Role of Shh in Disc Maintenance Figure 2. The effects of Shh blockade in P4 cultured discs and of Cre-mediated Shh targeting at P5. (A – C) show the whole disc (A), the NP (B), and AF (C) respectively of a normal disc immediately after explantation (P4 t0). (D–G) show the NP and AF respectively after 2 days culture in vehicle only (D and E), or 250 mM cyclopamine (F and G). (H–K) show the NP and AF respectively 5 days after doxycycline treatment at P5 of Shhflx/flx; R26rtTA mice (H and I) or of Shhflx/flx; (tetO)7-Cre; R26rtTA mice resulting in Cre-mediated targeting of Shh in (J and K). Both cyclopamine in vitro and Shh targeting in vivo have similar effects: the aggregation of NP cells into the center of the disc (F and J) and loss of polarity of the AF cells (G and K). Scale bars indicate magnifications used. NP = nucleus pulposus, AF = annulus fibrosus (dense peripheral eosin positive collagen layers are arrowed in C), IAF = inner AF, OAF = outer AF, EP = endplate. doi:10.1371/journal.pone.0035944.g002 Loss of Shh signaling affects differentiation markers of EP cells The EP is continuous around the NP with the AF, and so can be considered part of the IVD, but is also immediately adjacent to the proliferative zone of the growth plate, which is known to be controlled by Ihh released from the hypertrophic zone. Thus EP cells could respond to either Shh from the NP, or Ihh from the growth plate, or both. To study this, we assayed the expression of differentiation markers in the EPs after conditional targeting of Shh in vivo. In these specimens, Ihh from the growth plate is still present, but Shh from the NP will be reduced or absent. No major histological differences can been seen in EP of the control versus Shh deleted mice (Fig. 5A). However, Gli1 expression was reduced in the EP, but not in the growth plate chondrocytes, of these animals (Fig. 5B) suggesting that activation of Hh downstream targets in the EP at this stage is at least partially due to Shh from the NP. Sox9 expression was also reduced in the EP cells following loss of Shh, whilst the growth plate chondrocytes showed normal levels of Sox9 expression (Fig. 5C). Collagen 2 expression was lost in the EP cells following loss of Shh signaling (Fig. 5D). This suggests that for these components of their differentiation, the EP cells are controlled by Shh signals from the NP, rather than Ihh signals from the growth plate. Ihh expression is maintained in the growth plates of the Shh-targeted mice (Fig. 5E), and continues to control cell proliferation in the growth plate, as shown by BrdU incorporation (Fig. 5F). This data suggests that the EP cell differentiation is at least partially under the control Shh secreted by the NP cells. It has been shown previously [20] that the EP is affected by targeting of Ihh in the growth plate. It is therefore likely that the EP is controlled by both Hh ligands, as would be expected by its position between the two sources of signaling. Absence of the Ihh-secreting hypertrophic zone of the growth plate in vitro does not affect NP cell differentiation in vitro Cultured IVD’s contained attached cells from the adjacent growth plates. The hypertrophic zone of the growth plate is known to be a source of Ihh, and cyclopamine treatment of cultured IVD’s will thus inhibit both Shh from the NP and Ihh from the growth plate. The in vivo targeting specifically of Shh had the same effects as those of cyclopamine in culture, suggesting that the effects seen in the cultured IVD are specifically due to Shh inhibition. However, in an attempt to dissect the functions of the two Hh’s in vitro, we carefully dissected the hypertrophic zones from isolated P4 IVD’s before culture. After culture for two days, the histological structure of the disc was normal (Fig. 6A). Immunostaining using an antibody against both Shh and Ihh showed the presence of both ligands in the whole IVD before PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35944