Sox9 expression during chondrogenesis in micromass cultures of embryonic limb mesenchyme.

Sox9 plays a crucial role in chondrogenesis. It encodes an HMG-domain transcription factor that activates an enhancer in the gene for type II collagen (Col2a1), a principal cartilage matrix protein. We have characterized the temporal pattern of Sox9 RNA expression in micromass culture, a widely used in vitro model for the analysis of embryonic cartilage differentiation. Cultures were prepared from distal subridge mesenchyme of the stage 24/25 chick embryo wing bud, which undergoes uniform chondrogenic differentiation in vitro. The early "prechondrogenic" phase of culture was characterized by the activation of Sox9 RNA expression, which preceded detectable upregulation of Col2a1 transcription. Sox9 RNA levels peaked between 20 and 65 h of culture, a phase of progressive Col2a1 transcript accumulation, then declined in the mature cartilage of 120-h cultures. Staurosporine treatment enhanced chondrogenesis in micromass culture by inducing a rapid quantitative increase in Sox9 transcript levels. However, PMA, a phorbol ester that inhibits Col2a1 expression and chondrocyte differentiation, had an unexpectedly modest effect on Sox9 RNA accumulation.

Alcohol promotes in vitro chondrogenesis in embryonic facial mesenchyme.

Ethanol is a well-recognized teratogen in vertebrates that can perturb the development of the facial primordia and various other embryonic structures. However,the mechanisms underlying alcohol's effects on embryogenesis are currently unclear. Recent evidence suggests that the cranial neural crest, which forms the entire facial skeleton, may be a particularly sensitive target of ethanol teratogenicity. In the present study we have examined the influence of in vitro ethanol exposure on cartilage differentiation in micromass cultures of mesenchymal cells isolated from the various facial primordia (maxillary, mandibular, frontonasal, and hyoid processes) of the stage 24 chick embryo. In all four populations of facial mesenchyme, exposure to 1-1.5% ethanol promoted marked increases in Alcian blue-positive cartilage matrix formation, a rise in 35SO4 accumulation into matrix glycosaminoglycans, and enhanced expression of cartilage-characteristic type II collagen and aggrecan gene transcripts. In frontonasal and mandibular mesenchyme cultures, which undergo extensive spontaneous cartilage formation, ethanol treatment quantitatively elevated both matrix production and cartilage-specific gene transcript expression. In cultures of maxillary process and hyoid arch mesenchyme, which form little or no cartilage spontaneously, ethanol exposure induced the formation of chondrogenic cell aggregates and the appearance of aggrecan and type II collagen mRNAs. These actions were not restricted to ethanol, since tertiary butanol treatment also enhanced cartilage differentiation in facial mesenchyme cultures. Our findings demonstrate a potent stimulatory effect of alcohol on the differentiation of prechondrogenic mesenchyme of the facial primordia. Further analysis of this phenomenon might yield insight into the developmental mechanisms underlying the facial dysmorphologies associated with embryonic ethanol exposure.

Ethanol exposure stimulates cartilage differentiation by embryonic limb mesenchyme cells.

