Fibronectin gene expression during limb cartilage differentiation

A critical event in limb cartilage differentiation is a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely juxtaposed and interact with one another prior to initiating cartilage matrix deposition. Fibronectin (FN) has been suggested to be involved in regulating the onset of condensation and chondrogenesis by actively promoting prechondrogenic aggregate formation during the process. We have performed a systematic quantitative study of the expression of the FN gene during the progression of chondrogenesis in vitro and in vivo. In high-density micromass cultures of limb mesenchymal cells, FN mRNA levels increase about 5-fold coincident with the crucial condensation process, and remain relatively high during the initial deposition of cartilage matrix by the cells. Thereafter, FN mRNA levels progressively decline to relatively low levels as the cultures form a virtually uniform mass of cartilage. The changes in FN mRNA levels in vitro are paralleled closely by changes in the relative rate of FN synthesis as determined by pulse-labeling and immunoprecipitation analysis. The relative rate of FN synthesis increases 4- to 5-fold at condensation and the onset of chondrogenesis, after which it progressively declines to low levels as cartilage matrix accumulates. High levels of FN gene expression also occur at the onset of chondrogenesis in vivo. In the proximal central core regions of the limb bud in which condensation and cartilage matrix deposition are being initiated, FN mRNA levels and the relative rates of FN synthesis become progressively about 4-fold higher than in the distal subridge region, which consists of undifferentiated mesenchymal cells that have not yet initiated condensation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Retinoic acid promotes proliferation and chondrogenesis in the distal mesodermal cells of chick limb bud

Distal and proximal mesoderm of chick limb bud was respectively dissociated and cultured in the medium containing various concentrations of retinoic acid (RA). At low concentrations (5-50 ng/ml), RA promoted proliferation and chondrogenesis in the distal mesodermal cells. The distal cells of stage 20-24 limb buds were responsive to RA, although those of stages 25-27 were unresponsive. Both the cells of anterior and posterior regions of the distal mesoderm were responsive to RA, while the cells of proximal mesoderm were unresponsive. At higher concentrations, the growth-promoting effect of RA was reduced and chondrogenesis in the distal cells was rather inhibited. These results were discussed in relation to the role of RA as the morphogen in normal limb development and experimental duplicate formation.     

Hyaluronan in limb morphogenesis

Hyaluronan (HA) is a large glycosaminoglycan that is not only a structural component of extracellular matrices, but also interacts with cell surface receptors to promote cell proliferation, migration, and intracellular signaling. HA is a major component of the extracellular matrix of the distal subapical mesenchymal cells of the developing limb bud that are undergoing proliferation, directed migration, and patterning in response to the apical ectodermal ridge (AER), and has the functional potential to be involved in these processes. Here we show that the HA synthase Has2 is abundantly expressed by the distal subridge mesodermal cells of the chick limb bud and also by the AER itself. Has2 expression and HA production are downregulated in the proximal central core of the limb bud during the formation of the precartilage condensations of the skeletal elements, suggesting that downregulation of HA may be necessary for the close juxtaposition of cells and the resulting cell-cell interactions that trigger cartilage differentiation during condensation. Overexpression of Has2 in the mesoderm of the chick limb bud in vivo results in the formation of shortened and severely malformed limbs that lack one or more skeletal elements. Skeletal elements that do form in limbs overexpressing Has2 are reduced in length, exhibit abnormal morphology, and are positioned inappropriately. We also demonstrate that sustained HA production in micromass cultures of limb mesenchymal cells inhibits formation of precartilage condensations and subsequent chondrogenesis, indicating that downregulation of HA is indeed necessary for formation of the precartilage condensations that trigger cartilage differentiation. Taken together these results suggest involvement of HA in various aspects of limb morphogenesis.     

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.     

Temporal and spatial analysis of hyaluronidase activity during development of the embryonic chick limb bud

