulators. For example in Drosophila multi-dendritic neurons the Dscam1 receptors are important for neurite self-recognition as well as for controlling self-avoidance (Figures 1c and 2a). In contrast, dendrite self-avoidance in C. elegans PVD neurons is regulated by DCC, Netrin, and UNC-5 interactions, molecules that are not involved in selfrecognition (Figure 1e). We would therefore like to discuss first two molecular systems that underlie the process of self-recognition where direct links to self-avoidance can be made. In a Current Opinion in Neurobiology 2013, 23:983–989
984 Development of neurons and glia
Figure 1
(b)
(a) wild type
HmLAR2 RNAi
(c)
Self-avoidance wild type
Dscam1 null
(d)
Loss of self-avoidance
magnification
(e)
2° 1° 3° 4° wild type wild type
UNC-6 (Netrin) null
Pcdh-γ null Current Opinion in Neurobiology
Self-avoidance is important for neuronal wiring. (a) Self-avoidance in leech comb cell. Comb cell extends processes in parallel without overlapping (left). However, processes often cross over each other by HmLAR2 RNAi (right) [42]. (b) Self-avoidance in leech mechanosensory neuron. Mechanosensory Pv neuron extends axons in major field (blue) and minor field (pink). Sister branches do not overlap in both fields. In addition, major field branches do not invade minor field, and vice versa (left). Ablation of an axon of a minor field allows axons from the major field axon to spread into minor field territory (right) [2]. (c) Self-avoidance in Drosophila dendritic arborization neurons (da neurons). Wild type dendrites show self avoidance and do not cross each other. However, Dscam1 null dendrites show self-crossing. See arrows in magnified image [3,4,5]. (d) Self-avoidance in mouse retinal SACs. Wild type SAC denrdrites exhibit self-avoidance (left). Pcdh-g deficient SAC dendrites show self-crossing and form bundles (right) [10]. (e) Self-avoidance in C. elegans PVD neuron. PVD dendrites form a non-overlapping tree pattern (left). Arrowheads indicate self-avoidance between tertiary (38) dendrites. UNC-6 (Netrin) mutant shows overlap (arrows) of the adjacent 38 dendrites (right) [41].
second part we discuss several cellular and molecular systems, which are primarily dedicated to self-avoidance.
Molecular systems that control selfrecognition Drosophila Dscam1
In Drosophila Dscam1 is required for both, self-recognition and self-avoidance. Dscam1 is a member of the immunoglobulin super family (IgSF) class of surface receptors. The large extracellular domain contains three Ig domains (Ig2, Ig3, Ig7), which are hypervariable due to the existence of many tandemly arrayed alternative exons coding for Ig2, Ig3, Ig7, respectively [12]. Combinatorial Current Opinion in Neurobiology 2013, 23:983–989
but mutually exclusive use of the alternative exons allows the generation of maximally 18 612 protein isoforms. Several studies have examined the significance of this diversity and found that the remarkably high preference of homophilic binding [13–16] provides the molecular specificity for discriminating between cell-surfaces displaying matching (identical or highly similar) or nonmatching Dscam1 isoforms. As such, the molecular diversity of Dscam1 provides the molecular basis for selfrecognition [3–5,17–19]. Genetic studies were instrumental in showing that Dscam1 specificity and function provide the molecular www.sciencedirect.com
Role of self-avoidance in neuronal wiring Kise and Schmucker 985
Figure 2
(a) Repulsion response
Neurite 1
Ig7 Ig2, Ig3 and Ig7
Ig3 Ig2
Ig2 Ig3 Ig7
Repulsion response
(b) Repulsion response
Neurite 2
Neurite 1
EC2 and EC3 EC1 EC2 EC3
Repulsion response
Neurite 2 Current Opinion in Neurobiology
Isoform specific homophilic binding can provide a basis for self–non-self recognition: (a) Homophilic interaction of Drosophila melanogaster Dscam1. Trans interactions of Dscam1 isoforms with identical Ig 2, 3 and 7 domains support stable homophilic binding, whereas Dscam1 molecules with nonmatching domains fail to interact. Only the first eight Ig domains were shown [26]. (b) Homophilic interaction of clustered protocadherins in vertebrates. Protocadherin gamma isoforms are capable of forming tetrameric cis-clusters, which are thought to interact homophilically in trans. The specificity of the trans-interactions are likely mediated by the second and third extracellular domains. However, as a cautionary note, currently no structural data are available confirming these proposed interactions. Similarly, the importance of tetramer formation for the in vivo function of protocadherins has not yet been tested [10,27].
