Role of Shh in Disc Maintenance Figure 1. The normal early postnatal disc. The normal structure of the postnatal day four (P4) lumbar IVD are shown. All sections are mid- coronal. (A) shows the general structure of the disc, stained with H&E. B-M show the distribution of molecular markers stained with specific antibodies (red in each case): Brachyury in cell nuclei of the NP (B), Sox 9 in cell nuclei of the NP (C), AF and EP (D), Keratin 19 in the cytoplasm of NP cells (E and E9), collagen 1 (Col1) in NP and outer part of the AF (F), collagen 2 (Col2) in the superficial region of the EP and outer AF (G), chondroitin sulfate (Ch SO4) in both the NP, AF and EP (H), keratin sulfate (Ker SO4) in the outer AF (I), aggrecan in the NP only (J), Shh in the NP only (K), patched-1 (L) and Gli1 (M) in many regions of the IVD and growth plate. Scale bars indicate magnifications used. Green = wheat germ agglutinin, blue = nuclei stained with POPO-3, NP = nucleus pulposus, AF = annulus fibrosus, IAF = inner AF, OAF = outer AF, EP = endplate. For details, see text. doi:10.1371/journal.pone.0035944.g001 and five days in vivo, showing that the treatments were effective. Once again, the reduction was greatest in vitro (Fig. 4A–D). The AF does not express Bra, but the expression of Sox9 was reduced by both treatments (Fig. 4E and F). Expression of the glycoproteins collagen 1 (Fig. 4G and H) and collagen 2 (Fig. 4I and J) were reduced, as was the expression of the two proteoglycans, chondroitin sulfate (Fig. 4K and L) and keratan sulfate (Fig. 4M and N). These data suggest that AF differentiation is also regulated either directly or indirectly by Shh from NP cells. PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35944