The cis side of juxtacrine signaling: a new role in the development of the nervous system

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1 Review The cis side of juxtacrine signaling: a new role in the development of the nervous system Avraham Yaron 1 and David Sprinzak 2 1 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel 2 Department of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel Cell cell communication by juxtacrine signaling plays a key role in the development of the nervous system, from cell fate determination through axonal guidance to synaptogenesis. Interestingly, several juxtacrine signaling systems exhibit an inhibitory interaction between receptors and ligands in the same cell, termed cis inhibition. These include the Notch, semaphorin and ephrin signaling systems. Here we review the role of cis inhibition in these signaling systems in the development of the nervous system. We compare and contrast cis inhibition mechanisms and discuss their potential cellular function as a threshold-generating mechanism. The prevalence of cis inhibition suggests that these interactions and their functional regulatory roles may serve as a general design principle for juxtacrine signaling-mediated processes during and beyond neurodevelopment. Introduction In juxtacrine signaling systems, signals are transduced through the interaction of receptors and ligands in neighboring cells. Studies over the past several years have provided a growing body of evidence in support of a novel type of interaction, in which ligands inhibit their cognate receptor in the same cell or vice versa. This interaction is termed cis inhibition and has now been observed in three major signaling systems, Notch, ephrin and semaphorin, during neurogenesis and axonal guidance. The interesting fact that a similar regulatory process occurs across very different signaling systems raises several questions: are there common regulatory roles for cis inhibition in fundamentally different neurodevelopmental processes? How does this mode of regulation differ from other modes of regulation such as local translation, trafficking, endocytosis or shedding? Do biochemical mechanisms underlying cis inhibition in the different signaling systems share common features? Here we review the role of cis inhibition in neuronal guidance and neurogenesis, its biochemical basis and the distinctiveness of cis inhibition as a regulatory mechanism. Furthermore, we discuss recent concepts based on mathematical models that suggest that cis inhibition has roles in setting a threshold response to external signals, Corresponding authors: Yaron, A. (avraham.yaron@weizmann.ac.il); Sprinzak, D. (davidsp@post.tau.ac.il). facilitating faster signaling dynamics, and reducing errors in cell decisions. Regulation of neurogenesis by cis inhibition in the Notch signaling pathway The differentiation of cells during neurogenesis is largely controlled by direct cell cell interactions that coordinate the differentiation of adjacent cells into distinct cell types. The key signaling pathway that mediates these interactions in metazoans is the Notch signaling pathway, which was the first juxtacrine signaling system to be identified and is one of the most studied [1]. It is often involved in mediating developmental processes such as lateral inhibition [2,3], boundary formation [4] and asymmetric cell division [5,6]. Notch signaling is required for neurogenesis in both the central nervous system (CNS) and peripheral nervous system (PNS) [7,8] and has recently been implicated in the mediation of neuronal plasticity in the adult nervous system [9 11]. Notch receptors and the Delta, Serrate, LAG-2 (DSL) ligands are type I transmembrane proteins that are highly conserved across metazoans. Upon interaction between a Notch receptor in one cell and a DSL ligand in a neighboring cell, the Notch receptor is cleaved and its intracellular domain translocates to the nucleus, activating downstream transcriptional targets (Figure 1a) [12]. Cis inhibition in the Notch signaling pathway has been demonstrated in diverse organisms ranging from Caenorhabditis elegans (C. elegans) through mammals, both in vitro and in vivo (Figure 1, Table 1) [13,14]. Cis inhibition was initially observed in the context of the dorsal ventral (DV) boundary formation in the Drosophila wing imaginal disk [15 18] and has since been implicated in neurogenesis processes mostly in the context of lateral inhibition in both invertebrates and vertebrates [19 22]. Cell culture experiments in Drosophila, Xenopus and mammals provided additional evidence for cis inhibition and allowed direct probing of its underlying mechanisms [23 28]. During neurogenesis, single cells are often selected for a specific neural fate from an initially equivalent group of cells. Lateral inhibition, a process in which neighboring cells inhibit each other s differentiation, plays a key role in this process [29]. Examples of lateral inhibition are found in sensory organ precursor (SOP) fate determination in /$ see front matter ß 2011 Elsevier Ltd. All rights reserved. doi: /j.tins Trends in Neurosciences, April 2012, Vol. 35, No. 4

