Figure 1.
Skeletal staining with alcian blue and alizarin red and microCT of limb length.
Skeletal staining was attempted with the PBS- and 5E1-injected mice. (A) 6-week old mice exposed to 5E1 at E12.5 exhibit limb, skull, tail and trunk bones that are reduced in size and length, but mice exposed to PBS show the same bone length as wild-type mice. (B) The forelimbs and hindlimbs of E14.5, one-day-old, one-week-old, three-week-old and six-week-old mice were stained with alcian blue and alizarin red. Mice injected with 5E1 have both shorter forelimbs and hindlimbs, and their bones grow similarly to those of PBS-injected mice. (C) The length of the humerus, ulna, and radius in 5E1-injected mice are shorter than in the PBS-injected mice at one-week old, three-week old, and 6-week old; the skeletal staining results agree with those of the microCT analysis. Student’s t-test was employed for statistical analysis, with the level of statistical significance set at (*) p<0.01 or (**) p<0.05 (Sc; Scapula, S; Stylopod, Z; Zeugopod, A; Autopod, scale bar = 3 mm).
Figure 2.
(A) A table showing the fold change in the known chondrogenesis-related genes in limb between the groups injected with either PBS or 5E1 at E12.5. As expected, Gli1, Hhip, and Ptch1 were down-regulated. Interestingly, Runx2, Runx3, and Bmp5 were down-regulated. Sox9, which is the master gene in chondrogenesis, was not altered by 5E1 treatment. (B) The results of the RT-qPCR are consistent with the microarray data. The amount of each of the RT-qPCR products was normalized using β-2-microglobulin (B2m) as an internal control. Student’s t-test was used for statistical analysis with the level of statistical significance set at (*) p<0.01 or (**) p<0.05.
Figure 3.
Expression of Ihh, Ptch1, Gli1, Runx2, Runx3 and Sox9.
(A and A’) Ihh expressed in prehypertrophic chondrocytes. (B and B’) The Ihh expression pattern was not altered when hedgehog signaling one day of 5E1 injection at E12.5. The Ihh-expressing regions in second and third phalanges were connected following the injection of 5E1, but not after injecting PBS. (C and C’, E and E’) Ptch1 and Gli1 were expressed in the mesenchymal cells surrounding the prehypertrophic chondrocytes. (D and D’, F and F’) Ptch1 and Gli1 were down-regulated due to blocking the activity of the IHH protein. (G and G’, I and I’) Runx2 and Runx3 were expressed in mesenchymal cells, similar to Ptch1 and Gli1. (H and H’, J and J’) Runx2 and Runx3 were down-regulated when 5E1 was transferred. (K–L’) Sox9 expression did not differ between the PBS- and 5E1-treated groups. p1 (green box), condensation of phalange 1; p2/3 (red and yellow box), unseparated primordium of phalanges 2 and 3; p2/3* (orange box), connecting region of Ihh expression; m, metacarpals; A, anterior; D, distal part of the limb; (A–L, scale bar = 1 mm. A’–L’; longitudinal section view, scale bar = 100 µm.).
Figure 4.
The exogenous IHH protein induced proliferation and differentiation of E12.5 limb bud mesenchymal cells and ectopic expression of Runx2 and Runx3.
(A, B) Cells were cultured at a density of 2×107 cells/ml with 500 ng/ml of the IHH protein and 130 µg/ml of 5E1. The addition of exogenous IHH protein leads to the production of more cartilage nodules, whereas 5E1 treatment leads to a slight decrease in the formation of cartilage nodules. The value was quantified by measuring the absorbance of the bound alcian blue at 570 nm. (C) After the beads soaked in 1 mg/ml of IHH were implanted into 1.5×106 mesenchymal cell pellets of limb buds at E12.5, followed by incubation for 3 or 24 hours, ectopic Gli1, Runx2 and Runx3 expression appear strongly around the IHH protein-soaked beads. (D) The Gli1, Runx2 and Runx3 expression levels are up-regulated by the exogenous IHH protein. Student’s t-test was employed for statistical analysis, with the level of statistical significance set at (*) p<0.01 or (**) p<0.005 (scale bar = 1 mm in the whole view, and scale bar = 100 µm in the section view of the cell pellets).
Figure 5.
The Ihh-Gli pathway positively induces Runx2 and Runx3.
(A) Compared to the pGL3-basic, treatment with 10 ng of the Gli1 expression vector leads to a significant increase of wild-type Runx2 containing Gli-binding site (pGL3-Runx2 WT, 500 ng), but the luciferase activity of Runx2 containing mutated Gli-binding site (pGL2-Runx2 Mut, 500 ng) is decreased, compared to pGL3-Runx2 WT. (B) pGL3-Runx3 WT luciferase activity increases significantly with the Gli expression vector, but pGL3-Runx3 Mut luciferase activity decreases than pGL3-Runx3 WT. Student’s t-test was employed for statistical analysis, with the level of statistical significance set at (**) p<0.005.
Figure 6.
The differentiation of perichondrial cells was induced by Runx2 and Runx3 via the Ihh-Gli pathway.
(A) The cartilage in the Ihh −/− mutant limbs is shorter than that in the wild-type limbs at E14.5. This phenotype becomes progressively more severe by the time of birth, and the mice die at birth. When Ihh is blocked temporarily at E12.5, the cartilage primordium is shorter and similar to the Ihh−/− mouse limbs at E14.5. Then, because Ihh is restored to the epiphyseal plate, the bone is well formed but short. Nevertheless, these short-boned mice can survive. (B) The binding of Ihh, which is the signal from prechondrocyte to the Ptch1 receptor of perichondrial cells, relieves Smo inhibition, leading to the activation of Gli. GliA then translocates to the nucleus to control the transcription of the Ihh target genes Runx2 and Runx3 to promote perichondrocyte differentiation.