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Regulation of skeletal progenitor differentiation by the BMP and retinoid signaling pathways.

Weston AD, Rosen V, Chandraratna RA, Underhill TM - J. Cell Biol. (2000)

Bottom Line: Underhill. 1997.These findings show that BMP and RAR-signaling pathways appear to operate independently to coordinate skeletal development, and that retinoid signaling can function in a BMP-independent manner to induce cartilage formation.Thus, retinoid signaling appears to play a novel and unexpected role in skeletogenesis by regulating the emergence of chondroblasts from skeletal progenitors.

View Article: PubMed Central - PubMed

Affiliation: Division of Oral Biology, School of Dentistry, The University of Western Ontario, London, Ontario, Canada.

ABSTRACT
The generation of the paraxial skeleton requires that commitment and differentiation of skeletal progenitors is precisely coordinated during limb outgrowth. Several signaling molecules have been identified that are important in specifying the pattern of these skeletal primordia. Very little is known, however, about the mechanisms regulating the differentiation of limb mesenchyme into chondrocytes. Overexpression of RARalpha in transgenic animals interferes with chondrogenesis and leads to appendicular skeletal defects (Cash, D.E., C.B. Bock, K. Schughart, E. Linney, and T.M. Underhill. 1997. J. Cell Biol. 136:445-457). Further analysis of these animals shows that expression of the transgene in chondroprogenitors maintains a prechondrogenic phenotype and prevents chondroblast differentiation even in the presence of BMPs, which are known stimulators of cartilage formation. Moreover, an RAR antagonist accelerates chondroblast differentiation as demonstrated by the emergence of collagen type II-expressing cells much earlier than in control or BMP-treated cultures. Addition of Noggin to limb mesenchyme cultures inhibits cartilage formation and the appearance of precartilaginous condensations. In contrast, abrogation of retinoid signaling is sufficient to induce the expression of the chondroblastic phenotype in the presence of Noggin. These findings show that BMP and RAR-signaling pathways appear to operate independently to coordinate skeletal development, and that retinoid signaling can function in a BMP-independent manner to induce cartilage formation. Thus, retinoid signaling appears to play a novel and unexpected role in skeletogenesis by regulating the emergence of chondroblasts from skeletal progenitors.

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Transgene-expressing cells are excluded from cartilage nodules. (a and b) Wild-type fore limb cultures were fixed and stained with alcian blue on days 2 and 4. (d and e) Transgenic fore limb cultures were stained with magental-gal on days 2 and 4, followed by alcian blue staining. (c) Transgenic hind limb cultures were stained with magenta-gal on day 4, followed by alcian blue staining. (f) Higher magnification of day 4 transgenic fore limb cultures. Transgene-expressing cells condense (white arrow), but are excluded from the cartilage nodules (black arrow). Bar: (a, b, d, and e) 1 mm; (c and f) 0.2 mm.
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Figure 1: Transgene-expressing cells are excluded from cartilage nodules. (a and b) Wild-type fore limb cultures were fixed and stained with alcian blue on days 2 and 4. (d and e) Transgenic fore limb cultures were stained with magental-gal on days 2 and 4, followed by alcian blue staining. (c) Transgenic hind limb cultures were stained with magenta-gal on day 4, followed by alcian blue staining. (f) Higher magnification of day 4 transgenic fore limb cultures. Transgene-expressing cells condense (white arrow), but are excluded from the cartilage nodules (black arrow). Bar: (a, b, d, and e) 1 mm; (c and f) 0.2 mm.

Mentions: RARα expression is normally downregulated during chondroblast differentiation in vitro (Cash et al. 1997) and in vivo (Dolle et al. 1989). The continued activity of RARα inhibits chondroblast differentiation leading to cessation of cartilage formation and to skeletal deficiencies that are reminiscent of those observed in RA teratogenicity. To examine the cell fate of transgene-expressing cells, limb mesenchyme from the fore and hind limbs of E11.5 transgenic embryos was used to set up high density primary limb bud cultures. Under these conditions, condensation and differentiation of limb mesenchyme to cartilage mimics those events occurring in vivo (Ahrens et al. 1977). Fig. 1 shows cartilage nodule formation at day 2 and 4 in wild-type (Fig. 1, a and b) and transgenic (Fig. 1d and Fig. e) fore limb cultures. Consistent with previous observations, there were fewer alcian blue–stained nodules in the transgenic derived cultures than in the wild-type cultures. To precisely examine the distribution of transgene-expressing cells in cultures, a combination of magenta-gal and alcian blue staining was used to stain transgene-expressing cells and cartilage matrix, respectively. Transgene-expressing cells (purple stained) formed condensations but were mostly excluded from cartilage nodules (Fig. 1 f). These transgene-expressing condensations are still evident after 8 d in culture, suggesting that the transgene is not being downregulated during cartilage nodule formation. Furthermore, by day 4 in hind limb cultures there is extensive transgene expression that almost completely precludes the formation of cartilage nodules (Fig. 1 c). This is consistent in vivo in that the transgene-expressing cells are not found within the cartilaginous elements (data not shown). In the transgenic embryos, fore limb expression of the transgene is restricted primarily to the posterior region, thus, only a portion of the cells in these cultures stain with magenta gal. In the hind limb, the transgene is expressed throughout the limb mesenchyme. This results in hind limb defects that are more dramatic than those observed in the fore limb (Cash et al. 1997) and coincides with an almost complete lack of cartilage formation in hind limb cultures (Fig. 1 c).


