Expression of collagens in control and constitutively active Smo (CA-Smo) patellar tendons at E17.5.

<p>Panels A–D show immunohistochemistry of COL1 in control (A and C) and CA-Smo (B and D) sagittal sections of knee joints. Panels F–I show immunohistochemistry of COL2 in control (F and H) and CA-Smo (G and I). Activation of Smo in the tendon does not affect the expression of COL1 but causes ectopic expression of COL2 in the midsubstance. Panel E and J show pixel intensity measurements of COL1 and COL2 staining. COL2 was up-regulated in the CA-Smo mice throughout whole tendon. White dotted line marks the region of midsubstance. White dotted circle marks the insertion site. Error bar represents standard error and the asterisk represents statistical significance (<i>p</i><0.05). Blue staining shows nuclear staining using DAPI. T = Tibia. Scale bar = 100 µm.</p

List of analyzed genes, RPKM value, fold difference and brief summary of function.

<p>RPKM, reads per kilobase exon per million mapped sequences</p

Alcian blue staining is decreased in the tibial insertion sites of patellar tendons of <i>ScxCre;Smo^f/−(</i>Smo tKO).

<p>Shows alcian blue staining in insertion sites of control (A, C, and E) and Smo tKO (B, D, and F) patellar tendons at 2 wks (A and B), 3 wks (C and D) and 12 wks (E and F). See text for details. Dotted line outlines the insertion site. Blue dotted line shows the boundary between the tendon and tibial cartilage. See text for details. Cell nuclei are stained using fast red. Scale bar = 100 µm.</p

Expression of tenocyte and insertion site markers after administration of 500 ng/ml rIHH in PTT organ culture.

<p>Panel A–B show immunohistochemistry of TNC on PTT culture in PBS control (A) and rIHH (B). Panel E–F show immunohistochemistry of BGN in PTT culture in PBS control (E) and rIHH (F). Panel I–J show immunohistochemistry of COL2 in PTT culture in PBS control (I) and rIHH (J). Panel M–N show immunohistochemistry of COL1 in PTT culture in PBS control (M) and rIHH (N). Panel Q–R show immunohistochemistry of GLI1 in PTT culture in PBS control (Q) and rIHH (R). Panels C, G, K and O show pixel intensity measurements of TNC(C), BGN(G), COL2(K) and COL1(Q) staining. Panels D, H, L, P and S show expression levels of <i>Tnc</i> (D), <i>Bgn</i> (H), <i>Col2a1</i>(L), <i>Col1a1</i> (P), <i>Gli1</i> (S) mRNAs by Q-PCR. The expression of TNC, BGN, COL2 and GLI1 was up-regulated in tendons after rIHH treatment in culture, but there was no significant difference in COL1 expression between the control and rIHH treatment groups. See text for details. MS = midsubstance. T = Tibia. Arrow = insertion site. Scale bar = 100 µm. Error bar represents standard error and the asterisk represents statistical significance (<i>p</i><0.05).</p

IHH protein expression in the tibial insertion of the patellar tendon <i>in vivo</i>, and expression of tenocyte and insertion site markers in patellar-tendon-tibia (PTT) organ culture.

<p>(A) beta-gal staining (blue) of ShhCre; R26 knee joints. (B) Immunohistochemistry to show absence of SHH protein in P1 patellar tendon insertion site. Inset shows positive staining of SHH in the epithelium of a blood vessel in the same section as (B). (C) Immunohistochemistry of IHH protein in P1 patellar tendon insertion site. Inset shows higher magnification of IHH staining. (D–E) Tenascin-C staining before (D) and after (E) culture of P3 tendons. (F–G) Collagen I staining before (F) and after (G) culture. Expression of both COL1 and TNC was maintained in PTT organ culture. See text for details. P = Patella. PT = Patellar tendon. T = Tibia. Arrow = insertion site. Scale bar = 100 µm.</p

H&E staining of the tibial insertion site of patellar tendon of control and <i>ScxCre;Smo^f/−(</i>Smo tKO).

<p>Panel A–B show H&E staining of the sagittal sections of 2 weeks (2 wks) old control (A) and Smo tKO (B). Panel C–D show H&E staining of the sagittal sections of 3 wks old control (C) and Smo tKO (D). Panel E–F show H&E staining of the sagittal sections of 12 wks old control (E) and SmotKO (F). There are fewer chondrogenic cells (yellow arrow) in the tibial insertion site of patellar tendons in Smo tKO animals, and the tidemark of the Smo tKO mice is absent. See text for details. Yellow arrow = advanced differentiated cells. Blue arrow = tidemark. Scale bar = 100 µm.</p

Hedgehog signaling (Hh) is down-regulated at the insertion in <i>ScxCre;Smo^f/−(</i>Smo tKO).

<p>(A) Control mice (B) Smo tKO. The reduction of beta-gal staining (blue) in the Smo tKO indicated that Hh signaling was decreased in the insertion site. See text for details. Cell nuclei are stained using fast red. Dotted line circle = insertion site. Arrow = articular cartilage. Scale bar = 100 µm.</p

Expression of insertion site markers in the control and constitutively active Smo (CA-Smo) mice at E17.5.

<p>Panels A–D show double immunohistochemistry of GLI1 (purple in nucleolus) and TNC (green) in control (A and C) and CA-Smo (B and D) sagittal sections of knee joints. Panel F–I show the immunohistochemistry of BGN in control (F and H) and CA-Smo (G and I) sagittal sections of knee joints. Panel E and J show semi-quantitation of TNC and BGN staining by measurement of pixel intensity. The expression of GLI1 and TNC was ectopically present in the midsubstance of CA-Smo mice whereas the expression of BGN was up-regulated in the midsubstance of CA-Smo mice at E17.5. The Inset in D shows a higher magnification image of GLI1 staining in the nucleolus (purple, arrowed). White dotted lines outline the midsubstance in A,B, F and G, and the insertion site in C,D, H and I. Error bars represent standard errors of the mean and the asterisk represents statistical significance (<i>p</i><0.05). Blue staining shows nuclear staining using DPAI. T = Tibia. Scale bar = 100 µm.</p

Mechanical failure curves of patellar tendons of the control and <i>ScxCre;Smo^f/−(</i>Smo tKO) at 12 wks old.

<p>Adult Smo tKO patellar tendons displayed impaired structural (A) and material (B) properties compared to control animals. Smo tKO tendons showed decreased linear stiffness, increased displacement and increased strain (<i>p</i><0.05). Linear modulus trended towards a decrease in Smo tKO animals; however, results were not statistically significant.</p