Role of Shh in Disc Maintenance Figure 7. The effects of cyclopamine phenotype in NP cells can be rescued and are reversible in vitro. The panels are arranged as a table. Each row shows the staining pattern, and each vertical 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. H&E staining (A) shows collapsed NP cells are reversed after replacing cyclopamine with rShh. (B)–(D) show that the downstream targets of Shh, Gli1, Bra, and Sox9 are all reversed. (E) and (F) show that expression of chondrotin sulfate, and collagen 1 is also reversed. The vehicle treated and cyclopamine treated samples for similar time points have previously shown in (Figure 3K) (chondroitin sulfate) and 3M (collagen 1). 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. See text for details. doi:10.1371/journal.pone.0035944.g007 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 [20]. 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 [24–26] and in part due to its control of other signaling pathways in the IVD. Blockade of Shh caused the down-regulation of TGFb signaling, and up-regulation of BMP and Wnt signaling. In the future, it will be important to identify precisely which other PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35944