Role of Shh in Disc Maintenance Figure 8. The molecular markers of the AF are also reversed by rShh after cyclopamine treatment. Each row shows the staining pattern, and each column shows the treatment. P4 t5 Veh = five day vehicle treated control, P4 t5 CycA = five days in cyclopamine, P4 t5 CycA+ rShh = two days in cyclopamine followed by three days in rShh. (A) shows by H&E staining that loss of AF cell polarity is reversed by replacement of cyclopamine with rShh. (B)–(F) shows that expression of Gli1, Sox9, collagen 1, collagen 2, and chondroitin sulfate, respectively, are also reversed. Scale bars indicate magnifications used. IAF = inner annulus fibrosus, OAF = outer annulus fibrosus. Red = expression of the specific protein, Blue = cell nuclei stained with POPO-3, green = general counterstain with wheat germ agglutinin. See text for details. doi:10.1371/journal.pone.0035944.g008 pathways are controlled by Shh, how it does this, and how the major signaling pathways intersect to control the holistic postnatal growth and differentiation of the disc. For example, we show here that BMP and Wnt signaling are up-regulated by Shh blockade, and it has been shown that, during avian somite patterning, the BMP inhibitor noggin acts downstream of both Shh and Wnt signaling to inhibit BMP4 activity [27]. It will be interesting to see if the same mechanism is acting in the IVD. It will also be important in the future to elucidate the links between systemic growth signals and the local signals produced in the disc. It is likely, for example, that Shh expression by the NP cells, or its action on other signaling pathways, or both, are controlled by systemic signals which coordinate all the NP’s along the vertebral column so that the discs all grow and differentiate at the same rates and times [exemplified by the two discs shown in (Fig. 11)]. Most importantly, these data show that it may be PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e35944