basis for self-avoidance in Drosophila [3–7,18–20]. Given the numerous examples of developmental defects in axonal as well as dendritic branch patterning, it seems possible that Dscam1 is required for self-avoidance in all Drosophila neurons with branched neurite compartments. www.sciencedirect.com
The central model for Dscam1 function in self-avoidance has been most thoroughly established in the studies of the four classes of multidendritic sensory neurons termed dendritic arbor (da) neurons [3–5]. These studies showed that cell autonomous loss of Dscam1 results in aberrant Current Opinion in Neurobiology 2013, 23:983–989
986 Development of neurons and glia
self-crossing of dendrites or even dendrite fasciculation (Figure 1c). Within the distinct area of a dendritic field of da neurons the growing sister dendrites occasionally encounter each other. As these neurites emanate from the same cell body they express identical sets of Dscam1 isoforms. Upon contact of neurite membranes the matching Dscam1 isoforms engage in homophilic binding and trigger signaling events, which result in repulsion (Figure 2a). In contrast, contact with neurites from other cells does rarely juxtapose identical isoforms and therefore does not trigger repulsion. Indeed, expression profiling of several neuronal cell types are consistent with each cell expressing a unique combination of Dscam1 isoforms, thereby endowing each neuron with a distinct cell-surface identity [20,21]. Moreover, analysis of mutants encoding reduced numbers of isoforms established that thousands of isoforms are required to discriminate self and of non-self [18,19]. These Drosophila studies provide an example in which the neurite repulsion of sister neurites is executed by molecularly coupling self-recognition to repulsion. Important evidence that direct binding between Dscam1 isoforms is indeed the molecular basis for self-avoidance in vivo has recently been provided by use of de novo engineered chimeric isoforms [22]. In these experiments pairs of chimeric isoforms that bind each other but not to themselves were tested in transgenic animals. It was found that isoforms that lack homophilic binding did support self-avoidance only weakly, yet coexpression of complementary chimeric isoforms within the same neuron was sufficient to mediate self-avoidance. Depending on the neuron number and the extent of neurite-neurite contacts in a developing nervous system, the specificity of self-recognition depends on mechanisms excluding expression of matching isoform sets between different neurons. Theoretical modeling suggested that in the case of Dscam1 a minimum of some 5000 different isoforms might be required for excluding the likelihood of expression of matching isoforms between cells and thereby limiting Dscam mediated repulsion to sister neurites [19]. However, it should be noted that many questions related to this model are currently open: How much isoform matching between cells is required for triggering repulsion, how is repulsion triggered upon homophilic binding, what controls the number of isoforms per cell, is there a reason for the high cell-type specific bias in isoform expression? [21,23]. Clustered protocadherins in vertebrates
Given the profound requirement of Dscam1 in selfrecognition and self-avoidance in flies it seems puzzling that DSCAM genes in vertebrates did not reveal any sign of a large molecular diversity. This left it unclear how neurite self-recognition might be achieved in vertebrate neurons [24]. It was even more surprising that the recent Current Opinion in Neurobiology 2013, 23:983–989