2 Notch Ephrin Semaphorin (a) (c) PDZ binding motif (e) GPI anchor p Cis-inhibition Trans-activation EGF repeats DSL DSL domain LNR domain Activating complex (b) DSL X EGF repeats Notch S2 cleavage S3 cleavage Notch ICD Notch EphrinA Cysteine rich (d) EphA,B PDZ binding motif EphA EphrinA p p p Fibronectin type III repeats Kinase domain EphB EphrinB SAM domain EphrinB PSI domain Sema domains (f) Plexin A Plexin A X Semaphorin class 6 PSI domains IPT domains C1 domain C2 domain Semaphorin class 6 X TRENDS in Neurosciences Figure 1. Illustration of trans activation and cis inhibition in the Notch, ephrin and semaphorin juxtacrine signaling pathways. (a) Binding of the DSL ligand to the Notch receptor in the opposing cell leads to its cleavage at two sites (S3 and S2) and translocation of the Notch intracellular domain (ICD) to the nucleus. A protein complex that includes the Notch ICD activates a downstream gene target [1]. (b) Binding of DSL to Notch receptors in the same cell leads to cis inhibition of Notch signaling. (c) Ephrin-As and ephrin-bs bind and activate the tyrosine kinase domain in the EphA and EphB receptors, respectively, and these in turn regulate cytoskeleton dynamics [46,102]. (d) Binding of the same receptors and ligands in cis leads to inhibition of the kinase activity of the Eph receptors. (e) Members of the class 6 semaphorins bind to receptors from class A of the Plexin family, leading to conformational changes in their cytoplasmic domain, through which they regulate cytoskeleton dynamics[66]. (f) As in the two other signaling systems, signaling is inhibited on binding of Sema6 to Plexin-A receptors. Black X symbols denote the consequences of cis inhibition in the respective signaling systems. Schemes presented here show conserved features of the Notch signaling pathway (a,b) and mammalian ephrin and semaphorin pathway (c f). Relevant structural features of the different receptors and ligands are provided. Abbreviations: DSL, Delta, Serrate, LAG-2; EGF epidermal growth factor; GPI, glycosylphosphatidylinositol; IPT, Ig-like, Plexins, transcription factor; LNR, Lin-12 and Notch repeats; ICD, intracellular domain; PDZ, PSD95, DlgA and ZO-1; SAM, sterile alpha motif; PSI, Plexin semaphorin integrin domain. flies [2,21], neurogenesis in zebrafish [22] and neuroepithelial differentiation in the mammalian inner ear [30]. Classically, lateral inhibition is based on a positive feedback mechanism in which Notch signaling downregulates DSL in each of the cells. Now, however, several studies have highlighted the role of cis inhibition during lateral inhibition. An elegant demonstration of the role of cis inhibition during neurogenesis was recently reported in the context of photoreceptor differentiation in the compound eye of Drosophila [31]. Photoreceptor precursors R1 R8 in the Drosophila ommatidia go through an organized sequential process of cell fate determination. Three-way lateral inhibition between the R1, R6 and R7 precursors determines their fate and results in two of the cells adopting an R1/R6 fate and the remaining one an R7 fate (Figure 2a). Two alternative models could, in principle, generate the wildtype differentiation patterns: one based on feedback regulation and one based on cis inhibition. However, careful genetic analysis showed that only the cis inhibition model was able to account for the phenotype observed when Delta was removed from one of the prospective R1/R6 cells 231

3 Table 1. Members of the Notch, ephrin and semaphorin pathways that exhibit cis inhibition a Notch Ephrin Semaphorin Receptors Notch (Drosophila [15 21]) EphA3 [53,54] Plexin-A4 [65] Notch (chick [23]) EphA4 [53,56] Notch (zebrafish [22]) EphA7 [56] Lin-12 (C. elegans [33]) EphB2 [56] Notch1 (Xenopus [24]) EphB3 [56] Notch1-2 (mammals [24,27,28]) Ligands Delta, Serrate (Drosophila [15 21]) Ephrin-A2 [56] Sema6A, Sema6B [65] Delta (chick [23]) Ephrin-A5 [53,54,56] LAG-2 (C. elegans [33]) Ephrin-B2 [56] Delta1 (Xenopus [24]) DeltaD, DeltaA (zebrafish [22]) Dll1, Dll3 (mammals [24,27,28]) a Bold denotes members whose roles in cis inhibition have been demonstrated in the nervous system. Abbreviations: Lin-12, C. elegans Notch homolog; Dll1, Delta-like 1 and Dll3, Delta-like 3 (two mammalian Delta homologs); LAG-2, C. elegans Delta homolog. (Figure 2a). On the basis of these findings, it was suggested that cis inhibition in this system prevents reverse signaling from R7 to R1 and R6, and thus maintains the direction of the final pattern. Whereas cis inhibition usually refers to inhibition of the receptor by its ligand, the reciprocal direction, where the ligand inhibits the receptor, has also been observed in Drosophila and C. elegans [32,33]. A similar behavior has been suggested to occur during zebrafish neurogenesis, in which its DSL ligand, DeltaD is sequestered by Notch receptors and is removed from the plasma membrane [22]. This sequestration has been interpreted as cis inhibition of DeltaD by Notch; however, its functional consequences remain to be determined. Regulation of axon guidance by