2034 Research Article The stimulation of dendrite 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 axon and dendrite growth depend on facilitate the stimulation of dendrite extension. Conditional multiple guidance signals and growth factors. Semaphorin 3A inactivation of the genes encoding β1 integrin or FAK blocks (Sema3A) acts as a repellent for axons and attractant for the growth-promoting effect of Sema3A but not the collapse of dendrites. 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 neurons. 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 proteins and semaphorins 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 plexin 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). Plexins 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 growth cone. (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.