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At least 19 recordsLinked to original sources
PubMed1996

N-cadherin is involved in myoblast migration and muscle differentiation in the avian limb bud.

Limb muscle formation involves invasion of the limb bud mesoderm by myogenic precursor cells from the dermomyotomes at limb bud level. Directed cell migration, homing, and differentiation of myogenic cells are controlled by the stationary cells of the limb bud mesoderm. At the level of the extracellular matrix, the molecular basis of migration control has been suggested to be exerted by the distribution of hyaluronan. Here, we demonstrate that N-cadherin-mediated interactions play a role at cell-membrane level in myoblast distribution and differentiation. N-cadherin is strongly expressed by myogenic cells in the chick limb bud and more moderately expressed by stationary mesodermal cells in the myogenic zones and progress zone. After in vivo injection of antibodies and Fab-fragments against the homophilic binding site of N-cadherin into the wing bud mesoderm, aggregates of myoblasts are found predominantly in the dorsal myogenic zone 36 hr after injection apparently due to immobilization. In the same position, areas of myf-5-positive cells are also observed. In injected limb buds, Pax-3-positive cells are less evenly distributed than in uninjected limbs. They are found to spread up to the epidermis and also form loosely arranged aggregates. After prolonged reincubation periods, injected limbs show ectopic myoblasts that are rich in desmin and areas of strongly desmin-expressing myoblasts within muscle blastemas. These effects were not observed after application of antibodies against other parts of the N-cadherin molecule. We conclude that N-cadherin is involved in myoblast migration in the limb buds via homophilic interactions and that it plays a role in signal transduction during myogenesis.

Animals↗
PubMed1995

Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud.

Limb muscles develop from cells that migrate from the somites. The signal that induces migration of myogenic precursor cells to the limb emanates from the mesenchyme of the limb bud. Here we report that the c-met-encoded receptor tyrosine kinase is essential for migration of myogenic precursor cells into the limb anlage and for migration into diaphragm and tip of tongue. In c-met homozygous mutant (-/-) mouse embryos, the limb bud and diaphragm are not colonized by myogenic precursor cells and, as a consequence, skeletal muscles of the limb and diaphragm do not form. In contrast, development of the axial skeletal muscles proceeds in the absence of c-met signalling. The specific ligand of the c-met protein, the motility and growth factor scatter factor/hepatocyte growth factor, is expressed in limb mesenchyme and can thus provide the signal for migration which is received by c-met. We have therefore identified a paracrine signalling system that regulates migration of myogenic precursor cells.

Animals↗
PubMed1988

A gradient of responsiveness to the growth-promoting activity of ZPA (zone of polarizing activity) in the chick limb bud.

Limb bud mesoderm of stage 22-23 embryos was dissected into four pieces along the anteroposterior axis and dissociated cells of each region were separately cultured under various conditions. When the cells were cultured in medium containing 0.1% fetal calf serum (serum-poor medium) only a slight increase in cell number occurred in the cultures of all four regions. However, when the cells were cultured in medium containing 10% FCS, only cells of two central regions proliferated rapidly, and no growth promotion was observed in cells in the most anterior and posterior regions. Using the serum-poor medium, we examined the growth-promoting effects of cocultured limb bud fragments and of some growth factors on the cells of four regions. Anterior, distal, and proximal fragments promoted cell proliferation and their promotive effect on the cells of each region was equal. On the other hand, posterior fragments (containing ZPA) showed stronger promotive effects on preaxial cells than on postaxial cells. For comparison with the growth-promotive effect of the posterior fragment, fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin, and retinoic acid were tested in cell culture. FGF showed position-dependent growth promotion, while EGF and insulin promoted growth in the cells of all four regions to a similar degree. Retinoic acid showed no effect on cell growth at low concentrations, and was rather toxic at high concentrations. These results suggest that the cells of the posterior region secrete an FGF-like growth factor(s), which controls normal limb development and experimental duplicate formation.

Animals↗
PubMed1989

Thalidomide may inhibit proliferation of mesenchyme in human limb buds.