The glycosaminoglycan hyaluronate (HA) appears to play an important role in limb cartilage differentiation. The large amount of extracellular HA accumulated by prechondrogenic mesenchymal cells may prevent the cell-cell and/or cell-matrix interactions necessary to trigger chondrogenesis, and the removal of extracellular HA may be essential to initiate the crucial cellular condensation process that triggers cartilage differentiation. It has generally been assumed that HA turnover during chondrogenesis is controlled by the activity of the enzyme hyaluronidase (HAase). In the present study we have performed a temporal and spatial analysis of HAase activity during the progression of limb development and cartilage differentiation in vivo. We have separated embryonic chick wing buds at several stages of development into well-defined regions along the proximodistal axis in which cells are in different phases of differentiation, and we have examined HAase activity in each region. We have found that HAase activity is clearly detectable in undifferentiated wing buds at stage 18/19, which is shortly following the formation of a morphologically distinct limb bud rudiment, and remains relatively constant throughout subsequent stages of development through stage 27/28, at which time well-differentiated cartilage rudiments are present. Moreover, HAase activity in the prechondrogenic distal subridge regions of the limb at stages 22/23 and 25 is just as high as, or even slightly higher than, it is in proximal central core regions where condensation and cartilage differentiation are progressing. We have also found that limb bud HAase is active between pH 2.2 and 4.5 and is inactive above pH 5.0. This suggests that limb HAase is a lysosomal enzyme and that extracellular HA would have to be internalized to be degraded. These results indicate that the onset of chondrogenesis is not associated with the appearance or increase in activity of HAase. We suggest that possibility that HA turnover may be regulated by the binding and endocytosis of extracellular HA in preparation for its intracellular degradation by lysosomal HAase. Finally, we have found that the apical ectodermal ridge (AER)-containing distal limb bud ectoderm possesses a relatively high HAase activity. We suggest the possibility that a high HAase activity in the AER may ensure a rapid turnover and remodeling of the disorganized HA-rich basal lamina of the AER that might be essential for limb outgrowth.     

Cell sorting and chondrogenic aggregate formation in micromass culture

A fundamental feature of cartilage differentiation in the developing limb is the formation of a prechondrogenic cell condensation. An apparently similar process of prechondrogenic cell aggregation occurs in micromass cultures of limb bud mesenchyme with the formation of cellular aggregates which often differentiate into cartilage nodules. We have investigated the process of aggregate formation in micromass culture using chimaeric mixtures of potentially chondrogenic and nonchondrogenic cell types. Two systems were studied: mixtures of distal and proximal limb mesenchyme cells and mixtures of distal limb cells with avian tendon fibroblasts. In both cases cultures of varying proportions of each cell type have been prepared. The results demonstrate that aggregate formation in vitro is the consequence of a cell sorting process which can involve prechondrogenic cells of widely different spatial origins within the developing limb. This contrasts with in vivo prechondrogenic condensation in which there is no evidence of cell sorting (Searls, R.L. (1967), J. Exp. Zool. 166, 39-50). However, our findings do indicate that cell surface differences occur in apparently undifferentiated limb mesenchyme. The results also suggest that mesenchymal cell aggregates must achieve a threshold size before chondrogenesis can proceed. In addition, the results show that under some culture conditions nonchondrogenic cells will form aggregates.     

Bioassay of retinoids using cultured human conjunctival keratinocytes

The biological action of retinoids has been assayed using the differentiated properties of cultured human conjunctival keratinocytes. The effects measured were the suppression of envelope cross-linking and the promotion of synthesis of a keratin of molecular weight 40,000. Among the retinoids tested, the most powerful was the arotinoid Ro 13-6298, which reduced envelope formation detectably at 10(-11) M and by 90% at a concentration of 2 X 10(-10) M. The arotinoid was about 15 times more potent than trans-retinoic acid. The order of effectiveness of the retinoids in suppressing envelope cross-linking was the same as the order of effectiveness in promoting the synthesis of the 40-kd keratin.     

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.     

Role of chondrogenic tissue in programmed cell death and BMP expression in chick limb buds

In the developing chick leg bud, massive programmed cell death occurs in the interdigital region. Previously, we reported the inhibition of cell death by separation of the interdigital region from neighboring digit cartilage. In this study, we examined the relationship between cell death and cartilaginous tissue in vitro. First, cell fate was observed with DiI that was used to examine cell movement in the distal tip of leg bud. Labeled cells in the prospective digital region were distributed only in the distal region as a narrow band, while cells in the prospective interdigital region expanded widely in the interdigit. In coculture of monolayer cells and a cell pellet tending to differentiate into cartilage, monolayer cells migrated into the cell pellet. These results suggested that digit cartilage tends to recruit neighboring cells into the cartilage during limb development. Next, we observed the relationship between cell death and chondrogenesis in monolayer culture. Apoptotic cell death that could be detected by TUNEL occurred in regions between cartilaginous nodules in mesenchymal cell culture. More apoptotic cell death was detected in the cell culture of leg bud mesenchyme of stage 25/26 than that of leg bud mesenchyme of stage 22 or that of stage 28. The most developed cartilaginous nodules were observed in the cell culture of stage 25/26. Finally, we observed Bmp expression in vitro and in vivo. Bmp-2, Bmp-4 and Bmp-7 were detected around the cartilage nodules. When the interdigit was separated from neighboring digit cartilage, Bmp-4 expression disappeared near the cut region but remained near the digit cartilage. This correlation between cell death and cartilaginous region suggests that cartilage tissue can induce apoptotic cell death in the developing chick limb bud due to cell migration accompanying chondrogenesis and Bmp expression.     