loss-of-function studies in mice revealed that the vertebrate DSCAM and DSCAML1 genes have a role in self-avoidance of dendrites in the retina [8,9]. This raised the question which molecular system is responsible for self and non-self recognition in vertebrates [25], and suggested the possibility that — unlike Dscam1 in flies — molecularly self-recognition in vertebrates may not be directly coupled to self-avoidance [26]. Much clarification of these issues and questions were provided by two recent studies on clustered protocadherin genes in vertebrates. In a nutshell these studies provided firstly, evidence for a previously underappreciated molecular diversity of protocadherins [27]; secondly, evidence for homophilic binding specificity of diverse protocadherin clusters [27]; thirdly, evidence for a role of Pcdh-g in self-avoidance [10]. Therefore, these studies strongly support the hypothesis first proposed by Sanes and Zipursky that in vertebrates the clustered Protocadherins (Pcdhs) have an analogous role to Dscam1 in invertebrates and are important for both, self-recognition and self-avoidance [25]. In the mouse genome, there are three Pcdh gene clusters called Pcdh-a, Pcdh-b, and Pcdh-g which generate 14, 22, and 22 cadherin-like receptors, which are widely expressed in the nervous system. The first exon of each Pcdh-a and Pcdh-g mRNA encodes the entire extracellular domain and very short part of the cytoplasmic domain. This ‘variable’ part of the receptor is cis-spliced to constant exons encoding a common cytoplasmic domain for the alpha and gamma cluster. Each Pcdh-b gene consists of a single exon, which encodes a transmembrane receptor that lacks a cytoplasmic domain. Each variable Pcdh exon has its own promoter. Expression of different Pcdh receptors appears to be stochastic and different express different Pcdh combinations neurons [28,29,30,31–38]. A recent breakthrough finding by Schreiner and Weiner [27], showed that the recognition specificity of Pcdhs might be much higher than expected considering the relatively simple exon arrangement at the Pcdh locus. Specifically, they used biochemistry and binding assays to show that Pcdhs can form tetrameric cis-complexes when overexpressed (Figure 2b). Cell-binding assays further showed that tetramers can serve as ‘binding units’ for homophilic trans-interactions and that the specificity of those homophilic interactions depends on the subunit composition (i.e. type of monomeric Pcdh). As a result, the number of possible diverse Pcdh ‘tetramer-units’ amounts to thousands of different homophilic binding units even for just the Pcdh-g cluster alone. Whether these proposed tetrameric binding units are functionally important in vivo remains to be tested rigorously. Recent findings using immuno-precipitations from zebrafish www.sciencedirect.com
Role of self-avoidance in neuronal wiring Kise and Schmucker 987
tissue provide the first encouraging support of potential multimeric receptor units in showing that Pcdh-g and Pcdh-a can be co-immunoprecipitated [39]. One very recent genetic study of the role of Pcdh-g revealed a striking requirement of Pcdh-g for self-recognition as well as self-avoidance in the mouse retina [10]. Pcdh-g is expressed in retinal starburst amacrine cells (SACs) whose dendrites exhibit self-avoidance, but overlap between neighboring SACs. In a conditional Pcdh-g mutant retina where the common cytoplasmic domain for all Pcdh-g is deleted, SAC dendrites show self-crossing and fasciculation defects (Figure 1d). The same phenotype is observed even when the Pcdh-g gene is deleted in single SACs, indicating that Pcdh-g is cell-autonomously required for dendrite self-avoidance. Expression of a single isoform in mutant cells can rescue the Pcdh-g self-avoidance defects. However, if a single isoform is uniformly expressed, overlap of dendrites between neighboring SACs is significantly decreased, suggesting ectopic heteroneuronal repulsion. It is important to note that this phenotype is weak, presumably because Pcdh-a and Pcdh-b are also expressed in these cells and counteract the ectopic repulsion. Nevertheless, these results indicate that the diversity of Pcdh-g is required for self and nonself recognition. Therefore, invertebrates and vertebrates use a highly similar strategy for self-recognition and selfavoidance during neuronal wiring.