Limb buds from 4- and 4.5-week-old human embryos were cultured on agar medium consisting of Medium 199, chick embryo extract and horse serum for 4 days with or without thalidomide (1-1.5 microgram/ml), and the direct effect of thalidomide was examined morphologically in histological preparations. In the explants treated with thalidomide, mitotic figures of mesenchymal cells were significantly decreased both in overall explant and in mesenchymal cell aggregates, but the extracellular matrix in the mesenchymal cell aggregates was seen in the experimental and control explants. These findings suggest that thalidomide affects undifferentiated and differentiated mesenchymal cell proliferation but not the chondrogenic capacity of the mesenchyme.

Cartilage↗
PubMed1989

Elevations in the endogenous levels of the putative morphogen retinoic acid in embryonic mouse limb-buds associated with limb dysmorphogenesis.

Retinoic acid, an endogenous metabolite of vitamin A (retinol), possesses striking biological activity akin to a morphogen in developing and regenerating vertebrate limbs. Systemic administration of retinoic acid (RA) to pregnant mammals during the period of limb organogenesis invariably results in dose-dependent dysmorphogenesis. In an attempt to uncover the mode of action of RA in the developing limb bud we analyzed, by HPLC methods, the levels of RA and its metabolic precursor, retinol, in embryonic mouse tissues prior to and following maternal exposure to a teratogenic dose of RA. Detectable levels of both RA and its isomer 13-cis-retinoic acid were found in the limb buds of Day 11 mouse embryos (40 +/- 2 somites). Although retinol was the major retinoid found in ethanolic extracts of either whole embryo or the limb buds, the latter is enriched in RA compared to the whole embryo. This indicated either a higher degree of retinol metabolism or a sequestration of RA in the limb bud compared to the rest of the embryo at this stage of development. A study of the time course of retinoid levels in treated embryos showed that changes occur rapidly, are stable for several hours, and then begin to return to pretreatment levels. After a maternal dose of 10 mg/kg RA, which resulted in a mild degree of limb anomalies, peak RA levels in the limb bud increased 50-fold over the endogenous level; a full 300-fold increase was found after a 100 mg/kg dose which results in 100% incidence of phocomelia. Interestingly, a dose-dependent depression in retinol levels was observed after RA treatment both in maternal plasma as well as the embryo. Studies are in progress to trace the intracellular disposition of both retinol and RA as well as any further active metabolite of RA in the limb buds and other embryonic tissues.

Abnormalities, Drug-Induced↗
PubMed1991

Ghox 4.7: a chick homeobox gene expressed primarily in limb buds with limb-type differences in expression.

Homeobox genes play a key role in specifying the segmented body plan of Drosophila, and recent work suggests that at least several homeobox genes may play a regulatory role during vertebrate limb morphogenesis. We have used degenerate oligonucleotide primers from highly conserved domains in the homeobox motif to amplify homeobox gene segments from chick embryo limb bud cDNAs using the polymerase chain reaction. Expression of a large number of homeobox genes (at least 17) is detected using this approach. One of these genes contains a novel homeobox loosely related to the Drosophila Abdominal B class, and was further analyzed by determining its complete coding sequence and evaluating its expression during embryogenesis by in situ hybridization. Based on sequence and expression patterns, we have designated this gene as Ghox 4.7 and believe that it is the chick homologue of the murine Hox 4.7 gene (formerly Hox 5.6). Ghox 4.7 is expressed primarily in limb buds during development and shows a striking spatial restriction to the posterior zone of the limb bud, suggesting a role in specifying anterior-posterior pattern formation. In chick, this gene also displays differences in expression between wing and leg buds, raising the possibility that it may participate in specifying limb-type identity.

Animals↗
PubMed1998

Ectopic expression of Msx-2 in posterior limb bud mesoderm impairs limb morphogenesis while inducing BMP-4 expression, inhibiting cell proliferation, and promoting apoptosis.

