2034 Research Article The stimulation of growth by Sema3A requires integrin engagement and focal adhesion kinase

Uwe Schlomann1,*, Jens C. Schwamborn1,‡, Myriam Müller1, Reinhard Fässler2 and Andreas W. Püschel1,§ 1Institut für Allgemeine Zoologie und Genetik, Westfalische Wilhelms-Universität Münster, Schlossplatz 5, D-48149 Münster, Germany 2Department of Molecular Medicine, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany *Present address: Department of Biochemistry, Kingʼs College London, London SE1 9NH, UK ‡Present address: ZMBE, Institute of Cell Biology, Stem Cell Biology and Regeneration Group, Westfalische Wilhelms-Universität Münster, Von-Esmarch-Str. 56, 48149 Münster, Germany §Author for correspondence (e-mail: [email protected])

Accepted 9 March 2009 Journal of Cell Science 122, 2034-2042 Published by The Company of Biologists 2009 doi:10.1242/jcs.038232

Summary The rate and direction of and dendrite growth depend on facilitate the stimulation of dendrite extension. Conditional multiple guidance signals and growth factors. 3A inactivation of the encoding β1 integrin or FAK blocks (Sema3A) acts as a repellent for and attractant for the growth-promoting effect of Sema3A but not the collapse of . Here, we show that the requirement for integrin axonal growth cones. Our results demonstrate that different engagement distinguishes the response of axons and dendrites pathways mediate the stimulation of dendrite growth and the to Sema3A in hippocampal . Sema3A promotes the collapse of axonal growth cones by Sema3A. extension of hippocampal dendrites by a pathway that requires focal adhesion kinase (FAK). The stimulation of dendrite Supplementary material available online at growth and FAK phosphorylation by Sema3A depend on http://jcs.biologists.org/cgi/content/full/122/12/2034/DC1 integrin engagement. Unlike their function as a target of Sema3A during the collapse of axonal growth cones, integrins Key words: Axon guidance, Hippocampus, Semaphorin

Introduction neurons of cortical layer 5 (Polleux et al., 1998; Polleux et al., 2000; The ephrins, netrins, Slit and are four families Sasaki et al., 2002). In Sema3a–/– mice, apical dendrites deviate from of guidance signals that direct the extension of axons and dendrites, their normal orientation perpendicular to the pial surface (Polleux et and serve as attractive or repellent cues during the establishment of al., 2000). In vitro assays suggest that a Sema3A gradient attracts neuronal connections (Dickson, 2002; Goldberg, 2004; Huber et al., apical dendrites to the pial surface by a signalling pathway that 2003; Jan and Jan, 2003; Kim and Chiba, 2004). The effect of these depends on cGMP while directing axons in the opposite direction signals depends on the composition of receptor subunits and the through repulsion (Polleux et al., 1998; Polleux et al., 2000). Sema3A intracellular concentration of second messengers such as cAMP and acts through the same receptor complex to repel axons and attract cGMP (Corset et al., 2000; Höpker et al., 1999; Nishiyama et al., dendrites but it is unknown if the same signalling pathways are 2003; Song et al., 1998; Song and Poo, 1999; Yu and Bargmann, involved for both effects. 2001). Sema3A acts as a repellent for many types of axons and induces Another important factor that determines the response to guidance the collapse of growth cones by initiating the depolymerisation of signals is the extracellular matrix. The integrin ligand laminin actin filaments, promoting endocytosis and reducing integrin- converts the attractive effect of netrin-1 on Xenopus spinal-cord axons mediated adhesion (Fournier et al., 2000; Jin and Strittmatter, 1997; to a repulsive response (Höpker et al., 1999). The genetic interaction Kuhn et al., 1999; Mikule et al., 2002). Manipulating the concentration between mutants for integrins or their ligands and Slit suggests that of cGMP can convert the repellent Sema3A into an attractive cue integrin-dependent signalling influences the sensitivity of axons to (Campbell et al., 2001; Song et al., 1998). The repellent effects of the repellent guidance signal Slit in the Drosophila CNS (Stevens the secreted class-3 semaphorins are mediated by a receptor complex and Jacobs, 2002). A modulatory role of laminin and fibronectin that contains neuropilin-1 (Nrp-1) or Nrp-2 as the ligand-binding was also reported for mammalian Slit-2 (Nguyen-Ba-Charvet et al., subunit and an A-type as the signal-transducing subunit (Kruger 2001). The tyrosine kinase focal adhesion kinase (FAK) is a central et al., 2005; Püschel, 2007; Tran et al., 2007). act as GTPase- component of the signalling pathway regulated by integrins (Mitra activating proteins (GAPs) for R-Ras and antagonise integrin- et al., 2005; Parsons, 2003). Integrin activation leads to an increase dependent cell adhesion by reducing the local concentration of GTP- in FAK activity by the ordered phosphorylation at multiple tyrosine bound R-Ras (Kinbara et al., 2003; Kruger et al., 2005; Oinuma et residues. FAK activation is initiated by the autophosphorylation at al., 2004a; Oinuma et al., 2004b). In addition to its repellent effects, Y397, which enables the binding and activation of Src-family kinases Sema3A also acts as an attractive or growth-promoting signal for the (SFKs). SFKs in turn phosphorylate the kinase domain of FAK at dendrites of pyramidal neurons from the mouse cortex (Fenstermaker Y576/577. In addition to integrins, FAK is also regulated by axon- et al., 2004; Gu et al., 2003; Polleux et al., 2000; Sasaki et al., 2002). guidance signals such as netrin-1 and ephrin-A1 (Bourgin et al., 2007; Sema3A directs the orientation of axons and dendrites by pyramidal Carter et al., 2002; Li et al., 2004; Liu et al., 2004; Miao et al., 2000; Sema3A, FAK and integrins 2035

