Distinct cytoplasmic domains in Plexin-A4 mediate diverse responses to semaphorin 3A in developing mammalian neurons.

RESEARCH ARTICLE DEVELOPMENTAL NEUROBIOLOGY

Distinct Cytoplasmic Domains in Plexin-A4 Mediate Diverse Responses to Semaphorin 3A in Developing Mammalian Neurons Guy Mlechkovich,1* Sheng-Shiang Peng,2* Vered Shacham,1 Edward Martinez,2 Irena Gokhman,1 Adi Minis,1 Tracy S. Tran,2† Avraham Yaron1†

INTRODUCTION

During development, neurons extend their axons and dendrites to establish proper connections. This wiring process is largely controlled by extracellular cues that activate receptors on the responding neurons, which initiate signaling cascades that ultimately alter the cytoskeleton, generating diverse cellular responses (1, 2). Multiple studies show that guidance cues are multifunctional and can elicit distinct cellular responses in different neurons (2). However, the mechanistic basis for this multifunctionality is unclear. The semaphorin family is composed of 27 members across eight subclasses, of which the most studied are the class 3 secreted semaphorins. Semaphorins play important roles in a wide range of physiological processes; in the nervous system, they act as strong repellents on various growing axons and as pruning factors for hippocampal neurons, they alter synaptic transmission in the hippocampus and neocortex, and they promote basal-oriented dendritic elaborations in cortical pyramidal neurons (3–8). Receptors for the class 3 semaphorins, including semaphorin 3A (Sema3A), are composed of a heteromeric complex containing a ligand-binding moiety in the neuropilin receptor family and a signaling moiety in the type-A Plexin receptor family. The cytoplasmic domain of type-A Plexins contains two segments with sequence and structural similarity to RAS guanosine triphosphatase (GTPase)–activating proteins (GAPs)—the C1 and C2 domains, which are separated by a Hinge or Rho GTPase binding domain (H/RBD) that connects the two GAP segments (9). These domains interact with numerous proteins, including members of the small GTPase family (3, 10). Previously, we and others have shown both in cells and in vivo that Plexin-A4 (encoded by PlxnA4) is activated by Sema3A and can mediate growth cone collapse and axon repulsion as well as dendrite morphogenesis in different

neuronal populations during development (11–13). Homozygous mice with mutations in Nrp1 that inhibit its ability to bind Sema3A (Nrp1Sema−) or mice carrying null alleles for PlxnA4 or both PlxnA3 and PlxnA4 [PlxnA3/ PlxnA4 double knockout (DKO)] exhibit severe axonal guidance errors from dorsal root ganglion (DRG) sensory neurons during early embryonic ages (12), whereas in later developmental stages, mice carrying the Nrp1Sema− mutation or lacking PlxnA4 display profound basal dendritic defects in deep layer cortical neurons (11), but the mechanistic basis for these disparate functions mediated by Sema3A-Nrp1/Plexin-A4 signaling was not identified. Axonal repulsion induced by Sema3A binding to Nrp1 is mediated by the association and activation of a Plexin receptor with FARP2 [FERM, Rho guanine nucleotide exchange factor (RhoGEF), and pleckstrin domain–containing protein 2] (14). Downstream effectors for Sema3A-mediated dendritic arborization are largely unknown. Sema6A can directly stimulate Plexin-A4 and activate the prebound FARP1 association to influence dendritic length, but not branching, in spinal motor neurons (15). Together, these reports suggest that the nature of the cellular response may be dependent on the specific semaphorin ligand activating different GEFs that associate with the same Plexin-A signaling receptor. However, it remains unclear as to how the same ligand (Sema3A) signaling through the same receptor complex (Nrp1/Plexin-A4) can mediate both growth cone collapse and dendritic growth. Therefore, we conducted structure-function analysis of the Plexin-A4 cytoplasmic domains across three different cellular systems (sensory neurons, cortical neurons, and nonneuronal cells) to investigate the mechanisms that underlie the ability of Sema3A to induce diverse cellular responses through Plexin-A4 signaling. RESULTS

1

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel. 2Department of Biological Sciences, Rutgers University, 195 University Avenue, Newark, NJ 07102, USA. *These authors contributed equally to this work. †Corresponding author. E-mail: [email protected] (A.Y.); [email protected] (T.S.T.)

Plexin-A4 signaling induces distinct responses in DRG and cortical neurons Studies of axonal guidance receptors signaling in vertebrates have been mostly limited to heterologous systems or the use of chimeric receptors.

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Guidance receptor signaling is crucial for neural circuit formation and elicits diverse cellular events in specific neurons. We found that signaling from the guidance cue semaphorin 3A diverged through distinct cytoplasmic domains in its receptor Plexin-A4 to promote disparate cellular behavior in different neuronal cell types. Plexin-A4 has three main cytoplasmic domains—C1, Hinge/RBD, and C2—and interacts with family members of the Rho guanine nucleotide exchange factor FARP proteins. We show that growth cone collapse occurred in Plexin-A4–deficient dorsal root ganglion sensory neurons reconstituted with Plexin-A4 containing either the Hinge/RBD or C2 domain, whereas both of the Hinge/RBD and C1 domains were required for dendritic arborization in cortical neurons. Although knockdown studies indicated that both the collapse and arborization responses involved FARP2, mutations in the cytoplasmic region of Plexin-A4 that reduced its interaction with FARP2 strongly inhibited semaphorin 3A–induced dendritic branching but not growth cone collapse, suggesting that different degrees of interaction are required for the two responses or that developing axons have an indirect path to FARP2 activation. Thus, our study provided insights into the multifunctionality of guidance receptors, in particular showing that the semaphorin 3A signal diverges through specific functions of the modular domains of Plexin-A4.

RESEARCH ARTICLE Sema3A induces growth and branching of basal dendrites in cortical pyramidal neurons in culture (16). Mice harboring a knock-in mutation that expresses an Nrp1 incapable of binding to Sema3A (Nrp1Sema−) exhibit reduced basal dendrite growth and branching in both developing postnatal day 14 and adult layer V cortical neurons (11, 17), and mice deficient in Plexin-A4 phenocopy the basal dendrite arborization defects seen in adult Nrp1Sema− mutant animals (11), indicating that Sema3ANrp1/Plexin-A4 signaling promotes dendrite growth and branching. We tested the ability of exogenous Plexin-A4 variants to restore dendritic branching in PlxnA4−/− neurons in response to Sema3A. We dissociated and cultured E13.5 cortical neurons from Plexin-A4–deficient embryos and transfected the cultures with either a control green fluorescent protein (GFP) construct or Myc-tagged PlxnA4 FL; we split each culture into two cultures, with one culture treated with Sema3A, and stained the cells for Myc and microtubule-associated protein 2 (MAP2) to analyze dendritic morphology. Similar to our published in vivo results (11), PlxnA4−/− neurons transfected with control GFP, regardless of exposure to Sema3A, exhibited a markedly decreased number of dendritic intersections, decreased total dendritic length, and decreased dendritic branching complexity (Fig. 2, A to D). Overexpression of the full-length Plexin-A4 receptor restored dendritic growth, and branching was observed only with the addition of Sema3A (Fig. 2, A to D). These results demonstrate that dendritic elaboration in PlxnA4−/− cortical neurons is mediated by Sema3A-stimulated Nrp1/Plexin-A4 signaling.

Sema3A activates specific Plexin-A4 cytoplasmic domains for growth cone collapse in DRG axons

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Having established that Sema3A signaling through the same holoreceptor complex (Nrp1/Plexin-A4) controls diverse cellular functions in our cultured sensory and cortical neuron systems, we investigated the minimal Plexin-A4 cytoplasmic domain or domains required for either growth cone collapse or dendritic elaboration. We genA erated an array of N-terminal Myc-tagged Plexin-A4 expresPlxnA3/A4 DKO sion constructs containing the full extracellular domain and −Sema3A +Sema3A a cytoplasmic region that had various truncation or deletion domains (Fig. 3A). By several approaches, we detected all B C F G Plexin-A4 variants on the cell surface of transfected COS7 cells (fig. S2, A to C) and in dissociated cortical dendrites (fig. S3). We then tested their ability to rescue the growth cone D E H I collapse response in DRG neurons from PlxnA3/A4 DKO mouse embryos. Strong growth cone collapse was observed in neurons expressing Plexin-A4 constructs containing either B Fig. 1. Plexin-A4 is sufficient to restore the H/RBD or the C2 domains (Fig. 3, B and C), demon*** *** 100 Sema3A-induced growth cone collapse strating that these domains are sufficient to restore sensi−Sema3A 90 in PlxnA3/A4 DKO sensory neurons. tivity to Sema3A-induced signaling. However, expression of +Sema3A 80 (A) Representative immunofluoresthe DC1 construct, which contained both H/RBD and C2 do70 cence images of dissociated DRG mains, did not reveal a synergistic or additive effect on the 60 neurons from E13.5 PlxnA3/A4 DKO degree of growth cone collapse (Fig. 3C), suggesting that eiembryos transfected with Myc-tagged ther H/RBD or C2 is sufficient to mediate Sema3A signaling. 50 PlxnA4 FL or Myc-tagged PlxnA4 DCT In contrast, the activity of the Plexin-A4 construct contain40 and treated with or without AP-Sema3A ing the C1 domain alone was weak in our assay, suggesting 30 for 30 min. The transfected PlxnA4 that this domain has a minimal role in the collapse response 20 construct was detected by an antibody (Fig. 3, B and C). The C1 domain markedly reduced growth 10 against Myc (green), and dendrites cone collapse when coexpressed with either the H/RBD or the 0 CT PlxnA4 FL were visualized by rhodamine-phalloidin C2 domains (Fig. 3, B and C), suggesting that the C1 doto detect actin (red). White arrows, colmain has an inhibitory effect on growth cone collapse relapsed growth cones; white arrowheads, noncollapsed growth cones. Scale bar, 50 mm. sponse to Sema3A. Together, our results indicate that the (B) Quantification of collapsed growth cones described in (A). Data are mean percent of H/RBD or the C2 domains of Plexin-A4 are the minimal docollapsed growth cones ± SE from 400 (PlxnA4 FL) or 100 (DCT) Myc-positive growth mains that mediate Sema3A-induced growth cone collapse in cones obtained from at least three independent experiments; ***P < 0.001 by c2 test. DRG neurons.