During early stages of chick limb development, the homeobox-containing gene Msx-2 is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproximal posterior margin. These domains of Msx-2 expression roughly demarcate the anterior and posterior boundaries of the progress zone, the highly proliferating posterior mesodermal cells underneath the apical ectodermal ridge (AER) that give rise to the skeletal elements of the limb and associated structures. Later in development as the AER loses its activity, Msx-2 expression expands into the distal mesoderm and subsequently into the interdigital mesenchyme which demarcates the developing digits. The domains of Msx-2 expression exhibit considerably less proliferation than the cells of the progress zone and also encompass several regions of programmed cell death including the anterior and posterior necrotic zones and interdigital mesenchyme. We have thus suggested that Msx-2 may be in a regulatory network that delimits the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed. In the present study we show that ectopic expression of Msx-2 via a retroviral expression vector in the posterior mesoderm of the progress zone from the time of initial formation of the limb bud severely impairs limb morphogenesis. Msx-2-infected limbs are typically very narrow along the anteroposterior axis, are occasionally truncated, and exhibit alterations in the pattern of formation of skeletal elements, indicating that as a consequence of ectopic Msx-2 expression the morphogenesis of large portions of the posterior mesoderm has been suppressed. We further show that Msx-2 impairs limb morphogenesis by reducing cell proliferation and promoting apoptosis in the regions of the posterior mesoderm in which it is ectopically expressed. The domains of ectopic Msx-2 expression in the posterior mesoderm also exhibit ectopic expression of BMP-4, a secreted signaling molecule that is coexpressed with Msx-2 during normal limb development in the anterior limb mesoderm, the posterior necrotic zone, and interdigital mesenchyme. This indicates that Msx-2 regulates BMP-4 expression and that the suppressive effects of Msx-2 on limb morphogenesis might be mediated in part by BMP-4. These studies indicate that during normal limb development Msx-2 is a key component of a regulatory network that delimits the boundaries of the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed, thus restricting the outgrowth and formation of skeletal elements and associated structures to the progress zone. We also report that rather large numbers of apoptotic cells as well as proliferating cells are present throughout the AER during all stages of normal limb development we have examined, indicating that many of the cells of the AER are continuously undergoing programmed cell death at the same time that new AER cells are being generated by cell proliferation. Thus, a balance between cell proliferation and programmed cell death may play a very important role in maintaining the activity of the AER.

Animals↗
PubMed1999

Distinct signaling molecules control Hoxa-11 and Hoxa-13 expression in the muscle precursor and mesenchyme of the chick limb bud.

The limb muscles, originating from the ventrolateral portion of the somites, exhibit position-specific morphological development through successive splitting and growth/differentiation of the muscle masses in a region-specific manner by interacting with the limb mesenchyme and the cartilage elements. The molecular mechanisms that provide positional cues to the muscle precursors are still unknown. We have shown that the expression patterns of Hoxa-11 and Hoxa-13 are correlated with muscle patterning of the limb bud (Yamamoto et al., 1998) and demonstrated that muscular Hox genes are activated by signals from the limb mesenchyme. We dissected the regulatory mechanisms directing the unique expression patterns of Hoxa-11 and Hoxa-13 during limb muscle development. HOXA-11 protein was detected in both the myogenic cells and the zeugopodal mesenchymal cells of the limb bud. The earlier expression of HOXA-11 in both the myogenic precursor cells and the mesenchyme was dependent on the apical ectodermal ridge (AER), but later expression was independent of the AER. HOXA-11 expression in both myogenic precursor cells and mesenchyme was induced by fibroblast growth factor (FGF) signal, whereas hepatocyte growth factor/scatter factor (HGF/SF) maintained HOXA-11 expression in the myogenic precursor cells, but not in the mesenchyme. The distribution of HOXA-13 protein expression in the muscle masses was restricted to the posterior region. We found that HOXA-13 expression in the autopodal mesenchyme was dependent on the AER but not on the polarizing region, whereas expression of HOXA-13 in the posterior muscle masses was dependent on the polarizing region but not on the AER. Administration of BMP-2 at the anterior margin of the limb bud induced ectopic HOXA-13 expression in the anterior region of the muscle masses followed by ectopic muscle formation close to the source of exogenous BMP-2. In addition, NOGGIN/CHORDIN, antagonists of BMP-2 and BMP-4, downregulated the expression of HOXA-13 in the posterior region of the muscle masses and inhibited posterior muscle development. These results suggested that HOXA-13 expression in the posterior muscle masses is activated by the posteriorizing signal from the posterior mesenchyme via BMP-2. On the contrary, the expression of HOXA-13 in the autopodal mesenchyme was affected by neither BMP-2 nor NOGGIN/CHORDIN. Thus, mesenchymal HOXA-13 expression was independent of BMP-2 from polarizing region, but was under the control of as yet unidentified signals from the AER. These results showed that expression of Hox genes is regulated differently in the limb muscle precursor and mesenchymal cells.

