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BMP and Ihh/PTHrP signaling interact to coordinate chondrocyte proliferation and differentiation

¹ Otto Warburg-Laboratory, Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany
² Harvard University, Department of Molecular and Cellular Biology, Cambridge, MA 02138, USA
³ Western Regional Research Center, ARS, USDA, Albany CA 94710, USA

View larger version (103K): [in a new window]   Fig. 1. BMP signaling regulates chondrocyte differentiation. (A-D) Forelimbs of E14.5 mouse embryos were cultured for 4 days and photographed before (A) and after (B-D) culture. Serial sections of these limbs were hybridized with riboprobes for Ihh (E-H) or ColX (I-L). Uncultured limbs (A) display the characteristic expression domains of Ihh in prehypertrophic (E) and ColX in hypertrophic (I) chondrocytes. Limbs cultured for 4 days increase in size (C) but display the normal distribution of Ihh- (G) and ColX- (K) expressing cells. Treatment with BMP2 results in a further enlargement of the cartilage elements (D) and an increased size of the Ihh (H) and ColX (L) expression domains. By contrast, Noggin treatment blocks limb growth (B) and results in reduced expression of Ihh (F) and ColX (J). (A-D) Ruler indicates relative size units. In E-L ulna is upwards and radius is downwards.

View larger version (141K): [in a new window]   Fig. 2. Overexpression of Ihh results in upregulation of Bmp genes. Forelimbs of wild-type (A-D) and ColII/Ihh mouse embryos (E-H) at stage E14.5 were sectioned and hybridized with riboprobes for Ihh (A,E), Bmp3 (B,F), Bmp4 (C,G) or Bmp7 (D,H). In limbs of ColII/Ihh mice, endogenous Ihh expression is reduced compared with that in the wild-type mice (A,E). Bmp3 (F), Bmp4 (G) and Bmp7 (H) are upregulated in perichondrium of ColII/Ihh mouse limbs compared with that of wild-type limbs (B-D). In addition, in ColII/Ihh transgenic mice Bmp4 (C,G) and Bmp7 (D,H) are upregulated in proliferating chondrocytes. All panels show sections through the radius.

View larger version (111K): [in a new window]   Fig. 3. A block of BMP signaling cannot overcome the Ihh-induced delay in hypertrophic differentiation. Forelimbs of E14.5 embryos from wild-type (A,B) or transgenic ColII/Ihh mice (C-F) were cultured for 4 days in control medium (A-D) or treated with Noggin protein (E,F). Serial sections of these limbs were hybridized with riboprobes for Ihh (A,C,E) or ColX (B,D,F). (A-D) Untreated limbs of ColII/Ihh embryos display a reduced expression domain of Ihh (C) and ColX (D), compared with untreated limbs of wild-type embryos (A,B). (E,F) Forelimbs of ColII/Ihh embryos treated with Noggin show a further reduction of Ihh (E) and ColX (F) expression. All panels show sections through the humerus.

View larger version (78K): [in a new window]   Fig. 4. BMP2 does not rescue the advanced onset of hypertrophic differentiation induced by a loss of Ihh signaling. Forelimbs of E14.5 embryos were cultured for 4 days in control medium (A,B) or in medium supplemented with PTHrP (C,D), PTHrP and cyclopamine (E,F), cyclopamine (G,H), BMP2 and cyclopamine (I,J) or BMP2 (K,L). Serial sections were hybridized with riboprobes for Ihh (A,C,E,G,I,K) or ColX (B,D,F,H,J,L). (A-D) Treatment with PTHrP results in a delay of hypertrophic differentiation, as seen by the reduced expression of Ihh (A,C) and ColX (B,D), and the increased distance between the Ihh expression domain and the joint region. Treatment with cyclopamine results in an advanced onset of hypertrophic differentiation as can been seen by the enlarged domain of Ihh and ColX expression (G,H). (E,F) PTHrP can rescue the advanced onset of hypertrophic differentiation in explants co-treated with PTHrP and cyclopamine. (I,J) Co-treatment with BMP2 and cyclopamine does not rescue the advanced onset of hypertrophic differentiation induced by cyclopamine, but leads to a further enlargement of the Ihh expression domain (I) compared with limbs treated with either cyclopamine (G) or BMP2 (K). Limbs treated with cyclopamine and BMP2 plus cyclopamine were derived from the same embryo. In all panels ulna is upwards and radius is downwards. cycl, cyclopamine.

View larger version (131K): [in a new window]   Fig. 5. BMP2 signaling does not act as a secondary signal of Ihh to induce PTHrP expression. Forelimbs of E14.5 mouse embryos were cultured for 4 days in control medium (A,D), or in medium supplemented with Noggin (B,C), BMP2 (E), BMP2 and cyclopamine (F), or cyclopamine (G). Limbs in A,B,E-G were derived from wild-type embryos and limbs in C,D from ColII/Ihh embryos. Sections were hybridized with a riboprobe for PTHrP. (A,B) Treatment of wild-type limbs with Noggin results in a reduction of PTHrP expression (B, arrow) compared with untreated control limbs (A). (C,D) High expression of PTHrP in limbs of ColII/Ihh embryos (D) is not reduced after Noggin treatment (C). (E) BMP2-treated limbs show normal expression of PTHrP. (F,G) Cyclopamine treatment results in a block of PTHrP expression (G), which cannot be rescued by BMP2 in double treated cultures (F). In all panels, ulna is upwards and radius is downwards. cycl, cyclopamine; Nog, Noggin.