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We took advantage of the fact that DRG sensory neurons that lack both Plexin-A3 and Plexin-A4 or cortical neurons that lack Plexin-A4 are completely unresponsive to stimulation by Sema3A (11, 12), and we performed a comparative structure-function analysis of the Plexin-A4 cytoplasmic domains in these neurons. This complementation approach has two major advantages: First, although the transfected Plexin-A4 receptor cytoplasmic domain variants may be overexpressed, the endogenous number of Nrp1 co-receptors in the neurons exerts a physiological limit on the abundance of functional holoreceptor complexes in this system (fig. S1A), meaning that overexpression of Plexin-A4 alone is not sufficient to effect a change, as shown by the overexpression of Plexin-A4 in wild-type neurons (fig. S1B). Second, these functional experiments enable the isolation of the minimal subdomains that are necessary and sufficient to restore the neuronal response to Sema3A. Therefore, if the cellular response is reestablished, then the Plexin-A4 variant, in theory, is part of a functional Sema3A holoreceptor. We first tested whether the reintroduction of Plexin-A4 would restore the growth cone collapse response to Sema3A in developing axons of DRG sensory neurons. DRGs were dissected from embryonic day 13.5 (E13.5) PlxnA3/A4 DKO mice, dissociated into culture, and transfected with wild-type full-length Plexin-A4 (PlxnA4 FL) or Plexin-A4 that contains only the first nine amino acids of the cytoplasmic domain (DCT). Both contained an N-terminal Myc tag to enable identification of the transfected neurons by immunocytochemistry alongside phalloidin to visualize the actin cytoskeleton within the growth cones. Marked growth cone collapse in response to Sema3A was detected in the majority (94%) of DRG neurons that expressed the PlxnA4 FL receptor, whereas no collapse was detected in neurons that expressed the DCT receptor variant compared with untreated cultures (Fig. 1, A and B). These results demonstrate that Sema3A-mediated axonal growth cone collapse can be restored with PlxnA4 FL in embryonic DRG neurons from PlxnA3/A4 DKO mice.

RESEARCH ARTICLE PlxnA4 FL) separately in PlxnA4−/− dissociated cortical neurons isolated from E13.5 PlxnA4−/− embryos, at a develop−Sema3A +Sema3A mental stage where the majority of deep layer pyramidal neurons are born. We analyzed their dendritic morphology by Myc and MAP2 staining after 5 to 6 days in culture and 24 hours of Sema3A exposure (Fig. 4A) to assess dendritic arborization [by Sholl analysis (11)] and the total dendritic length and to calculate the dendritic complexity index (DCI). Compared with overexpression of PlxnA4 FL, overexpression of the DCT, DC1, or DH/RBD constructs was unable to rescue the dendritic arborization defect in the PlxnA4−/− neurons (Fig. 4, B and C). However, PlxnA4−/− neurons transfected with the PlxnA4 DC2 construct exhibited a similar number of dendritic intersections, similar total dendritic length, and a similar DCI to those transfected with PlxnA4 FL (Fig. 4, B and C), suggesting that the combination of the C1 and H/RBD domains is required for fully rescuing the dendritic B 8 P lxnA 4 FL +S em a3A growth and branching defects. To determine if, indeed, both P lxnA 4 FL −S em a3A 7 the C1 and H/RBD are required to mediate Sema3A-induced G F P +S em a3A 6 Nrp1/Plexin-A4–mediated dendrite development, we singly G F P −S em a3A transfected PlxnA4−/− neurons with PlxnA4 C1-, H/RBD-, 5 or C2-only constructs (fig. S4A). Sholl analysis revealed that 4 PlxnA4−/− neurons transfected with any of these three con3 structs (fig. S4A) or the DC1 or DH/RBD construct (Fig. 4A) had a significant decrease in the number of dendritic in2 tersections at 15- to 40-mm distances from the center of the 1 cell body compared with PlxnA4 FL, DCT, or DC2 (fig. S4, 0 B and C), consistent with previous in vivo results (11). Neu10 15 20 25 30 35 40 45 50 55 60 0 rons transfected with the PlxnA4 C2 construct additionally Distance from center of cell body (µm) had a marked decrease in the number of dendritic intersections at 10- to 35-mm distances from the center of cell body C D −S em a3A −S em a3A +S em a3A (fig. S4C), and the dendritic branching complexity and length +S em a3A appeared to be decreased compared with the PlxnA4 C1– or H/RBD–transfected neurons (Fig. 4, D and E). This suggests * * * that the C2 domain, which is important for axonal growth cone collapse, may have an inhibitory effect on dendrite mor* * * phogenesis. These findings indicate that the promotion of dendritic growth and branching by Sema3A requires both the cytoplasmic C1 and H/RBD domains of Plexin-A4, in conPlxnA4 FL GFP GFP PlxnA4 FL trast to growth cone collapse, for which the H/RBD or the Fig. 2. Plexin-A4 rescues dendritic growth and branching defects of PlxnA4−/− cortical C2 domains of Plexin-A4 were sufficient. These compleneurons in culture. (A) Confocal micrographs of dissociated cortical neurons from E13.5 mentary experiments in DRG and cortical neurons also serve PlxnA4−/− embryos transfected with either Myc-tagged PlxnA4 FL or GFP and treated as counter controls for each other, validating the Plexin-A4 with 5 nM AP (−Sema3A) or AP-tagged Sema3A (+Sema3A) for 24 hours and stained cytoplasmic domain deletion mutants as functional receptors. for Myc (green) and MAP2 (red). Scale bar, 20 µm. (B to D) Sholl analysis quanti- For example, the fact that Plexin-A4 lacking the C2 domain was dysfunctional for growth cone collapse in DRGs but was fied dendritic growth and branching in PlxnA4−/− cortical neurons, measuring the number of dendritic intersections (B), the total dendritic length (C), and the DCI (D). still functional for dendritic morphology shows that the change Data are means ± SEM from three to four independent cultures of each construct, in in function was not due to protein misfolding or altered membrane trafficking that deletions may cause. which a total of 40 neurons each were analyzed. Compared with PlxnA4 FL + Sema3A: Moreover, we tested the effects of gain of function of these in (B), P < 0.01 for PlxnA4 FL + AP (*), GFP + Sema3A (†), and GFP + AP (#); in (C) Plexin-A4 mutant receptors on dendritic and axonal growth/ and (D), *P < 0.001; one-way analysis of variance (ANOVA) followed by post hoc branching in wild-type cortical neurons. We found that overTukey test. expression of PlxnA4 FL receptors was similar to control GFP-transfected neurons after Sema3A treatment (fig. S4, D Distinct Plexin-A4 cytoplasmic domains control cortical to G) because of the limitation in the endogenous amounts of Nrp1 in the neuron dendrite growth and branching cell, which restricts the formation of the active Nrp1/Plexin-A4 holorecepTo determine which Plexin-A4 cytoplasmic domain components were tor complex (fig. S1). However, wild-type neurons expressing DCT, DC1, necessary and sufficient for cortical axonal growth and/or dendrite elabo- or DH/RBD exhibited a significant decrease in the number of dendritic ration, we overexpressed the different Plexin-A4 deletion constructs used intersections compared with those expressing PlxnA4 FL or DC2 (Fig. 5A in the axon growth cone collapse assay (DCT, DC1, DH/RDB, DC2, or and fig. S4H). In addition, both the total dendritic length and DCI were PlxnA4–/–

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Fig. 3. Specific Plexin-A4 cytoplasmic signaling domains control axonal growth cone collapse in sensory neurons. 90 (A) Schematic of the PlxnA4 deletion variants. D denotes 80 mutant constructs, either through mutation or deletion of 70 a single domain or through truncation of the entire cyto60 plasmic domain, as depicted. (B) Myc-tagged PlxnA4 50 deletion variants were transfected into PlxnA3/A4 DKO 40 neurons taken from E13.5 DRGs, PlxnA4 DC1 PlxnA4 30 DH/RBD, PlxnA4 DC2, PlxnA4 cytoplasmic domain 20 containing C1 only, H/RBD only, or C2 only. After Sema3A 10 treatment, dissociated neurons were stained for Myc 0 (green) to detect the transfected PlxnA4 variants and PlxnA4 FL CT C1 H/RBD C2 C1 H/RBD C2 rhodamine-phalloidin (red) to visualize actin. White arrows, collapsed growth cones; white arrowheads, noncollapsed growth cones. Scale bar, 50 mm. (C) Quantification of collapsed growth cones. Data represent percent of collapsed growth cones ± SE in 53 to 158 growth cones pooled from at least three experiments per condition; ***P < 0.001 by c2 test; n.s., no significance. +Sema3A