Animals↗
PubMed2003

A role for hairy1 in regulating chick limb bud growth.

Limb growth in higher vertebrate embryos is initially due to the outgrowth of limb buds and later continues as a result of elongation of the skeletal elements. The distal limb mesenchyme is crucial for limb bud outgrowth. Members of the Hairy/Enhancer of Split family of DNA binding transcriptional repressors can be effectors of Notch signaling and often act to maintain cell populations in an undifferentiated, proliferating state, properties predicted for the distal limb mesenchyme. We find that a member of this family, c-hairy1, is expressed in this region and that two alternatively spliced isoforms, c-hairy1A and c-hairy1B, of this gene are produced, predicting proteins that differ in their basic, DNA binding, domains. Viral misexpression of c-hairy1A causes a reduction in size of the limb and shortened skeletal elements, without affecting the chondrocyte differentiation program. Misexpression of c-hairy1B leads to a significantly lesser shortening of the bones, implying functional differences between the two isoforms. We conclude that c-hairy1 regulates the size of the limb, suggesting a role for Notch signaling in the distal mesenchyme.

Alternative Splicing↗
PubMed1996

BMP-2/-4 mediate programmed cell death in chicken limb buds.

During limb development, the mesenchymal cells in restricted areas of limb bud, anterior necrotic zone, posterior necrotic zone, opaque zone and interdigital necrotic zones, are eliminated by programmed cell death. The transcripts of bone morphogenetic protein (Bmp)-2 and -4 were first detected in the areas where cell death was observed, then showed overlapping expression with the programmed cell death zones except the opaque zone. To investigate the function of BMP-2 and BMP-4 during limb pattern formation, the dominant negative form of BMP receptor was overexpressed in chick leg bud via a replication-competent retrovirus to block the endogenous BMP-2/-4 signaling pathway. This resulted in excess web formation at the anterior and posterior regions of limb buds in addition to marked suppression of the regression of webbing at the interdigital regions. Significant reductions in the number of apoptotic cells in these three necrotic zones were found in the limb buds which received the virus carrying dominant negative BMP receptor. This indicates that extra tissue formation is due to suppression of programmed cell death in the three necrotic zones. Moreover, BMP-2/-4 protein induced apoptosis of mesenchymal cells isolated from the interdigital region in vitro. Other TGFbeta family proteins as TGFbeta1 and Activin did not show this effect. These results suggest that BMP-2 and BMP-4 are the apoptotic signal molecules of the programmed cell death process in the chick limb buds.

Animals↗
PubMed1997

Distribution of nestin in the developing mouse limb bud in vivo and in micro-mass cultures of cells isolated from limb buds.

Early skeletal muscle development is accompanied by changes in the composition of the cytoskeleton. In this report we analyze the distribution of the intermediate filament nestin in the developing mouse limb buds in vivo and in mesenchymal cells isolated from limb buds in vitro. The subcellular distribution of nestin mRNA and protein in muscle cells was also analyzed. We find a shift in nestin expression during early limb bud development. At embryonic day 11 (E11), low levels of nestin (protein) were expressed in the mesenchymal cells of the developing limb bud. Later, nestin mRNA and protein were down-regulated in the mesenchymal condensations undergoing chondrogenesis (E12 and E13), but remained expressed predominantly in the ectodermal cells and in the differentiating myoblasts. At E18, only muscle fibres, endothelial cells and nerves were nestin positive. This shift in expression was reproduced in vitro, in micro-mass cultures of mesenchymal cells. In E11 cultures, nestin protein was initially expressed in all cells. Upon formation of cartilage foci (after 2-3 days in culture), nestin immunoreactivity was not observed in cartilage, and low levels were detected in the cells located between the foci. A subpopulation of mono- and multinucleated cells, peripheral to the cartilage nodules, expressed the muscle-specific intermediate filament desmin protein together with high levels of nestin protein. The proportion of nestin-expressing cells could be changed by addition of specific signalling molecules. Insulin-like growth factors I and II (IGF I and II) increased the number of nestin-positive cells, while basic fibroblast growth factor (FGF) reduced the number of nestin-expressing cells. Finally, we present evidence for a different subcellular localization of nestin protein and mRNA: the mRNA is predominantly located in the ends of the muscle cell, whereas the protein is found in the central region. Intracellular localization of nestin mRNA may constitute an additional level of regulation of the cytoskeleton during muscle development.