cis inhibition During development, axons navigate over extremely long distances to reach their final destinations. The navigation is operated through receptors on the growing axons that detect specific guidance cues from the extracellular environment [34]. Among the most important signaling systems in axon guidance are the ephrin and semaphorin families. An increasing body of evidence in recent years shows that cis inhibition between receptors and ligands in both families is important for regulation of the axonal response to these guidance cues (Figure 1, Table 1). Cis inhibition in the ephrin signaling pathway The first evidence for the regulation of axon guidance by cis inhibition emerged from studies on the ephrin family of guidance cues [35 37]. Ephrins are membrane-bound ligands that are divided into two classes: ephrin-as are anchored to the membrane through a glycosylphosphatidylinositol (GPI) tail and ephrin-bs are transmembrane proteins with an intracellular cytoplasmic tail [38]. In many cases ephrins act as repellents, setting barriers that (a) Drosophila ommatidia (b) Mammalian sensory neurons Feedback model wt R1 R6 cis-inhibition model wt R1 R6 Wildtype: sema6a+/+ R7 Mutant R7 Mutant Mutant: sema6a-/- R1 Dl-/- R7 R6 R1 Dl-/- R7 R6 Key: Sema6A expressing cells Control cells TRENDS in Neurosciences Figure 2. Evidence of cis inhibition in the Notch and semaphorin pathways during nervous system development. (a) Cis inhibition in the Drosophila ommatidia [31]. During ommatidium differentiation, a group of three cells differentiates into either R7 fate (blue) or R1/R6 fate (red) in a Notch-dependent manner [31] Two alternative models can account for the differentiation pattern observed: The feedback model (left) in which Delta (red rods) is indirectly downregulated through intracellular feedback of Notch (blue rods) signaling in each cell. In this model, signaling from R7 to R1 and R6 is inhibited since Delta in these cells is turned on earlier. In the alternative cis inhibition model (right), Notch signaling from R7 to R1 and R6 is prevented by cis inhibition of Notch by the high (endogenous) levels of Delta in these cells. The two models can be distinguished by a null Dl / mutant in R1 (bottom panels), which would be expected to produce different patterns. In the feedback model (bottom left), signaling from R6 would still be expected to downregulate Delta in R7, preventing it from signaling to R1. By contrast, in the cis inhibition model, Delta in R7 would not be downregulated, and therefore could signal R1, which then subsequently adopts the R7 fate. Experimentally, the latter mutant behavior (the cis inhibition model) is the one that has been observed [31] (b) Cis inhibition by Sema6A in mammalian sensory neurons. Sensory axons are poorly repelled by exogenous Sema6A in vitro, as demonstrated by growing them on Sema6A-expressing cells. By contrast, Sema6A / sensory neurons were found to exhibit reduced axonal growth compared to wild-type neurons, suggesting that endogenous Sema6A blocks the response, presumably by cis inhibition [65]. 232

4 (a) Wildtype Ephrin overexpression Cleavage of membrane ephrins (i) Stripe assay (ii) (iii) (iv) Temporal Nasal Mouse retina EphrinA2/5 Temporal Nasal Temporal Nasal Temporal Nasal EphA4/5 Key: With exogenous ephrin w/o exogenous ephrin (b) Wildtype EphrinA5 KD EphrinB2 KD Motor axons Limb Key: M-LMC: ephrina5 EphAs EphBs Ventral limb: expressing ephrina5 L-LMC: ephrinb2 Dorsal limb: expressing ephrinb2 TRENDS in Neurosciences Figure 3. Evidence for cis inhibition in the ephrin signaling pathway during mammalian nervous system development. (a) Cis inhibition by ephrins in mouse retinal ganglia cells (RGCs). (i) In the retina, EphA4 and A5 are uniformly expressed by RGCs, whereas ephrina2 and A5 are expressed in a nasal to temporal gradient. (ii) Using an in vitro stripe assay in which RGCs can choose to grow axons on streaks with or without exogenous ephrin, it was shown that neurons from the temporal part of the retina have a strong inhibitory response to exogenous ephrin, whereas neurons from the nasal part do not [103]. (iii) When the same stripe assay was performed using RGCs that overexpress ephrin-a5, rather than wild-type cells, the response of the temporal neurons was similar to that of nasal neurons. (iv) By contrast, cleaving off the membrane ephrin-as sensitized nasal neurons to exogenous ephrin [53]. (b) In vivo evidence of cis inhibition in motor axons of rodents. At the choice point in the limb, axons of the medial lateral motor columns (M-LMCs) grow to the ventral limb part, whereas axons of the lateral LMCs (L-LMCs) grow to the dorsal part. In wild-type axons (left), EphAs in the M-LMCs are cis-inhibited by co-expressed ephrin-a5, making them insensitive to ephrin-a5 expressed in the limb. Similarly, EphBs in L-LMCs are cis-inhibited by coexpressed ephrin-b2, making them insensitive to ephrin-b2 expressed in the limb. Knockdown (KD) of ephrin-a5 in M-LMCs (center) or ephrin-b2 in L-LMCs (right) removes the cis inhibition and leads to guidance errors[56]. prevent axons from traveling