Ren et al., 2004). FAK and SFKs directly interact with the netrin parallel to each other in the stratum radiatum until they reached the receptor DCC and are activated by netrin-1. Their activity is required stratum lacunosum-moleculare, where they spread out. In Sema3a- for the attraction of axons by netrin-1. deficient mice, this well-ordered organisation was disrupted (Fig. Here we show that Sema3A promotes dendrite growth by 1A). The majority of apical dendrites was stunted and branched stimulating FAK phosphorylation and increasing the intracellular soon after entering the stratum radiatum. When the relative position concentration of cGMP. The activation of FAK and the stimulation of the first branch point of apical dendrites in the stratum radiatum of dendrite extension but not the collapse of axonal growth cones was measured, a significant difference to wild-type hippocampus by Sema3A depend on the simultaneous activation of integrins. The was obvious (Fig. 1B). In total, 81% of the apical dendrites branched stimulatory effect of Sema3A but not growth-cone collapse is in the stratum radiatum of Sema3a–/– mice, whereas branches in blocked by conditional deletion of the β1 integrin (Itgb1) and FAK the stratum radiatum were seen only in 45% of wild-type neurons (Fak) genes. Thus, integrin activation facilitates the effect of (n=108 neurons each; three animals). In addition, branches were Sema3A on dendrites through a signalling pathway that is not significantly closer to the stratum pyramidale in Sema3a mutants required for collapse of the . (Fig. 1). Although the orientation of apical dendrites appeared to be normal, their branches formed a disorganised dendritic arbour. Results The branches of apical dendrites did not extend parallel to each Aberrant development of hippocampal dendrites in Sema3a- other as in wild-type hippocampi and showed more irregular knockout mice trajectories in the stratum radiatum of Sema3a mutants. It is To investigate the role of Sema3A in the development of unlikely that the extensive disorganisation of apical dendrites in the hippocampal neurons, the Thy1-YFPH transgene, which drives YFP Sema3a mutant is an indirect effect of axonal misprojections. In expression in pyramidal neurons of the hippocampus (Feng et al., wild-type mice, entorhino-hippocampal projections are restricted 2000), was crossed into a Sema3a-knockout strain (Taniguchi et to the stratum lacunosum-moleculare. In Sema3a mutants, only a al., 1997). Expression of the Thy1-YFPH transgene was detectable few entorhino-hippocampal fibres extend into inappropriate layers only in a few cells at early postnatal stages but became visible in such as the stratum radiatum (Pozas et al., 2001). These minor a larger number of neurons at postnatal day 15 (P15) (Fig. 1). The guidance defects are unlikely to cause the pronounced morphology of pyramidal neurons was analysed in 60-μm sections disorganisation of dendrites observed in the mutant hippocampus. from brains of P15 mice. The structure of the hippocampus was normal in Sema3a–/– mice, as was reported previously (Pozas et al., Sema3A stimulates dendrite growth 2001). However, the morphology of apical dendrites showed defects The abnormal morphology of apical dendrites in Sema3a-deficient in Sema3a mutants. In wild-type mice, pyramidal neurons extended mice might reflect an inhibitory function of Sema3A that prevents apical dendrites from the stratum pyramidale through the stratum premature branching in the stratum radiatum. Alternatively, Sema3A radiatum and formed branches mainly in the stratum lacunosum- could influence the growth of dendrites as an attractive or growth- moleculare or in the stratum radiatum close to the border with the stimulating signal as described for cortical neurons (Fenstermaker et stratum lacunosum-moleculare. The dendrites usually extended al., 2004; Gu et al., 2003; Polleux et al., 2000; Sasaki et al., 2002). To investigate how Sema3A regulates the morphology of dendrites, we used cultures of dissociated hippocampal neurons from embryonic day 18 (E18) mouse or rat embryos with fibronectin as a substrate. At 3 days after plating [3 days in vitro (d.i.v.)], Sema3A was added for 16-20 hours and the development of dendrites was analysed after staining with an anti-MAP2 antibody. When added to cultured neurons from mouse embryos, Sema3A increased the average length of dendrites by more than 50%, from 51±4.5 μm in controls to 79±2 μm (Fig. 2) (n=30–50 neurons, three experiments). By contrast, Sema3a–/– mouse hippocampal neurons formed shorter dendrites than wild-type neurons, with an average length of 37±1 μm (Fig. 2B). Incubation of Sema3a–/– neurons with Sema3A stimulated the growth of dendrites and rescued the loss of Sema3A. The branching of dendrites was not affected by Sema3A treatment during the observed time period (data not shown). We also investigated the effect of Sema3A on neurons prepared from embryonic rat hippocampus (supplementary material Fig. S1A,B). Compared with mouse neurons, the average length of dendrites from rat neurons was slightly shorter Fig. 1. Abnormal morphology of apical dendrites in the hippocampus of at 4 d.i.v. (control: 33±1 μm) and was increased by the addition of Sema3a-deficient mice. (A) The