Tissue localization of Bmp2CreER activity during the course of limb development.

<p>Upper panels: 24-hour collection (A-J). Tissue localization of Bmp2CreER activity at 24 hours after tamoxifen (Tam) treatment on consecutive days from E10.5–14.5. Panels A,C,E,G,I (forelimb, FL) and B,D,F,H,J (hindlimb, HL) show longitudinal sections through limb dorsoventral axis oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Hatch marks in panels C-J show the levels at which cross-sections below panels C-J were taken (distal to proximal). Cross-sections are all oriented with dorsal at top and anterior-to-posterior (digit 1–5) from left-to-right. Lower panels: E13.5 collection (K-N). Tissue distribution of LacZ activity at E13.5 after tamoxifen (Tam) treatment at E10.5 (K,L) or E11.5 (M,N). For each treatment time, whole mount and adjacent longitudinal and cross-sections of forelimb (FL) or hindlimb (HL) are shown. Hatch marks in panels of longitudinal sections show the levels at which cross-sections through digit region were taken (distal to proximal). Longitudinal and cross-sections are oriented as for Upper Panels. Whole mount images are oriented the same as longitudinal sections.</p

Tissue distribution of LacZ activity at E15.5 following tamoxifen (Tam) treatment at different stages from E10.5—E14.5.

<p>Panels A,C,E,G (forelimb, FL) and B,D,F,H (hindlimb, HL) show longitudinal sections through limb dorsoventral axis oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Hatch marks in panels A-H show the levels at which cross-sections below panels A-H were taken (distal, proximal). Cross-sections are all oriented with dorsal at top and anterior-to-posterior (digit 1–5) from left-to-right. Note that tamoxifen treatment at E13.5 results in LacZ staining restricted to collateral ligament. Lower panel cartoon shows a diagram of an E15.5 digit cross-section with cartilage (red), tendon (blue) and ligament (green) structures annotated for reference.</p

Bmp2CreER activity 24 hours after tamoxifen (Tam) treatment surveyed at daily intervals during the course of limb development.

<p>A. LacZ activity at 48 hours after tamoxifen treatment at E9.5 with arrow pointing to weak activation in distal forelimb (FL inset). Only trace expression is seen in hindlimb (HL inset). (No appreciable LacZ staining was observed at 24 hours after tamoxifen treatment; data not shown). B. LacZ activity assayed at 24 hrs after tamoxifen treatment at times indicated above each panel. All insets to the right show forelimb (FL) and hindlimb (HL) buds oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Note that no LacZ staining is evident in tissues outside of the limb.</p

Bmp2CreER, Osr1Cre and Gdf5Cre have distinct and partly complementary distributions of recombinase activity.

<p>Bmp2CreER activity after single dose tamoxifen (Tam) administration at 2 different times (E11.5, A-B or E13.5, C-D) compared to Osr1Cre (E-F) and Gdf5Cre (G-H) in E15.5 limbs. In each case, whole mount and adjacent longitudinal sections through dorsoventral axis of forelimb (FL) or hindlimb (HL) are oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Hatch marks in panels of longitudinal sections show the levels at which cross-sections were taken (rightmost panel sets; distal to proximal). Cross-sections are all oriented with dorsal at top and anterior-to-posterior (digit 1–5) from left-to-right. Note that Bmp2CreER labels LacZ-positive descendants mainly in interdigital mesenchyme, including collateral and joint ligaments, whereas Osr1Cre descendent cells reside in subcutaneous tissue and joint ligaments, and Gdf5Cre labels cells lining the joint regions and immediately adjacent subarticular and perichondrial cells at E15.5.</p

Comparison of Bmp2CreER activity levels and distribution assayed at E15.5 in LacZ-positive descendants, following tamoxifen (Tam) treatment between stages E11.25 to E11.75.

<p>For each tamoxifen (Tam) treatment time, whole mount and adjacent longitudinal sections through dorsoventral axis of forelimb (FL) or hindlimb (HL) are oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Hatch marks in panels of longitudinal sections show the levels at which cross-sections through digit region were taken (rightmost panel sets; distal to proximal). Cross-sections are all oriented with dorsal at top and anterior-to-posterior (digit 1–5) from left-to-right. Note that Cre activity transitions from digit condensations and interdigital mesenchyme to primarily interdigits and collateral ligaments during this time window of tamoxifen administration.</p

Expression of <i>Ccn2</i> in developing vasculature.