Specialized systems that control selfavoidance Although fly Dscam and vertebrate clustered Pcdhs have a role in both self-recognition and self-avoidance, selfavoidance does not strictly depend on self–non-self discrimination. In principle specific expression of repellent molecules within a neuron, but not the surrounding cells may suffice to avoid crisscrossing of self-neurites. However, the demands on the specificity of such an exclusive system are high and therefore may only be found in systems with sparse neuronal populations and little neurite overlap. One example of ‘pre-specified’ cell-autonomous repulsive interactions are the transmembrane Sema6A and PlexA4 interactions, which have been shown to be required for horizontal cell dendritic self-avoidance in mouse retina [40]. Horizontal cells in PlexA4 or Sema6A knockout retina exhibit abnormal dendrite elaboration with reduced overall coverage of neurites in the outer plexiform layer (OPL). The analysis at single cell level revealed that PlexA4 -/- horizontal cells exhibited reduced dendritic self-avoidance. However, in contrast to the phenotype observed in DSCAM mutant retina, horizontal cell mosaic spacing is normal. Another ligand/receptor pair has been found to mediate self-avoidance in PVD nociceptive neurons in C. elegans www.sciencedirect.com
[41]. In this case UNC-6 (Netrin) and its receptors UNC-40 (DCC) and UNC-5 are required for dendritic self-avoidance. UNC-6 is secreted from ventral cells and UNC-40 captures UNC-6 at the surface of PVD dendrites. Sequestered UNC-6 interacts with UNC-5 in trans to mediate repulsive responses. It is likely that UNC-40 also mediates self-avoidance independent of UNC-6 and UNC-5 because expression of an UNC-40 mutant form, which lacks an intracellular domain cannot rescue the selfavoidance defect in UNC-40 mutants, suggesting that there is an additional UNC-40 ligand for self-avoidance. The comb cell in leech is one of the classical models for the study of neurite self-avoidance. Comb cells are found in the peripheral body wall of leech embryo and extend approximately 70 parallel processes bilaterally without overlapping with one another ([42], Figure 1a). The LARlike receptor tyrosine phosphatase, HmLAR2 is highly expressed in comb cells and the inhibition of HmLAR2 results in crossing-over of sibling processes [42]. In addition, HmLAR2 ectodomain can bind to itself, suggesting that HmLAR2 may have a role in homophilic binding mediated self-avoidance in leech. Moreover, the example of da neurons in Drosophila larvae illustrates that neurites often grow restricted to a 2D space. The Drosophila da neurons establish their dendritic field between an epithelial cell layer and the extracellular matrix (ECM) at the larval body wall. High-resolution confocal imaging, electron microscopy, and markers for enclosed dendrites revealed that loss of integrin a (mew) or b (mys) subunit increases the dendrite enclosure leading to apparent dendrite crossing in class IV da neurons [43,44]. However, these aberrant dendrite crossings are not the result of defective self-avoidance. Moreover, dendrite crossing phenotypes previously observed in mutants of fry, trc, and the Torc2 complex, which were suggested to be signaling components responsible for hetero-neuronal tiling, may not be caused by a lack of homotypic dendrite-dendrite repulsion, but rather due to a lack of growth restriction to 2D space (i.e. lack of direct dendrite-dendrite contact). Because integrin overexpression in these mutant backgrounds rescues dendrite-crossing phenotypes, it is likely that there exists a contact-mediated homotypic repulsion (i.e. tiling) pathway, which has not yet been identified. As evident from these examples, cell surface receptors play a key role in self-avoidance in general. Control of surface receptor expression by transcription factors may in some cases be an important regulatory step in modulating aspects of self-recognition and/or self-avoidance. The transcriptional factor SatB2 is expressed in the layer II/ III pyramidal neurons of the mouse cerebral cortex at early postnatal stages during which their specific dendritic arborization occurs. While SatB2 loss-of-function causes Current Opinion in Neurobiology 2013, 23:983–989
988 Development of neurons and glia
cell-fate specification defects, a precisely timed SatB2 shRNA knock-down in layer II/III neurons by in utero electroporation results in dendrite self-crossing, dendrite fasciculation, and soma clumping between neighboring neurons [45]. As these defects have striking similarity with phenotypes observed in retinal cells of DSCAM/ DSCAML1 mutant mice, the expression of surface receptors such as DSCAM/DSCAML1, clustered Pcdhs or other related receptors in cortical neurons may be regulated by SatB2, revealing a potentially direct link of self-avoidance components to cell type specific differentiation programs of cortical neurons.