morphology of pyramidal neurons was Sema3A to 57±3 μm (Fig. 3) (n=3 with 75 neurons each). As already μ analysed in 60- m sections from the CA1 region of P15 wild-type and observed for the stimulation of neurite extension by PC12 cells Tg(Thy1-YFPH)2Jrs/J+/0;Sema3a–/– mice. Arrowheads indicate branching points of apical dendrites in the stratum radiatum (sr). Apical dendrites branch (Schwamborn et al., 2004), a prolonged incubation with Sema3A shortly after they enter the sr in Sema3a–/– mice (arrowheads). slm, stratum was required to stimulate dendritic growth. When Sema3A was lacunosum-moleculare; sp, stratum pyramidale. (B) The relative position of the replaced by control medium after incubation for only 1 hour and the first branch point was determined. The relative length of the sr was set to 1 culture continued for 16 hours, no effect on dendritic growth was (position 0 is the border between sp and sr, position 1 the border between sr and slm). The position at which apical dendrites branch is significantly closer observed (data not shown). When neurons were cultured on the to the sp in Sema3a–/– mice (S3a–/–; means ± s.e.m.; n=70 neurons from three nonspecific adhesive poly-L-ornithine (PO), Sema3A was unable to different animals; *P<0.001 compared with wild type). Scale bar: 100 μm. stimulate dendrite growth and the length of dendrites from wild-type 2036 Journal of Cell Science 122 (12)

Fig. 3. The stimulation of dendrite growth by Sema3A depends on integrin activation. (A) Hippocampal neurons were plated on poly-L-ornithine (PO) or Fig. 2. Sema3A stimulates dendrite elongation of hippocampal neurons. fibronectin and incubated with control medium (control) or medium –/– (A,B) Hippocampal neurons from wild-type or Sema3a mice were cultured containing Sema3A (+Sema3A) in the absence (–) or presence of 1 mM MnCl2 on fibronectin (FN, shown in A) or poly-L-ornithine (PO, not shown in A) as a or 200 μM RGD peptide as indicated at 3 d.i.v. for 16-20 hours. Neurons were substrate and incubated at 3 d.i.v. with control medium or Sema3A for 16-20 stained at 4 d.i.v. with an anti-MAP2 antibody to visualise dendrites (n=75 hours. Neurons were stained with an anti-MAP2 (green) and the Tau-1 (red) neurons, three experiments). Scale bar: 30 μm. (B) The length of the dendrites antibody as dendritic and axonal markers, respectively. Scale bar: 100 μm. was determined (means ± s.e.m.; n=30-50 neurons, three experiments; (B) The length of dendrites after incubation with control medium (white bars) *P<0.001 compared with control medium without Sema3A). or Sema3A (black bars) is shown as means ± s.e.m.; n=30-50 neurons, six experiments for FN; n=15-30 neurons, three experiments for PO; ***P<0.001, *P<0.05 compared to control (WT neurons, –Sema3A) or between the values indicated by the bracket. result suggests that the stimulation of dendrite extension by Sema3A depends on the ligation of integrins by fibronectin. To and Sema3a–/– neurons did not differ significantly (Fig. 2B). These test the role of integrins, we used Mn2+ ions as an activator of results indicate that Sema3A does not act as an inhibitory signal for integrins, which stabilises the active conformation, and an RGD dendrite growth or branching but stimulates dendrite elongation in a peptide as a specific inhibitor (Gailit and Ruoslahti, 1988; Mould substrate-dependent manner. et al., 1995; Takagi et al., 2002). Incubation of neurons with Mn2+ ions had no effect on dendrite length by itself but could replace The stimulation of dendrite growth by Sema3A depends on fibronectin when Sema3A was added to neurons cultured on PO integrin engagement (Fig. 3). Blocking the interaction of fibronectin and integrins by Sema3A increased the length of dendrites only when neurons were an RGD peptide prevented the stimulatory effect of Sema3A. This cultured on fibronectin but not on the nonspecific adhesive PO indicates that Sema3A stimulates dendrite elongation only when (Fig. 2B; Fig. 3). When rat hippocampal neurons were cultured on integrins are activated. PO, the addition of Sema3A did not have any significant effect on The repellent activity of Sema3A is mediated by a receptor the extension of dendrites (control: 32±1 μm; Sema3A: 33±1 μm; complex that consists of Nrp-1 and A-type plexins (Tran et al., n=4 with 75 neurons each). In the presence of control medium, 2007). To test whether these receptor subunits are also required for dendrites had an average length of approximately 30 μm on PO the stimulatory activity of Sema3A on dendrites, we transfected rat or fibronectin (control: 32±1 μm; n=4 with 75 neurons each). This hippocampal neurons at 2 d.i.v. with vectors for dominant-negative Sema3A, FAK and integrins 2037