into inappropriate targets. In other cases, they serve as adhesion molecules and branching factors [39 41]. The tyrosine kinase receptors EphAs and EphBs transmit ephrin-a and ephrin-b signaling, respectively (Figure 1c,d) [38]. Reverse signaling within the ligand-expressing cell has also been observed. Such signaling is transduced either directly (in the case of ephrin-bs) or by interaction with a co-receptor (as has been observed with ephrin-as) [42 46]. Ephrins have been extensively studied in the retinotectal topographic map, which allows the projection of an image caught by the retina to the CNS [35,36,47 51]. This map is achieved by stereotypic wiring of axons from specific regions in the retina to the corresponding regions in the tectum. During the formation of this map, neurons from the temporal part of the retina project axons to the rostral tectum, whereas neurons from the nasal part project axons to the caudal tectum. It has been shown that the former process is largely controlled by EphA3 receptors, which are highly expressed in retinal ganglion cells (RGCs) on the temporal side [35,52]. Signaling from these receptors, induced by ephrin-a expressed in the caudal tectum, causes repulsion of temporal axons. By contrast, nasal axons are not repelled from the caudal tectum even though they do express EphA receptors. So why do the EphA receptors in the nasal RGCs not respond in a like manner to the same ephrin signal? The lack of response is most likely to be caused by cis inhibition of EphA receptors by ephrinas, which are co-expressed in nasal RGCs [53]. Indeed, overexpression of ephrin-a5 in temporal neurons abrogated their response to membranes extracted from the rostal tectum [53,54], and thus supports such a proposition. Ligand overexpression also caused axon guidance errors in vivo that are consistent with a lack of response to ephrin-as [53,55]. More importantly, it was shown that clipping off membrane ephrins using phosphatidylinositol phospholipase C (which cleaves GPI-anchored proteins) sensitized nasal axons to exogenous recombinant ephrin- As (Figure 3a) [53]. It should be noted that these observations could also be explained by a feedback mechanism that involves reverse signaling via ephrin-as, which would subsequently result in indirect inactivation of EphAs. The retinotectal system is not the only axon guidance system in which cis inhibition involving ephrins has been observed. A recent elegant study focused on the role of ephrins in the guidance of motor axons and the function of cis inhibition in this system [56]. During development, motor axons of the lateral motor column (LMC) select their 233

5 growth trajectory between the dorsal and ventral parts of the limb (Figure 3b) [57,58]. This selection is highly stereotypic and is partly controlled by both EphAs and -Bs in a complementary manner. This model system consists of a specific choice point for the growing axon, allowing accurate dissection of binary guidance decisions in a relatively simple manner. Previous studies have shown that lateral-lmcs (L- LMCs) are repelled to the dorsal part by ventral ephrin- A5, and medial-lmcs (M-LMCs) are repelled to the ventral part by dorsal ephrin-b2 (Figure 3b) [59 62]. However, both types of neuron express both EphAs and EphBs, so it is unclear how the specificity of the axon response is achieved. Overexpression and sirna knockdown of ephrin-as and ephrins-bs were used to show that cis inhibition is required for the proper trajectory selection by motor axons to the dorsal and ventral parts of the limb (Figure 3b) [56]. Interestingly, cis inhibition in this system involves both EphAs and EphBs: Eph-Bs, which are expressed in L-LMCs, are cis-inhibited by ephrin-b2, whereas Eph-As, expressed in M-LMCs, are cis-inhibited by ephrin-a5. This finding is the first in vivo evidence of cis inhibition by the ephrin-bs [63]. The concern that reverse signaling, rather than cis inhibition, may have been the major regulatory process involved was also addressed in this work. Overexpression of an ephrin-a5 mutant, known to be a poor trans-activator, or an ephrin-b2 mutant that cannot signal resulted in the same phenotype as overexpression of wild-type ephrins, a finding that is consistent with the cis inhibition model. Interestingly, the levels of ephrins in cultured neurons shifted the balance between cis inhibition and parallel signaling (both forward and reverse signaling occurred concurrently) [56,64]. This raises the question as to whether such a shift occurs in motor axons once they pass the limb-choice point. This could allow the axons to respond to new signals as they navigate to their final target. Cis inhibition in the semaphorin signaling pathway The second axonal guidance cue in which cis inhibition has been observed is the semaphorin signaling pathway [65]. The highly conserved semaphorin family is comprised of 20 secreted and membrane-bound proteins that are divided into eight classes according to structural homology [66]. The membrane-bound semaphorins signal through the Plexin receptor family (Figure 1e,f), whereas the secreted