Concluding remarks A substantial body of work has firmly established the importance of self-recognition and self-avoidance as an essential mechanism contributing to the wiring of the nervous system. It demonstrated the central role of surface receptors such as Dscam1 in flies and clustered Pcdhs in mammals. However, important questions relating to the molecular principles of receptor expression, receptorreceptor interactions, and receptor signal transduction pathways remain challenges for future work. It is currently entirely unclear how each neuron avoids expressing all isoforms of Dscam1 or clustered Pcdhs and why there is a strong splicing bias even though the canonical model of Dscam1 or Pcdh function implies sufficiency of stochastic expression. What is the molecular mechanism of converting homophilic binding into repulsion signaling? Or what drives and controls oligomeric assembly of Pcdh receptors? Are tetrameric receptor units indeed the main functional unit in vivo? Furthermore, we anticipate that future studies will reveal molecular cross-talk between self-recognition mechanisms and other neuron-specific pathways patterning dendritic or axonal branching.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Kramer AP, Kuwada JY: Formation of the receptive fields of leech mechanosensory neurons during embryonic development. J Neurosci 1983, 3:2474-2486.
2.
Kramer AP, Stent GS: Developmental arborization of sensory neurons in the leech Haementeria ghilianii. II. Experimentally induced variations in the branching pattern. J Neurosci 1985, 5:768-775.
3.
Hughes ME, Bortnick R, Tsubouchi A, Ba¨umer P, Kondo M, Uemura T, Schmucker D: Homophilic Dscam interactions control complex dendrite morphogenesis. Neuron 2007, 54:417-427.
4.
Matthews BJ, Kim ME, Flanagan JJ, Hattori D, Clemens JC, Zipursky SL, Grueber WB: Dendrite self-avoidance is controlled by Dscam. Cell 2007, 129:593-604.
5.
Soba P, Zhu S, Emoto K, Younger S, Yang SJ, Yu HH, Lee T, Jan LY, Jan YN: Drosophila sensory neurons require Dscam for dendritic self-avoidance and proper dendritic field organization. Neuron 2007, 54:403-416.
Current Opinion in Neurobiology 2013, 23:983–989
6.
Zhu H, Hummel T, Clemens JC, Berdnik D, Zipursky SL, Luo L: Dendritic patterning by Dscam and synaptic partner matching in the Drosophila antennal lobe. Nat Neurosci 2006, 9:349-355.
7.
Wang J, Zugates CT, Liang IH, Lee CH, Lee T: Drosophila Dscam is required for divergent segregation of sister branches and suppresses ectopic bifurcation of axons. Neuron 2002, 33:559-571.
8.
Fuerst PG, Koizumi A, Masland RH, Burgess RW: Neurite arborization and mosaic spacing in the mouse retina require DSCAM. Nature 2008, 451:470-474.
9.
Fuerst PG, Bruce F, Tian M, Wei W, Elstrott J, Feller MB, Erskine L, Singer JH, Burgess RW: DSCAM and DSCAML1 function in selfavoidance in multiple cell types in the developing mouse retina. Neuron 2009, 64:484-497.
10. Lefebvre JL, Kostadinov D, Chen WV, Maniatis T, Sanes JR: Protocadherins mediate dendritic self-avoidance in the mammalian nervous system. Nature 2012, 488:517-521. This study shows for the first time that Pcdh-g is required for dendritic self-avoidance in retinal starburst amacrine cells. It also provides evidence that its isoform diversity is important to allow for heteroneuronal interactions. The data strongly support the notion that Dscam in arthropods and Protocadherin in vertebrates serve analogous roles during dendrite patterning. 11. Grueber WB, Sagasti A: Self-avoidance and tiling: mechanisms of dendrite and axon spacing. Cold Spring Harb Perspect Biol 2010, 2:a001750. 12. Schmucker D, Clemens JC, Shu H, Worby CA, Xiao J, Muda M, Dixon JE, Zipursky SL: Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 2000, 101:671-684. 13. Wojtowicz WM, Flanagan JJ, Millard SS, Zipursky SL, Clemens JC: Alternative splicing of Drosophila Dscam generates axon guidance receptors that exhibit isoform-specific homophilic binding. Cell 2004, 118:619-633. 14. Wojtowicz WM, Wu W, Andre I, Qian B, Baker D, Zipursky SL: A vast repertoire of Dscam binding specificities arises from modular interactions of variable Ig domains. Cell 2007, 130:1134-1145. 15. Meijers R, Puettmann-Holgado R, Skiniotis G, Liu JH, Walz T, Wang JH, Schmucker D: Structural basis of Dscam isoform specificity. Nature 2007, 449:487-491. 