medium. Second, we carried out a conditional targeting of Shh during the early postnatal stages, to provide in vivo confirmation of the results seen in cultured discs. The results show that in vitro cultures of the IVD offer a rapid assay for the functions of specific signals during the postnatal stages. They also show that Shh signaling is required in the IVD for many components of its postnatal growth and differentiation. Results The normal early postnatal disc First, we characterized the expression at P4 of several markers of NP, AF, and EP differentiation, since we wish to experimentally manipulate Shh signaling at this stage. (Fig. 1A) shows the histological structure of a normal P4 mouse lumbar IVD (all sections are mid-coronal unless otherwise noted), as well as differentiation markers used in this study. The NP contains large well spread cells, whilst the AF cells are undergoing polarization into layers, and collagens are being laid down in the outermost region [stained pink with eosin in (Fig 1A)]. The AF is continuous with the EP where it meets the growth plate of the vertebra [arrowed in (Fig 1A)]. The T box transcription factor Brachyury (Bra) is expressed only in the NP (Fig. 1B). The HMG box transcription factor Sox9 is expressed by NP, AF and EP cells (Fig. 1C and D). Cytokeratin 19 (K19) is expressed only by the NP cells (Fig. 1E and E9). Collagen 1 is expressed at this stage in the NP, and the outer regions of the AF (Fig. 1F), whilst collagen 2 is expressed in the cells lining the EP and outer regions of the AF (Fig. 1G). Chondroitin sulfate is expressed by all components of the IVD (Fig. 1H), whilst keratan sulfate is expressed in the outer layers of the AF (Fig. 1I). Aggrecan is expressed only in the NP at this stage (Fig. 1J). Shh is expressed at high levels in the NP only (Fig. 1K), whilst the receptor Ptch1 and the downstream target Gli1 are expressed to different extents in all components of the disc (Fig. 1L and M). These data show that there exist good spatially localized cellular and extracellular markers of disc differentiation during the first week after birth that can be used to assay the roles of specific signaling pathways. The role of Shh signaling in the postnatal IVD Two series of experiments were carried out. First, we established a simple in vitro assay for function by excising single lumbar IVDs at P4, and placing them in organ culture conditions. At this stage, the IVDs are small [,1 mm across, see (Fig. 1A)] and are avascular in vivo, and so nutrients, oxygen, and carbon dioxide, are transported in and out of the disc by diffusion in vivo. (Fig. 2A– C) show the histology of an IVD fixed immediately after removal (P4 t0). The NP cells are spread to form a reticular network that fills the disc cavity (Fig. 2B), and the AF cells are already forming concentric layers as shown by the polarized and elongated cell nuclei (Fig. 2C). Dense eosin staining at the periphery of the AF [arrowed in (Fig. 2C)] indicates the laying down of collagens between the AF cells. (Fig. 2D and E) show that the normal histological structures of both the NP and AF are retained in culture for two days (P4 t2). In fact, normal histology is maintained for at least five days [see later section, (Figs. 7 and 8)]. Since the discs were cultured without serum, all intercellular signaling required for maintenance of the differentiated structure of the disc up to P4 must be intrinsic to the IVD. Since the NP expresses high levels of Shh, we blockaded Hh signaling in vitro using cyclopamine. (Figs. 2F and G) show sections through the NP and AF regions respectively of IVD’s maintained for two days in 250 mM cyclopamine. This was the lowest dose of cyclopamine that completely inhibited Gli1 expression by the NP cells Role of Shh in Disc Maintenance (Figure S1). There were dramatic changes to the histological structures of both the NP and the AF, compared to the control discs (incubated in vehicle only) (Fig. 2D and E). The NP cells lost their characteristic reticular network and collapsed into the center of the disc space (Fig. 2F), and the AF cells lost their obvious polarity and layered structure (Fig. 2G). To confirm these results in vivo, and to show that the effects of cyclopamine in vitro were due to the specific blockade of Shh secreted by the NP cells, and not due to blockade of Ihh secreted by the hypertrophic zone of the adjacent vertebral growth plates (which were also present in the cultures), we generated mice carrying floxed alleles of Shh, doxycycline-inducible Cre transgene (Shhflx/flx; (tetO)7-cre; R26rtTA) as well as mice carrying Cre transgene only. Doxycycline was administered on P5, and the IVD’s harvested on P10. NP and AF from doxycycline-treated Shhflx/flx; R26rtTA mutant mice which served as controls (Fig. 2H and I), and the data from doxycycline treated Cre control mice were identical (Figure S2). The IVDs had the normal reticular structure of the NP cells (Fig. 2H), and the layered, polarized structure of the AF (Fig. 2I). However, IVDs taken five days after doxycycline treatment from Shhflx/flx;(tetO)7-cre; R26rtTA com- pound mutant mice resulting in deletion of Shh showed a histology (Fig. 2J and K) very similar to that caused by cyclopamine in vitro. The NP cells were collapsed into the center of the disc cavity, and the AF cells lost their polarity and characteristic layered structure. These data suggest that Shh secreted by the NP cells is required for the maintenance of normal differentiation of both NP and AF cells during early life. Loss of Shh signaling results in loss of differentiation markers of NP cells Histological sections were stained with differentiation markers of NP (Fig. 3), AF (Fig. 4), and EP (Fig. 5). First, to confirm that Shh signaling was blocked by the two treatments, we stained for Shh, Gli1, and Ptch1, which are all known targets of Shh signaling [15–19]. Gli1, Ptch1, and Shh expression in the NP cells was dramatically reduced within two days (P4 t2) following cyclopa- mine treatment in vitro (Fig. 3A, C and E), and five days after doxycycline treatment in vivo (Fig. 3B, D and F), showing that Shh signaling was blocked. Shh expression was reduced after conditional deletion in vivo (Fig. 3F), but not as completely as by cyclopamine treatment in vitro (Fig. 3E), suggesting that Cre- mediated excision of the Shh gene was not complete, or that some perduring Shh protein was still present. Next, we analyzed the effects of Shh blockade in vitro and in vivo on the expression of the transcription factors Bra and Sox9 (Fig. 3G–J). Expression of Bra was reduced to levels that were undetectable using the same staining and imaging protocols as the controls following cyclopa- mine treatment in vitro (Fig. 3G), and reduced but still detectable after in vivo Shh targeting (Fig. 3H). Expression of Sox9 showed the same pattern (Fig. 3I and J). Extracellular matrix glycopro- teins, GAGs, and proteoglycans were also reduced in the NP by both treatments (Fig. 3K–N). However, this took longer; so that a much larger reduction in staining intensity was seen after five days in culture [P4 t5, (Fig. 3K and M)]. These results show that the loss of histological structure of NP cells coincides with the changes in gene expression. Loss of Shh signaling affects differentiation markers of AF cells The expression of AF differentiation markers was also reduced by Hh blockade in vitro and Shh targeting in vivo (Fig. 4). Gli1 and Ptch1 expression were both reduced after two days in vitro PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35944