semaphorins require neuropilins as binding moieties [67 70]. Semaphorins play a significant role in multiple aspects of the wiring of the nervous system during development in both vertebrates and flies. Members of the class 6 transmembrane semaphorins (Sema6A D) act as repulsive guidance cues for various axonal tracts and migrating cells, and govern axonal lamination in the hippocampus and the retina [71 77]. Indirect evidence of cis inhibition by class 6 semaphorins was first observed in the developing CNS. Studies in granule cells suggested that Sema6A might act as a cis inhibitor of its receptor, Plexin-A2, because the absence of Sema6A caused overactivation of the receptor [78]. Studies on laminar termination in the hippocampus suggested a reciprocal action of Plexin-A2 in inhibiting Sema6A. In this system, co-expression of Plexin-A2 with Sema6A proved to be essential for proper invasion of the mossy fibers into the stratum lucidum; in the absence of Plexin-A2, mossy fibers were repelled in a Sema6A-dependent manner [71]. More direct evidence of cis inhibition in the Sema6A signaling pathway was recently reported [65]. It was shown that the lack of response of sensory neurons expressing high levels of Plexin-A4 receptor [65] to its ligand, Sema6A, could be attributed to cis inhibition. Knockout of Sema6A from sensory neurons enhanced their response to exogenous Sema6A (Figure 2b). Additional biochemical support for this interaction was revealed by detailed analysis in a heterologous cell system. It should be noted that unlike Notch and ephrins, cis inhibition by the class-6 semaphorins was not tested in gain-of-function experiments, and conclusive evidence of this regulatory mechanism in vivo is still lacking. Interestingly, sympathetic neurons respond to Sema6A in a Plexin-A4-dependent manner, but do not express Sema6A and 6B [79]. These neurons may serve as a good platform for future studies of cis inhibition by the class-6 semaphorins. The soluble class 3 semaphorins also exhibit modulation of signaling when co-expressed with their receptors; however, it is unclear whether this type of regulation shares the same regulatory properties as cis inhibition by membrane-anchored ligands [80]. Biochemical mechanisms of cis inhibition The biochemical mechanisms that underlie cis inhibition have been studied in the three signaling pathways described above. Strikingly, although not related to each other, they share many features in their cis inhibition mechanisms. Below, we discuss similarities and differences between the three cis inhibition mechanisms. Studies using transgenic animals and transfected cells demonstrated that co-expression of DSL, ephrin, or Sema6A with their cognate receptors prevents the binding of ligands presented in trans [23,24,28,54,65,81,82]. Furthermore, pull-down and co-immunoprecipitation experiments have shown a direct association between the ligands and the receptors in cis, mediated by their extracellular domains [23,24,28,54,65,81,82]. Several studies have examined where this direct association takes place, whether in the secretory pathway or on the cell surface. Although the former option seemed initially appealing, several experiments on cis inhibition of Notch by DSL demonstrated that the amount of surface Notch receptors is not affected by co-expression of the ligands [17,24]. Similarly, studies of ephrins and semaphorins have also pointed to cell-surface cis inhibition. Biochemical analysis did not reveal any change in the membrane expression of Plexin-A4 or EphA3 when they were expressed with or without their ligands [54,65]. This is further supported by the observation that shedding of the membrane-bound ephrins alone was sufficient to abolish cis inhibition [53]. As mentioned earlier, the reciprocal action in which Notch receptors cis-inhibit DSL ligands has also been observed. Interestingly, DSL ligands in this process are removed from the cell surface by Notch-mediated endocytosis [22,32]. By contrast, cis inhibition of Notch by DSL is endocytosis-independent [31]. Are the biochemical mechanisms of these two modes truly independent or somehow 234

6 coupled? Most reports discuss either one direction or the other. Further studies in developmental systems in which both are observed may help to resolve this issue. The possibility of cis inhibition of ligands by receptors has also been discussed for semaphorins and ephrins [56,71]. In particular, such interactions may affect reverse signaling to the ligand-expressing cell. How can ligand receptor interaction in cis and in trans produce opposite effects? And why does the binding of a ligand in cis not result in activation of the receptor? One possible solution is that there are different structural conformations for binding in cis and in trans. This idea is supported by nuclear magnetic resonance experiments that suggest parallel and anti-parallel binding conformations of Notch and its ligands [83]. Furthermore, cis inhibition of Notch also prevents ligand-independent activation by EDTA, suggesting that cis inhibition is not simply caused by competition between cis