Role of Shh in Disc Maintenance Figure 6. The effects of removal of the hypertrophic zone of the vertebral growth plates (-HZGP) from IVDs before culture. (A) shows their histology after two days in culture. (B–B9) shows low and high magnification immunostaining for both Shh and Ihh, using an antibody that stains both, in the growth plate chondrocytes (GP) and EP before culture. (C–C9) shows low and high magnification images respectively of 48 hour cultures to show that Hh staining is still seen in the EP in the absence of the growth plate. (D–I) show normal levels of expression of Shh, Gli1, Bra, Sox9, collagen 2, and chondroitin sulfate, in discs cultured for 48 hour in the absence of the growth plate. Scale bars indicate magnifications used. Red = expression of the specific protein, Blue = cell nuclei stained with POPO-3, green = general counterstain with wheat germ agglutinin. NP = nucleus pulposus, AF = annulus fibrosus, EP = end plate, GP = growth plate. See text for details. doi:10.1371/journal.pone.0035944.g006 described above, either in vehicle only, or cyclopamine, for five days (P4 t5). We consistently see a reduction in TOPGAL expression during culture (not shown), suggesting that canonical Wnt signaling requires systemic signaling, which will be absent in culture. However, when Hh signaling was blockaded, Wnt signaling was upregulated in both AF and EP cells (Fig. 10G). These data suggest that Shh signaling regulates other major cell signaling pathways in the IVD during active growth and differentiation. Discussion The data presented here show that cell signaling intrinsic to the IVD controls both cell proliferation in the NP, and the expression of disc differentiation markers in the IVD. The culture periods were not systematically extended to see how long normal disc structure could be maintained in the absence of exogenous signals. However, it was interesting to note that, although disc differen- tiation was maintained in culture, it did not progress as it would have done in vivo. Discs cultured for five days maintained the structure they had at P4 t0, rather than attaining the structure and size they would have reached by P9. In addition, cell proliferation in the NP’s of cultured discs did not continue past five days in vitro (the equivalent of P9 in vivo), but normally extends to P14 before ceasing in vivo [3]. These data suggest that signaling pathways within the disc are in turn controlled by signals from the circulation to control the continued progression of disc growth and differentiation. (Fig. 11) shows a schema of this proposed mechanism. There is also a possibility that the reduced growth of the IVD in longer duration of culture can be due to lack of nutritional factors in the culture medium. Second, Shh is a major signaling pathway controlling IVD growth and differentiation. Shh has been shown previously by our lab and others to be synthesized in the NP [3,7–10]. Shh is also importnat for the formation of the notochord sheath and patterning of embryonic nucleus pulposus cells in the embryo [23]. However, neither its role, nor the extent of its importance in IVD postnatal development have been previously appreciated. A particularly interesting finding was that NP cells signal to each PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35944