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Fig. 4. Specific Plexin-A4 cytoplasmic domains regulate the dendritic growth and branching of PlxnA4−/− cortical neurons. (A) Myc (green) and MAP2 (red) double staining in PlxnA4−/− cortical neurons dissociated from E13.5 embryos, transfected with Myctagged PlxnA4 variants, and treated with 5 nM Sema3A for 24 hours. White arrows, PlxnA4 FL expression in the cell body and dendrites. Scale bar, 20 µm. (B to E) Quantification of total dendritic length (B and D) and DCI (C and E) in the neurons described in (A). Data are means ± SEM from three to four independent experiments for each construct. *P < 0.001, versus PlxnA4 FL; †P < 0.05, versus PlxnA4 DCT; one-way ANOVA followed by post hoc Tukey test.


significantly reduced in wild-type neurons overexpressing DCT, DC1, or DH/RBD (Fig. 5, B and C). Expression of the H/RBD-alone, but not the C1- or C2-alone, construct had a partially stimulatory effect on dendritic intersection number (fig. S4, I and J), but neither the H/RBD nor the C1 or C2 construct promoted the growth or branching of cortical neurons (Fig. 5, D and E). This result was consistent with our findings in PlxnA4−/− cortical neurons where both C1 and H/RBD domains were necessary for Sema3A-mediated dendrite elaboration in developing cortical neurons. Because it has been shown that Sema3A signaling can affect certain aspects of cortical axon guidance in vivo (18), we wondered if the same PlxnA4 variants might also influence cortical axonal development. First, we confirmed that the PlxnA4 FL, DCT, DC1, DH/RBD, and DC2 constructs are expressed in axonal processes (fig. S5). In contrast to dendritic branching and growth, we observed no significant changes in axon length, branching, or branch length in wild-type cortical neurons overexpressing these PlxnA4 variant constructs (fig. S6). Together, these results further validate and support our complementary culture systems with signaling events observed in vivo (11, 12, 19).

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Previous studies suggest that several members of the Rho family of GTPases, including Rac and Rnd1, are involved downstream in Sema3A signaling and are important L 1 2 T D C L T C C1 RBD C2 4F C RB 4F C H/ nA H/ nA for its induction of axon guidance (23, 24). Plx Plx In particular, the LVP motif in Plexin-A1, Fig. 5. Plexin-A4 cytoplasmic domains control dendritic which is related to short motifs found in elaboration of wild-type cortical neurons. (A) Confocal proteins that bind small GTPases, has been micrographs of representative primary cortical neurons shown to be necessary for receptor activafrom E13.5 embryos transfected with Myc-tagged tion, which in turn stimulates several downPlxnA4 FL and mutants. Overexpression of these stream signaling pathways, resulting in PlxnA4 variants was detected in the cell body and denCOS7 cell collapse (23, 25, 26). However, drites (white arrows) as seen with MAP2 (red) and Myc it remains unclear whether this mechanism (green) labeling. Scale bar, 20 µm. WT, wild type. (B to of Plexin-A1 activation also regulates PlexinE) Quantification of total dendritic length (B and D) and A4 and how it is activated in axons and DCI (C and E). For every construct, n = 3 to 4 independent cultures in which a total of 50 neurons were dendrites in response to Sema3A. Therefore, analyzed. Data are means ± SEM. *P < 0.001, versus PlxnA4 FL; †P < 0.05, versus DCT; one-way ANOVA we investigated whether the corresponding followed by post hoc Tukey test. GTPase binding motif in Plexin-A4, LVS (located within the H/RBD), was required for growth cone collapse and dendritic branching in developing mammalian neurons. Expression of a mutant Analysis of the Plexin-A4 cytoplasmic domain in Plexin-A4, in which the LVS motif was mutated to GGA (DLVS), in senfibroblasts reveals structure and function COS7 cells do not secrete semaphorin or have the Nrp1/Plexin co-receptor, sory neurons from PlxnA3/A4 DKO mice abolished the growth cone colbut previous studies have demonstrated that COS7 cells cotransfected with lapse response to Sema3A (Fig. 6, A and B). In support of previous Nrp1 and type-A Plexin will contract in response to Sema3A (20). Be- studies, these results suggest that the binding of small GTPases to Plexincause Sema3A signaling has emerged in nonneuronal cell types as well, A4 is critical for its stimulation in growth cone collapse activity. Looking the COS7 system may model such cell types (21). However, because at dendritic growth and branching, we found that expression of DLVS in signaling by guidance receptors in neurons and other cell types might wild-type neurons reduced the number of dendritic intersections and debe different (22), we investigated whether the domains required for growth creased the total dendritic length and DCI in response to Sema3A comcone collapse were also necessary for contraction in this nonneuronal pared with those expressing the PlxnA4 FL construct (Fig. 6, C to E). In model. We coexpressed each of the Plexin-A4 variants together with PlxnA4−/− cortical neurons, expression of DLVS was unable to completeNrp1 in COS7 cells and assessed their whole cell contractility in response ly rescue the dendritic defects observed compared with expression of to alkaline phosphatase (AP)–tagged Sema3A. Similar to our observations PlxnA4 FL (Fig. 6F). Similar to the observations for growth cone colin DRG neurons, we found that Plexin-A4 constructs containing either the lapse, the LVS-mutant receptor appeared to retain partial function as seen H/RBD or the C2 domains were sufficient to generate a contractile re- with Sholl analysis (Fig. 6, F and G), but the total dendritic length and sponse to Sema3A (fig. S7). Plexin-A4 variants that contained either just DCI were similar to those of the DCT mutant (Fig. 6H). Together, our the C1 domain on the cytoplasmic side or the C1 and either the H/RBD or results suggest that the structural integrity of the H/RBD and its potential C2 domain reduced the Sema3A-induced contractions in COS7 cells (fig. to bind small GTPases are important for Sema3A-induced Plexin-A4 activaS7). Thus, our results demonstrate that the Plexin-A4 mutant receptors are tion in mediating both growth cone collapse and dendritic elaboration. functional, can form holoreceptor complexes with Nrp1, and respond to Sema3A in a heterologous system. Furthermore, our findings suggest that The KRK motif is required for dendritic branching but not the same domains in the cytoplasmic part of Plexin-A4 required for for growth cone collapse Sema3A-induced axonal collapse in neurons are also sufficient for The conserved triplet of basic amino acids, KRK, in the juxtamembrane Sema3A-induced contraction in nonneuronal cells. Two of our Plexin-A4 region of the Plexin receptor is a binding motif for FERM domains variants, those containing just the C1 domain or DH/RBD, did not transmit (14, 27). Two FERM domain–containing GEFs, FARP2 and FARP1, are *

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Fig. 6. The LVS motif is required for Plexin-A4– mediated growth cone collapse and dendritic branching. (A) PlxnA3/ A4 DKO DRG neurons transfected with PlxnA4 FL and PlxnA4-DLVS (LVS substitution to GGA). White arrows, collapsed growth cones; white arrowheads, noncollapsed growth cones. Scale bar, 50 mm. (B) Data are mean percent of collapsed growth cones ± SE from Myc-positive growth cones in (A) combined from at least three experiments (PlxnA4 FL, 400; PlxnA4-DLVS, 71). ***P < 0.001 by c2 test. (C) Dissociated WT cortical neurons were transfected with PlxnA4 FL, DCT, or DLVS. Scale shown in (F). (D) Sholl analysis of the number of dendritic intersections of WT neurons. (E) Quantification of total dendritic length and DCI of WT neurons. (F) Representative micrographs of PlxnA4−/− primary cortical neurons transfected with PlxnA4 FL, DCT or DLVS. Scale bar, 20 mm. (G) Quantification of dendritic arborization of Plxn−/− neurons. (H) Quantification of total dendritic length and DCI in Plxn−/− cortical neurons. Data are means ± SEM from four to five independent cultures in which a total 35 neurons were analyzed per PlxnA4 variant. In (D) and (G), *P < 0.01 for PlxnA4 FL versus DLVS; †P < 0.01 for DCT versus DLVS. In (E) and (H), ***P < 0.001 versus PlxnA4 FL; oneway ANOVA followed by post hoc Tukey test.

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RESEARCH ARTICLE implicated in semaphorin-induced axon repulsion and spinal motor neuron dendritic growth, respectively (14, 15). Therefore, we tested the ability of a Plexin-A4 KRK-mutant variant, where the basic KRK motif is mutated to AAA (DKRK), to restore the growth cone collapse and dendritic branching responses in our neuronal systems. Expression of the DKRK construct restored Sema3A-induced growth cone collapse in sensory neurons from PlxnA3/A4 DKO mice (Fig. 7, A and B). However, compared with either PlxnA4 FL or DCT, expression of DKRK in wild-type cortical neurons blocked the stimulatory effect of Sema3A on dendritic growth and branching (Fig. 7, C to E), and it failed to rescue the dendritic defects in PlxnA4−/− cortical neurons (Fig. 7, F to H). Overall, our data indicate that the KRK motif is essential for dendritic branching but is dispensable for growth cone collapse, supporting the idea that distinct elements within Plexin-A4 signaling domains are required for the two processes.