Studies of neural, hepatic, and other cells have demonstrated that in vitro ethanol exposure can influence a variety of membrane-associated signaling mechanisms. These include processes such as receptor-kinase phosphorylation, adenylate cyclase and protein kinase C activation, and prostaglandin production that have been implicated as critical regulators of chondrocyte differentiation during embryonic limb development. The potential for ethanol to affect signaling mechanisms controlling chondrogenesis in the developing limb, together with its known ability to promote congenital skeletal deformities in vivo, prompted us to examine whether chronic alcohol exposure could influence cartilage differentiation in cultures of prechondrogenic mesenchyme cells isolated from limb buds of stage 23-25 chick embryos. We have made the novel and surprising finding that ethanol is a potent stimulant of in vitro chondrogenesis at both pre- and posttranslational levels. In high-density cultures of embryonic limb mesenchyme cells, which spontaneously undergo extensive cartilage differentiation, the presence of ethanol in the culture medium promoted increased Alcian-blue-positive cartilage matrix production, a quantitative rise in 35SO4 incorporation into matrix glycosaminoglycans (GAG), and the precocious accumulation of mRNAs for cartilage-characteristic type II collagen and aggrecan (cartilage proteoglycan). Stimulation of matrix GAG accumulation was maximal at a concentration of 2% ethanol (v/v), although a significant increase was elicited by as little as 0.5% ethanol (approximately 85 mM). The alcohol appears to directly influence differentiation of the chondrogenic progenitor cells of the limb, since ethanol elevated cartilage formation even in cultures prepared from distal subridge mesenchyme of stage 24/25 chick embryo wing buds, which is free of myogenic precursor cells. When limb mesenchyme cells were cultured at low density, which suppresses spontaneous chondrogenesis, ethanol exposure induced the expression of high levels of type II collagen and aggrecan mRNAs and promoted abundant cartilage matrix formation. These stimulatory effects were not specific to ethanol, since methanol, propanol, and tertiary butanol treatments also enhanced cartilage differentiation in embryonic limb mesenchyme cultures. Further investigations of the stimulatory effects of ethanol on in vitro chondrogenesis may provide insights into the mechanisms regulating chondrocyte differentiation during embryogenesis and the molecular basis of alcohol's teratogenic effects on skeletal morphogenesis.

Cloning and characterization of a cDNA encoding the collagen-binding stress protein hsp47 in zebrafish.

Hsp47 is a major stress-inducible protein that is localized to the endoplasmic reticulum of avian and mammalian cells and is thought to act as a molecular chaperone specific for the processing of procollagen. Although hsp47 is coordinately expressed together with several collagen types, and vertebrate embryos are known to express collagen genes in complex spatial and temporal patterns, limited information is available regarding the function or regulation of hsp47 during early embryonic development. We have initiated an examination of hsp47 in the zebrafish, Danio rerio, which offers a number of features that make it attractive as a model developmental system with which to examine the expression and function of hsp47. A polymerase chain reaction (PCR)-based cloning strategy was used to isolate a hsp47 cDNA from an embryonic zebrafish cDNA library. The deduced translation product of the cDNA is a 404-amino-acid polypeptide that is 72% identical to chicken, 64% identical to mouse and rat, and 69% identical to human hsp47. The protein contains a typical hydrophobic signal sequence, an RDEL endoplasmic reticulum retention signal, and a serine protease inhibitor signature sequence, all of which are characteristic of hsp47 in higher vertebrates. Thus, it is likely that hsp47 in zebrafish is also localized to the endoplasmic reticulum and may play a similar role to its counterpart in higher vertebrates. Northern blot analysis revealed that the hsp47 gene is expressed at relatively low levels in embryos during normal development but is strongly induced following exposure to heat shock at the gastrula, midsomitogenesis, 2-day, and 3-day larval stages. The level of induction was much higher than has previously been reported in chicken and mouse cells.

Staurosporine, a protein kinase inhibitor, stimulates cartilage differentiation by embryonic facial mesenchyme.

We have examined the effects of staurosporine, a potent inhibitor of protein kinase C, on cartilage differentiation in cultured mesenchyme of embryonic facial primordia. Mesenchymal cells from the frontonasal, maxillary, and mandibular processes and hyoid arches of stage 24/25 chicken embryos were maintained in high density micromass cell cultures in the presence or absence of 5 nM staurosporine. In cultures of frontonasal and mandibular process mesenchyme, which spontaneously developed numerous chondrogenic cell aggregates, staurosporine treatment enhanced Alcian blue-positive matrix accumulation, increased pericellular sulfated glycosaminoglycan (GAG) deposition by 5.8- and 2.7-fold, respectively, and elevated cytoplasmic levels of cartilage-specific proteoglycan mRNA. In maxillary process mesenchyme, which formed little cartilage matrix under control culture conditions, staurosporine treatment stimulated extensive cartilage nodule formation, promoted a 5.4-fold rise in matrix GAG accumulation, and increased expression of both type II collagen and cartilage proteoglycan mRNA. Moreover, staurosporine treatment initiated chondrocyte differentiation and induced the expression of type II collagen and cartilage proteoglycan gene transcripts in hyoid arch mesenchyme, which exhibited no spontaneous chondrogenesis in control cultures. The results demonstrate that staurosporine promotes cartilage formation in embryonic facial mesenchyme, and suggest the possibility that protein kinase C might function as an inhibitory modulator of chondrocyte differentiation in the neural crest-derived progenitor cells of the embryonic facial skeleton.