Nrp-1 or plexin-A1, which interfere with signalling through Nrp- 1 and A-type plexins, respectively. At 3 d.i.v., neurons were treated with Sema3A for 16 hours. Both constructs blocked the effect of Sema3A on dendrites (supplementary material Fig. S1C). By contrast, expression of a dominant-negative mutant for plexin-B1, the receptor for Sema4D, did not interfere with the stimulation of neurite extension by Sema3A. Sema3A has a potent growth cone collapsing activity for most types of neurons. It also induced the collapse of growth cones from hippocampal axons (supplementary material Fig. S2). When hippocampal neurons were cultured on fibronectin, 19±4% (n=3) of the axonal growth cones were collapsed under control conditions. Incubation with Sema3A for 1 hour increased the percentage of collapsed growth cones to 60±7%. The collapse of axonal growth cones by Sema3A was independent of the substrate and was not affected by the addition of RGD peptides or Mn2+ ions (supplementary material Fig. S2). Thus, the response of axonal growth cones to Sema3A is not influenced by integrin engagement. Nearly all fibronectin-binding integrins belong to the β1- or αv- integrin subfamilies (Hynes, 2002). Hippocampal neurons express β1 integrin (Belvindrah et al., 2007; Huang et al., 2006). Staining of cultured hippocampal neurons for β1 integrin showed a punctate distribution in axons, dendrites and growth cones (supplementary material Fig. S3A). The immunofluorescence signals were weak compared with non-neuronal cells present in these cultures. We failed to detect αv integrins in hippocampal using different antibodies, fixation protocols and blocking methods. Non-neuronal cells that are also present in these cultures showed a staining pattern typical for focal-adhesion complexes with some of the tested antibodies (data not shown). Thus, αv integrin is not expressed in neurons or present only at low levels, and is not concentrated at sites of adhesion. To investigate the role of fibronectin receptors including the β1- integrin subunit, we used hippocampal neurons prepared from a conditional knock-out (Itgb1flox/flox) (Potocnik et al., 2000). The cells were cultured on fibronectin and transfected at 3 d.i.v. with an Fig. 4. The stimulation of dendrite growth by Sema3A depends on β1 expression vector for EGFP-Cre (pBS505) to inactivate Itgb1 (Fig. integrins. (A) Neurons from Itgb1flox/flox embryos were cultured on fibronectin 4). After 24 hours, the cells were incubated with Sema3A or control and transfected at 3 d.i.v. with vectors for EGFP (green) to visualise neuronal β morphology or EGFP and EGFP-Cre (pBS505; localised to the nucleus). At 24 medium for 16 hours. The staining with an antibody specific for 1 hours after the transfection, the cells were incubated with Sema3A for 16 hours integrin showed that the immunofluorescence signal was strongly and stained with an anti-MAP2 antibody (red). Scale bar: 100 μm. (B) The reduced in cells expressing EGFP-Cre (supplementary material Fig. length of dendrites is shown as means ± s.e.m.; n=30-50 neurons, three S3B). The length of dendrites extended by these neurons was experiments; *P<0.001 compared with control (GFP, –Sema3A) or between significantly shorter (49±2 μm) compared with control neurons (59±1 the values indicated by the bracket. μm; n=15-25 neurons, three experiments) and the stimulatory effect of Sema3A on dendrite growth was completely blocked (Fig. 4). The length of axons deficient for β1 integrins was similar to that of a]quinoxalin-1-one (ODQ). ODQ completely blocked the wild-type neurons (supplementary material Fig. S4). Expression of stimulation of dendrite growth by Sema3A (Fig. 5A,B). The EGFP-Cre by itself did not affect neuronal differentiation. The length average length of dendrites after treatment with Sema3A for 16 of dendrites formed by wild-type or Ingb+/flox neurons transfected hours was 45±13 μm (n=100 neurons, three experiments). Addition with the vector for EGFP-Cre did not differ from neurons transfected of ODQ together with Sema3A reduced dendrite length to that of with vector for EGFP (data not shown). control neurons (~24±6 μm). An inhibitor for NO synthase (L- NAME) also blocked the effect of Sema3A on dendrites. ODQ and The stimulation of dendrite growth by Sema3A depends on L-NAME did not affect the extension of dendrites on PO or cGMP production fibronectin by themselves. Thus, an increase in the cGMP It has been shown previously that activation of the nitric oxide (NO)- concentration is required for the stimulation of dendrite extension. dependent guanylate cyclase can switch the repellent activity of To test whether cGMP is responsible for the modulation of the Sema3A to an attractive effect (Castellani et al., 2000; Polleux et response to Sema3A by integrins, we directly measured the cGMP al., 2000). To test whether NO production and cGMP synthesis are concentration after addition of Sema3A. Sema3A induced a tenfold required for the stimulatory effect of Sema3A, we inhibited NO- increase in the intracellular cGMP level within 5 minutes that lasted dependent guanylate cyclase pharmacologically by incubation of for at least 1 hour (Fig. 5C). After 12 hours of culture in the presence rat hippocampal neurons with 1H-[1,2,4]oxadiazolo[4,3- of Sema3A, the cGMP concentration returned to control levels. The 2038 Journal of Cell Science 122 (12)

Fig. 6. Sema3A stimulates FAK phosphorylation. (A) Hippocampal neurons from mouse embryos were cultured on fibronectin and incubated at 3 d.i.v. with control medium or medium containing Sema3A for 20 minutes. Neurons were lysed and FAK or phosphorylated proteins immunoprecipitated (IP) using anti-FAK or anti-phospho-tyrosine (pY) antibodies. Precipitated proteins were analysed by western blot (WB) using anti-FAK or anti-pY antibodies. (B) Hippocampal neurons from mouse embryos were cultured on fibronectin Fig. 5. The stimulation of dendrite growth by Sema3A depends on cGMP and incubated at 3 d.i.v. with control medium (–) or medium containing production. (A,B) Hippocampal neurons were plated on PO (B) or fibronectin Sema3A for 20 minutes. FAK phosphorylation was analysed by western blot (A,B). At 3 d.i.v., control medium (–) or medium containing Sema3A was using antibodies specific for phosphorylation at Y397 or Y576/577. An anti- added for 16-20 hours. ODQ (5 