FARP1 and FARP2 associate with Plexin-A4

FARP2 is required for both dendritic arborization and growth cone collapse in response to Sema3A

Because our findings thus far showed that the DKRK construct can mediate growth collapse in DRGs after Sema3A treatment, we wanted to investigate the role of FARP1 and FARP2 in cortical and DRG neurons. A publicly available database showed by in situ hybridization that whereas FARP2 mRNA exhibited broad localization in the developing mouse embryo, FARP1 mRNA showed a less dispersed expression pattern (fig. S9, A to D) but appeared to be present in the same areas. We were unable to reliably detect FARP2 protein, or Plexin-A4 protein, with currently available commercial antibodies in the colocalization assays, but the FARP1 protein abundance in the cortex was in agreement with the mRNA expression (fig. S9, E to H). Nevertheless, both FARP1 and FARP2 appear to be expressed at the transcript level in DRGs and the cortex in a profile that appears similar to that published for PlxnA4 mRNA (28, 29), suggesting that both FARP1 and FARP2 may play a role downstream of Plexin-A4 signaling in DRG and cortical neurons; however, the colocalization remains to be investigated in the same tissue. We tested the direct requirement of FARP1 and FARP2 for growth cone collapse and dendritic branching in our neuronal systems (Fig. 9A). Wildtype DRG neurons were cotransfected with a GFP plasmid and either control small interfering RNA (siRNA) or siRNA against FARP2 or FARP1 (fig. S10, A and B; siRNA specificity confirmed in fig. S10C) and stimulated with Sema3A. Consistent with a suggested role of FARP2 in Sema3A-Nrp1/

DISCUSSION

Various guidance cues, including semaphorins, use several receptor complexes for mediating different responses during neural development (30–35), and auxiliary receptors may also modify axonal responses (36, 37). Thus, one mechanism of how the same guidance cue can exert both attraction and repulsion is through the diversity of different receptor complexes in specific populations of cells. However, it is not clear how the same ligand/receptor pair can elicit different cellular processes (11, 38–40). Recently, the Rac GTPase-activating protein b2-Chimaerin has been shown to be required and to selectively bind to the Sema3F receptor Nrp2, leading to hippocampal axon pruning, but not axonal repulsion and spine morphogenesis (41). It seems unlikely that solely the presence or absence of an intracellular molecule dictates which signaling cascade, and thus physiological outcome, the receptor activates, because many of the identified intracellular effectors that are ubiquitously present in the cell cytoplasm can be found in both axons and dendrites and play important roles in various physiological processes. Moreover, manipulating the abundance of cyclic nucleotides or interfering with kinase activities can convert attraction to repulsion and vice versa for multiple guidance cues (42, 43). Here, we demonstrated across three different cell types that the instructive signal mediating Sema3A-Nrp1/PlxnA4–induced growth cone collapse or cellular repulsion versus dendritic arborization is embedded in distinct cytoplasmic domains. Our results indicate that Sema3A can activate PlexinA4 in at least two different ways: (i) through signaling by the H/RBD or C2 domains to mediate axonal repulsion, growth cone collapse, or cell contraction, which requires cytoskeleton rearrangement, or (ii) through signaling by both the H/RBD and C1 domains to mediate dendritic branching and elaboration, which requires cytoskeleton synthesis and thus possibly hindering growth cone collapse. It will be of interest and importance for future studies to determine how the differential mechanisms are regulated in specific neurons and whether a default pathway dominates over the other. The consensus motif LVS in the H/RBD, which interacts with several Rho GTPases (23, 25, 44), was necessary for both growth cone collapse and dendritic branching, in the context of the full-length receptor. This suggests that the transduction of Plexin-A4 signaling by small GTPases and guanosine triphosphate–guanosine diphosphate exchange may be a common mechanism for both growth cone collapse and dendritic branching. However, our deletion analysis argues that it is not absolutely required for growth cone collapse because the C2 domain was sufficient for this activity. Therefore, the LVS motif may be required for conformational change in the receptor cytoplasmic domain after activation of the receptor (9), which is bypassed by our deletion variants. Two FERM domain–containing GEF proteins, FARP1 and FARP2, bind Plexin-A4 and Plexin-A1, respectively (14, 15). Our studies demonstrated that these two related GEF proteins used different modes of interaction


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The differential requirement of the KRK motif for dendritic branching versus growth cone collapse prompted us to further investigate the role of this motif in the interaction of Plexin-A4 with its potential immediate downstream effectors, FARP1 and FARP2. In transfected human embryonic kidney (HEK) 293T cells, hemagglutinin (HA)–tagged FARP2 coimmunoprecipitated with the full-length Plexin-A4 construct (Fig. 8A), but its interaction with DKRK was comparably decreased by about half (Fig. 8B), suggesting that the association of FARP2 with Plexin-A4 may only be partially dependent on the binding of the FERM domain to the KRK motif. In contrast, HA-tagged FARP1 coimmunoprecipitated with both fulllength and KRK-mutant Plexin-A4 constructs similarly (Fig. 8B). However, deletion of most of the cytoplasmic domain in combination with mutations in the KRK motif (DCT + DKRK) or DCT in combination with a deletion of nine amino acids including the KRK located C-terminal to the transmembrane region (DCT + DTMC9) decreased their interaction to near or below detection by Western blot (Fig. 8C). These experiments suggest that the two GEFs, FARP1 and FARP2, associate with the FERM domain with the KRK motif in Plexin-A4 to different extents, and may possibly interact with additional regions of the Plexin-A4 cytoplasmic domain.

Plexin-A1 signaling for axonal repulsion (14), we detected reduced growth cone collapse in the GFP-positive FARP2-deficient axons compared with controls (Fig. 9, A and B), but not in FARP1-deficient axons (Fig. 9, A and C). These data suggest that FARP2, but not FARP1, is required for growth cone collapse in response to Sema3A. Next, we looked at the effect of FARP1 and FARP2 knockdown on dendritic elaboration in Sema3A-treated cortical neurons. We observed reduced dendritic complexity, length, and number of intersections only in FARP2-deficient neurons as compared with control knockdown or FARP1deficient neurons (Fig. 10, A, B, and C), similar to that of untreated neurons. Overall, our results suggest that FARP2 is a common effector of Sema3APlexin signaling, but that FARP2 does not appear to be the mechanism that specifies the distinct cellular responses.

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Fig. 7. The KRK motif is required for Plexin-A4– mediated dendritic arborization. (A) PlxnA3/A4 DKO sensory neurons transfected with PlxnA4 FL and PlxnA4-DKRK (KRK substitution to AAA). White arrows, collapsed growth cones; white arrowheads, noncollapsed. Scale bar, 50 mm. (B) Data are mean percent of collapsed growth cones ± SE from Mycpositive growth cones combined from at least three experiments (PlxnA4 FL, 400; PlxnA4-DKRK, 64); ***P < 0.001 by c2 test. (C) Dissociated WT cortical neurons were transfected with PlxnA4 FL, DCT or DKRK. Scale shown in (F). (D) Sholl analysis of dendritic intersections from WT cortical neurons. (E) Quantification of total dendritic length and DCI from WT cortical neurons. (F) PlxnA4−/− primary cortical neurons were transfected with PlxnA4 FL, DCT or DKRK. Scale bar, 20 mm. (G) Sholl analysis of dendritic intersections in PlxnA4−/− cortical neurons. (H) Quantification of total dendritic length and DCI in PlxnA4−/− cortical neurons, visualized by staining for MAP2 (red) and Myc (green). Data are means ± SEM from four to five independent cultures in which a total of 35 neurons were analyzed. In (D) and (G), *P < 0.01 for PlxnA4 FL versus DKRK; †P < 0.01 for DCT versus DKRK. In (E) and (H), ***P < 0.001 versus PlxnA4 FL by oneway ANOVA followed by post hoc Tukey test.

RESEARCH ARTICLE with Plexin-A4. The binding of FARP2 was largely but not completely dependent on the KRK motif, suggesting that FARP2 might bind to additional sequences in the Plexin-A4 cytoplasmic domain, which are not yet defined. In contrast, the binding of FARP1 to Plexin-A4 was not affected by mutations in the KRK motif, but truncation of most of the Plexin-A4 cytoplasmic domain did reveal an interaction between FARP1 and this motif. The reason for this differential interaction among the two