16. Sawaya MR, Wojtowicz WM, Andre I, Qian B, Wu W, Baker D, Eisenberg D, Zipursky SL: A double S shape provides the structural basis for the extraordinary binding specificity of Dscam isoforms. Cell 2008, 134:1007-1018. 17. Chen BE, Kondo M, Garnier A, Watson FL, Pu¨ettmann-Holgado R, Lamar DR, Schmucker D: The molecular diversity of Dscam is functionally required for neuronal wiring specificity in Drosophila. Cell 2006, 125:607-620. 18. Hattori D, Demir E, Kim HW, Viragh E, Zipursky SL, Dickson BJ: Dscam diversity is essential for neuronal wiring and selfrecognition. Nature 2007, 449:223-227. 19. Hattori D, Chen Y, Matthews BJ, Salwinski L, Sabatti C, Grueber WB, Zipursky SL: Robust discrimination between self and non-self neurites requires thousands of Dscam1 isoforms. Nature 2009, 461:644-648. 20. Zhan XL, Clemens JC, Neves G, Hattori D, Flanagan JJ, Hummel T, Vasconcelos ML, Chess A, Zipursky SL: Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies. Neuron 2004, 43:673-686. 21. Neves G, Zucker J, Daly M, Chess A: Stochastic yet biased expression of multiple Dscam splice variants by individual cells. Nat Genet 2004, 36:240-246. 22. Wu W, Ahlsen G, Baker D, Shapiro L, Zipursky SL: Complementary chimeric isoforms reveal Dscam1 binding specificity in vivo. Neuron 2012, 74:261-268. This paper provides direct evidence for the importance of Dscam1 homophilic binding for self-avoidance in vivo by using complementary pairs of chimeric Dscam isoform that bind to each other but not homophilically. Pairwise but not single expression within the same neuron supports self-avoidance. www.sciencedirect.com
Role of self-avoidance in neuronal wiring Kise and Schmucker 989
23. Sun W, You X, Gogol-Do¨ring A, He H, Kise Y, Sohn M, Chen T, Klebes A, Schmucker D, Chen W: Ultra-deep profiling of alternatively spliced Drosophila Dscam isoforms by circularization-assisted multi-segment sequencing. EMBO J 2013, 32:2029-2038. This paper provides the first quantitative expression analysis of Dscam1 isoform and shows that most of possible isoforms are expressed in vivo. It also demonstrates that there is a strong tissue- and developmental stagespecific selectivity in isoform expression. The ‘how’ and ‘why’ of such a significant splicing bias is currently unknown. 24. Schmucker D, Flanagan JG: Generation of recognition diversity in the nervous system. Neuron 2004, 44:219-222. 25. Zipursky SL, Sanes JR: Chemoaffinity revisited: dscams, protocadherins, and neural circuit assembly. Cell 2010, 143:343-353. 26. Schmucker D, Chen B: Dscam and DSCAM: complex genes in simple animals, complex animals yet simple genes. Genes Dev 2009, 23:147-156. 27. Schreiner D, Weiner JA: Combinatorial homophilic interaction between gamma-protocadherin multimers greatly expands the molecular diversity of cell adhesion. Proc Natl Acad Sci U S A 2010, 107:14893-14898. This breakthrough publication provides for the first time evidence that the recogniton specificity of Pcdhs might be much higher than previously thought. It describes that Pcdhs can form tetramers, which can bind homophilically. Specificity of homophilic binding was found to be controlled by Pcdh isoform composition of the respective tetrameric complexes (see Figure 2b). 28. Weiner JA, Jontes JD: Protocadherins, not prototypical: a complex tale of their interactions, expression, and functions. Front Mol Neurosci 2013, 6:4. 29. Yagi T: Molecular codes for neuronal individuality and cell assembly in the brain. Front Mol Neurosci 2012, 5:45. 30. Hirayama T, Tarusawa E, Yoshimura Y, Galjart N, Yagi T: CTCF is required for neural development and stochastic expression of clustered Pcdh genes in neurons. Cell Rep 2012, 2:345-357. This paper characterizes the stochastic expression of Pcdh genes and identifies CTCF as an important activator of clustered Pcdh expression. 31. Golan-Mashiach M, Grunspan M, Emmanuel R, Gibbs-Bar L, Dikstein R, Shapiro E: Identification of CTCF as a master regulator of the clustered protocadherin genes. Nucleic Acids Res 2012, 40:3378-3391. 32. Monahan K, Rudnick ND, Kehayova PD, Pauli F, Newberry KM, Myers RM, Maniatis T: Role of CCCTC binding factor (CTCF) and cohesin in the generation of single-cell diversity of protocadherin-a gene expression. Proc Natl Acad Sci U S A 2012, 109:9125-9130. 33. Guo Y, Monahan K, Wu H, Gertz J, Varley KE, Li W, Myers RM, Maniatis T, Wu Q: CTCF/cohesin-mediated DNA looping is required for protocadherin a promoter choice. Proc Natl Acad Sci U S A 2012, 109:21081-21086.