μM) or L-NAME (1 μM) were added as tubulin antibody was used to confirm the loading of equivalent amounts of indicated. At 4 d.i.v., the neurons were stained with an anti-MAP2 antibody . (C) Quantification of FAK phosphorylation shown in A (pFAK) and B and the average length of dendrites was determined (means ± s.e.m.; n=50 (Y397 and Y576/577; n=3, *P<0.001 compared with control medium). neurons, three experiments; *P<0.001 compared with control medium without Sema3A). Scale bar: 30 μm. (C,D) Hippocampal neurons were plated on poly- L-ornithine (PO) or fibronectin (FN). At 3 d.i.v., control medium or medium containing Sema3A was added for the indicated times. L-NAME (LN; 1 μM) on which neurons were grown (Fig. 5E). cGMP could rescue the or ODQ (O; 5 μM) were added as indicated. The cells were lysed and the block of neurite growth by ODQ, confirming the specificity of the intracellular concentration of cGMP measured by the cGMP Direct Biotrak inhibitor. These results show that the production of cGMP is a direct EIA system. *P<0.001 compared with control medium without Sema3A. effect of Sema3A and independent of the substrate. cGMP is required (E) Hippocampal neurons were incubated with control medium or medium containing Sema3A for 16-20 hours and 0.1 mM 8-Bromo-cGMP (cGMP) or for the stimulation of dendrite growth by Sema3A but does not 5 μM ODQ added as indicated. Neurons were fixed at 4 d.i.v., stained with an appear to be responsible for mediating the effect of integrins on the anti-MAP2 antibody and the length of dendrites determined (means ± s.e.m.; Sema3A signalling pathway. n=3, *P<0.001 compared with control medium without Sema3A). Sema3A stimulates FAK activity Activated integrins mediate the transmission of signals from the increase in cGMP was completely blocked by L-NAME and ODQ. extracellular matrix to the cytoskeleton during cell adhesion, spreading To analyse the time course of cGMP production in more detail, the and migration (Giancotti and Ruoslahti, 1999; Mitra et al., 2005). A level of cGMP was measured between 1 minute and 1 hour after central component in this pathway is FAK, which is phosphorylated the addition of Sema3A (Fig. 5D). The intracellular cGMP at several tyrosine residues upon recruitment to focal adhesions (Mitra concentration increased within 1 minute after application of Sema3A and Schlaepfer, 2006). The axon-guidance signals netrin-1 and and remained constant for at least 1 hour. However, the induction ephrin-A1 can also stimulate FAK phosphorylation (Carter et al., of cGMP production was independent of integrin engagement. We 2002; Li et al., 2004; Liu et al., 2004; Ren et al., 2004). To investigate observed a similar increase in cGMP levels when neurons were a possible involvement of FAK, Sema3A was added at 3 d.i.v. to cultured on PO or fibronectin. Staining with an antibody specific mouse hippocampal neurons cultured on fibronectin (Fig. 6). After for cGMP showed that Sema3A stimulated cGMP production in 20 minutes, cells were lysed and protein phosphorylation investigated both dendrites and axons (supplementary material Fig. S5). with an anti- phospho-tyrosine (anti-pY) antibody. Sema3A increased Treatment only with the cell-permeable cGMP analogue 8-bromo- the phosphorylation of several proteins, including a major band at a cGMP had no effect on dendrite length, irrespective of the substrate molecular weight of 125 kDa (Fig. 6A). Immunoprecipitation of Sema3A, FAK and integrins 2039

Fig. 7. Integrin activation is necessary for the increase in FAK phosphorylation by Sema3A. (A,B) Hippocampal neurons from mouse embryos were cultured on PO and incubated at 3 d.i.v. with control medium or medium containing Sema3A for 20 minutes. MnCl2 (1 mM) was added together with the medium as indicated. Neurons were lysed and FAK phosphorylation was analysed by western blot (WB) using antibodies specific for FAK phosphorylated at Y397 or Y576/577. An anti-FAK antibody was used to confirm the loading of equivalent amounts of protein. (B) The quantification of FAK phosphorylation is shown as means ± s.e.m.; *P<0.001 compared with control medium without MnCl2; n=3. phosphorylated proteins with the anti-pY antibody showed an increase in FAK phosphorylation by more than twofold in western blots with an anti-FAK antibody (Fig. 6A,C). The same result was observed when FAK was immunoprecipitated and phosphorylated proteins Fig. 8. The stimulation of dendrite growth but not growth-cone collapse detected with an anti-pY antibody. depends on FAK. (A) Neurons from wild-type or Fakflox/flox embryos were FAK autophosphorylation at Y397 initiates the binding of SFKs cultured on fibronectin, transfected at 3 d.i.v. with vectors for EGFP-Cre (Mitra et al., 2005). The subsequent phosphorylation of FAK at (green) and mCherry (red) to visualise neuronal morphology, incubated 24 hours after the transfection with control medium or medium containing Y576/577 is required for its full activation. Using antibodies Sema3A for 16 hours, and stained with an anti-MAP2 antibody (blue). Scale specific for individual phosphorylation sites, FAK phosphorylation bar: 100 μm. (B) The length of the dendrites is shown [means ± s.e.m.; n=40- can be detected at both Y397 and Y576/577 in neuronal cultures 80 neurons, three experiments; *P<0.001 compared with control (GFP, on fibronectin as a substrate (Fig. 6B,C). Incubation with Sema3A –Sema3A) or between the values indicated by the bracket]. (C) Neurons from flox/flox increased autophosphorylation at Y397 and phosphorylation