Regulation of skeletal progenitor differentiation by the BMP and retinoid signaling pathways.

Weston AD, Rosen V, Chandraratna RA, Underhill TM - J. Cell Biol. (2000)

Transgene-expressing cells are excluded from cartilage nodules. (a and b) Wild-type fore limb cultures were fixed and stained with alcian blue on days 2 and 4. (d and e) Transgenic fore limb cultures were stained with magental-gal on days 2 and 4, followed by alcian blue staining. (c) Transgenic hind limb cultures were stained with magenta-gal on day 4, followed by alcian blue staining. (f) Higher magnification of day 4 transgenic fore limb cultures. Transgene-expressing cells condense (white arrow), but are excluded from the cartilage nodules (black arrow). Bar: (a, b, d, and e) 1 mm; (c and f) 0.2 mm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169377&req=5

Figure 1: Transgene-expressing cells are excluded from cartilage nodules. (a and b) Wild-type fore limb cultures were fixed and stained with alcian blue on days 2 and 4. (d and e) Transgenic fore limb cultures were stained with magental-gal on days 2 and 4, followed by alcian blue staining. (c) Transgenic hind limb cultures were stained with magenta-gal on day 4, followed by alcian blue staining. (f) Higher magnification of day 4 transgenic fore limb cultures. Transgene-expressing cells condense (white arrow), but are excluded from the cartilage nodules (black arrow). Bar: (a, b, d, and e) 1 mm; (c and f) 0.2 mm.
Mentions: RARα expression is normally downregulated during chondroblast differentiation in vitro (Cash et al. 1997) and in vivo (Dolle et al. 1989). The continued activity of RARα inhibits chondroblast differentiation leading to cessation of cartilage formation and to skeletal deficiencies that are reminiscent of those observed in RA teratogenicity. To examine the cell fate of transgene-expressing cells, limb mesenchyme from the fore and hind limbs of E11.5 transgenic embryos was used to set up high density primary limb bud cultures. Under these conditions, condensation and differentiation of limb mesenchyme to cartilage mimics those events occurring in vivo (Ahrens et al. 1977). Fig. 1 shows cartilage nodule formation at day 2 and 4 in wild-type (Fig. 1, a and b) and transgenic (Fig. 1d and Fig. e) fore limb cultures. Consistent with previous observations, there were fewer alcian blue–stained nodules in the transgenic derived cultures than in the wild-type cultures. To precisely examine the distribution of transgene-expressing cells in cultures, a combination of magenta-gal and alcian blue staining was used to stain transgene-expressing cells and cartilage matrix, respectively. Transgene-expressing cells (purple stained) formed condensations but were mostly excluded from cartilage nodules (Fig. 1 f). These transgene-expressing condensations are still evident after 8 d in culture, suggesting that the transgene is not being downregulated during cartilage nodule formation. Furthermore, by day 4 in hind limb cultures there is extensive transgene expression that almost completely precludes the formation of cartilage nodules (Fig. 1 c). This is consistent in vivo in that the transgene-expressing cells are not found within the cartilaginous elements (data not shown). In the transgenic embryos, fore limb expression of the transgene is restricted primarily to the posterior region, thus, only a portion of the cells in these cultures stain with magenta gal. In the hind limb, the transgene is expressed throughout the limb mesenchyme. This results in hind limb defects that are more dramatic than those observed in the fore limb (Cash et al. 1997) and coincides with an almost complete lack of cartilage formation in hind limb cultures (Fig. 1 c).

Bottom Line: Underhill. 1997.These findings show that BMP and RAR-signaling pathways appear to operate independently to coordinate skeletal development, and that retinoid signaling can function in a BMP-independent manner to induce cartilage formation.Thus, retinoid signaling appears to play a novel and unexpected role in skeletogenesis by regulating the emergence of chondroblasts from skeletal progenitors.

View Article: PubMed Central - PubMed

Affiliation: Division of Oral Biology, School of Dentistry, The University of Western Ontario, London, Ontario, Canada.

ABSTRACT
The generation of the paraxial skeleton requires that commitment and differentiation of skeletal progenitors is precisely coordinated during limb outgrowth. Several signaling molecules have been identified that are important in specifying the pattern of these skeletal primordia. Very little is known, however, about the mechanisms regulating the differentiation of limb mesenchyme into chondrocytes. Overexpression of RARalpha in transgenic animals interferes with chondrogenesis and leads to appendicular skeletal defects (Cash, D.E., C.B. Bock, K. Schughart, E. Linney, and T.M. Underhill. 1997. J. Cell Biol. 136:445-457). Further analysis of these animals shows that expression of the transgene in chondroprogenitors maintains a prechondrogenic phenotype and prevents chondroblast differentiation even in the presence of BMPs, which are known stimulators of cartilage formation. Moreover, an RAR antagonist accelerates chondroblast differentiation as demonstrated by the emergence of collagen type II-expressing cells much earlier than in control or BMP-treated cultures. Addition of Noggin to limb mesenchyme cultures inhibits cartilage formation and the appearance of precartilaginous condensations. In contrast, abrogation of retinoid signaling is sufficient to induce the expression of the chondroblastic phenotype in the presence of Noggin. These findings show that BMP and RAR-signaling pathways appear to operate independently to coordinate skeletal development, and that retinoid signaling can function in a BMP-independent manner to induce cartilage formation. Thus, retinoid signaling appears to play a novel and unexpected role in skeletogenesis by regulating the emergence of chondroblasts from skeletal progenitors.

Show MeSH
Related in: MedlinePlus