To reveal the mechanism governing the neurovascular congruency in the whisker pad system, we first considered the contributions from the target tissues on their coordinated patterning. Emerging evidence suggests that the neural and vascular systems share many similar mechanisms and molecular cues to regulate their development (Adams and Eichmann, 2010, Carmeliet and Tessier-Lavigne, 2005 and Gelfand et al., 2009). Therefore, we next asked whether guidance cues could be found in the whisker follicle, and whether these cues could

function as an organizing signal to coordinately pattern the nerves and vessels into the congruent double ring structure. We first performed expression screening of known guidance molecules in the TG and whisker targets, and found a striking complementary expression pattern of Sema3e and its receptor Plxnd1 in these KU 55933 areas ( Figures 3A–3H). Moreover, the spatiotemporal expression of these molecules coincides with the developmental profile of whisker follicles. Plxnd1 mRNA was expressed at very low levels in the TG neurons at an early stage (E12.5), when axons arrive at the whisker target ( Figure 3A) ( van der Zwaag et al., 2002). Its expression level is significantly increased by E14.5, when trigeminal axons and blood vessels begin to organize the double ring structure

in the whisker follicles ( Figure 3B) and continues through E16.5 and E18.5, when target innervation is refined and a clear double ring structure is apparent ( Figure 3C; Vorinostat cost data not shown). In a manner similar to the temporal profile of Plxnd1 expression in the TG, Sema3e is not expressed at E12.5 in the whisker follicles ( Figure 3E). too Sema3e mRNA is found in the mesenchymal sheath surrounding the hair follicles starting

at E14.5 and continuing to E16.5 and E18.5 ( Figures 3F–3H). To characterize the precise location of Sema3e within the follicle, we performed immunostaining of axons after Sema3e in situ hybridization (ISH) on the same tissue sections and demonstrated that Sema3e was expressed inside the nerve ring ( Figures 3I and 3J). Finally, to examine whether Plxnd1 is also expressed in the outer blood vessel ring, we performed double fluorescence ISH of Plxnd1 and the endothelial cell marker Flk-1. As shown in Figure 3L, mRNA expression of Plxnd1 and Flk-1 completely overlaps within the vessel ring. Together, these expression data suggest that, in the whisker pad, Sema3E expression surrounds the hair follicle ( Figures 3K and 3M), while Plexin-D1 is expressed in the blood vessels and may also expressed in the innervating trigeminal axons based on its mRNA expression in the TG ( Figure 3M). This complementary expression pattern in the target area suggests that Sema3E-Plexin-D1 signaling plays a role in coordinating trigeminal target innervation and the formation of blood vessels into the double ring structure.