<p>(A) β-galactosidase activity in <i>Ccn2-lacZ</i> transgenic mice reveals <i>Ccn2</i> promoter activity throughout the vasculature in E16.5 embryos. (B) <i>Ccn2-lacZ</i> expression in dermal microvessels at E13.5. (C–E) EGFP fluorescence in CCN2-EGFP BAC transgenic mice demonstrates CCN2 expression in the endothelium of arterial elements (C and E), venous elements (C), and developing capillary networks (D). Arrowheads in (C) and (E) demarcate arterial element. Arrow in (C) identifies endothelial cells of a venous element. Arrowhead in (E) highlights EGFP expression in mural cells in the arterial element. Arrow in (E) highlights expression in endothelial cells in the arterial element. (F) Immunofluorescence and (G,H) immunohistochemical staining with an αCCN2 antibody on paraffin sections through dermis, demonstrating CCN2 expression in endothelial cells. Arrows in (F) highlight endothelial cells in E18.5 microvasculature. Specificity of the αCCN2 antibody is demonstrated by the absence of reactivity in the <i>Ccn2−/−</i> section (H). Arrows in (G) and (H) demarcate abluminal surface of the endothelium. Asterisks in (G) and (H) identify blood cells within the vessels. αCCN2 staining in (G) shows punctate intracellular expression, presumably with the Golgi, in addition to the surface expression marked by the arrow.</p

Defective endothelial-pericyte interactions in <i>Ccn2</i> mutants.

<p>(A, B) Co-immunofluorescence staining for desmin and PECAM in E18.5 dermis from (A) WT and (B) <i>Ccn2−/−</i> mice analyzed by confocal microscopy. (C) Quantification of vessel coverage by pericytes in E18.5 dermis; asterisk, p<0.05. (D, E) Co-immunofluorescence staining for NG2 and PECAM in E16.5 lung from (D) WT and (E) <i>Ccn2−/−</i> mice analyzed by confocal microscopy. (F) Quantification of vessel coverage by pericytes in E16.5 lung; asterisk, p<0.05. (G,H) Confocal analysis of NG2 and PECAM immunostaining in (G) WT and (H) <i>Ccn2−/−</i> E16.5 dermis. Pericytes are elongated around the microvessel in (G), whereas in mutants (H), pericytes (arrows) are associated with the endothelium, but are rounder, and fewer of them have elongated along the endothelial surface. (I–L) Confocal sections through E16.5 dermis analyzed for desmin (green) and PECAM (red) immunofluorescence. (I,J) WT desmin positive pericytes appear elongated and cover most of the surface of the microvessels. (K.L) <i>Ccn2−/−</i> desmin-positive pericytes have a rounder appearance and show less extensive coverage of the surface of the endothelium.</p

Endothelial basement membrane defects in <i>Ccn2</i> mutants.

<p>Electron microcopic images of endothelial basement membranes in dermal capillaries of E16.5 (A) WT and (B) <i>Ccn2−/−</i> littermates. Arrows demarcate the plasma membrane (bottom arrow) and top of the interstitial matrix (top arrow). (C,D) Confocal images of dermis of E16.5 WT (C) and <i>Ccn2−/−</i> (D) mice analyzed by immunofluorescence for fibronectin (FN) and PECAM. Arrows identify an arteriole. The arteriole in (C) is surrounded by several layers of FN. The arteriole in (D) is incompletely invested with FN. (E,F), Lower magnification confocal images through (E) WT and (F) <i>Ccn2−/−</i> E16.5 dermis, illustrating less fibronectin throughout the dermis in mutants. (G,H) Confocal images of dermis of E16.5 (G) WT and (H) <i>Ccn2−/−</i> mice analyzed by immunofluorescence for ColIV (Col4α2) and PECAM. Arrows identify an arteriole. ColIV coverage of the mutant vasculature is incomplete. (I,J) Confocal images of lungs of E16.5 (I) WT and (J) <i>Ccn2−/−</i> mice analyzed by immunofluorescence for ColIV and PECAM. Most of the vascular elements in the WT lung are surrounded by ColIV. Coverage is incomplete in the <i>Ccn2</i> mutant lung. Arrows in (J) identify vessels lacking coverage by ColIV. (K) CCN2 induces expression of FN and ColIV in HUVECS. HUVECs were infected with Ad-CCN2-GFP or Ad-control. Lysates were collected at the indicated time points post-infection. Levels of FN are elevated 8 hours after infection, concomitant with accumulation of CCN2. There appeared to be an increase in FN levels at 12 hours in the presence of CCN2 in the blot shown, but this was not seen in every experiment and the result did not reach statistical significance at this time point. Similarly, there was a trend towards increased expression of Col IV at 12 hr, but this increase did not reach statistical significance (p = 0.065). The experiment was repeated three times. A representative blot is shown. Quantification of levels of FN and Col IV are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030562#pone.0030562.s007" target="_blank">Figure S6</a>.</p