and trans ligands [25]. Another interesting possibility is that cis binding of DSL to Notch cannot produce the pulling force, believed to be required for activation, in a parallel binding conformation [84]. A possible mechanism for cis inhibition in the ephrin pathway is that the cis ligands simply intercalate between the receptors, reducing their clustering potential, which is required for Eph activation [38]. The idea that different confirmations underlie cis versus trans interactions implies that different protein domains may be involved in each of the interactions. Indeed, a detailed structure function analysis of ephrin-a5 has identified a mutation that abolishes the trans but not the cis interactions, although this does not rule out the possibility that the ligand binding domain in EphA is involved in cis interactions [54]. Similarly, preliminary analysis has proposed different domain requirements for Plexin-A4 Sem6A interaction in trans versus in cis [65]. Interestingly, cis-specific mutations have not yet been reported for Notch or its ligands. Does co-expression of ligands and receptors necessarily result in cis inhibition? Studies in several systems demonstrated that receptors and their ligands are able to segregate into different membrane subdomains. Such segregation may serve as a mechanism for regulation of cis inhibition. As noted above, this was clearly shown for a subset of motor neurons in which EphA and ephrin-a are relegated to different domains on the axon growth cone, mediating parallel signaling [64]. By contrast, in motor neurons or transfected cells in which cis inhibition occurs, EphA and ephrin-a are localized to the same domain [56,82]. Segregation of Notch receptors and their ligands has been observed in neuroepithelial cells [85,86] and in the immune synapse [87], but it remains to be seen if this segregation regulates cis inhibition. Cellular and biological roles of cis inhibition The similarity of cis inhibition across different signaling systems raises questions about the biological role of this mode of regulation. Why should ligands have dual roles as trans activators and cis inhibitors? Some have suggested that cis inhibition of the receptor by its ligand is yet another layer of modulation of the receptor activity; however, it is not clear why such modulation is needed and what its benefits are over other types of regulation. Several recent studies have addressed these questions using a combination of mathematical modeling and experimental work [28,88,89]. One emerging model for cis interaction, termed the mutual inactivation model, assumes Box 1. Modeling of cis inhibition between receptors and ligands Mathematical models of cis inhibition between receptors and ligands provide insights into the role of cis inhibition in various differentiation processes. A relatively simple model suggested recently [28] shows how cis interaction can generate a sharp threshold response to a graded input (Figure I). Consider a simplified model of a single cell expressing both receptors and ligands. We assume that cis interaction occurs through the irreversible formation of an inactive complex described by the following simplified interaction: ½RŠ þ ½LŠ! k ½RLŠ where [L], [R] and [RL] denote the concentration of unbound ligands, unbound receptors and bound receptor ligand complexes, respectively, and k denotes the strength of the interaction. The dynamic equations for the receptor and ligand are then given by d½rš ¼ b dt R g½rš k½rš½lš d½lš ¼ b dt L g½lš k½rš½lš; where b R and b L are the rates of production for receptors and ligands, respectively, and g is the degradation rate for both receptors and ligands (taken to be the same for simplicity). The steady-state solution for [L] and [R] (obtained by solving d½rš dt ¼ 0; d½lš dt ¼ 0) is plotted in Figure I as a function of ligand production b L, for fixed receptor production b R. When cells express more receptors than ligands (Figure I, left-hand side), most of the ligands are bound and there is an excess of free receptors. Conversely, if there are more ligands than receptors (Figure I, right-hand side), then most of the receptors are bound and there is an excess of free ligands. The transition between the two regimes (indicated by the center line in Figure I) can be very sharp, depending on the binding strength (k) of the cis interaction. Thus, strong cis interaction leads to distinct sender and receiver cellular states. Concentration [a.u.] Cell Receiver Cell Sender β L =β R Ligand production, β L [a.u.] Key: Receptor Ligand Free ligand Free receptor Total ligand Total receptor TRENDS in Neurosciences Figure I. Mathematical model of cis inhibition between ligands (red) and receptors (blue) exhibiting a sharp switch from a sender state (right) to a receiver state (left). Inserts depict the cellular state at the limits of lower (left) and higher (right) ligand expression. Abbreviation: a.u., arbitrary units. This figure is loosely adapted from [28]. 235

