In addition to the DH-PH domain that is responsible for its GEF activity, Arhgef7 also contains a CH (calponin homology) and an SH3 (Src homology 3) domain at the N-terminus and proline-rich, GIT1-binding and coiled coil (CC) domains at the C-terminus (Fig.?1a)11,14,17,18. developing cortex. Arhgef7 is a guanine-nucleotide exchange factor (GEF) for Cdc42, a GTPase that has a central role in directing the formation of axons during brain development. However, active Cdc42 was not able to rescue the knockdown of Arhgef7. We show that Arhgef7 interacts with the GTPase TC10 that is closely related to Cdc42. Expression of active TC10 can restore the ability to extend axons in Arhgef7-deficient neurons. Our results identify an essential role of Arhgef7 during neuronal development that promotes axon formation upstream of TC10. Introduction The characteristic six-layered structure of the mammalian neocortex arises by the sequential generation of neurons from neural progenitors located in the ventricular (VZ) and subventricular zone (SVZ) of the embryonic cortex and their subsequent radial migration into the cortical plate1. When they move from the VZ/SVZ into the intermediate zone, the newborn neurons initially have a multipolar morphology with several dynamic neurites1. After forming an axon and a leading process, neurons become bipolar and migrate into the cortical plate. A similar process can be observed in cultures of neurons from the embryonic brain2. Initially unpolarized neurons attach to the culture substrate (stage 1) and extend several neurites (stage 2). Neurons polarize by selecting one of these undifferentiated neurites as the axon (stage 3), which undergoes a rapid extension PPP2R1B and acquires axon-specific markers. Small GTPases play Clenbuterol hydrochloride a crucial role during the transition from a multipolar to a bipolar morphology and the formation of axons1,3,4. Their activity is regulated by GEFs and GTPase activating proteins. The Rap1 GTPases are central regulators of the multi-to-bipolar transition5. They act upstream of Rho family GTPases like Cdc42 that is essential for the establishment of neuronal polarity and axon formation6,7. A knockout of Cdc42 results in an almost complete loss of axons in the cortex. Cdc42 directly regulates actin dynamics through cofilin6. The Par3/Par6 complex in addition couples Cdc42 to the GEFs Tiam1 and Tiam2/Stef that promote axon growth by activating Rac2. Despite its central role for neuronal development and axon formation, very little is known about the GEFs that regulate Cdc42 during neuronal differentiation. The Rho guanine nucleotide exchange factor 7 (Arhgef7) also called Pix (Pax-interacting exchange factor beta) or Cool1 (Cloned out of library 1) and the closely related Arhgef6 (Pix) belong to the Dbl family of Rho GEFs. They contain a DH-PH (DBL and plekstrin homology) domain and activate Rac1 and Cdc428C14. While a knockout of Arhgef6 is viable and shows defects in the immune system, the knockout of Arhgef7 is embryonically Clenbuterol hydrochloride lethal at early stages of development precluding an analysis of its function in the nervous system15,16. In addition to the DH-PH domain that is responsible for its GEF activity, Arhgef7 also contains a CH (calponin homology) and an SH3 (Src homology 3) domain at the N-terminus and proline-rich, GIT1-binding and coiled coil (CC) domains at the C-terminus (Fig.?1a)11,14,17,18. These domains mediate the interaction with multiple binding partners that include p21 activated kinases (Paks), Git1 (G-protein-coupled receptor-interacting protein 1), and Scribble11,14,19,20. Arhgef7 has been implicated in multiple processes including the regulation of focal adhesion maturation, actin dynamics, the remodeling and trafficking of membranes and exocytosis11C14,18,19,21C27. Open in a separate window Figure 1 Expression of Arhgef7 in neurons. (a) A schematic representation of the Arhgef7 domain structure and the major isoforms is shown. DH: DBL homology domain, PH: plekstrin homology domain, CH: calponin homology domain, SH3: Src homology 3 domain, GBD: Git binding domain, CC: Coiled coil domain. (b) The expression of Arhgef7 in Clenbuterol hydrochloride the cortex of E17 mouse embryos and postnatal mice (P0 or P2) was analyzed by Western blot (WB) using an anti-Arhgef7 antibody. The loading of comparable amounts of proteins was verified using and anti-actin antibody. Molecular weights are indicated in kDa. (c) Hippocampal neurons from E18 rat embryos were analyzed at 24?h (stage 2) or 72?h (stage 3) of culture by staining with an anti-Arhgef7 (green), the Tuji 1 or Tau-1 (red) antibodies and CellTracker Blue CMAC (blue) as a volume marker. The scale bars are 20?m and 2 (upper panel) or 5?m (lower panels), respectively. In the nervous system, Arhgef7 acts as regulator of dendrite branching, the formation of dendritic spines28C31 and synaptic structure and function14,32C35. Arhgef7 is localized to presynaptic sites and the postsynaptic density by its interaction with Scribble, Git1/2 and Shank (SH3 and multiple ankyrin repeat domains).