, 1998). Thus, VEGF chemoattracts commissural axons www.selleckchem.com/products/iwr-1-endo.html through Flk1, with a negligible or redundant role for other Flk1 ligands (VEGF-C) or activators (Sema3E), or VEGF receptors (Npn1). Curiously, motor columns express Vegf mRNA, yet neither vessels nor commissural axons invade these structures ( James et al., 2009). It is possible that motor neurons make the Vegf message (mRNA), but do not secrete the protein from their cell body, and target it to their axonal compartment (as is thought to occur for Slit2; M.T.-L. and A. Jaworski, unpublished data). Another alternative

explanation is that additional signals prevent blood vessels and commissural axons from entering the motor columns. VEGF was originally discovered as a key angiogenic factor. Only subsequent studies revealed that this factor can affect neurons directly, independently of its angiogenic activity (Rosenstein et al., 2010, Ruiz de Almodovar et al., 2009, Ruiz de Almodovar et al., 2010 and Tam and Watts, 2010). In the developing spinal cord, VEGF orchestrates the formation of the neurovascular plexus and subsequent vessel sprouting from this plexus into the avascular AT13387 neural tube (James et al., 2009). Interestingly, however, even though vascularization of the neural tube occurs at the same time as commissural axon midline crossing, our conditional Flk1 inactivation

studies in commissural neurons and in vitro turning assays establish that VEGF chemoattracts

these axons independently of any VEGF-related vascular activity. To the best of our knowledge, this is the first report documenting an angiogenesis-independent effect of VEGF on axon guidance. The VEGFLacZ mouse line was kindly provided by A. Nagy and was previously described about ( Miquerol et al., 1999). VEGF-C knockout mice were previously described ( Karkkainen et al., 2004), and the VEGFlox/lox mouse line was kindly provided by D. Anderson and previously described ( Gerber et al., 1999). The transgenic Wnt1-Cre mouse line was kindly provided by A. McMahon. The transgenic Hoxa1-Cre mouse line was generated by A. Chedotal using a previously described cDNA ( Li and Lufkin, 2000). The Flk1lox/LacZ mouse line was generated by crossing Flk1lox/lox mice ( Haigh et al., 2003) with Flk1LacZ/+ mice ( Maes et al., 2010). For each transgenic line, WT littermate embryos were used. Wistar or Sprague Dawley rat embryos (E13) were used for explant outgrowth assays, purification of commissural neurons and for immuno-histochemistry. All animals were treated according to the guidelines approved by the Animal Care Committees of the K.U.Leuven (Belgium) and of the IRCM (Canada). Commissural neurons were prepared from the dorsal fifth of E13 rat neural tubes as described (Langlois et al., 2010 and Yam et al., 2009).