Type IX collagen gene expression during limb cartilage differentiation.

Changes in the steady-state levels of mRNAs for the alpha 1(IX) and alpha 2(IX) polypeptide chains of cartilage-characteristic type IX collagen were examined during the course of chick limb chondrogenesis in vitro and in vivo. Cytoplasmic type IX collagen mRNAs begin to accumulate at the onset of overt chondrogenesis in high density micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of type IX collagen mRNA accumulation at condensation coincides with the initiation of accumulation of cartilage proteoglycan core protein mRNA and with a striking increase in type II collagen mRNA accumulation. Following condensation in vitro, there is a concomitant progressive increase in cytoplasmic type IX collagen, core protein, and type II collagen mRNA levels which parallels the progressive accumulation of cartilage matrix. Type IX collagen mRNAs also begin to accumulate at the initiation of overt chondrogenesis in vivo in the chondrogenic central core of the developing limb bud. In contrast, little, or no type IX collagen mRNAs are detectable in the nonchondrogenic peripheral regions of the developing limb bud.

Promotion of embryonic limb cartilage differentiation in vitro by staurosporine, a protein kinase C inhibitor.

Phorbol 12-myristate 13-acetate (PMA), a protein kinase C-activating phorbol ester, is known to inhibit chondrogenic differentiation by embryonic limb mesenchyme cells in vitro. The present study demonstrates that staurosporine, a potent inhibitor of protein kinase C, conversely stimulates cartilage differentiation in cultures of limb mesenchyme cells isolated from whole wing buds of stage 23/24 chick embryos or from the distal subridge region of stage 25 wing buds. In high density micromass cultures, in which limb mesenchyme cells undergo extensive spontaneous cartilage differentiation, exposure to 5-20 nM staurosporine promotes an accelerated accumulation of type II collagen and cartilage proteoglycan mRNA transcripts and a 2- to 3-fold increase in matrix glycosaminoglycan deposition. Even in low density, monolayer cultures in which the mesenchymal cells do not normally form cartilage, treatment with 5 nM staurosporine induces extensive Alcian blue-positive matrix production, a striking 4- to 18-fold rise in sulfated glycosaminoglycan accumulation, and a dramatic elevation of cartilage-characteristic gene transcript expression. Moreover, concurrent treatment with staurosporine overcomes the inhibitory effects of PMA on in vitro limb cartilage differentiation. The results suggest the hypothesis that protein kinase C might function as a negative modulator of chondrogenic differentiation during embryonic limb development.

Stimulation of limb cartilage differentiation by cyclic AMP is dependent on cell density.

Cyclic AMP (cAMP) has been implicated in the regulation of limb cartilage differentiation. This study represents an attempt to clarify potential mechanisms by which cAMP might regulate chondrogenesis. We have found that the ability of cAMP to stimulate limb cartilage differentiation in vitro is dependent on cell density. Dibutyryl cAMP (dbcAMP) elicits a striking increase in the accumulation of Alcian blue, pH 1.0-positive cartilage matrix, and a corresponding three- to fourfold increase in the accumulation of 35S-labeled glycosaminoglycans (GAG) by limb mesenchymal cells cultured in low serum medium at densities greater than confluence (i.e. micromass cultures established with 1-2 x 10(5) cells in 10 microliters of medium). Moreover, dbcAMP causes a striking (two- to fourfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific sulfated proteoglycan in these high density, supraconfluent cultures. In contrast, cAMP does not promote the chondrogenesis of limb mesenchymal cells cultured at subconfluent densities (i.e. cultures initiated with 2.5-5 x 10(4) cells in 10 microliters of medium). In these low density cultures, dbcAMP does not promote the formation of cartilage matrix, sulfated GAG accumulation or the accumulation of cartilage-specific mRNAs. These observations suggest that cAMP may exert its regulatory effect in part by facilitating cell-cell communication during the critical condensation phase of chondrogenesis.