www.sciencedirect.com
34. Ribich S, Tasic B, Maniatis T: Identification of long-range regulatory elements in the protocadherin-alpha gene cluster. Proc Natl Acad Sci U S A 2006, 103:19719-19724. 35. Kehayova P, Monahan K, Chen W, Maniatis T: Regulatory elements required for the activation and repression of the protocadherin-alpha gene cluster. Proc Natl Acad Sci U S A 2011, 108:17195-17200. 36. Yokota S, Hirayama T, Hirano K, Kaneko R, Toyoda S, Kawamura Y, Hirabayashi M, Hirabayashi T, Yagi T: Identification of the cluster control region for the protocadherin-beta genes located beyond the protocadherin-gamma cluster. J Biol Chem 2011, 286:31885-31895. 37. Hirano K, Kaneko R, Izawa T, Kawaguchi M, Kitsukawa T, Yagi T: Single-neuron diversity generated by Protocadherin-b cluster in mouse central and peripheral nervous systems. Front Mol Neurosci 2012, 5:90. 38. Kawaguchi M, Toyama T, Kaneko R, Hirayama T, Kawamura Y, Yagi T: Relationship between DNA methylation states and transcription of individual isoforms encoded by the protocadherin-alpha gene cluster. J Biol Chem 2008, 283:12064-12075. 39. Biswas S, Emond MR, Jontes JD: The clustered protocadherins Pcdha and Pcdhg form a heteromeric complex in zebrafish. Neuroscience 2012, 219:280-289. 40. Matsuoka RL, Jiang Z, Samuels IS, Nguyen-Ba-Charvet KT, Sun LO, Peachey NS, Che´dotal A, Yau KW, Kolodkin AL: Guidance-cue control of horizontal cell morphology, lamination, and synapse formation in the mammalian outer retina. J Neurosci 2012, 32:6859-6868. 41. Smith CJ, Watson JD, VanHoven MK, Colo´n-Ramos DA, Miller DM 3rd: Netrin (UNC-6) mediates dendritic self-avoidance. Nat Neurosci 2012, 15:731-737. This study reveals a novel mechanism controlling self-avoidance in C. elegans nociceptice neurons. It demonstrates that the diffusible guidance cue Netrin (Unc-6) and its receptors interact in trans on sister dendrites and thereby triggering repulsion (i.e. self-avoidance) in C. elegans. 42. Baker MW, Macagno ER: The role of a LAR-like receptor tyrosine phosphatase in growth cone collapse and mutualavoidance by sibling processes. J Neurobiol 2000, 44:194-203. 43. Kim ME, Shrestha BR, Blazeski R, Mason CA, Grueber WB: Integrins establish dendrite–substrate relationships that promote dendritic self-avoidance and patterning in Drosophila sensory neurons. Neuron 2012, 73:79-91. 44. Han C, Wang D, Soba P, Zhu S, Lin X, Jan LY, Jan YN: Integrins regulate repulsion-mediated dendritic patterning of Drosophila sensory neurons by restricting dendrites in a 2D space. Neuron 2012, 73:64-78. 45. Zhang L, Song NN, Chen JY, Huang Y, Li H, Ding YQ: Satb2 is required for dendritic arborization and soma spacing in mouse cerebral cortex. Cereb Cortex 2012, 22:1510-1519.
Current Opinion in Neurobiology 2013, 23:983–989