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 E′), 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.
Figure 2.
The effects of Shh blockade in P4 cultured discs and of Cre-mediated Shh targeting at P5.
(A – C) show the whole disc (A), the NP (B), and AF (C) respectively of a normal disc immediately after explantation (P4 t0). (D–G) show the NP and AF respectively after 2 days culture in vehicle only (D and E), or 250 μM cyclopamine (F and G). (H–K) show the NP and AF respectively 5 days after doxycycline treatment at P5 of Shhflx/flx; R26rtTA mice (H and I) or of Shhflx/flx; (tetO)7-Cre; R26rtTA mice resulting in Cre-mediated targeting of Shh in (J and K). Both cyclopamine in vitro and Shh targeting in vivo have similar effects: the aggregation of NP cells into the center of the disc (F and J) and loss of polarity of the AF cells (G and K). Scale bars indicate magnifications used. NP = nucleus pulposus, AF = annulus fibrosus (dense peripheral eosin positive collagen layers are arrowed in C), IAF = inner AF, OAF = outer AF, EP = endplate.
Figure 3.
The effects of Shh blockade in vitro and Shh targeting in vivo of molecular markers of NP differentiation.
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). Each horizontal row shows the expression, by immunocytochemistry, of Gli 1 (A) and (B), Ptch1 (C) and (D), Shh (E) and (F), Brachyury (G) and (H), Sox9 (I) and (J), chondroitin sulfate (K) and (L), and collagen 1 (M) and (N). All the above markers show reduced expression after treatment with cyclopamine in vitro, or targeting of Shh in vivo. Scale bars indicate magnifications used. Blue = nuclei stained with POPO-3, green = counterstain with wheat germ agglutinin. See text for details.
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.
Figure 5.
The effects of deletion of Shh signaling on the end plate of the IVDs in vivo.
(A) shows H&E staining and histology of EP following doxycycline treatment of the control [P5 d5 Shhflx/flx; R26rtTA] and Shh mutant mice [P5 d5; Shhd/d(tetO)7-Cre; R26rtTA]. (B)–(D) shows reduced expression of Gli1, Sox9 and collagen 2 expression in the EP cells and the inner layer of AF following Shh deletion in vivo. (E) shows that Ihh continues to be expressed in the growth plate chondrocytes of the Shh mutant as well as in the control mice, while hedgehog (Hh) expression was undetectable in the EP cells. (F) shows that there is no effect on BrdU incorporation (Brown = BrdU positive cells, cell nuclei stained with nuclear fast red) in the growth plate following Shh deletion, compared to the control mice. Scale bars indicate magnifications used. Red = expression of the specific protein, Blue = cell nuclei stained with POPO-3, green = general counterstain with wheat germ agglutinin. EP = end plate, GP = growth plate. See text for details.
Figure 6.
The effects of removal of the hypertrophic zone of the vertebral growth plates (-HZGP) from IVDs before culture.
(A) shows their histology after two days in culture. (B–B′) shows low and high magnification immunostaining for both Shh and Ihh, using an antibody that stains both, in the growth plate chondrocytes (GP) and EP before culture. (C–C′) shows low and high magnification images respectively of 48 hour cultures to show that Hh staining is still seen in the EP in the absence of the growth plate. (D–I) show normal levels of expression of Shh, Gli1, Bra, Sox9, collagen 2, and chondroitin sulfate, in discs cultured for 48 hour in the absence of the growth plate. Scale bars indicate magnifications used. Red = expression of the specific protein, Blue = cell nuclei stained with POPO-3, green = general counterstain with wheat germ agglutinin. NP = nucleus pulposus, AF = annulus fibrosus, EP = end plate, GP = growth plate. See text for details.
Figure 7.
The effects of cyclopamine phenotype in NP cells can be rescued and are reversible in vitro.
The panels are arranged as a table. Each row shows the staining pattern, and each vertical column shows the treatment (P4 t5 Veh = five day vehicle treated control, P4 t5 CycA = five days in cyclopamine, P4 t5 CycA+ rShh = two days in cyclopamine followed by three days in rShh. H&E staining (A) shows collapsed NP cells are reversed after replacing cyclopamine with rShh. (B)–(D) show that the downstream targets of Shh, Gli1, Bra, and Sox9 are all reversed. (E) and (F) show that expression of chondrotin sulfate, and collagen 1 is also reversed. The vehicle treated and cyclopamine treated samples for similar time points have previously shown in (Figure 3K) (chondroitin sulfate) and 3M (collagen 1). Scale bars indicate magnifications used. Red = expression of the specific protein, Blue = cell nuclei stained with POPO-3, green = general counterstain with wheat germ agglutinin. See text for details.
Figure 8.
The molecular markers of the AF are also reversed by rShh after cyclopamine treatment.
Each row shows the staining pattern, and each column shows the treatment. P4 t5 Veh = five day vehicle treated control, P4 t5 CycA = five days in cyclopamine, P4 t5 CycA+ rShh = two days in cyclopamine followed by three days in rShh. (A) shows by H&E staining that loss of AF cell polarity is reversed by replacement of cyclopamine with rShh. (B)–(F) shows that expression of Gli1, Sox9, collagen 1, collagen 2, and chondroitin sulfate, respectively, are also reversed. Scale bars indicate magnifications used. IAF = inner annulus fibrosus, OAF = outer annulus fibrosus. Red = expression of the specific protein, Blue = cell nuclei stained with POPO-3, green = general counterstain with wheat germ agglutinin. See text for details.
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.
Figure 10.
Shh is the key regulator of other major cell signaling pathways.
Figure 10 shows that cyclopamine treatment in vitro, and targeting of Shh in vivo both cause alterations in activities of other signaling pathways. (A) and (B) show increases in BMP signaling and (C) shows the graph for the intensity of immunostaining from both the experiments. (D) and (E) show decreases in TGFβ signaling represented by graph plotted for the intensity of immunostanining (E). (F) shows an increase in canonical Wnt signaling. In (A)–(D), red = expression of the specific protein. In E, blue = β-gal positive cells, pink = nuclei stained with nuclear fast red.
Figure 11.
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.