However, when Sema3E and VEGF were added together, almost no migration was observed (Figures 4K and 4L), indicating that Sema3E inhibits VEGF-induced migration. Moreover, Sema3E also blocked the basal level of migration (Figure 4I). Together, these results support the idea that Sema3E can dominantly block the attractive effects exerted upon Plexin-D1-expressing endothelial cells. The appealing hypothesis that Protein Tyrosine Kinase inhibitor emerged, based on these in vitro results, is that Sema3E acts as a repulsive guidance cue for both trigeminal axons and blood vessels and serves to organize the double ring neurovascular structure

surrounding the follicle. If this hypothesis is true, what accounts for the relative position of the nerves and vessels, with the nerve ring consistently positioned inside of the vessel ring? In particular, it is puzzling how trigeminal axons expressing Plexin-D1 are able to innervate an area that is so close to a secreted repulsive cue. One potential possibility is that the abundance of Plxnd1 mRNA transcripts in the

TG may not reflect protein levels at the nerve terminal. To test this idea, we performed AP-Sema3E binding on tissue sections to detect Plexin-D1 protein along the trigeminal nerve ( Chauvet et al., 2007 and Gu et al., 2005). As shown in Figure 5A, although Plexin-D1 protein is highly expressed in the trigeminal nerves projecting to the whisker follicle, surprisingly, Plexin-D1 protein is very low in the nerve terminals (white arrowheads Tyrosine Kinase Inhibitor Library order in Figures 5B and 5C). This difference is not due to our inability to detect protein binding at the nerve terminal, because Npn-1 protein is equally well detected in both the projecting axons and the nerve terminal by AP-Sema3A binding (arrowheads in Figures 5D and 5E). The absence of AP-Sema3E binding on the vessel ring (red arrow in Figure S3C) of Plxnd1 null mice further next confirmed that Plexin-D1 is the only receptor for Sema3E in the vessel ring ( Figures S3A–S3D). The absence of AP-Sema3E binding on the nerve ring in Sema3e

null mice also ruled out the possibility that the lack of AP-Sema3E binding may be due to the sequestration of Plexin-D1 by endogenous Sema3E ( Figures S3E–S3H). To further confirm the selective downregulation of Plexin-D1 protein in the TG nerve terminals, we also performed anti-Plexin-D1 immunohistochemistry ( Chauvet et al., 2007). Consistent with the AP-Sema3E binding result, TG nerve terminal exhibited extremely low Plexin-D1 immunoreactivity ( Figures 5F and 5G). Therefore, the Plexin-D1 protein is selectively downregulated in the TG nerve terminals, which enables the nerves to innervate areas close to the Sema3E-expressing region and form the inner ring. In stark contrast to the low expression of Plexin-D1 in the nerve ring, the Plexin-D1 protein level visualized by both AP-Sema3E and anti-Plexin-D1 antibodies in the blood vessels is remarkably high (white arrows in Figures 5B, 5C, 5H, and 5I).