Animals↗
PubMed1998

Hox gene expression, AV-1 antigen expression, and cartilage pattern formation in chick recombinant limb buds.

A recombinant limb bud composed of dissociated and reaggregated mesenchyme and an ectodermal jacket develops a limblike structure with bifurcated and segmented cartilage. We compared the cartilage structure formed from recombinants that were composed of mesenchymal cells derived from the limb bud progress zone at various stages. In the case of recombinants containing distal mesenchyme of early-stage limb buds (stage 18 or 20), long and thick cartilage structures were formed in the proximal region, and segmented digitlike structures were formed in the distal region. On the contrary, in the case of recombinants containing distal mesenchyme of late-stage limb buds (stage 25 or 27), only poorly developed cartilage structures were formed. Next, we analyzed expression patterns of the position-specific genes HoxA11, A13 and D12 and the position-specific antigen AV-1 protein in recombinants containing distal mesenchyme of stage 20 limb buds (stage 20 recombinants) or stage 25 limb buds (stage 25 recombinants). In stage 20 recombinants, HoxA11 was expressed throughout the mesenchyme, but HoxA13 was expressed only in the distal half of the mesenchyme. In stage 25 recombinants, HoxA13 was expressed throughout the mesenchyme, but HoxA11 was only faintly expressed. The expression pattern of HoxD12 was similar to that of HoxA13 in both stage 20 and stage 25 recombinants, and no asymmetric expression pattern, which is observed in normal limb buds, was detected. AV-1 antigen was expressed in the core region of stage 20 recombinants, and anteroposterior asymmetry, which is observed in the anterior-ventral-distal region of normal limb buds, was not found. No AV-1 expression was observed in stage 25 recombinants. These results suggest that the mesenchyme in recombinants shows spatially controlled gene/protein expressions along the proximodistal axis, and that these differences in gene/protein expressions may affect cartilage pattern formation in recombinants.

Animals↗
PubMed1999

Involvement of Frzb-1 in mesenchymal condensation and cartilage differentiation in the chick limb bud.

In developing limb bud, mesenchymal cells form cellular aggregates called "mesenchymal condensations". These condensations show the prepattern of skeletal elements of the limb prior to cartilage differentiation. Roles of various signaling molecules in chondrogenesis in the limb bud have been reported. One group of signaling factors includes the Wnt proteins, which have been shown to have an inhibitory effect on chondrogenesis in the limb bud. Therefore, regulation of Wnt activity may be important in regulating cartilage differentiation. Here we show that Frzb-1, which encodes a secreted frizzled-related protein that can bind to Wnt proteins and can antagonize the activity of some Wnts, is expressed in the developing limb bud. At early stages of limb development, Frzb-1 is expressed in the ventral core mesenchyme of the limb bud, and later Frzb-1 expression becomes restricted to the central core region where mesenchymal condensations occur. At these stages, a chondrogenic marker gene, aggrecan, is not yet expressed. As limb development proceeds, expression of Frzb-1 is detected in cartilage primordial cells, although ultimately Frzb-1 expression is down-regulated. Similar results were obtained in the recombinant limb bud, which was constructed from dissociated and re-aggregated mesenchymal cells and an ectodermal jacket with the apical ectodermal ridge. In addition, Frzb-1 expression preceded aggrecan expression in micromass cultures. These results suggest that Frzb-1 has a role in condensation formation and cartilage differentiation by regulating Wnt activity in the limb bud.

Aggrecans↗
PubMed1990

Comparison between ectoderm-conditioned medium and fibronectin in their effects on chondrogenesis by limb bud mesenchymal cells.