at Fak embryos were cultured on fibronectin, transfected at 3 d.i.v. with vectors for EGFP-Cre or EGFP and mCherry, and incubated 48 hours after the Y576/577 approximately twofold (Fig. 6C) (Y397: increase by a transfection with control medium (–) or medium containing Sema3A (+) for 30 factor of 1.9±0.2; Y576/577: 1.8±0.2; n=3). The increase in FAK minutes. The percentage of collapsed axonal growth cones is shown as means phosphorylation at Y397 was also observed by immunofluorescence ± s.e.m.; n=40-80 neurons, three experiments; *P<0.001 compared with (supplementary material Fig. S6). To investigate whether the control (GFP, –Sema3A). increase in FAK phosphorylation depends on integrin activation, neurons were cultured on PO. Sema3A had no effect on FAK phosphorylation when PO was used as a substrate (Fig. 7). When neurons from Fakflox/flox mice (Beggs et al., 2003) were cultured neurons were cultured on PO in the presence of Mn2+ ions, Sema3A on fibronectin and transfected with an expression vector for significantly increased phosphorylation at Y397 and Y576/577. FAK EGFP-Cre (pBS505) at 3 d.i.v. to generate FAK-deficient cells and phosphorylation at Y576/577 was only slightly increased when the a vector for mCherry to visualise neuronal morphology (Fig. 8). neurons were treated with Mn2+ ions alone. These results show that After 24 hours, Sema3A was added for 16 hours and cells were the stimulation of FAK activity by Sema3A depends on the stained with an anti-MAP2 antibody. The development of dendritic simultaneous activation of integrins. arbours was not reduced in untreated FAK-deficient neurons in comparison to wild-type neurons (control: 58±2 μm; Fak–/–: 54±5 Sema3A requires FAK to stimulate dendrite growth μm). However, the stimulation of dendrite growth by Sema3A was To test whether FAK is required for the extension of dendrites or blocked in FAK-deficient neurons (Fig. 8) (control: 73±4 μm; the stimulation of dendrite growth by Sema3A, hippocampal Fak–/–: 51±5 μm). To investigate whether FAK is also required for 2040 Journal of Cell Science 122 (12) the collapse of axonal growth cones by Sema3A, the percentage FAK is mediated by SFKs such as Fyn (Mitra et al., 2005). Sema3A of collapsed axonal growth cones was determined after incubation activates SFKs and Cdk5, which could mediate the activation of of FAK-deficient neurons with Sema3A for 1 hour at 3 d.i.v. (Fig. FAK also in hippocampal neurons (Brown et al., 2004; Mitsui et al., 8C; supplementary material Fig. S7). Sema3A induced the collapse 2002; Morita et al., 2006; Sasaki et al., 2002; Uchida et al., 2005). of FAK-deficient hippocampal growth cones to an extent Fyn is also required for the effect of Sema3A on cortical dendrites comparable to that of control neurons. It has been previously and Cdk5 has been shown to phosphorylate FAK in neurons (Morita reported that FAK is required for the collapse of growth cones et al., 2006; Sasaki et al., 2002; Xie et al., 2003). A stimulation of from cortical axons (Bechara et al., 2008). These results were based FAK has also been reported for netrin-1 and ephrin-A1 (Carter et on the effect of overexpressing an inactive FAK mutant (FAK- al., 2002; Li et al., 2004; Liu et al., 2004; Ren et al., 2004). However, R454). When we expressed this mutant in murine hippocampal the effect of netrin-1 did not require integrin function. neurons, we also observed that FAK-R454 blocked growth-cone The adhesion molecule L1 can serve as an alternative signalling collapse by Sema3A (supplementary material Fig. S8). Because receptor for Sema3A (Castellani et al., 2002). Recently, it was neurons from Fak-knockout mice still show a collapse of growth reported that Sema3A activates FAK in an L1-dependent manner cones after incubation with Sema3A, FAK-R454 does not appear in cortical neurons during the collapse of axonal growth cones to act by inhibiting endogenous FAK. Thus, FAK is not required (Bechara et al., 2008). In these experiments, expression of an for the effect of Sema3A on axonal growth cones but is essential inactive FAK mutant in cortical neurons blocked growth-cone for the stimulation of dendrite growth by Sema3A. collapse by Sema3A. Our results show that, although expression of the inactive mutant FAK-R454 blocks growth-cone collapse also Discussion in hippocampal neurons, inactivation of the Fak by a Sema3A acts as a repellent for axons and an attractant or growth- conditional knockout does not. The difference in the effects of FAK- promoting signal for dendrites (Campbell et al., 2001; Fenstermaker R454 expression and genetic inactivation of Fak demonstrates that et al., 2004; Gu et al., 2003; Polleux et al., 1998; Polleux et al., FAK-R454 does not block the function of endogenous FAK. The 2000; Sasaki et al., 2002; Song et al., 1998). However, it has been effect of FAK-R454 might result from sequestering and inhibiting unclear so far whether both activities of Sema3A impinge on the the function of FAK-interacting proteins that have a second, FAK- same pathway, or whether different signalling cascades are involved independent function to mediate growth-cone collapse downstream in its effects on axons and dendrites (Kruger et al., 2005). Here we of the Sema3A receptor. show that the requirement for integrin engagement distinguishes It has been shown previously that an increase in the concentration the response of axons and dendrites to Sema3A in hippocampal of cGMP can convert the repellent effect of Sema3A to an attraction neurons. Sema3A induces the collapse