7 that the ligand and the receptor in the same cell inactivate each other [28]. In this model, a cell becomes either a sender cell, which can send signals but cannot receive them, or a receiver cell, which can receive signals but cannot send them, depending on the relative expression levels of receptor and ligand in that cell. For sufficiently strong cis interaction (i.e. with high binding affinity), these two states are mutually exclusive and the switch between these states is very sharp (Box 1). This sharp switch between sender and receiver states was observed experimentally in a quantitative cell culture assay in which the Notch signaling response to both cis- and trans-delta was mapped [28]. It has been suggested that an analogous molecular sequestration model underlies the sharp switch-like behavior in gene expression regulation [90 92]. The mutual inactivation model has important implications in the coordination of differentiation between neighboring cells [28]. In this model, a small difference between ligand levels (or receptor levels) in neighboring cells can be amplified to a large difference in downstream signaling. One scenario for such a situation is when the ligand is expressed in a gradient across a tissue in which the receptor is expressed uniformly. In this case, the tissue divides into two distinct regions with a sharp boundary between them: one region in which all the cells are senders and the other region in which all the cells are receivers (Figure 4a). It has been suggested that this model underlies the formation of the sharp boundaries defining the wing veins in Drosophila (Figure 4b). One strong prediction of this boundary model is that the position of the boundary depends on the ratio between ligand and receptor expression. Indeed, double heterozygous mutants for Notch and Delta in the wing vein system exhibit wild-type veins, whereas single heterozygous mutation of either Notch or Delta results in a vein phenotype [93]. This ratiometric dependence is a unique feature of the model and it will be interesting to see if it is also applicable to other cis inhibition interactions. How does cis inhibition affect lateral inhibition patterning (Figure 4c,d)? It was recently reported that cis inhibition facilitates robust lateral inhibition patterning during PNS development in Drosophila [88], where patterns of evenly spaced SOPs are formed. Interestingly, certain mutants cause the formation of a dual SOP phenotype, in which two neighboring sensory organs appear instead of one (Figure 4e) [94]. In a combined modeling and experimental study, it was shown that such errors are likely to occur in classical models of lateral inhibition (Figure 4c), which are sensitive to time delays in the intracellular transcriptional feedback underlying lateral inhibition [88]. However, wild-type SOP patterns in vivo seem to show extremely low error rates. The proposed hypothesis was that the cis inhibition mechanism (Figure 4d) circumvents the slower feedback dynamics and reduces the effective time delay in the system. This was supported by a comprehensive theoretical comparison between lateral inhibition models with and without cis inhibition [89] that showed that patterning in a model that includes cis inhibition occurs much faster (Figure 4f) and over a broader range of parameters than a model without cis inhibition. Our new understanding of cis inhibition during neurogenesis also raises the question as to whether cis inhibition exhibits similar behavior during axonal guidance. Guidance cues are largely expressed in gradients extended over long distances; however, axons need to be targeted to precise positions. Could cis inhibition be involved in converting the response to these gradients into an all-ornothing decision? A clue for such threshold behavior can be observed in retinal axons in vitro (Figure 4b). In this system, all axons express EphA4/5 uniformly, but ephrina2 is expressed in a gradient along the nasal temporal axis. The growth response to exogenous ephrins seems to exhibit a threshold behavior. It is yet to be determined whether this behavior is indeed caused by mutual inactivation and whether a sender receiver transition actually occurs. Cis inhibition may also serve as a local sequestration mechanism for guidance cue receptors. The growth cone, which leads axon navigation, is located far from the cell body. Cis inhibition may serve as mechanism to sequester receptors in an inactive state until needed, dispensing with the necessity of energy-demanding synthesis and transport from the soma. Regulation by the cis inhibition itself would need to be controlled dynamically and locally, conditions that could potentially be achieved through the localization of receptors and ligands as discussed above. Future prospects for cis inhibition in the nervous system The importance of cis inhibition as a regulatory mechanism in the nervous system is only starting to emerge. So far it has been shown that cis inhibition controls cell fate specification and axonal guidance. However, taking into account the unique features described above, we can speculate about other possible roles in the nervous system. One unexplored function for cis inhibition is the regulation of