The effects of removal of the hypertrophic zone of the vertebral growth plates (-HZGP) from IVDs before culture.

<p>(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.</p

The molecular markers of the AF are also reversed by rShh after cyclopamine treatment.

<p>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.</p

Shh is the key regulator of other major cell signaling pathways.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035944#pone-0035944-g010" target="_blank">Figure 10</a> 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.</p

Shh signaling is required for proliferation of NP cells.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035944#pone-0035944-g009" target="_blank">Figure 9</a> 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.</p

The normal early postnatal disc.

<p>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.</p

Par6b Regulates the Dynamics of Apicobasal Polarity during Development of the Stratified <i>Xenopus</i> Epidermis

<div><p>During early vertebrate development, epithelial cells establish and maintain apicobasal polarity, failure of which can cause developmental defects or cancer metastasis. This process has been mostly studied in simple epithelia that have only one layer of cells, but is poorly understood in stratified epithelia. In this paper we address the role of the polarity protein Partitioning defective-6 homolog beta (Par6b) in the developing stratified epidermis of <i>Xenopus laevis</i>. At the blastula stage, animal blastomeres divide perpendicularly to the apicobasal axis to generate partially polarized superficial cells and non-polarized deep cells. Both cell populations modify their apicobasal polarity during the gastrula stage, before differentiating into the superficial and deep layers of epidermis. Early differentiation of the epidermis is normal in Par6b-depleted embryos; however, epidermal cells dissociate and detach from embryos at the tailbud stage. Par6b-depleted epidermal cells exhibit a significant reduction in basolaterally localized E-cadherin. Examination of the apical marker Crumbs homolog 3 (Crb3) and the basolateral marker Lethal giant larvae 2 (Lgl2) after Par6b depletion reveals that Par6b cell-autonomously regulates the dynamics of apicobasal polarity in both superficial and deep epidermal layers. Par6b is required to maintain the “basolateral” state in both epidermal layers, which explains the reduction of basolateral adhesion complexes and epidermal cells shedding.</p></div

The expression pattern of <i>par6b</i> mRNA and the subcellular localization of Par6b protein during development.

<p>(<b>A</b>) Sagittal view of <i>par6b</i> expression pattern in a st9 blastula embryo by whole mount ISH. (<b>B</b>) Sense <i>par6b</i> probe control in a st9 embryo. (<b>C</b>) Transverse view of <i>par6b</i> expression pattern in a st17 neurula embryo. (<b>D</b>) <i>par6b</i> expression pattern in a transverse section of st17 embryo. (<b>E</b>) Staining of E-cadherin in the epidermis in a transverse section of st17 embryo. (<b>I–N</b>) Staining with anti-HA of Par6b (red) and anti-β-catenin (green) antibodies on st9 or st17 ectoderm. (<b>D–N</b>). Double-headed arrows (black in panel D and E, white in panel I–N) respectively indicate boundaries of the superficial and deep layers. Scale bars, 50 µm.</p

Par6b depletion reduces protein levels but not the mRNA levels of E- and C-cadherin.

<p>(<b>A</b>) Western blot analysis of total protein extracts from uninjected, 20 ng and 40 ng Par6b-MO injected embryos with anti-C-cadherin and E-cadherin antibodies. (<b>B</b>) Quantitative RT-PCR results of <i>e-cadherin</i> and <i>c-cadherin</i> mRNA levels by two doses of Par6b-MO injection at st9, 12, 16 and 19.</p

Par6b depletion reduces epidermal cadherins.

<p>(<b>A–C’</b>) Embryos were injected with Par6b-MO together with GFP mRNA into one animal ventral blastomere at the 8-cell stage. Staining of E-cadherin (A, A’) or C-cadherin (C, C’) (red) and GFP (green) in sections of st15 embryos. Surface magnified view of whole-mount staining of E-cadherin and GFP of st15 injected embryos (B, B’). (<b>D–F’</b>) Phenotypes of uninjected, Par6b-MO injected alone, and Par6b-MO with <i>Xt par6b</i> mRNA injected embryos at st32 and st39. (<b>G, </b><b>H, I</b>) Corresponding whole-mount staining of E-cadherin on st30 epidermis.</p

The dynamics of Crb3 and Lgl2 subcellular localization in the developing <i>Xenopus</i> stratified epidermis.

<p>(<b>A, B, C</b>) Schematic representations of the epidermis development at blastula (st9), gastrula and neurula (st17) stages, respectively. Blue arrowheads in panel B illustrate the radial interdigitation of deep cells in the gastrula ectoderm. The black boxes mark the imaging area with the apical surface facing up. (<b>D, F, G, H</b>) Crb3-GFP (green) distribution in st9, 10.5, 12, 17 ectodermal cells, respectively. Red arrowheads indicate dynamic subcellular changes of Crb3-GFP. (<b>E, I</b>) GFP-Lgl2 (white) distribution in st9 and st17 ectodermal cells. (<b>A–I</b>) Double-headed black arrows on the left side of each panel show the boundaries of the superficial and deep layers. (<b>D–I</b>) Scale bars, 50 µm.</p