View larger version (82K): [in a new window]   Fig. 6. Interaction of BMP and Ihh signaling in chick embryos. Wings of HH32 chick embryos were cultured for 2 days in control medium (A,B) or medium supplemented with Noggin (C,D), Shh and Noggin (E,F), Shh (G,H), BMP2 (I,J), BMP2 and cyclopamine (K,L) or cyclopamine (M,N). Parallel sections were hybridized with riboprobes for chick Ihh (A,C,E,G,I,K,M) or chick PTHrP (B,D,F,H,J,L,N). Noggin treatment results in small cartilage elements that show a reduced level of Ihh (C) and PTHrP (D) expression compared with untreated cultures (A,B). Treatment with Shh leads to a delay in hypertrophic differentiation as seen by the smaller domain of Ihh expression and the increased distance between the Ihh expression domain and the joint region (G) if compared with untreated explants (A). Shh-treated limbs show high expression of PTHrP in periarticular chondrocytes (H). Co-treatment of limbs with Shh and Noggin leads to a further reduction of Ihh expression (E) and does not block the expression of PTHrP (F). BMP2-treated limbs show high expression of Ihh (I) and PTHrP (J). Treatment with cyclopamine results in an advanced onset of hypertrophic differentiation, as seen by the expanded domain of Ihh expression and reduced distance between the Ihh expression domain and the joint region (M) compared with control limbs (A). Furthermore, PTHrP expression is blocked by cyclopamine treatment (N). Both effects cannot be rescued by BMP2 in explants co-treated with BMP2 and cyclopamine (K,L). Arrows point to the PTHrP expression domain. All panels display sections through metatarsals. cycl, cyclopamine; Nog, Noggin.

View larger version (120K): [in a new window]   Fig. 7. BMP and Ihh signaling regulate chondrocyte proliferation in parallel pathways. Forelimbs of E14.5 mouse embryos were cultured for 2 days in control medium (A,D) or in medium supplemented with Noggin (B,C), BMP2 (E), BMP2 and cyclopamine (F) or cyclopamine (G). Limbs in A,B,E-G were derived from wild-type mice, and limbs in C,D from ColII/Ihh embryos. Proliferating cells were labeled with BrdU and detected by antibody staining. (A,B) Noggin treatment results in a block of chondrocyte proliferation (B) compared with untreated limbs (A). Explants from ColII/Ihh embryos show a high level of chondrocyte proliferation (D), which is blocked after Noggin treatment (C). (E-G) BMP2 treatment results in an increased zone of proliferating cells (E) but cannot overcome the cyclopamine-induced block of chondrocyte proliferation (G) in explants double treated with BMP2 and cyclopamine (F). In all panels radius is upwards and ulna is downwards. cycl, cyclopamine; Nog, Noggin.

View larger version (77K): [in a new window]   Fig. 8. BMP signaling delays terminal hypertrophic differentiation independent of the Ihh/PTHrP system. Forelimbs of E14.5 mouse embryos were cultured for 2 days in control medium (A,B) or in medium supplemented with cyclopamine (C,D), Noggin and cyclopamine (E,F), Noggin (G,H), Noggin and PTHrP (I,J) or PTHrP (K,L). Serial sections were hybridized with riboprobes for ColX (A,C,E,G,I,K) or Osp (B,D,F,H,J,L). (A-D) Cyclopamine treated limbs show increased expression of ColX (C) but normal expression of the marker for terminally differentiated cells, Osp (D) compared with untreated limbs (A,B). (E-H) Noggin treatment results in reduced expression of ColX (G) and significantly increased expression of Osp (H), similar to co-treatment with Noggin and cyclopamine (E,F). (I-L) Limbs treated with PTHrP show reduced expression of ColX (K), which is further reduced in explants double treated with Noggin and PTHrP (I). The low expression of Osp after PTHrP treatment (L) is increased in limbs co-treated with Noggin and PTHrP (J). In all panels, ulna is upwards and radius is downwards. cycl, cyclopamine; Nog, Noggin.

View larger version (56K): [in a new window]   Fig. 9. Interaction of BMP and Ihh signaling. During endochondral ossification Ihh is expressed in the differentiating chondrocytes (red). Ihh induces the expression of PTHrP in periarticular chondrocytes (yellow) independently of BMP signaling. The range of PTHrP signaling keeps chondrocytes in a proliferating state and determines the distance from the joint region at which chondrocytes can undergo hypertrophic differentiation. Ihh in addition regulates the expression of Bmp genes in the perichondrium/periosteum and in part of the proliferating chondrocytes (green). BMP and Ihh signaling together upregulate chondrocyte proliferation, thereby pushing cells out of the range of PTHrP signaling. These chondrocytes are released from the block of differentiation and upregulate Ihh expression upon BMP signaling. BMP signaling furthermore negatively regulates the development of terminally differentiated hypertrophic chondrocytes (blue). By regulating chondrocyte proliferation, Ihh expression and terminal hypertrophic differentiation, BMP signaling integrates the different steps of endochondral ossification.