synaptogenesis. In view of the importance of ephrins and some transmembrane semaphorins in synapse formation and maintenance, this seems a likely possibility [66,95]. Two principles of cis inhibition threshold response and the ability to reduce errors may come into play. For example, the initial contact between axon and dendrite might be stabilized by adhesive interaction between ephrin-b (on the axon) and its receptor EphB (on the dendrite), followed by a series of signaling events [95 98]. Co-expression of EphB and ephrin-b on either side of the synapse may ensure that adhesive interactions can only occur above a certain threshold. This could enhance the precision and efficiency of connectivity formation. It is well established that changes in synaptic plasticity involve interaction between neighboring synapses. Some synaptic connections are reinforced, whereas others, in a process similar to lateral inhibition, are eliminated. In adult animals, it has been shown that synapse potentiation is regulated by forward and reverse signaling of EphB ephrin-b [99 101]. It would be interesting to see if similar concepts to those observed in lateral inhibition during cell fate decisions also apply to the synaptogenic system. Concluding remarks In summary, we have reviewed the evidence, mechanisms and roles of cis inhibition in neuron development across three juxtacrine signaling systems: Notch, ephrins and 236

8 (a) (b) (i) Drosophila wing veins Ligand expression Unbound receptors and ligands Receptor expression (ii) Mammalian retinal ganglion cells Receivers Senders? Sharp boundary Temporal Nasal (c) Classical lateral inhibition (d) Lateral inhibition+cis-inhibition Cell 1 Cell 2 Cell 1 Cell 2 Nucleus Nucleus Nucleus Nucleus Key: Notch DSL (e) (i) Lateral inhibition +time delay (f) with errors: double SOPs Time (ii) Lateral inhibition +time delay+cis-inhibition w/o errors: single SOPs DSL concentration [a.u.] 10 2 high DSL cells cis-inhibion -cis-inhibition 10-2 low DSL cells Time (hours) TRENDS in Neurosciences Figure 4. Proposed models of potential biological roles of cis inhibition. (a) Cis inhibition between receptor and ligand can facilitate the formation of a sharp boundary in response to a graded input. For example, if the ligand is expressed in a gradient (red, left) across a field of cells and the receptor is expressed uniformly (blue, left), the concentration of free receptors and ligands (right) will split into two regions (senders and receivers), with a sharp boundary at their transition point. This is according to the mutual inactivation model discussed in Box 1. (b) This mechanism is believed to underlie (i) the formation of sharply defined wing veins in Drosophila [15 18]. (ii) Such a mechanism may also explain the sharp transition between inhibited and non-inhibited axonal growth that is observed in mammalian retinal ganglion cells using the stripe assay described in Figure 3. Solid black arrows mark the boundaries observed. (c,d) Alternative models of lateral inhibition circuits with (d) or without (c) cis inhibition. In both cases, Notch signaling in each of the cells inhibits the DSL activity (blunt arrows) in that cell. This type of circuit can generate checkerboard-like patterning from an initially uniform field of cells (see e and f below) such as Drosophila sensory organ precursor (SOP) patterns [2]. (e) A lateral inhibition model of the formation of SOP patterning (SOP, red; regular epithelia, green) that includes a time delay in the feedback mechanism (e.g. owing to transcription and translation) may generate errors, such as (i) double SOP errors. (ii) However, in a model that includes cis inhibition, such errors are reduced. Thus, a model with cis inhibition provides a more robust patterning mechanism [88]. (f) Simulations of the dynamics of patterning into cells expressing high DSL levels (red) and low DSL levels (green) [89] in the two models above (c and d) reveal much faster patterning dynamics for the model that includes cis inhibition. Adapted from [89]. Abbreviations: a.u., arbitrary units; DSL, Delta Serrate LAG-2 ligands. 237

9 semaphorins. Surprisingly, although very different, the three systems share many common features, both in their biochemical mechanisms and in the functional consequences of cis inhibition. Many open questions still remain. What are the detailed biochemical interactions underlying cis inhibition? How is cis inhibition regulated or modulated within each cell and between cells? And finally, what additional roles of cis inhibition can be identified in the nervous system? These questions should be addressed both through the development of quantitative experimental techniques and through novel mathematical modeling approaches. Acknowledgements We would like to thank Oren Schuldiner, Avigdor Eldar and Iftach Nachman for their insightful comments on the manuscript. A Human Frontier Science Program (HFSP) Career Development Award, a grant from Marla Schaefer and the Israel Science Foundation supported research related to this review in the laboratory of A.Y. 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