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. 2010 Feb;46(2):472-8.
doi: 10.1016/j.bone.2009.09.009. Epub 2009 Sep 15.

Partial rescue of postnatal growth plate abnormalities in Ihh mutants by expression of a constitutively active PTH/PTHrP receptor

Yukiko Maeda  1 , Ernestina Schipani, Michael J Densmore, Beate Lanske
Affiliations

Partial rescue of postnatal growth plate abnormalities in Ihh mutants by expression of a constitutively active PTH/PTHrP receptor

Yukiko Maeda et al. Bone. 2010 Feb.

Abstract

Indian hedgehog (Ihh) is essential for chondrocyte proliferation/differentiation and osteoblast differentiation during prenatal endochondral bone formation. Ihh expression in postnatal chondrocytes has a non-redundant role in maintaining a growth plate and sustaining trabecular bone after birth. Loss of Ihh in postnatal chondrocytes results in fusion of the growth plate and a decrease in trabecular bone. In order to normalize this abnormal chondrocyte phenotype and to investigate whether a putative rescue of the growth plate anomalies is sufficient to correct the severe alterations in the bone, we expressed a constitutively active PTH/PTHrP receptor (an Ihh downstream target) in the chondrocytes of Col2 alpha 1-Cre ER; Ihh(dld) mice by mating Col2 alpha 1-Cre ER; Ihh(fl/fl) mice with Col2 alpha 1-constitutively active PTH/PTHrP receptor transgenic mice (Jansen, J). Col2 alpha 1-Cre ER; Ihh(f/f); J mice were then injected with tamoxifen at P0 to generate Col2 alpha 1-Cre ER; Ihh(d/d); J mice. In contrast with the previously reported growth plate phenotype of Col2 alpha 1-Cre ER; Ihh(d/d) mice that displayed ectopic chondrocyte hypertrophy at P7, growth plates of Col2 alpha 1-Cre ER; Ihh(d/d); J double mutants were well organized, and exhibited a gene expression pattern similar to the one of control mice. However, expression of osteoblast markers and Dkk1, a Wnt signaling target, remains decreased in the bone collar of Col2 alpha 1-Cre ER; Ihh(d/d); J mice when compared to control mice despite the rescue of abnormal chondrocyte differentiation. Moreover, proliferation of chondrocytes was still significantly impaired in Col2 alpha 1-Cre ER; Ihh(d/d); J mice, and this eventually led to the fusion of the growth plate at P14. In summary, we have demonstrated that expression of a Jansen receptor in chondrocytes was able to rescue abnormal chondrocyte differentiation but not impaired chondrocyte proliferation and the bone anomalies in mice lacking the Ihh gene in chondrocytes after birth. Taken together, our findings suggest that Ihh has both PTHrP-dependent and -independent functions during postnatal endochondral bone development.

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Figures

Fig. 1
Fig. 1
Growth plate morphology and mRNA expression of Ihh signaling at P7. Hematoxylin/eosin staining (a–d) and in situ hybridization (e–l) for Ihh (e–h), Ptch (i–l). At P7, the growth plate of double mutants (d) was restored when compared to the one of Ihh mutants (c) and appears similar to the one of control and Jansen control mice (a and b). Ihh expression is detected in prehypertrophic chondrocytes in control and Jansen control mice (e and f). Significant decrease of Ihh expression was confirmed in Ihh mutants and double mutants (g and h). The expression of Ptch was also found to be significantly decreased in Ihh mutants and double mutants (k and l). Magnification bar =0.5mm.
Fig. 2
Fig. 2
mRNA expression and proliferation of chondrocytes. In situ hybridization for Col2 (a–d), and ColX (e–h). Expression of Col2 was found to be normal in double mutants (d). Ectopic hypertrophic chondrocytes in Ihh mutant completely lacked Col2 expression (c, arrow). Expression of ColX in double mutants (h), was similar to the one in control and Jansen control mice (e and f), despite the narrowing of the space between the two expression centers in double mutants (h, arrow). Ihh mutants showed ColX expression in abnormal ectopic hypertrophic chondrocytes (g, arrow). Proliferation was analyzed by PCNA staining (i–l) and was significantly decreased in Ihh mutants and double mutants when compared with control and Jansen control mice (m) (*, P<0.05; n =5 mice, one section each.) All chondrocytes were counted in the boxed area and the percentage of PCNA positive (brown nuclei) chondrocytes was calculated (lower panels represent magnification of the boxed areas. Magnification bar = 0.05mm, Magnification bar for a-I = 0.5mm).
Fig. 3
Fig. 3
mRNA expression of osteoblast markers. Expression of Col1 and OCN was decreased in the bone collar of both Ihh mutants and double mutants (c, d, g, h, boxed area) when compared to controls (a and e, boxed area). Magnification bar = 0.1mm. Magnification of the boxed area =0.06 X 0.04mm. Bar graph is showing mRNA expression of Dkk1 in the bone collar and primary spongiosa of controls and mutants at P7 (i). Data are expressed as fold over control. The expression of Dkk1 was significantly reduced in both Ihh mutants and double mutants when compared to controls (*, P<0.05; n = 5 mice, measured in triplicates each). No other significant changes were found.
Fig. 4
Fig. 4
Body size and histology of bones at P14. Macroscopic image of the double mutant (a). Magnification bar =1 cm. Double mutants appear smaller in size when compared to the control and Jansen control mice, however are 8 % larger by body length than Ihh mutant littermates (*, #, P<0.05) (n = 6, 15, 6, and 3 mice in each group, respectively from left to right) (a and b). In situ hybridization showed normal ColX expression pattern in the growth plate of control and Jansen control mice at P14 (c and d). In both Ihh mutants and the double mutants, however, the growth plate has disappeared (e and f). Magnification bar =0.1mm.
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