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FARP GEFs is not clear. One possible explanation might be the requirement of FARP2 to dissociate from Plexin-A4 upon activation, and therefore, tight binding is not as needed in comparison to FARP1, which functions while it is still bound to the receptor (14). In addition, the KRK motif is adjacent to the TM domain and is hence present in all the Plexin-A4 variants we examined, including DCT, which is inactive. Therefore, the recruitment of FARP2 to DCT through this motif was not sufficient to induce either growth cone collapse or dendritic branching, suggesting that FARP2 is acting as a perA C missive factor for receptor activation and downstream HA-FARP2 HA-FARP1 signaling through the distinct cytoplasmic domain(s) _ _ _ _ PlxnA4 FL + + + + Myc-PlxnA4 required for generating the cellular response. This _ + _ _ _ + _ _ KRK + + + + + + HA-FARP1 may explain the ability of the PlxnA4 DKRK, in Blot: IP Blot: IP HA msIgG HA msIgG HA msIgG which there is residual binding of FARP2, to bind Myc Myc enough receptors to transduce growth cone collapse Co-IP Co-IP signaling. In contrast, during prolonged or repeated HA HA activation, such as what occurs during axonal repulIgG IgG sion or dendritic growth and branching, this residual Myc binding was insufficient. We hypothesized that even Myc Input if reduced association of FARP2 with the PlxnA4 Input HA HA KRK mutant could mediate the fast growth cone collapse (which can occur within minutes), it is inadequate * 0.4 B Fig. 8. Differential modes of interaction of FARP1 and for the slow dendrite growing/branching or axonal re0.3 FARP2 with Plexin-A4. (A and B) Western blot (A) and pulsion and retraction (which requires up to hours and quantification (B) of coimmunoprecipitation (co-IP) be- days), which follows in vivo developmental time scales. 0.2 tween Myc-tagged PlxnA4 FL or DKRK and HA-tagged Furthermore, the degree of association of FARP2 with 0.1 FARP1 or FARP2 in HEK293T cells. Blot is representaPlexin-A4 may also contribute to the downstream 0.0 tive of and data are means ± SEM from at least four ex- signaling mechanisms to additionally regulate the outperiments. *P < 0.05, two-tailed Student’s t test. (C) Western come of Sema3A-activated Plexin-A4 in various neublot of coimmunoprecipitation between Myc-tagged rons. Alternatively, it might well be that FARP2 is PlxnA4 constructs with HA-tagged FARP1 in HEK293T active in DRG axons in a Plexin-A4–independent manner, and although it is required for growth cone cells. Blot is representative of three experiments. collapse, its direct association with the receptor is not needed. A −Sema3A +Sema3A Our findings that FARP1 did not play a role in Plexin-A4–dependent dendrite growth and branching in cortical neurons differed from that reported in spinal motor neurons (15), and FARP1 was also shown to control excitatory synapse number and spine morphology in hippocampal neurons by activating the GTPase Rac1 downstream of the synaptogenic adhesion molecule SynCAM 1 (45). Thus, it remains to be determined whether FARP1 plays a role in dendritic differentiation in other neuronal populations or B −Sema3A C −Sema3A Fig. 9. FARP2 is required for growth at later developmental stages such as during +Sema3A +Sema3A cone collapse. (A) Sensory neuron aggredendritic remodeling. Furthermore, we are 100 *** gates transfected with siRNA-Control + unable to rule out that there could yet be un90 80 GFP, siFARP2 + GFP, and siFARP1 + known FERM-containing proteins inter70 GFP were treated with Sema3A for acting with Plexin-A4 through the KRK 60 30 min, fixed, and stained with phalloidin motif that may be important for Sema3A50 40 for actin staining (red) or with anti-GFP induced dendritic growth and branching in 30 (green). Arrows, collapsed growth cortical neurons. 20 10 cones; arrowheads, preserved growth Although some studies are beginning to 0 cones. Scale bar, 50 mm. (B and C) identify critical molecules in specifying densiC siFARP1 siC siFARP2 Quantification of the growth cone coldritic morphology of pyramidal neurons lapse in cells deficient in (B) FARP2 or (C) FARP1. Data are mean percent collapsed growth cones ± in vivo (46, 47), little is known about the SD from GFP-positive growth cones from at least three experiments [(B) siC, 123; siFARP2, 156; (C) siC, molecular cues that specify basal dendritic 102; siFARP1, 127]; ***P < 0.001 by c2 test. morphology in developing cortical neurons.

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Fig. 10. FARP2 is required for dendrite arborization. (A) Cortical neurons were dissected form E13.5 WT embryo and cotransfected with GFP and scramble control siRNA, FARP1 siRNA, or FARP2 siRNA. All neurons analyzed were labeled with MAP2 (red) and co-imaged with GFP (green). Scale bar, 20 µm. (B) Dendritic intersections were quantified using Sholl analysis. (C) Quantification of total dendritic length and DCI for control and knockdown neurons. Data are means ± SEM from three independent cultures and a total of 35 neurons for each condition. In (B), *P < 0.05; in (C), *P < 0.001; one-way ANOVA followed by post hoc Tukey test.

Thus far, the main body of work showing how neurons acquire their specific dendritic morphology comes from genetic studies in Drosophila melanogaster, which reveal a complex network of transcription factors and cell surface receptor ligands that regulate dendritic arborization in fly neurons (48). Molecular mechanisms and intracellular signaling cascades that control dendritic morphogenesis in the mammalian central nervous system are far less understood. A previous study in mouse cortical neurons demonstrated that signaling activated by the interaction of Slit with its receptor Robo influences both apical and basal dendrite branching in outer layer pyramidal neurons (49). In addition, Sema3A promotes the growth and branching of cortical neuron dendrites in brain slices (16), and in support of the results in cultured cells, both Nrp1Sema− knock-in and PlxnA4 null mutants exhibit reduced dendrite arborization in layer V pyramidal neurons (11, 17). Together, these previous findings suggest that guidance cues and their receptors, first identified to influence axon guidance, can later signal to promote cortical dendrite growth and branching. Our results corroborate with these previous studies and demonstrate how downstream Sema3A signaling promotes dendrite elaboration through the distinct activation of both the H/RBD and C1 domain of the PlexinA4 receptor. In contrast to the cellular or axon collapse effect, H/RBD and C1 appear to work synergistically to promote dendritic growth and branching in cortical neurons. This raised the question: Do downstream interactors of the H/RBD and C1 domain also work together to support dendrite morphogenesis? Previously, differential abundances of cyclic nucleotides after Sema3A treatment were found to influence initial neurite polarization in rat cortical neurons (50, 51). More recently, the intracellular TaoK2–Jun N-terminal kinase (JNK) signaling pathway has been shown to be downstream of Sema3A-mediated basal dendrite

morphogenesis in cortical pyramidal neurons (52). Nrp1 appears to directly interact with TaoK2, and Sema3A treatment can activate both TaoK2 and JNK in both axons and dendrites of cultured cortical neurons. However, whether Plexin-A4 also feeds into, or signals independently but parallel to, this intracellular cascade remains to be determined. In summary, our study demonstrates the modular nature of guidance cue receptors in regulating growth cone collapse and dendrite morphology in cultures of two different neuronal cell types. Future studies with genetically modified animals will enable further testing and validation of the roles of distinct Plexin-A4 signaling domains in the developing nervous system and possibly other cellular processes (53).

MATERIALS AND METHODS

Plexin-A4 constructs

All the Plexin-A4 variants were constructed on the basis of the wild-type mouse PlexinA4, Myc-tagged on the N terminus, and driven by the chicken b-actin promoter. Domains were annotated as C1: 1267L-1497L, H/RBD: 1498V-1653N, and C2: 1654H-1890S. The Plexin-A4 variants were designed as follows (numbers correspond to amino acids in the full-length Plexin-A4): PlxnA4 FL: 1–1890; DCT: 1–1266; DCT(KRK): 1–1266 (KRK-AAA); DCT-TM: 1–1257; DC1: 1–1266 + 1498–1890; DH/RBD: 11497 + 1654–1890; DC2: 1–1653; C1: 1–1497; H/RBD: 1–1266 + 1498–1653; C2: 1–1266 + 1654–1890.

Immunocytochemistry and Western blotting Primary DRG and cortical neurons and COS7 cells were transfected with PlxnA4 FL, a cytoplasmic domain deletion variant, or a GFP control construct; grown on glass coverslips; and fixed for immunocytochemistry as previously described (11). Primary antibodies used for immunofluorescence staining were against monoclonal mouse Myc (D9 hybridoma, Weizmann Institute, 1:2000 dilution; or 9E10, Sigma, 1:1000), chicken MAP2 (1:1000, Millipore), polyclonal rabbit GFP (1:500, Millipore), mouse monoclonal SMI312 (1:500, Covance), and polyclonal rabbit FARP1 (Santa Cruz Biotechnology, 1:500). Secondary fluorescently conjugated antibodies were Alexa Fluor 488 donkey anti-mouse immunoglobulin G (IgG) (1:500, Jackson ImmunoResearch Laboratories), Alexa Fluor 546 goat anti-chicken IgG (1:500, Life Technologies), and Cy3 donkey anti-rabbit (1:800, Jackson ImmunoResearch Laboratories). After immunofluorescence labeling, coverslips were affixed to microscope slides with Vectashield fluorescence mounting medium (Vector Laboratories Inc.), and confocal stack images were taken with a Zeiss AxioExaminer Z1, Yokogawa spinning disc microscope. For Western blots, the antibodies used were against Myc (D9 hybridoma, Weizmann Institute, 1:2000), actin (MP Biomedicals, 1:5000), HA (Santa Cruz Biotechnology, 1:2000), FARP1 (Santa Cruz Biotechnology, 1:250), FARP2 (Santa Cruz Biotechnology, 1:250), and Plexin-A4 (polyclonal rabbit, Abcam, 1:100). Immunoblots were developed with horseradish peroxidase–linked anti-mouse IgG (GE Healthcare, 1:2000),


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siRNA FARP1

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RESEARCH ARTICLE followed by detection with chemiluminescence. For FARP1 staining of DRG neurons, 4-mm paraffin sections of E13.5 embryos were stained with antiFARP1 (1:100) as described (54). For FARP1 staining of the cortex, E17.5 and P0/P1 coronal brain sections were cut at 20-mm thickness and stained with anti-FARP1 (1:500).

Myc-positive growth cones with few or no filopodia were considered collapsed, and the percentage was calculated. For each of the Plexin-A4 construct variants, experiments were performed at least three times.

