Regulation of skeletal progenitor differentiation by the BMP and retinoid signaling pathways.
(17/992)
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. (+info)
BMPs are required at two steps of limb chondrogenesis: formation of prechondrogenic condensations and their differentiation into chondrocytes.
(18/992)
Formation of the long bones requires a cartilage template. Cartilage formation (chondrogenesis) proceeds through determination of cells and their aggregation into prechondrogenic condensations, differentiation into chondrocytes, and later maturation. Several studies indicate that members of the bone morphogenetic protein (BMP) family promote cartilage formation, but the exact step(s) in which BMPs are involved during this process remains undefined. To resolve this issue, we have used a retroviral vector to misexpress the BMP antagonist Noggin in the embryonic chick limb. Unlike previous reports, we have characterized the resulting phenotype in depth, analyzing histological and early chondrogenic markers, as well as the patterns of cell death and proliferation. Misexpression of Noggin prior to the onset of chondrogenesis leads to the total absence of skeletal elements, as previously reported (J. Capdevila and R. L. Johnson, 1998, Dev. Biol. 197, 205-217). Noggin inhibits cartilage formation at two distinct steps. First, we demonstrate that mesenchymal cells do not aggregate into prechondrogenic condensations, and additional results suggest that these cells persist in an undifferentiated state. Second, we show that differentiation of chondroprogenitors into chondrocytes can also be blocked, concurrent with expanded expression of a presumptive joint region marker. In addition, we observed alterations in muscle and tendon morphogenesis, and the potential role of BMPs in these processes will be discussed. Our studies therefore provide in vivo evidence that BMPs are necessary for different steps of chondrogenesis: chondroprogenitor determination and/or condensation and subsequent differentiation into chondrocytes. (+info)
Conditional inactivation of VEGF-A in areas of collagen2a1 expression results in embryonic lethality in the heterozygous state.
(19/992)
VEGF-A has been implicated in regulating the initial angiogenic invasion events that are essential for endochondral bone formation. VEGF-A mRNA expression was indeed found in the sclerotome of the developing somite and in the limb-bud mesenchyme at E10.5 in mouse development but declined during chondrogenesis and became upregulated in hypertrophic chondrocytes prior to angiogenic invasion. To determine the functional importance of VEGF-A expression in the developing chondrogenic tissues, VEGF-A was conditionally inactivated during early embryonic development using Collagen2a1-Cre transgenic lines. Deletion of a single VEGF-A allele in Collagen2a1-Cre-expressing cells results in embryonic lethality around E10.5. This lethality is characterized by aberrant development of the dorsal aorta and intersomitic blood vessels, along with defects in the developing endocardial and myocardial layers of the heart. A small percentage of VEGF(Flox)/+, Collagen2a1-Cre fetuses survive until E17.5, show aberrant endochondral bone formation and develop a heart phenotype resembling a dilated form of ischemic cardiomyopathy. These results provide insights into the function of VEGF-A in heart and endochondral bone formation and underscore the importance of tightly controlled levels of VEGF-A during development. (+info)
Convergence of the BMP and EGF signaling pathways on Smad1 in the regulation of chondrogenesis.
(20/992)
Bone morphogenetic protein 4 (BMP4) induces, whereas epidermal growth factor (EGF) inhibits chondrogenesis. We hypothesize that BMP4 and EGF mediated intracellular signals are both coupled in the regulation of Meckel's cartilage development. Two chondrogenic experimental model systems were employed to test the hypothesis: (1) an ex vivo, serum-free, organ culture system for mouse embryonic mandibular processes, and (2) a micromass culture system for chicken embryonic mandibular processes. Chondrogenesis was assayed by alcian blue staining and expression of Sox9 and type II collagen. Exogenous EGF inhibited and BMP4 induced ectopic cartilage in a dose-dependent manner. When BMP4- and EGF-soaked beads were implanted in juxtaposition within embryonic day 10 mouse mandibular processes, the incidence and amount of ectopic cartilage, and Sox9 and type II collagen expression induced by BMP4, were significantly reduced as the concentration of EGF was increased. Similarly, in chicken serum-free micromass cultures, expression of a constitutively active BMP receptor type IB by replication competent avian retrovirus system promoted the rate and extent of chondrogenesis; however, exogenous EGF attenuated this effect. In micromass cultures, BMP signaling resulted in nuclear translocation and accumulation of the signaling molecule Smad1, whereas the addition of EGF inhibited this event. Our results suggest that BMP4 and EGF function antagonistically, yet are coupled in the regulation of initial chondrogenesis. Smad1 serves as a point of convergence for the integration of two different growth factor signaling pathways during chondrogenesis. (+info)
Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I.