of axonal growth cones but or block the collapse of sensory growth cones (Castellani et al., 2002; stimulates the extension of dendrites. Defects in the formation of Song and Poo, 1999). In addition, an involvement of cGMP synthesis dendritic arbours were observed in the hippocampus of Sema3a–/– by the guanylyl cyclase Gyc76C was demonstrated genetically in mice and in cultures of Sema3a–/– neurons. The addition of Sema3A Drosophila, in which Gyc76C facilitates the repulsion of plexin-A- to cultured neurons stimulates dendrite growth. This effect depends expressing axons by Sema1a (Ayoob et al., 2004). In cultured on β1 integrins and FAK, which both are dispensable for the collapse Xenopus spinal-neuron growth cones, Sema3A triggers cGMP of axonal growth cones by Sema3A. These results reveal a production, which activates Ca2+ channels (Togashi et al., 2008). bidirectional interaction of integrins and semaphorin signalling. β1 Our results show that Sema3A induces the production of cGMP in integrins facilitate the effect of Sema3A on dendrites, whereas both axons and dendrites from hippocampal neurons, which is integrins are inhibited by Sema3A during growth-cone collapse necessary but not sufficient for the response of hippocampal dendrites (Kruger et al., 2005; Mikule et al., 2002; Toyofuku et al., 2005) to Sema3A. However, cGMP production does not depend on integrin and in endothelial cells (Barberis et al., 2004; Serini et al., 2003). activation and, unlike Mn2+ ions, cGMP is not able to substitute for Sema3A stimulates dendrite extension only when neurons are integrin activation. Thus, cGMP acts as a second messenger but not cultured on the integrin ligand fibronectin. Fibronectin could be as a modulator of the response to Sema3A in dendrites. substituted by Mn2+ ions, which stabilise the active conformation In Sema3a–/– mice, apical dendrites branch prematurely in the of integrins (Gailit and Ruoslahti, 1988; Mould et al., 1995; Takagi stratum radiatum and these branches show irregular trajectories. et al., 2002), allowing Sema3A to promote dendrite extension on However, we did not observe a significant effect on the orientation the non-specific adhesive PO. A soluble RGD peptide that blocks of apical dendrites before they branch, suggesting that Sema3A the interaction with integrins and deletion of the Itgb1 gene probably does not act as an attractant in the hippocampus. The abrogated the stimulatory effect of Sema3A. By contrast, the abnormal organisation of apical dendrites could result from the collapse of axonal growth cones was independent of integrin absence of a repellent signal in the stratum radiatum that restricts engagement. Unlike the effect of cyclic nucleotides on the response branching to the stratum lacunosum-moleculare. However, we did to axon-guidance signals (Campbell et al., 2001; Song et al., 1998), not observe any inhibitory effect of Sema3A on dendritic branching β1-integrin engagement does not modulate the response of dendrites in cultured hippocampal neurons. In addition, Sema3A does not because Sema3A had no effect on dendrite length after inactivation show a layer-specific expression in the hippocampus (Chedotal et of Itgb1 or Fak. Our results show that the effect of Sema3A on al., 1998; Pozas et al., 2001; Skaliora et al., 1998). Therefore, we dendrite growth requires a signalling pathway that is distinct from think that the absence of Sema3A reduces the growth of apical that involved in collapse of the growth cone. dendrites resulting in a shift in the position of the branching point. Sema3A and β1-integrin signalling converge on FAK to stimulate The Sema3a–/– phenotype in the hippocampus is similar to that dendrite growth. Sema3A increases FAK phosphorylation at Y397 reported for cortical neurons in some aspects (Polleux et al., 1998; and Y576/577 only when integrins are activated simultaneously. Polleux et al., 2000; Sasaki et al., 2002). Apical dendrites are Deletion of Fak blocked the stimulation of dendrite growth but did disorientated and deviate from their normal orientation of being not prevent the collapse of growth cones. The phosphorylation of perpendicular to the pial surface in the cortex of Sema3a–/– mice Sema3A, FAK and integrins 2041

(Polleux et al., 2000). However, there is no evidence that Sema3A peroxidase and an immobilised anti-cGMP antibody. The concentration of intracellular acts as an attractant signal in the hippocampus. cGMP was calculated based on the amount of bound cGMP-coupled peroxidase. In Figs S1-S8 in the supplementary material, neurons were incubated at 3 d.i.v. In summary, our results provide evidence for a new integrin- and with medium containing CMFDA (16 µM, Invitrogen) for 30 minutes, fixed with FAK-dependent signalling pathway that mediates the stimulation 4% paraformaldehyde and stained with an anti-cGMP antibody (Chemicon). The of dendrite growth by Sema3A but is not required for growth-cone cGMP immunofluorescence signals for cGMP and CMFDA in the growth cones of dendrites and axons were measured using ImageJ (NIH) and the signal for cGMP collapse. normalised to that of CMFDA to determine the relative level of cGMP in arbitrary units (a.u.). To analyze FAK phosphorylation, neurons were stained with an anti- Materials and Methods phospho-FAK (Y397) antibody (BD Bioscience). Plasmids The vectors for dominant-negative Nrp-1, plexin-A1 or plexin-B1 were described Immunoprecipitation and western blot previously (Rohm et al., 2000b; Serini et al., 2003). Neurons were transfected with Mouse