N-Cadherin expression and signaling in limb mesenchymal chondrogenesis: stimulation by poly-L-lysine

Cellular condensation is a requisite step in the initiation of mesenchymal chondrogenesis in the embryonic limb bud. We have previously shown that cellular condensation of limb chondroprogenitor mesenchymal cells is accompanied by elevated expression of N-cadherin during chondrogenesis both in vivo and in vitro. N-Cadherin-mediated cell-cell interaction is also functionally required for proper mesenchymal chondrogenesis both in vivo and in vitro. In this report, we have further analyzed the functional importance of N-cadherin in the cellular condensation-chondrogenesis pathway by examining N-cadherin expression and related activities in high density micromass cultures of chick limb mesenchymal cells in which chondrogenesis is being stimulated with the cationic polymer, poly-L-lysine (PL). The chondrogenesis-promoting action of PL is thought to involve the clustering of cells via ionic cross-linking, perhaps mimicking the action of an endogenous matrix component. Immunohistochemistry, immunoblotting, and Northern blot analysis all show that PL treatment results in a time-dependent increase in N-cadherin expression at both the protein and mRNA levels. In addition, inhibition of N-cadherin function with a neutralizing monoclonal antibody directed to its extracellular domain inhibits the chondrogenesis-stimulating effect of PL. PL treatment also alters the tyrosine-phosphorylation state of the N-cadherin associated signaling protein, beta-catenin. These results suggest that N-cadherin-mediated cell adhesion is a requisite regulatory component of the limb mesenchymal chondrogenic differentiation program, involving at least in part beta-catenin tyrosine phosphorylation as a signaling step.     

The effects of local application of retinoids to different positions along the proximo-distal axis of embryonic chick wings

Summary Two retinoids, all-trans-retinoic acid and a synthetic analog, TTNPB, were locally applied to different positions along the proximo-distal axis of embryonic chick wing buds using controlled release carriers. Truncations or limbs with duplicated structures across the antero-posterior axis develop after retinoid application to distal positions in buds from stage 20–24 embryos. Phocomelic limbs develop when the retinoids are applied more proximally to buds of stage 23–24 embryos. Duplications of the pattern of structures along the proximo-distal axis never occur.Using TTNPB that is relatively stable, the amount of retinoid in the wing tissue when phocomelia is induced was measured. There is twice as much retinoid per cell in the proximal half of the bud as in the distal half of the bud. The concentration of TTNPB in proximal tissue is estimated to be three times higher than in distal tissue in which pattern formation and cartilage morphogenesis are relatively normal.At early stages in the development of phocomelia, the shape of the bud changes and the indentation that marks the elbow does not arise. Neither retinoid-induced cell killing nor effects on the pattern of programmed cell death were detected.The induction of phocomelia by retinoids appears to be based on effects on proximal cells, whereas retinoids produce pattern changes by acting on distal cells. Furthermore, compared with pattern changes, higher concentrations of retinoid in the bud tissue are required to produce phocomelia.     

Two distinct classes of prechondrogenic cell types in the embryonic limb bud

Differences are demonstrated in the chondrogenic potential of cells derived from the distal and proximal halves of chick wing buds from as early as stage 23, prior to the appearance of overt cartilage differentiation. In high cell density cultures, cells obtained from the distal portions of stage 23 or 24 limb buds are spontaneously chondrogenic in micromass cultures. Cells obtained from the proximal portions, however, become blocked in their differentiation as protodifferentiated cartilage cels, since these cells in micromass cultures make detectable type II collagen, but fail to synthesize significant levels of cartilage proteoglycan or to accumulate an extracellular matrix that will stain for sulfated glycosaminoglycans. Such cultures of proximal limb bud cells can be stimulated to form alcian blue staining nodules by the addition of 1 mM dbcAMP or 50 micrograms/ml ascorbate, or by mixing proximal cells with small numbers of distal cells (1 distal cell to 10 proximal cells). These results demonstrate the existence of two distinct stages among prechondrogenic mesenchyme cells. The earlier stage appears to be able to provide a chondrogenic stimulus to proximal cells.     

