Role of Shh in Disc Maintenance Figure 11. Postnatal regulation of IVD. Schema to show how systemic circulating signals control and coordinate the growth and differentiation of all the IVDs by controlling expression of local signals such as Shh. Two IVDs are shown for simplicity. The dotted line indicates the same events throughout the vertebral column. BV = blood vessel. doi:10.1371/journal.pone.0035944.g011 detects Shh), abcam; phospho Smad 1, 5 and 8, 9511, Cell Signaling; phospho Smad 2 and 3, sc-11769-R, Santa Cruz) overnight at 4uC in a humidified chamber, washed in PBST (three times), treated with 15 mg/mL of the appropriate Cy5-conjugated secondary antibody (Goat anti Mouse; Donkey anti Goat; Goat anti Rabbit; Goat anti Rat, from Jackson ImmunoResearch Laboratories, INC) in blocking buffer for an hour at room temperature, in the dark, and washed in PBS (twice). The sections were counterstained with POPO-3TM iodide (1:4000, Molecular Probes) and wheat germ agglutinin (1:1000, Molecular Probes) to stain the cell nuclei and cell membranes respectively, washed in 1x PBS (twice) and mounted in PBS containing 50% glycerol and 25 mg/mL of the anti-quenching agent DABCOTM (D27802, Sigma). Images were captured using a Zeiss LSM 510 confocal microscope. Control sections were incubated in the appropriate secondary antibody only, and were all negative (data not shown). X-gal staining Lumbar vertebrae were collected from P4 TOPGAL mice and the dissected IVDs were cultured as described above. At the end of culture period, the IVDs were embedded and snap frozen in OCT molds. b-galactosidase staining was carried out on 6 mm thick sections collected in the coronal plane following fixation in 2% PFA for two minutes and three washes in 1x PBS. Sections were washed twice for 10 minutes each in 2 mM MgCl2, permeabilized using 0.02% NP-40 and 2 mM MgCl2, stained for b-gal activity using X-gal in the staining solution (5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 0.01% NaDeoxycholate, 0.02% NP- 40, 1 mg/mL X-gal made in 1x PBS) at 37uC overnight. After washing twice in PBS the slides were counterstained with nuclear fast red (N3020, Sigma) dehydrated through an ethanol series and mounted. Sections were photographed using a Zeiss Axiovert microscope. Statistical analysis Statistical analysis was carried out by counting the percentage of BrdU-positive NP cells from the total NP cells on individual section of different treatment groups. The statistical analyses of the immunostaining studies was carried out by measuring the mean fluorescence intensities in the entire IVD from individual sections and different treatment groups. The means and s.d. of the values obtained from individual experiments were calculated. Charts show the s.d. of the mean for each data point. Significance was calculated using unpaired student’s t-test. Supporting Information Figure S1 Dose response of cyclopamine on Gli1 expression in P4 cultured IVDs. P4 IVDs were cultured for 5 five days (P4 t ) either in vehicle (A), or in increasing PLoS ONE | www.plosone.org 13 April 2012 | Volume 7 | Issue 4 | e35944