Figure 9. Shh signaling is required for proliferation of NP cells. Figure 9 shows that cyclopamine treatment in vitro, and targeting of Shh in vivo both cause loss of NP cell proliferation. (A) shows representative images of BrdU staining of NP from IVDs cultured for two days in vehicle (P4 t2 Veh) and cyclopamine (P4 t2 CycA) treated medium. The percentage of BrdU-positive NP cells from different experimental groups in the study is quantified in (B) which shows decrease in the percentage of BrdU positive NP cells from both the in vitro and in vivo experiments. The percentage of BrdU positive cells increased in the NP cells from rShh rescue experiment carried out in vitro. Graph shows the s.d. of the mean for each data point. Significance was calculated using unpaired student’s t-test, and *** represents p# 0.001. Brown = BrdU positive cells, cell nuclei stained with nuclear fast red. doi:10.1371/journal.pone.0035944.g009 possible to control both growth and differentiation of diseased discs, by the application of appropriate agonists and antagonists of the signaling pathways found to carry out specific functions in vivo. Materials and Methods Mice FVB mice were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all experiments were carried out in strict accordance with institutional guidelines under Institutional Animal Care and Use Committee (IACUC) approval at Cincinnati Children’s Hospital Research Foundation (CCHRF). IACUC at CCHRF approved the study described in this manuscript with Animal Use Protocol number 9A01004. All studies were carried out using at least four IVDs in each group and repeated three times. Generation of doxycycline inducible Shh flx/flx compound transgenic mice Shh flx/flx mice [28,29] were interbred with (tetO)7-Cre [30] and rosa26 rtTA IRES eGFP [31] mouse lines to obtain Shh flx/ flx; (tetO)7-Cre; R26 rtTA compound offspring and genotyped as previously described. The postnatal day five (P5) pups which were Role of Shh in Disc Maintenance intraperitoneally injected with 2 mg of doxycycline (D9891, Sigma- Aldrich) in a final volume of 50 ml, on P5 and P7 and the lumbar vertebrae were collected for analysis on P10. All the pups were administered 50 ml of BrdU 10 hours before sample collection. Organ culture of neonatal IVDs Lumbar vertebrae (LV) were dissected from four-day postnatal mice (P4) and placed in ice-cold 1x phosphate buffered saline (PBS) containing antibiotics (pen/strep). The IVDs were dissected out from the LV along with the adjacent vertebral growth plates under a dissecting microscope. One batch of IVDs was snap frozen at t0 as control in vivo samples (P4 t0). The IVDs were then placed on type-IV Collagen (354233; BD Biosciences) coated MillicellH-CM cell culture inserts (PICM01250; Millipore) and cultured in 24-well culture plates in DMEM Ham F-12 containing 1% pen/strep and supplemented with 1x Insulin-Transferrin- Sodium Selenite supplement (11074547001; Roche Diagnostics) The culture was maintained at 37uC and 5% CO2. To study the effect of loss of Shh signaling, one group of IVDs were treated with 250 mM cyclopamine (C-8700; LC Laboratories). The treatment was carried out for two-five days (t2 2 t5) depending on the culture duration. The vehicle -treated discs served as controls. In order to study the specificity of cyclopamine action on Shh signaling pathway, the IVDs were initially treated with cyclopamine for two days, then the medium was removed and the IVDs were thoroughly washed. The discs were further cultured for another three-days in medium supplemented with 100 ng/mL of recom- binant Shh [rmShh(C25II)-N; 464-Sh; Research Diagnostics] or medium containing vehicle only. At the end of the culture the IVDs were washed three times in 1x PBS and snap frozen in OCTTM molds. BrdU incoporation assay Bromo-deoxy-Uridine (BrdU) assays were carried out using AmershamTM Cell Proliferation Kit (RPN20; GE Healthcare) as per manufacture’s instructions. BrdU (1:1000) was added to the culture medium 24 hours before the termination of culture, at which the IVDs were washed three times in 1x PBS and snap frozen in OCTTM molds. Cryosections were collected from the IVDs of the in vitro and in vivo experiments at 6 mm thickness, fixed in 2% paraformaldehyde (PFA) and washed thrice in 1x PBS. Next, the sections were treated with 2% H202 diluted in methanol to quench the endogenous perxoxidase activity, which otherwise would interfere with the interpretation of the results. The sections were washed three times in 1x PBS and incubated with anti-BrdU antibody for 2 hours at room temperature, followed by 30 minutes incubation with HRP-conjugated secondary antibody. To visual- ize the BrdU positive cells under a microscope, the sections were treated with the DAB Plus Substrate Staining System (Thermo Scientific), and the nuclei were counter-stained pink with nuclear fast red (N3020; Sigma). The sections were dehydrated and mounted in xylene based mounting medium. Sections were photographed using a Zeiss axiovert microscope and NP cells were counted using the Axio vision software. Histology Cryosections of the cultured and t0-control IVDs, and from the Shh mutant mice were collected at a thickness of 6 mm in the coronal plane, using a Leica cryostat. Sections were processed immediately, or stored at -20uC for later use. Histology of the vertebrae was analyzed by fixing sections in 2% PFA and staining with hematoxylin and eosin (H&E). Sections were photographed using a Zeiss axiovert microscope. PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e35944