Promotion of embryonic chick limb cartilage differentiation by transforming growth factor-beta.

This study represents a first step in investigating the possible involvement of transforming growth factor-beta (TGF-beta) in the regulation of embryonic chick limb cartilage differentiation. TGF-beta 1 and 2 (1-10 ng/ml) elicit a striking increase in the accumulation of Alcian blue, pH 1-positive cartilage matrix, and a corresponding twofold to threefold increase in the accumulation of 35S-sulfate- or 3H-glucosamine-labeled sulfated glycosaminoglycans (GAG) by high density micromass cultures prepared from the cells of whole stage 23/24 limb buds or the homogeneous population of chondrogenic precursor cells comprising the distal subridge mesenchyme of stage 25 wing buds. Moreover, TGF-beta causes a striking (threefold to sixfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific proteoglycan. Only a brief (2 hr) exposure to TGF-beta at the initiation of culture is sufficient to stimulate chondrogenesis, indicating that the growth factor is acting at an early step in the process. Furthermore, TGF-beta promotes the formation of cartilage matrix and cartilage-specific gene expression in low density subconfluent spot cultures of limb mesenchymal cells, which are situations in which little, or no chondrogenic differentiation normally occurs. These results provide strong incentive for considering and further investigating the role of TGF-beta in the control of limb cartilage differentiation.

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)

In situ hybridization analysis of the expression of the type II collagen gene in the developing chicken limb bud.

In situ hybridization with [32P]- or [35S]-labeled double-stranded DNA or single-stranded RNA probes was used to investigate the temporal and spatial distribution of cartilage-characteristic type II collagen mRNA during embryonic chick limb development and cartilage differentiation in vivo. When the type II collagen probes were hybridized to sections through embryonic limb buds at the earliest stages of their development (stages 18-25), an accumulation of silver grains representing type II collagen mRNA first became detectable in the proximal central core of the limb coincident with the prechondrogenic condensation of mesenchymal cells that characterizes the onset of cartilage differentiation. At later stages of development (stage 32; 7 days) intense hybridization signals with the type II collagen probes were localized over the well differentiated cartilage rudiments, whereas few or no silver grains above background were observed over the non-chondrogenic tissues. In contrast, sections hybridized with a probe complementary to mRNA for the alpha 1 chain of type I collagen exhibited an intense hybridization signal over the perichondrium and little or no signal over the cartilage primordia. At all stages of development examined, [32P]-labeled double-stranded DNA probes or single-stranded RNA probes labeled with either [32P] or [35S] provided adequate hybridization signals. Several experimental protocols were employed to control for the potential cross-hybridization and non-specific hybridization of the type II collagen probes. These included the utilization of labeled noncomplementary "sense-strand" type II collagen RNA as a control probe for nonspecific background, and prehybridization with a large excess of appropriate unlabeled RNA to block sequences in heterologous collagen RNAs that might cross-hybridize to the specific labeled probe.

Hyaluronic acid production and hyaluronidase activity in the newt iris during lens regeneration.