Effects of vitamin D metabolites on axolotl limb regeneration

Vitamin D is essential for normal metabolism of phosphorus and calcium, and differentiation of skeletal elements. 1,25 dihydroxyvitamin-D₃, the biologically active metabolite, acts as an induction/proliferation switch in various cell types and promotes chondrogenesis of chick limb bud mesenchymal cells. The function of vitamin D is mediated through its nuclear receptor, the vitamin D receptor (VDR). The proliferative actions of 1,25(OH)₂-D₃ on limb bud mesenchymal cells are similar to the ones produced by retinoids, such as all- trans retinoic acid (RA) or 9- cis retinoic acid (9- cis ). The retinoids have been shown to be compounds of extreme importance in the field of limb development and regeneration. In order to examine possible roles of vitamin D metabolites on limb regeneration, the effects of 1,25(OH)₂-D₃, 24,25(OH)₂-D₃ and KH1060 (a more potent metabolite) alone or in conjunction with all- trans RA or 9- cis RA on the regenerating axolotl limb. Vitamin D affects limb morphogenesis by generating abnormalities in skeletal elements. Synergism of vitamin D with retinoic acid in affecting pattern formation is suggested by the results.     

Chondrogenesis in mouse limb bud in vitro. I. Effect of the ectoderm

The role of the ectoderm in the chondrogenesis of mouse limb bud mesoderm was investigated in vitro at several developmental stages by analysis of the evolution of DNA content, the accumulation of sulfated glycosaminoglycans and histochemical procedures. Young limb buds or the undifferentiated apex of older buds (stages 17 and 19 of Theiler's table) from which the ectoderm had been removed with trypsin treatment initiated a large chondrogenesis but not morphogenesis. When the ectoderm was present, these limb buds showed a polarized proximal to distal outgrowth and differentiated skeletal primordia. Mesodermal cells of stage 20 limb bud apex were able to differentiate autopodial skeletons with or without the presence of the ectoderm: cartilaginous areas of the limb skeleton seem determined at this developmental stage. These results, which show the importance of the ectoderm in limb bud morphogenesis, are compared with results obtained using other methods with mouse or bird buds.     

Ephrin-A2 regulates position-specific cell affinity and is involved in cartilage morphogenesis in the chick limb bud

In the developing limb bud, mesenchymal cells show position-specific affinity, suggesting that the positional identity of the cells is represented as their surface properties. Since the affinity is regulated by glycosylphosphatidylinositol (GPI)-anchored cell surface proteins, and by EphA4 receptor tyrosine kinase, we hypothesized that the GPI-anchored ligand, the ephrin-A family, also contributes to the affinity. Here, we describe the role of ephrin-A2 in the chick limb bud. Ephrin-A2 protein is uniformly distributed in the limb bud during early limb development. As the limb bud grows, expression of ephrin-A2 is strong in its proximal-to-intermediate regions, but weak distally. The position-dependent expression is maintained in vitro, and is regulated by FGF protein, which is produced in the apical ectodermal ridge. To investigate the role of ephrin-A2 in affinity and in cartilage morphogenesis of limb mesenchyme, we ectopically expressed ephrin-A2 in the limb bud using the retrovirus vector, RCAS. Overexpressed ephrin-A2 modulated the affinity of the mesenchymal cells that differentiate into autopod elements. It also caused malformation of the autopod skeleton and interfered with cartilage nodule formation in vitro without inhibiting chondrogenesis. These results suggest that ephrin-A2 regulates the position-specific affinity of limb mesenchyme and is involved in cartilage pattern formation in the limb.     

Retinoid suppression of transglutaminase activity and envelope competence in cultured human epidermal carcinoma cells

Growth of SCC-13 squamous carcinoma cultures in the presence of retinoids considerably reduced the expression of two differentiation markers, the cellular capability to form cross-linked envelopes, and the enzyme transglutaminase required for cross-linking. A limited survey of retinoids showed that all-trans retinoic acid, 13-cis retinoic acid, and arotinoid Ro 13-6298 were highly effective in the absence of hydrocortisone and were only slightly antagonized by its presence in the medium. In contrast, retinyl acetate, retinol, and retinol bound to its plasma binding protein were quite active in the absence of hydrocortisone but were essentially inactive in its presence. Dexamethasone was also highly effective in antagonizing the suppressive action of retinyl acetate on envelope formation, while the corticosteroid antagonists cortexolone and progesterone were inactive. These results suggest that there are separate pathways, which are differentially regulated by hydrocortisone, for either the metabolism or action of retinol and retinoic acid in SCC-13 cells.