Figure 5. The effects of deletion of Shh signaling on the end plate of the IVDs in vivo. (A) shows H&E staining and histology of EP following doxycycline treatment of the control [P5 d5 Shhflx/flx; R26rtTA] and Shh mutant mice [P5 d5; Shhd/d(tetO)7-Cre; R26rtTA]. (B)–(D) shows reduced expression of Gli1, Sox9 and collagen 2 expression in the EP cells and the inner layer of AF following Shh deletion in vivo. (E) shows that Ihh continues to be expressed in the growth plate chondrocytes of the Shh mutant as well as in the control mice, while hedgehog (Hh) expression was undetectable in the EP cells. (F) shows that there is no effect on BrdU incorporation (Brown = BrdU positive cells, cell nuclei stained with nuclear fast red) in the growth plate following Shh deletion, compared to the control mice. Scale bars indicate magnifica- tions used. Red = expression of the specific protein, Blue = cell nuclei stained with POPO-3, green = general counterstain with wheat germ agglutinin. EP = end plate, GP = growth plate. See text for details. doi:10.1371/journal.pone.0035944.g005 Role of Shh in Disc Maintenance Shh signaling is required for postnatal proliferation of NP cells To test the role of hedgehog signaling on cell proliferation discs cultured for two or five days with or without cyclopamine were treated with bromo-deoxyuridine (BrdU) 24 hours before being snap-frozen, sectioned, fixed and stained for incorporated BrdU. Many NP cells were BrdU-positive in vehicle-treated control IVDs after two days, but not after five days, in culture (Fig. 9A and B). In vivo, cell proliferation continues until P14–21 [3]. The discrep- ancy between in vitro and in vivo data suggests that continued cell proliferation in the NP requires continued systemic signals from the circulation. Few, if any labeled cells were found in the AF or EP (data not shown). (Fig. 9A) shows an example of the effect of cyclopamine after two-day culture compared to a vehicle-treated control disc. No BrdU incorporation in the NP was seen after cyclopamine treatment. (Fig. 9B) shows the percentage of BrdU- positive NP cells from the different experimental conditions, assayed by expressing the number of BrdU-positive cells as a percentage of the total number of nuclei in each section. A total of twelve sections were analyzed from four discs for each treatment. The error bars represent the standard deviation to show the variation in different experimental samples. The p-value was calculated between two groups to compare the effects of Shh blockade or rescue using the unpaired student’s t-test. BrdU incorporation was significantly (p#0.001) inhibited by incubation in cyclopamine, indicating that Shh signaling controls, either directly or indirectly, the proliferation of the NP cells. Replacing cyclopamine with rShh, followed by three days of further culture, caused significant increase (p#0.001) in the cell proliferation of NP compared to the NP cells from both the P4 t2 vehicle treated as well as P4 t2 cyclopamine treated IVDs. However, cell pro- liferation was not rescued back to control levels by Shh replacement in vitro. BrdU incorporation was also inhibited in the in vivo experiments using the Shh mutant mice [see P5 d5 Shh flx/flx compared with P5 d5 Shh d/d bars in (Fig. 9B), p#0.001]. However, the effect was not as great as seen by cyclopamine treatment in vitro [see P4 t2 Veh compared with P4 t2 CycA bars in (Fig. 9B)]. This could be due to the fact that some Shh protein remains after five days of doxycycline treatment [see (Fig. 3F)]. It could also be due to some level of control by Ihh from the growth plate in vivo. Double in vivo targeting would be required to resolve this point. Shh is the key regulator of other major cell signaling pathways Blockade of Shh signaling in vitro, or targeting in vivo, had a large range of effects on the IVD. This suggests that Shh may either act directly on a large number of downstream targets, or may act indirectly by controlling other signaling pathways. To distinguish between these possibilities, we assayed for the levels of activity of different signaling pathways in IVD’s treated with cyclopamine or from in vivo targeting studies, and compared these with treatment controls. For BMP signaling we used an antibody against the phosphorylated forms of Smad 1, 5, and 8 [21]. BMP signaling was increased in all components of IVD following both in vivo and in vitro loss of Shh signaling (Fig. 10A–C). For TGFb signaling, we used an antibody against the phosphorylated forms of Smad 2, 3 [22]. TGFb signaling was reduced in IVD cells following both in vitro and in vivo blockade of Shh signaling (Fig. 10D–F). To analyze the affects of loss of Shh signaling on canonical Wnt signaling pathway in vitro, IVDs from TOPGAL reporter mice were cultured under the same conditions and duration as PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35944