Mouse strains For timed pregnancies, embryonic day 0.5 (E0.5) was considered to be noon on the day the vaginal plug was observed. In all cases, genotyping was performed using polymerase chain reaction and DNA samples were generated from mouse ear or tail tissue biopsies. Pregnant dams were exposed to compressed carbon dioxide and sacrificed by cervical dislocation, and the embryos were removed by cesarean section. PlxnA4 KO and PlxnA3/A4 DKO mice were previously described (12). The Institutional Animal Care and Use Committees of the Weizmann Institute of Science and Rutgers University collectively approved the animal use protocols.

HEK293T cells were seeded on 10-cm plates 1 day before transfection with 8 mg of DNA of Plexin-A4 constructs using jetPEI reagent (Polyplus transfection). Forty-eight hours after transfection, cells were washed and lysed using radioimmunoprecipitation assay (RIPA) lysis buffer, and protein expression was evaluated by Western blot analysis. For surface staining of the different Plexin variants, COS7 cells were seeded on six-well plates 1 day before transfection with 3 mg of DNA of Plexin-A4 constructs using jetPEI reagent (Polyplus transfection). Forty-eight hours after transfection, cells were fixed for 60 min with 4% formaldehyde and stained with anti-Myc and 4′,6-diamidino-2-phenylindole, without permeabilization.

Production of AP-Sema3A

Cell surface biotinylation

HEK293T cells grown in Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM-F12) supplemented with 10% fetal bovine serum (Sigma) and 1% penicillin-streptomycin (Life Technologies) were transfected using either Lipofectamine (Life Technologies) or jetPEI reagent (Polyplus transfection) with an AP-Sema3A expression vector (55). Twenty-four hours after transfection, medium was replaced with serum-free DMEM-F12, which was collected after an additional 48 hours. The conditioned medium was concentrated using Vivaspin 6 tubes (GE Healthcare). AP-Sema3A concentration was determined by AP activity assay using AP substrate buffer para-nitrophenyl phosphate (Sigma). For each batch of Sema3A, the minimum concentration required for complete collapse of wild-type DRGs in 30 min was determined experimentally through dose-dependent collapse response assays on wild-type DRGs before use on the transfected neurons.

Cell surface biotinylation was done on transfected COS7 cells using the cell surface biotinylation and isolation kit (Pierce).

DRGs from E13.5 mice were aseptically removed and pelleted in Hanks’ balanced salt solution (Biological Industries) for 10 min and dissociated with 5% trypsin at 37°C for 5 min. The trypsin was neutralized with 10 ml of L15 medium supplemented with 5% fetal calf serum. Cells were centrifuged at 600g, 21°C, for 4 min and resuspended in neurobasal medium supplemented with B-27, glutamine, and neuronal growth factor (NGF; 12.5 ng/ml). Dissociated neurons were counted, and for each transfection reaction, 100,000 DRGs were centrifuged at 600g, 21°C, for 2 min and washed twice with phosphate-buffered saline without calcium and magnesium (Sigma). Cells were transfected using the Neon transfection system (Invitrogen) at 1400 V, 20-ms width, and 1 pulse. After transfection, cells were cultured on 13-mm poly-D-lysine (PDL)/laminin–coated coverslips in 24-well plates in neurobasal medium with NGF (12.5 ng/ml). Primary cortical neurons were dissected from E13.5 wild-type or PlxnA4−/− mice as previously described (11). Dissociated neurons were transfected with various Myc-tagged Plexin-A4 constructs using Nucleofector for mouse primary neurons (Lonza/Amaxa), plated onto glass coverslips, and cultured with neurobasal growth medium supplemented with B-27, penicillinstreptomycin, and Glutamax for 6 to 7 days. Cortical neurons were treated with 5 nM AP-Sema3A or AP for 24 hours, fixed in 4% paraformaldehyde at 4°C for 15 min, and processed for immunocytochemistry.

Growth cone collapse assay Cells were stimulated with 30 nM Sema3A for 30 min. After stimulation, neurons were fixed with 4% paraformaldehyde, and growth cones were visualized by staining with antibodies for Myc and rhodamine-phalloidin.

COS7 collapse assay COS7 cells were seeded onto gelatin-coated 12-well plates at about 3 × 104 cells per well 1 day before transfection with Plexin-A4 constructs and Nrp1 at a 7:1 ratio using jetPEI reagent. Twenty-four hours after transfection, cells were stimulated with 100 nM AP-Sema3A at 37°C or at room temperature for 90 min. Cells expressing Nrp1-bound AP-Sema3A were stained with the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate (Roche). For each of the Plexin-A4 variants, the experiment was repeated three times, and more than 200 cells were counted in each experiment.

siRNA transfection HEK293T cells were seeded on six-well plates 1 day before transfection with siRNA (80 nM) and a plasmid expressing HA-tagged FARP1 or FARP2 using DharmaFECT (Thermo Scientific). Forty-eight hours later, cells were washed and lysed using RIPA lysis buffer, and protein abundance was evaluated by Western blot. Dissociated E13.5 mouse DRG sensory neurons were transfected with a GFP-expressing plasmid and siRNA (Dharmacon) using the Neon transfection system (Invitrogen) at 1400 V, 20-ms width, and 1 pulse. After transfection, the neurons were centrifuged at 2400 rpm, 4 min, at 21°C; resuspended in 9 ml of growth medium [neurobasal medium with NGF (12.5 ng/ml)]; and transferred to 15 ml of growth medium as a hanging drop overnight to form cell aggregates. Aggregates were cut into four pieces and plated on PDL/laminin-coated glass slides. Forty-eight hours after transfection, DRG aggregates were stimulated with Sema3A, fixed, and stained with phalloidin, and GFP-positive growth cones were counted. E13.5 cortical neurons were cotransfected with a GFPexpressing plasmid and siRNA using the Nucleofector kit (Lonza/Amaxa) as described for mouse primary neurons. Transfected neurons were plated on PDL-coated coverslips and cultured with neurobasal medium for 6 days. Neurons were treated with 5 mM AP-Sema3A for 24 hours, then fixed in 4% PFA for 15 min, and processed for immunocytochemistry.

Coimmunoprecipitation HEK293T cells were cotransfected with plasmids expressing HA-tagged FARP1 or FARP2 and Myc-tagged Plexin-A4 full length or KRK mutant by using Lipofectamine 2000 (Invitrogen) and lysed after 48 hours with 20 mM tris-HCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, and 150 mM NaCl at pH 7.4 with proteinase inhibitor (Roche). Protein lysates were incubated with primary antibody against HA (Santa Cruz Biotechnology,


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Primary neuron cultures and transfections

Transfection of cell lines and preparation for immunoblotting

RESEARCH ARTICLE 1:1000) and protein G–Sepharose beads (GE Healthcare) overnight at 4°C. The beads were then washed with lysis buffer and boiled in sample buffer for Western blot analysis.

Dendritic morphology analyses

Total axonal length analysis and Plexin-A4 variant expression E13.5 mouse cortical neurons were dissociated and sparsely cultured for 6 days, followed by a short treatment (1.5 hour) or long treatment (24 to 30 hours) with 5 nM AP-Sema3A before immunocytochemistry for a pan-axonal neurofilament marker (SMI312) and Myc. Compiled confocal Z-stack data images were stitched using Adobe Photoshop CS5 software and analyzed using NeuronJ plugin available for ImageJ. Only SMI312/Myc co-staining axons from neurons resembling pyramidal morphology were measured. Total axonal length was measured in micrometers for a total of 10 to15 neurons per construct from two to three independent cultures. Statistical analyses were done using GraphPad Prism 5 software. ANOVA was performed, followed by post hoc Tukey test for both the short and long treatments; P < 0.05 was considered statistically significant. All data are means ± SEM. For expression of the Plexin-A4 variants, wild-type E13.5 dissociated cortical neurons were cultured for 3 to 4 days before immunocytochemistry. SUPPLEMENTARY MATERIALS www.sciencesignaling.org/cgi/content/full/7/316/ra24/DC1 Fig. S1. Overexpression of Plexin-A4 does not enhance growth cone collapse in response to Sema3A. Fig. S2. Plexin-A4 receptor variants are expressed on cell surface. Fig. S3. Cell surface detection of Plexin-A4 variants on cortical neuron soma and neurites. Fig. S4. Plexin-A4 cytoplasmic domain variants regulate dendritic morphogenesis of wild-type and PlxnA4−/− cortical neurons. Fig. S5. Axonal expression of Plexin-A4 variants in cortical neurons.