Limb bud ectoderm inhibits chondrogenesis by limb bud mesenchymal cells cultured at high density or on collagen gels. This ectodermal antichondrogenic influence has been postulated to function in vivo in regulating the spatial patterning of cartilage and soft connective tissue in the limb. We have developed a method for preparing ectoderm-conditioned medium containing antichondrogenic activity. Using a simple bioassay, we have investigated some characteristics of the ectodermal products and their effects on limb bud mesenchymal cells. Inhibition of chondrogenesis by ectoderm-conditioned medium was tested on limb bud mesenchymal cells cultured on collagen gels. The antichondrogenic influence involves enhanced cell spreading and is alleviated by agents, such as cytochalasin D, that induce cell rounding. Fibronectin resembles ectoderm-conditioned medium in its ability to inhibit chondrogenesis and promote cell spreading in collagen gel cultures of limb bud mesenchymal cells. However, Western blot analysis shows that the antichondrogenic activity of ectoderm-conditioned medium is not due to fibronectin in the medium. Peptides related to the fibronectin cell-binding domain block the antichondrogenic effect of fibronectin, but not that of ectoderm-conditioned medium. On the other hand, an antibody to an integrin, as well as heparan sulfate, alleviates the antichondrogenic effects of both fibronectin and ectoderm-conditioned medium. The antichondrogenic effect of ectoderm-conditioned medium may be mediated by an integrin and by a cell surface heparan sulfate proteoglycan, but it does not depend directly upon fibronectin-mediated cell spreading.

Amino Acid Sequence↗
PubMed1989

A study on skeletal myogenic cell movement in the developing avian limb bud.

Quail limb mesenchyme containing myogenic cells of somitic origin were transplanted into chick limb buds to determine whether cell movement might play a role in avian limb myogenesis. In general, cell displacement was not detected 1-day after implantation: all quail cells were found at the graft site. Migration was evident 2-days after implantation but not all cell types were capable of movement; myogenic cells were very invasive while chondrocytes were relatively immobile. The spreading of myogenic cells was discernible up to 4-days after implantation and specifically in a proximodistal direction towards the apex of the limb.

Animals↗
PubMed1982

Analysis of the intermediate size proteoglycans from the developing chick limb buds.

Limb-bud proteoglycans are heterogeneous molecules which vary in their chemical and physical properties with development. This report describes proteoglycan intermediates (PG-I) that predominate in stage-34 limbs, and compares them with proteoglycan aggregates (PG-A) in stage-38 limbs. We analysed proteoglycans and their components extracted with guanidinium chloride by subjecting them to density gradient centrifugation, molecular sieve chromatography, electrophoretic separation, and selective enzymatic degradation. PG-I and PG-A have similar chondroitin sulphate composition, amino sugars, chondroitin sulphate side-chain length, glycoprotein link factors, and hyaluronic acid binding capacity, and both cross react with antisera prepared against cartilage-specific chick sternal proteoglycans. However, PG-I has lower molecular weight, lower buoyant density, and fewer chondroitin sulphate side chains on the protein core. The PG-I in the developing limb can be considered a mixture of smaller aggregates and cartilage-specific large monomers in which the former predominate.

Animals↗
PubMed2003

Regulation of myogenic differentiation in the developing limb bud.

The limb myogenic precursors arise by delamination from the lateral dermomyotome in response to signals from the lateral plate mesoderm. They subsequently migrate into the developing limb bud where they switch on the expression of the myogenic regulatory factors, MyoD and Myf5, and coalese to form the dorsal and ventral muscle masses. The myogenic cells subsequently undergo terminal differentiation into slow or fast fibres which have distinct contractile properties determining how a muscle will function. In general, fast fibres contract rapidly with high force and are characterized by the expression of fast myosin heavy chains (MyHC). These fibres are needed for movement. In contrast, slow fibres express slow MyHC, contract slowly and are required for maintenance of posture. This review focuses on the molecular signals that control limb myogenic development from the initial delamination and migration of the premyogenic cells to the ultimate formation of the complex muscle pattern and differentiation of slow and fast fibres.

Animals↗