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 differentiation 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 
. 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. () 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 
. Shh is also importnat for the formation of the notochord sheath and patterning of embryonic nucleus pulposus cells in the embryo 
. 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 other (or themselves). Many of the effects of cyclopamine are on the NP cells that synthesize the Shh. Interestingly, cyclopamine treatment resulted in loss of Shh expression in the NP cells, suggesting that a feedback loop controls Shh protein expression in the disc. Although we did not identify all the functions of Shh in the IVD, nor all of its targets, we found that these include the maintenance of differentiation markers in both the NP and the AF, as well as cell proliferation in the NP. In the future it will be important to identify all the targets of Shh signaling.
Control of the EP is likely to be particularly interesting, as it is sandwiched between cells releasing Shh (the NP) and Ihh (the growth plate). In this study, we found that expression of Gli1, Sox9, and collagen 2 were reduced after in vivo targeting of Shh expression at P5. However, previous work has shown that the EP is also affected by Ihh targeting in the growth plate 
. Based on these combined data, it is likely that the EP is controlled by both signaling sources. Whether this is a quantitative or qualitative difference in signal strength will require further experiments.
Third, the pleiotropic effects of Shh signaling found here seem to be due in part to its regulation of transcription factors, such as Sox9, known to regulate the synthesis of several extracellular matrix components such as collagen 1, collagen 2 and chondroitin sulfate 
and in part due to its control of other signaling pathways in the IVD. Blockade of Shh caused the down-regulation of TGFβ signaling, and up-regulation of BMP and Wnt signaling. In the future, it will be important to identify precisely which other 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 
. 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 ()]. Most importantly, these data show that it may be possible to control both growth and differentiation of diseased discs, by the application of appropriate agonists and antagonists of the signaling pathways found to carry out specific functions in vivo.