hippocampal neurons from E18 were cultured on fibronectin (Roche) or PO pEGFP-N3 (Clontech) or pmCherry to visualise their morphology. The expression (Sigma). After 4 days in culture the cells were incubated with Sema3AP1b for 20 vector for monomeric Cherry (pmCherry) was constructed by introducing BamHI minutes. In some cases the neurons were incubated with 1 mM MgCl2 before and and NotI restriction sites into the sequence of mCherry by PCR using pRSET-b- during treatment with Sema3A. The neurons were lysed in 50 mM HEPES buffer, mCherry (Shaner et al., 2004) as template and cloning the resulting fragment into pH 7.4, 1% NP-40, 1% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 1.5 mM MgCl2 pEGFP-N3 (Clontech) to replace the coding sequence of EGFP. The vector for EGFP- and 10% glycerol including protease inhibitor cocktail (complete, Roche) and Cre (pBS505; Addgene plasmid 11955) has been described before (Le et al., 1999). phosphatase inhibitors (1 mM Na3VO4 and 50 mM NaF). The cleared lysates were The vector for FAK-R454 (see supplementary material Figs S1-S8) has been diluted 1:1 with 50 mM HEPES, pH 7.4, 1% NP-40, 150 mM NaCl, 1.5 mM MgCl2 described before (Sieg et al., 1999). and 10% glycerol including the inhibitors, and proteins were immunoprecipitated for 1 hour at 4°C with anti-FAK (BD Bioscience) or anti-phospho-tyrosine (Cell Mutant mice Signaling) antibodies, and analysed by western blot using anti-FAK, anti-phospho- Mouse strains with floxed alleles of the genes encoding FAK (Ptk2tm1Lfr; MMRRC FAK (Y397) (BD Bioscience), anti-phospho-FAK (Y576/577), anti-phospho-tyrosine 009967-UCD) and β1 integrin (Itgb1) have been described previously (Beggs et al., (Cell Signaling), anti-α-tubulin (Sigma) and horseradish-peroxidase-coupled 2003; Potocnik et al., 2000). Sema3a+/– mice were backcrossed to CD1 mice for more secondary antibodies (Dianova). Chemiluminescence (Uptilight HRP Blot than five generations and genotyped as described (Serini et al., 2003; Taniguchi et Chemiluminescent Substrate, Uptima) was detected using the LAS-1000 luminescent al., 1997). Although most Sema3a–/– mutants die around birth in the C57/B6 image analyser (Fuji). The signal intensity was determined for each band using the background, a few mutant mice survive for several weeks in the CD1 background. Image Gauge 3.12 software (Fujifilm). The background was measured for an area Tg(Thy1-YFPH)2Jrs/J mice (Thy1-YFPH) (Feng et al., 2000) were obtained from of identical size and subtracted from the value for each band. The corrected signal the Jackson Laboratory (Bar Harbor, MA) and crossed with Sema3a+/– mice to obtain intensity for phosphorylated FAK was normalised to the signal for total FAK or α- Thy1-YFPH+/+;Sema3a+/– mice. For the analysis of neuronal morphology, the brains tubulin. The value for the control experiment was set to 1.0 and changes in the level were dissected from P15 Thy1-YFPH+/0;Sema3a–/– mice, fixed overnight in 4% of phosphorylated FAK calculated relative to the value of the control. paraformaldehyde/phosphate-buffered saline (PBS), and coronal sections (60 μm) cut with a vibratome (Leica). Genotyping was performed as described (Beggs et al., 2003; Feng et al., 2000; Potocnik et al., 2000; Schwarting et al., 2000). We thank Takeshi Yagi and Masahiko Taniguchi for providing us with Sema3a+/– mice, Roger W. Tsien for providing the pREST- Neuronal cultures, transfection and immunofluorescence mCherry, David D. Schlaepfer for the FAK-R454 construct, and Lena Cultures of dissociated hippocampal neurons were prepared and transfected as Will for constructing pCherry. The GFP-Cre (pBS505) construct described previously (Schwamborn et al., 2006). Briefly, the hippocampus was generated by Brian Sauer was obtained through addgene. This work dissected from E18 rat or mouse embryos, dissociated, and neurons were plated onto was supported by grants from the Deutsche Forschungsgemeinschaft coverslips coated with PO (Sigma) or fibronectin (Roche) at a density of 40,000 cells per coverslip in a 24-well plate and cultured in Neurobasal medium (Invitrogen) (SFB 492-B14) to A.W.P. and a fellowship from the Boehringer supplemented with B27 (Invitrogen). A stably transfected HEK293 cell line (cell line Ingelheim Fonds (J.C.S.). 293-717) was used to produce AP-Sema3AP1b (Zanata et al., 2002). The 293-717 cells were incubated with serum-free medium for 48 hours and the conditioned References medium was concentrated with 100 kDa centrifugal filter devices (Millipore). The Ayoob, J. C., Yu, H. H., Terman, J. R. and Kolodkin, A. L. (2004). The Drosophila concentration of Sema3AP1b was measured by an alkaline phosphatase assay with receptor guanylyl cyclase Gyc76C is required for semaphorin-1a-plexin A-mediated para-nitrophenol (Sigma) as a substrate (Rohm et al., 2000a). Hippocampal neurons axonal repulsion. J. Neurosci. 24, 6639-6649. were incubated with equal amounts of Sema3AP1b as quantified with the phosphatase Barberis, D., Artigiani, S., Casazza, A., Corso, S., Giordano, S., Love, C. A., Jones, assay with a final concentration of approximately 10 nM. Medium conditioned by E. Y., Comoglio, P. M. and Tamagnone, L. (2004). Plexin signaling hampers integrin- HEK293T cells was used as control medium. DMSO, ODQ, Nω-Nitro-L-arginine based adhesion, leading to Rho-kinase independent cell rounding, and inhibiting methyl ester (L-NAME), 8-Br-cGMP, MnCl2 and the RGD peptide (all Calbiochem) lamellipodia extension and cell motility. 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