REFERENCES AND NOTES 1. A. B. Huber, A. L. Kolodkin, D. D. Ginty, J. F. Cloutier, Signaling at the growth cone: Ligand-receptor complexes and the control of axon growth and guidance. Annu. Rev. Neurosci. 26, 509–563 (2003). 2. A. L. Kolodkin, M. Tessier-Lavigne, Mechanisms and molecules of neuronal wiring: A primer. Cold Spring Harb. Perspect. Biol. 3, a001727 (2011). 3. T. S. Tran, A. L. Kolodkin, R. Bharadwaj, Semaphorin regulation of cellular morphology. Annu. Rev. Cell Dev. Biol. 23, 263–292 (2007). 4. R. J. Pasterkamp, Getting neural circuits into shape with semaphorins. Nat. Rev. Neurosci. 13, 605–618 (2012). 5. Y. Yoshida, Semaphorin signaling in vertebrate neural circuit assembly. Front. Mol. Neurosci. 5, 71 (2012). 6. S. Kang, A. Kumanogoh, Semaphorins in bone development, homeostasis, and disease. Semin. Cell Dev. Biol. 24, 163–171 (2013). 7. L. Tamagnone, Emerging role of semaphorins as major regulatory signals and potential therapeutic targets in cancer. Cancer Cell 22, 145–152 (2012). 8. A. Sakurai, C. L. Doçi, J. S. Gutkind, Semaphorin signaling in angiogenesis, lymphangiogenesis and cancer. Cell Res. 22, 23–32 (2012). 9. P. K. Hota, M. Buck, Plexin structures are coming: Opportunities for multilevel investigations of semaphorin guidance receptors, their cell signaling mechanisms, and functions. Cell. Mol. Life Sci. 69, 3765–3805 (2012). 10. Y. Zhou, R. A. Gunput, R. J. Pasterkamp, Semaphorin signaling: Progress made and promises ahead. Trends Biochem. Sci. 33, 161–170 (2008). 11. T. S. Tran, M. E. Rubio, R. L. Clem, D. Johnson, L. Case, M. Tessier-Lavigne, R. L. Huganir, D. D. Ginty, A. L. Kolodkin, Secreted semaphorins control spine distribution and morphogenesis in the postnatal CNS. Nature 462, 1065–1069 (2009). 12. A. Yaron, P. H. Huang, H. J. Cheng, M. Tessier-Lavigne, Differential requirement for Plexin-A3 and -A4 in mediating responses of sensory and sympathetic neurons to distinct class 3 Semaphorins. Neuron 45, 513–523 (2005). 13. F. Suto, K. Ito, M. Uemura, M. Shimizu, Y. Shinkawa, M. Sanbo, T. Shinoda, M. Tsuboi, S. Takashima, T. Yagi, H. Fujisawa, Plexin-A4 mediates axon-repulsive activities of both secreted and transmembrane semaphorins and plays roles in nerve fiber guidance. J. Neurosci. 25, 3628–3637 (2005). 14. T. Toyofuku, J. Yoshida, T. Sugimoto, H. Zhang, A. Kumanogoh, M. Hori, H. Kikutani, FARP2 triggers signals for Sema3A-mediated axonal repulsion. Nat. Neurosci. 8, 1712–1719 (2005). 15. B. Zhuang, Y. S. Su, S. Sockanathan, FARP1 promotes the dendritic growth of spinal motor neuron subtypes through transmembrane Semaphorin6A and PlexinA4 signaling. Neuron 61, 359–372 (2009). 16. V. Fenstermaker, Y. Chen, A. Ghosh, R. Yuste, Regulation of dendritic length and branching by semaphorin 3A. J. Neurobiol. 58, 403–412 (2004). 17. C. Gu, E. R. Rodriguez, D. V. Reimert, T. Shu, B. Fritzsch, L. J. Richards, A. L. Kolodkin, D. D. Ginty, Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development. Dev. Cell 5, 45–57 (2003). 18. L. K. Low, X. B. Liu, R. L. Faulkner, J. Coble, H. J. Cheng, Plexin signaling selectively regulates the stereotyped pruning of corticospinal axons from visual cortex. Proc. Natl. Acad. Sci. U.S.A. 105, 8136–8141 (2008). 19. S. M. Catalano, E. K. Messersmith, C. S. Goodman, C. J. Shatz, A. Chédotal, Many major CNS axon projections develop normally in the absence of semaphorin III. Mol. Cell. Neurosci. 11, 173–182 (1998). 20. T. Takahashi, A. Fournier, F. Nakamura, L. H. Wang, Y. Murakami, R. G. Kalb, H. Fujisawa, S. M. Strittmatter, Plexin-neuropilin-1 complexes form functional semaphorin-3A receptors. Cell 99, 59–69 (1999). 21. G. Neufeld, O. Kessler, The semaphorins: Versatile regulators of tumour progression and tumour angiogenesis. Nat. Rev. Cancer 8, 632–645 (2008). 22. M. V. Gelfand, S. Hong, C. Gu, Guidance from above: Common cues direct distinct signaling outcomes in vascular and neural patterning. Trends Cell Biol. 19, 99–110 (2009). 23. L. J. Turner, S. Nicholls, A. Hall, The activity of the plexin-A1 receptor is regulated by Rac. J. Biol. Chem. 279, 33199–33205 (2004). 24. Z. Jin, S. M. Strittmatter, Rac1 mediates collapsin-1-induced growth cone collapse. J. Neurosci. 17, 6256–6263 (1997). 25. S. M. Zanata, I. Hovatta, B. Rohm, A. W. Püschel, Antagonistic effects of Rnd1 and RhoD GTPases regulate receptor activity in Semaphorin 3A-induced cytoskeletal collapse. J. Neurosci. 22, 471–477 (2002).


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Dissociated E13.5 mouse cortical neurons were cultured for 6 to 7 days, and neurons resembling a pyramidal morphology with a long apical dendrite and shorter basal dendrites were selected for analysis. Compiled confocal Z-stack data images from four to five independent cultures were used for all wild-type and PlxnA4−/− analyses. Only dendrites expressing both MAP2 and Myc were used for measurements of total dendritic length and DCI and Sholl analysis. All Sholl analyses were performed using the ImageJ Sholl Analysis Plugin (http://www-biology.ucsd.edu/labs/ghosh/ software/), with the center of all concentric circles defined as the center of cell soma. Sholl analysis parameters were as follows: the smallest radius was 10 mm, the largest radius was 60 mm, and the interval between consecutive radii was 5 mm, as previously described for in vivo measurements (11). For each Plexin-A4 construct analyzed across all genotypes, a total of 40 to 50-plus dendrite intersections per radii were averaged. Data are means ± SEM, analyzed by one-way ANOVA followed by post hoc Tukey test; P < 0.01 was considered significant. Total dendritic length was measured using the ImageJ plugin NeuronJ (http://www.imagescience.org/ meijering/software/neuronj/), calculated in micrometers for a total of 35 neurons per construct and across all genotypes. Dendritic order was defined as follows: primary dendrites were traced from the cell soma to the tip of the entire dendritic length, and secondary and tertiary dendrites were traced from the tip to the dendritic branch point using Adobe Photoshop CS5 software. Dendrite lengths that were less than the diameter of the cell soma were disregarded. The DCI was measured by the following formula: (S branch tip orders + no. of branch tips)/(no. of primary dendrites) × (total arbor length) (56). DCI data are means ± SEM. Statistical analysis used GraphPad Prism 5 software for ANOVA followed by post hoc Tukey test; P < 0.001 was considered statistically significant.

Fig. S6. Sema3A-induced Plexin-A4 signaling does not alter total axonal length in wild-type cortical neurons. Fig. S7. Specific Plexin-A4 cytoplasmic domains regulate cellular collapse in COS7 cells. Fig. S8. Interaction of Plexin-A4 active and nonactive variants with neuropilin-1. Fig. S9. Expression of FARP1 and FARP2 in the mouse cortex and DRG. Fig. S10. Western blots illustrate FARP protein knockdown by specific siRNAs in primary cortical and DRG neuron cultures. Reference (57)

