Role of Shh in Disc Maintenance Figure 3. The effects of Shh blockade in vitro and Shh targeting in vivo of molecular markers of NP differentiation. The panels are arranged as in a table. Each vertical column shows a treatment (two or five day vehicle treated control [P4 t2 or 5 Veh] and two or five days in cyclopamine [P4 t2 or 5 CycA] from the in vitro experiments; and control Shh flox [P5 d5 Shhflx/flx; R26rtTA] and Shh mutant [P5 d5; Shhd/d(tetO)7-Cre; R26rtTA] mice from the in vivo experiments). Each horizontal row shows the expression, by immunocytochemistry, of Gli 1 (A) and (B), Ptch1 (C) and (D), Shh (E) and (F), Brachyury (G) and (H), Sox9 (I) and (J), chondroitin sulfate (K) and (L), and collagen 1 (M) and (N). All the above markers show reduced expression after treatment with cyclopamine in vitro, or targeting of Shh in vivo. Scale bars indicate magnifications used. Blue = nuclei stained with POPO-3, green = counterstain with wheat germ agglutinin. See text for details. doi:10.1371/journal.pone.0035944.g003 dissection (Fig. 6B and B9), and the presence of Hh staining in all components of the IVD after removal of the hyptertrophic zone and culture for 2 days (Fig. 6C and C9). The expression of Gli1, and the differentiation markers Bra, Sox9, collagen 2, and chondroitin sulfate continue to be at normal levels after culture for 2 days in the absence of the growth plate (Fig. 6D–I). Similar pattern of histology and expression levels of differentiation markers were observed in the P4 IVDs cultured for five day (P4 t5), from which the hypertrophic zone was dissected prior to culture (Figure S3). This data supports the conclusion from in vivo targeting of Shh that IVD’s are responding to Shh secreted by the NP. Of course we cannot exclude the possibility that some aspects of IVD differentiation are controlled by Ihh, since not all differentiation markers were assayed. Recombinant Shh rescues the cyclopamine phenotype in vitro As an additional control for non-Hh off-target effects of cyclopamine in vitro, we washed out cyclopamine after two days PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35944