Role of Shh in Disc Maintenance Figure 4. The effects of Shh targeting in vitro and in vivo on the expression of molecular markers in the AF. 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). The rows shows the expression, by immunocytochemistry, or Gli1 (A) and (B), Ptch1 (C) and (D), Sox9 (E) and (F), collagen 1 (G) and (H), collagen 2 (I) and (J), chondroitin sulfate (K) and (L), and keratin sulfate (M) and (N). The expression of all these markers was reduced after treatment with cyclopamine in vitro, or Shh targeting in vivo. Scale bars indicate magnifications used. IAF = inner annulus fibrosus, OAF = outer annulus fibrosus. Blue = cell nuclei stained with POPO-3, green = general counterstain with wheat germ agglutinin. See text for details. doi:10.1371/journal.pone.0035944.g004 culture, and replaced it with 100 ng/mL recombinant Shh (rShh) for three further days (P4 t5). The effects of Shh blockade on the NP were reversed by this treatment, including the reticular structure of the NP cells (Fig. 7A), expression of Gli1 (Fig. 7B) and Bra (Fig. 7C), Sox9 (Fig. 7D) chondroitin sulfate (Fig. 7E, compare with Fig. 3K), and collagen 1 [(Fig. 7F), compare with (Fig. 3M)]. Addition of recombinant Shh also reversed the effects of cyclopamine on the AF, including the histological structure (Fig. 8A), expression of Gli1 (Fig. 8B), Sox9 (Fig. 8C), collagen 1 (Fig. 8D), collagen 2 (Fig. 8E) and chondroitin sulfate (Fig. 8F). These data show that the observed effects were due to loss of Hh signaling, rather than non-specific effects of the cyclopamine treatment. Cultures from which cyclopamine was removed, but not replaced by Shh [(Figure S4); P4 t2 CycA t3–5 Veh], did not recover significantly within the three day period. PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35944