RESEARCH ARTICLE 48. Y. N. Jan, L. Y. Jan, Branching out: Mechanisms of dendritic arborization. Nat. Rev. Neurosci. 11, 316–328 (2010). 49. K. L. Whitford, V. Marillat, E. Stein, C. S. Goodman, M. Tessier-Lavigne, A. Chédotal, A. Ghosh, Regulation of cortical dendrite development by Slit-Robo interactions. Neuron 33, 47–61 (2002). 50. F. Polleux, T. Morrow, A. Ghosh, Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 404, 567–573 (2000). 51. K. L. Whitford, P. Dijkhuizen, F. Polleux, A. Ghosh, Molecular control of cortical dendrite development. Annu. Rev. Neurosci. 25, 127–149 (2002). 52. F. Calderon de Anda, A. L. Rosario, O. Durak, T. Tran, J. Gräff, K. Meletis, D. Rei, T. Soda, R. Madabhushi, D. D. Ginty, A. L. Kolodkin, L. H. Tsai, Autism spectrum disorder susceptibility gene TAOK2 affects basal dendrite formation in the neocortex. Nat. Neurosci. 15, 1022–1031 (2012). 53. H. Takamatsu, A. Kumanogoh, Diverse roles for semaphorin–plexin signaling in the immune system. Trends Immunol. 33, 127–135 (2012). 54. M. Maor-Nof, N. Homma, C. Raanan, A. Nof, N. Hirokawa, A. Yaron, Axonal pruning is actively regulated by the microtubule-destabilizing protein kinesin superfamily protein 2A. Cell Rep. 3, 971–977 (2013). 55. A. B. Huber, A. Kania, T. S. Tran, C. Gu, N. De Marco Garcia, I. Lieberam, D. Johnson, T. M. Jessell, D. D. Ginty, A. L. Kolodkin, Distinct roles for secreted semaphorin signaling in spinal motor axon guidance. Neuron 48, 949–964 (2005). 56. B. Lom, S. Cohen-Cory, Brain-derived neurotrophic factor differentially regulates retinal ganglion cell dendritic and axonal arborization in vivo. J. Neurosci. 19, 9928–9938 (1999). 57. G. Diez-Roux, S. Banfi, M. Sultan, L. Geffers, S. Anand, D. Rozado, A. Magen, E. Canidio, M. Pagani, I. Peluso, N. Lin-Marq, M. Koch, M. Bilio, I. Cantiello, R. Verde, C. De Masi, S. A. Bianchi, J. Cicchini, E. Perroud, S. Mehmeti, E. Dagand, S. Schrinner, A. Nürnberger, K. Schmidt, K. Metz, C. Zwingmann, N. Brieske, C. Springer, A. M. Hernandez, S. Herzog, F. Grabbe, C. Sieverding, B. Fischer, K. Schrader, M. Brockmeyer, S. Dettmer, C. Helbig, V. Alunni, M. A. Battaini, C. Mura, C. N. Henrichsen, R. Garcia-Lopez, D. Echevarria, E. Puelles, E. Garcia-Calero, S. Kruse, M. Uhr, C. Kauck, G. Feng, N. Milyaev, C. K. Ong, L. Kumar, M. Lam, C. A. Semple, A. Gyenesei, S. Mundlos, U. Radelof, H. Lehrach, P. Sarmientos, A. Reymond, D. R. Davidson, P. Dollé, S. E. Antonarakis, M. L. Yaspo, S. Martinez, R. A. Baldock, G. Eichele, A. Ballabio, A high-resolution anatomical atlas of the transcriptome in the mouse embryo. PLOS Biol. 9, e1000582 (2011). Acknowledgments: We would like to thank M. Fainzilber (Weizmann Institute of Science) and W. Friedman (Rutgers University) for critically reading the paper, and C. Rapadas (Rutgers University) for excellent technical support. We thank S. Sockanathans (The Johns Hopkins University) for the HA-FARP1 and FARP-2 plasmids and the MPI for Biophysical Chemistry for the use of the in situ hybridization data. Funding: This work is supported by grants from the Israel Science Foundation (1470/09) and the Joseph D. Shane Fund for Neurosciences (to A.Y.) and the Charles and Johanna Busch Biomedical Funds (to T.S.T.). Author contributions: A.Y. and T.S.T. conceived the project and wrote the manuscript. G.M. performed molecular cloning of all the Plexin-A4 variants and the experiments in DRG neurons. S.-S.P. performed most of the cortical dendritic experiments, including the siRNA FARP1 in cortical neurons, coimmunoprecipitation of Plexin-A4 variants with FARP1 and FARP2, and expression of Plexin-A4 variants in cortical axons. V.S. performed the COS cell collapse experiments and immunohistochemical analysis. E.M. performed some of the cortical dendritic experiments and immunohistochemistry for anti-FARP1 in brain sections. I.G. performed the cell surface biotinylation. A.M. assisted with the siRNA analysis. Competing interests: The authors declare that they have no competing financial interests. Data and materials availability: All plasmid constructs used in this study will be freely available upon request. Submitted 18 September 2013 Accepted 18 February 2014 Final Publication 11 March 2014 10.1126/scisignal.2004734 Citation: G. Mlechkovich, S.-S. Peng, V. Shacham, E. Martinez, I. Gokhman, A. Minis, T. S. Tran, A. Yaron, Distinct cytoplasmic domains in Plexin-A4 mediate diverse responses to semaphorin 3A in developing mammalian neurons. Sci. Signal. 7, ra24 (2014).


11 March 2014


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26. Y. Tong, P. Chugha, P. K. Hota, R. S. Alviani, M. Li, W. Tempel, L. Shen, H. W. Park, M. Buck, Binding of Rac1, Rnd1, and RhoD to a novel Rho GTPase interaction motif destabilizes dimerization of the plexin-B1 effector domain. J. Biol. Chem. 282, 37215–37224 (2007). 27. J. W. Legg, C. M. Isacke, Identification and functional analysis of the ezrin-binding site in the hyaluronan receptor, CD44. Curr. Biol. 8, 705–708 (1998). 28. F. Suto, Y. Murakami, F. Nakamura, Y. Goshima, H. Fujisawa, Identification and characterization of a novel mouse plexin, plexin-A4. Mech. Dev. 120, 385–396 (2003). 29. H. J. Cheng, A. Bagri, A. Yaron, E. Stein, S. J. Pleasure, M. Tessier-Lavigne, Plexin-A3 mediates semaphorin signaling and regulates the development of hippocampal axonal projections. Neuron 32, 249–263 (2001). 30. K. Hong, L. Hinck, M. Nishiyama, M. M. Poo, M. Tessier-Lavigne, E. Stein, A ligandgated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion. Cell 97, 927–941 (1999). 31. Y. Liu, J. Shi, C. C. Lu, Z. B. Wang, A. I. Lyuksyutova, X. Song, Y. Zou, Ryk-mediated Wnt repulsion regulates posterior-directed growth of corticospinal tract. Nat. Neurosci. 8, 1151–1159 (2005). 32. A. M. Schmitt, J. Shi, A. M. Wolf, C. C. Lu, L. A. King, Y. Zou, Wnt-Ryk signalling mediates medial-lateral retinotectal topographic mapping. Nature 439, 31–37 (2006). 33. A. I. Lyuksyutova, C. C. Lu, N. Milanesio, L. A. King, N. Guo, Y. Wang, J. Nathans, M. Tessier-Lavigne, Y. Zou, Anterior-posterior guidance of commissural axons by Wnt-frizzled signaling. Science 302, 1984–1988 (2003). 34. F. Charron, E. Stein, J. Jeong, A. P. McMahon, M. Tessier-Lavigne, The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance. Cell 113, 11–23 (2003). 35. D. Bourikas, V. Pekarik, T. Baeriswyl, A. Grunditz, R. Sadhu, M. Nardó, E. T. Stoeckli, Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord. Nat. Neurosci. 8, 297–304 (2005). 36. V. Castellani, A. Chédotal, M. Schachner, C. Faivre-Sarrailh, G. Rougon, Analysis of the L1-deficient mouse phenotype reveals cross-talk between Sema3A and L1 signaling pathways in axonal guidance. Neuron 27, 237–249 (2000). 37. S. Chauvet, S. Cohen, Y. Yoshida, L. Fekrane, J. Livet, O. Gayet, L. Segu, M. C. Buhot, T. M. Jessell, C. E. Henderson, F. Mann, Gating of Sema3E/PlexinD1 signaling by neuropilin-1 switches axonal repulsion to attraction during brain development. Neuron 56, 807–822 (2007). 38. L. Ma, M. Tessier-Lavigne, Dual branch-promoting and branch-repelling actions of Slit/Robo signaling on peripheral and central branches of developing sensory axons. J. Neurosci. 27, 6843–6851 (2007). 39. A. Bagri, H. J. Cheng, A. Yaron, S. J. Pleasure, M. Tessier-Lavigne, Stereotyped pruning of long hippocampal axon branches triggered by retraction inducers of the semaphorin family. Cell 113, 285–299 (2003). 40. A. Sahay, M. E. Molliver, D. D. Ginty, A. L. Kolodkin, Semaphorin 3F is critical for development of limbic system circuitry and is required in neurons for selective CNS axon guidance events. J. Neurosci. 23, 6671–6680 (2003). 41. M. M. Riccomagno, A. Hurtado, H. Wang, J. G. Macopson, E. M. Griner, A. Betz, N. Brose, M. G. Kazanietz, A. L. Kolodkin, The RacGAP b2-Chimaerin selectively mediates axonal pruning in the hippocampus. Cell 149, 1594–1606 (2012). 42. J. Falk, A. Bechara, R. Fiore, H. Nawabi, H. Zhou, C. Hoyo-Becerra, M. Bozon, G. Rougon, M. Grumet, A. W. Puschel, J. R. Sanes, V. Castellani, Dual functional activity of semaphorin 3B is required for positioning the anterior commissure. Neuron 48, 63–75 (2005). 43. H. Song, G. Ming, Z. He, M. Lehmann, L. McKerracher, M. Tessier-Lavigne, M. Poo, Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides. Science 281, 1515–1518 (1998). 44. H. Togashi, E. F. Schmidt, S. M. Strittmatter, RanBPM contributes to Semaphorin3A signaling through Plexin-A receptors. J. Neurosci. 26, 4961–4969 (2006). 45. L. Cheadle, T. Biederer, The novel synaptogenic protein Farp1 links postsynaptic cytoskeletal dynamics and transsynaptic organization. J. Cell Biol. 199, 985–1001 (2012). 46. A. P. Barnes, F. Polleux, Establishment of axon-dendrite polarity in developing neurons. Annu. Rev. Neurosci. 32, 347–381 (2009). 47. R. Hand, D. Bortone, P. Mattar, L. Nguyen, J. I. Heng, S. Guerrier, E. Boutt, E. Peters, A. P. Barnes, C. Parras, C. Schuurmans, F. Guillemot, F. Polleux, Phosphorylation of Neurogenin2 specifies the migration properties and the dendritic morphology of pyramidal neurons in the neocortex. Neuron 48, 45–62 (2005).

Distinct Cytoplasmic Domains in Plexin-A4 Mediate Diverse Responses to Semaphorin 3A in Developing Mammalian Neurons Guy Mlechkovich, Sheng-Shiang Peng, Vered Shacham, Edward Martinez, Irena Gokhman, Adi Minis, Tracy S. Tran and Avraham Yaron (March 11, 2014) Science Signaling 7 (316), ra24. [doi: 10.1126/scisignal.2004734]

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