(21/992)
Membrane-type matrix metalloproteinase I (MT1-MMP)-deficient mice were found to have severe defects in skeletal development and angiogenesis. The craniofacial, axial, and appendicular skeletons were severely affected, leading to a short and domed skull, marked deceleration of postnatal growth, and death by 3 wk of age. Shortening of bones is a consequence of decreased chondrocyte proliferation in the proliferative zone of the growth plates. Defective vascular invasion of cartilage leads to enlargement of hypertrophic zones of growth plates and delayed formation of secondary ossification centers in long bones. In an in vivo corneal angiogenesis assay, null mice did not have angiogenic response to implanted FGF-2, suggesting that the defect in angiogenesis is not restricted to cartilage alone. In tissues from null mice, activation of latent matrix metalloproteinase 2 was deficient, suggesting that MT1-MMP is essential for its activation in vivo. (+info)
Positionally-dependent chondrogenesis induced by BMP4 is co-regulated by Sox9 and Msx2.
(22/992)
Cranial neural crest cells emigrate from the posterior midbrain and anterior hindbrain to populate the first branchial arch and eventually differentiate into multiple cell lineages in the maxilla and mandible during craniofacial morphogenesis. In the developing mouse mandibular process, the expression profiles of BMP4, Msx2, Sox9, and type II collagen demonstrate temporally and spatially restrictive localization patterns suggestive of their functions in the patterning and differentiation of cartilage. Under serumless culture conditions, beads soaked in BMP4 and implanted into embryonic day 10 (E10) mouse mandibular explants induced ectopic cartilage formation in the proximal position of the explant. However, BMP4-soaked beads implanted at the rostral position did not have an inductive effect. Ectopic chondrogenesis was associated with the up-regulation of Sox9 and Msx2 expression in the immediate vicinity of the BMP4 beads 24 hours after implantation. Control beads had no effect on cartilage induction or Msx2 and Sox9 expression. Sox9 was induced at all sites of BMP4 bead implantation. In contrast, Msx2 expression was induced more intensely at the rostral position when compared with the proximal position, and suggested that Msx2 expression was inhibitory to chondrogenesis. To test the hypothesis that over-expression of Msx2 inhibits chondrogenesis, we ectopically expressed Msx2 in the mandibular process organ culture system using adenovirus gene delivery strategy. Microinjection of the Msx2-adenovirus to the proximal position inhibited BMP4-induced chondrogenesis. Over-expression of Msx2 also resulted in the abrogation of endogenous cartilage and the down-regulation of type II collagen expression. Taken together, these results suggest that BMP4 induces chondrogenesis, the pattern of which is positively regulated by Sox9 and negatively by Msx2. Chondrogenesis only occurs at sites where Sox9 expression is high relative to that of Msx2. The combinatorial action of these transcription factors appear to establish a threshold for Sox9 function and thereby restricts the position of chondrogenesis. (+info)
Platelet-derived growth factor A modulates limb chondrogenesis both in vivo and in vitro.
(23/992)
Cartilage formation in the chick limb follows rapid proliferation, condensation and differentiation of limb mesenchyme. The control of these early events is poorly understood. Platelet-derived growth factor receptor alpha (PDGFR-alpha) is present throughout the mesenchyme of early chick limb buds, while its ligand, PDGF-A, is expressed in the surrounding epithelium. PDGFR-alpha is down-regulated in areas that will not give rise to cartilage and is then lost from cartilage forming areas after they begin to differentiate. PDGF-A increases chondrogenesis in micromass cultures of stage-20-24 limb buds, but not stage 25, where it inhibits chondrogenesis. Ectopic PDGF-A in the chick wing can lead to either a localized increase in cartilage formation, or an inhibition. Inhibition of PDGF signalling in the chick limb results in the loss of cartilage. These data demonstrate that PDGF-A functions to promote chondrogenesis at early stages of limb development and suggest that it inhibits chondrogenesis at later stages. (+info)
Col2-GFP reporter marks chondrocyte lineage and chondrogenesis during mouse skeletal development.
(24/992)
Mice were generated in which a Col2-GFP transgene serves as a reporter for the chondrocyte lineage and for chondrogenesis in live embryos and newborn pups. Cells actively engaged in chondrogenesis were identified by confocal optical sectioning within their native environments in embryos and in thick tissue slices. Chondrocytes exhibiting GFP fluorescence were purified from rib cages by high-speed cell sorting of crude cell suspensions. Intensity of fluorescence correlated with biosynthesis of procollagen II in these cells. The use of these mice and their cells provides a novel approach for studying chondrocyte differentiation and chondrogenesis during skeletal development. (+info)