The process of lens regeneration in newts involves the dedifferentiation of pigmented iris epithelial cells and their subsequent conversion into lens fibers. In vivo this cell-type conversion is restricted to the dorsal region of the iris. We have examined the patterns of hyaluronate accumulation and endogenous hyaluronidase activity in the newt iris during the course of lens regeneration in vivo. Accumulation of newly synthesized hyaluronate was estimated from the uptake of [3H]glucosamine into cetylpyridinium chloride-precipitable material that was sensitive to Streptomyces hyaluronidase. Endogenous hyaluronidase activity was determined from the quantity of reducing N-acetylhexosamine released upon incubation of iris tissue extract with exogenous hyaluronate substrate. We found that incorporation of label into hyaluronate was consistently higher in the regeneration-activated irises of lentectomized eyes than in control irises from sham-operated eyes. Hyaluronate labeling was higher in the dorsal (lens-forming) region of the iris than in ventral (non-lens-forming) iris tissue during the regeneration process. Label accumulation into hyaluronate was maximum between 10 and 15 days after lentectomy, the period of most pronounced dedifferentiation in the dorsal iris epithelium. Both normal and regenerating irises demonstrated a high level of endogenous hyaluronidase activity with a pH optimum of 3.5-4.0. Hyaluronidase activity was 1.7 to 2 times higher in dorsal iris tissue than in ventral irises both prior to lentectomy and throughout the regeneration process. We suggest that enhanced hyaluronate accumulation may facilitate the dedifferentiation of iris epithelial cells in the dorsal iris and prevent precocious withdrawal from the cell cycle. The high level of hyaluronidase activity in the dorsal iris may promote the turnover and remodeling of extracellular matrix components required for cell-type conversion.

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.

Cartilage proteoglycan core protein gene expression during limb cartilage differentiation.

Changes in the steady-state cytoplasmic levels of mRNA for the core protein of the major sulfated proteoglycan of cartilage were examined during the course of limb chondrogenesis in vitro using cloned cDNA probes. Cytoplasmic core protein mRNA begins to accumulate at the onset of overt chondrogenesis in micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of core protein mRNA accumulation coincides with a dramatic increase in the accumulation of mRNA for type II collagen, the other major constituent of hyaline cartilage matrix. Following condensation, there is a concomitant progressive increase in cytoplasmic core protein and type II collagen mRNA accumulation which parallels the progressive accumulation of cartilage matrix by the cells. The relative rate of accumulation of cytoplasmic type II collagen mRNA is greater than twice that of core protein mRNA during chondrogenesis in micromass culture. Cyclic AMP, an agent implicated in the regulation of chondrogenesis elicits a concomitant two- to fourfold increase in both cartilage core protein and type II collagen mRNA levels by limb mesenchymal cells. Core protein gene expression is more sensitive to cAMP than type II collagen gene expression. These results suggest that the cartilage proteoglycan core protein and type II collagen genes are coordinately regulated during the course of limb cartilage differentiation, although there are quantitative differences in the extent of expression of the two genes.

Collagen gene expression during limb cartilage differentiation.

As limb mesenchymal cells differentiate into chondrocytes, they initiate the synthesis of type II collagen and cease synthesizing type I collagen. Changes in the cytoplasmic levels of type I and type II collagen mRNAs during the course of limb chondrogenesis in vivo and in vitro were examined using cloned cDNA probes. A striking increase in cytoplasmic type II collagen mRNA occurs coincident with the crucial condensation stage of chondrogenesis in vitro, in which prechondrogenic mesenchymal cells become closely juxtaposed before depositing a cartilage matrix. Thereafter, a continuous and progressive increase in the accumulation of cytoplasmic type II collagen mRNA occurs which parallels the progressive accumulation of cartilage matrix by cells. The onset of overt chondrogenesis, however, does not involve activation of the transcription of the type II collagen gene. Low levels of type II collagen mRNA are present in the cytoplasm of prechondrogenic mesenchymal cells at the earliest stages of limb development, well before the accumulation of detectable levels of type II collagen. Type I collagen gene expression during chondrogenesis is regulated, at least in part, at the translational level. Type I collagen mRNAs are present in the cytoplasm of differentiated chondrocytes, which have ceased synthesizing detectable amounts of type I collagen.