Vascular abnormalities in <i>Ccn2</i> mutant embryos.

<p>(A) E18.5 WT and (B) <i>Ccn2−/−</i> littermate, showing vessel dilation throughout the mutant embryo. (C, D) H&E-stained paraffin sections through the lumbar dorsal dermis of (C) E18.5 WT and (D) <i>Ccn2−/−</i> littermate. Arrowheads point to vessels. Bars highlight the enlarged distance between the hypodermal and epidermal layers in the mutant, indicative of local edema. (E,F) Hematoxylin and eosin-stained sections through E16.5 WT (E) and <i>Ccn2−/−</i> (F) descending aorta at thoracic level. Smooth muscle cells in the tunica media are spindle-shaped and arranged in layers in the WT embryo, but are more cuboidal and disorganized in the <i>Ccn2−/−</i> littermate. (G,H) Higher magnification images through aorta at lumbar level in E16.5 (G) WT and (H) <i>Ccn2−/−</i> littermate showing spindle-shaped smooth muscle cells (arrowheads) in WT that have a cuboidal shape in the mutant. (I,J) Confocal images of PECAM-stained dorsal dermal vasculature in (I) WT and (J) <i>Ccn2−/−</i> littermates. Arrows demarcate arterial elements; arrowheads demarcate venous elements; asterisks identify capillary beds. (K,L) Higher magnification confocal images of (K) WT and (L) <i>Ccn2−/−</i> dorsal dermal capillary beds, showing increased capillary density in the mutant. (M,N) High magnification confocal image of (M) WT and (N) <i>Ccn2−/−</i> dorsal dermal capillaries, showing numerous abluminal protrusions (arrows in (N)) on the mutant capillary. (O,P) Electron micrographs of newborn (P0) (O) WT and (P) <i>Ccn2−/−</i> dermal capillaries, showing abluminal and luminal (arrows in (P)) protrusions.</p

CCN2 potentiates PDGF-B signaling.

<p>(A) rCCN2 induces PDGF-B expression in HUVEC cells. Right panel, representative Western blot. Left panel, Quantification of relative expression levels of PDGF-B in cells treated with or without rCCN2 from three separate experiments. *, p<0.02. (B) Adenovirally expressed CCN2 induces PDGF-B expression in HUVECs compared to transfection with an empty adenoviral control. The extent of PDGF-B induction correlated with levels of CCN2 expression. As reported previously, a higher molecular weight isoform of CCN2, presumably a result of post-translational modification <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030562#pone.0030562-Brigstock1" target="_blank">[1]</a>, is detected 4 and 8 hours post-infection. Relative level of PDGF-B expression was assessed using ImageJ software. The experiment was repeated three times, with similar results each time. The induction of PDGF-B in the presence of CCN2 was statistically significant for each time point; p<0.05. A representative Western blot is shown. (C) Effects of rPDGF-B, and/or pcDNA3-CCN2-HA expression on activation of PDGF pathways in MOVAS cells. PDGF-B stimulated activation of Stat3, ERK, and Akt, whereas CCN2-HA on its own had no effect. However, combined treatment with PDGF-B and CCN2-HA led to prolonged Akt activation (arrows). Relative levels of pAKT expression were assessed using ImageJ software. All experiments were performed in triplicate and repeated three times, with similar results each time. The increase in pAKT levels in the presence of CCN2 was statistically significant at each time point; p<0.05. A representative Western blot is shown.</p