Stage-related chondrogenic potential of avian mandibular ectomesenchymal cells

We have examined the in vitro stage-related chondrogenic potential of avian mandibular ectomesenchymal cells using micromass cultures. Our results indicate that mandibular ectomesenchymal cells as early as stage 16, soon after the formation of the mandibular arches and well before the initiation of in vivo chondrogenesis, have chondrogenic potential which is expressed in micromass culture. There is an increase in the total area of the cultures occupied by cartilage when cells from increasing stages of development are used. The nodular pattern of chondrogenesis in these cultures indicates that mandibular ectomesenchymal cells are a heterogenous population from the time of mandibular arch formation. In addition, we studied the temporal expression of the genes for extracellular matrix proteins during in vitro chondrogenesis and correlated the morphological changes with the pattern of gene expression. Low levels of type II collagen mRNA are present in the cultures prior to detection of any stainable cartilage matrix and increase 5 fold just before the onset of chondrogenesis in vitro. On the other hand mRNA for cartilage proteoglycan core protein was not detected until the second day of culture when stainable cartilage matrix was present and progressively increased thereafter. Messenger RNA for type I collagen was present at the time of initiation of cultures and continuously increased during the culture period. Our experiments also indicated that embryonic epithelia can inhibit the in vitro chondrogenesis of mandibular ectomesenchymal cells and that the inhibitory effect of embryonic epithelia is independent of its age and site of origin.     

Separation of precursor myogenic and chondrogenic cells in early limb bud mesenchyme by a monoclonal antibody

We have addressed the problem of the segregation of cell lineages during the development of cartilage and muscle in the chick limb bud. The following experiments demonstrate that early limb buds consist of at least two independent subpopulations of committed precursor cells-- those in (a) the myogenic and (b) the chondrogenic lineage--which can be physically separated. Cells obtained from stage 20, 21, and 22 limb buds were cultured for 5 h in the presence of a monoclonal antibody that was originally isolated for its ability to detach preferentially myogenic cells from extracellular matrices. The detached limb bud cells were collected and replated in normal medium. Within 2 d nearly all of the replated cells had differentiated into myoblasts and myotubes; no chondroblasts differentiated in these cultures. In contrast, the original adherent population that remained after the antibody-induced detachment of the myogenic cells differentiated largely into cartilage and was devoid of muscle. Rearing the antibody-detached cells (i.e., replicating myogenic precursors and postmitotic myoblasts) in medium known to promote chondrogenesis did not induce these cells to chondrify. Conversely, rearing the attached precursor cells (i.e., chondrogenic precursors) in medium known to promote myogenesis did not induce these cells to undergo myogenesis. The definitive mononucleated myoblasts and multinucleated myotubes were identified by muscle- specific antibodies against light meromyosin or desmin, whereas the definitive chondroblasts were identified by a monoclonal antibody against the keratan sulfate chains of the cartilage-specific sulfated proteoglycan. These findings are interpreted as supporting the lineage hypothesis in which the differentiation program of a cell is determined by means of transit through compartments of a lineage.     

Chondrogenesis of limb bud mesenchyme in vitro: stimulation by cations

To analyze the nature of cell-cell interactions in chondrogenesis, two cations that influence these interactions, calcium and poly-L-lysine (PL), were tested for their effects on chondrogenesis in vitro. High density cultures of chick limb bud mesenchyme (Hamilton-Hamburger stages 23/24), were exposed to culture media containing calcium (0.6-3.3 mM) or PL (1-10 micrograms/ml). Both cations stimulated chondrogenesis in a dose-dependent manner, and also promoted cartilage formation in normally non-chondrogenic, low cell density cultures. Chondrogenesis was assayed based on cartilage nodule number, [35S]sulfate incorporation, and expression of type II collagen as detected by immunohistochemistry. The calcium effect was not mimicked by other divalent cations (Cd, Co, Ni, Mg, Mn, and Sr). The effect of PL was dependent on its Mr (greater than or equal to 14K) and charge, and was mimicked by poly-D-lysine but not by lysine or other analogs of PL or lysine (epsilon-amino caproic acid, lysozyme, poly-L-arginine, and spermidine). Calcium and PL probably act by different mechanisms since their effects were additive, and required their presence on different days of culture: calcium acted on Day 1, and PL on Day 2. It is proposed that calcium may play a role in the cell aggregation phase of chondrogenesis whereas PL, or a naturally occurring polypeptide of similar nature, may promote chondrogenesis by crosslinking specific anionic components of the cell surface or extracellular matrix.     

Activity of protein kinase C during the differentiation of chick limb bud mesenchymal cells

Abstract. To investigate the relationship between protein kinase C (PKC) and chondrogenesis, PKC activity was assayed in cultures of stage 23/24 chick limb bud mesenchymal cells under various conditions. PKC activities of cytosolic and particulate fractions were low in 1 day cultured cells. As chondrogenesis proceeds, cytosolic PKC activity increased more than twofold, while that of the particulate fraction increased only slightly. Three days' treatment of cultures with phorbol-12-myristate-13-acetate (PMA, 5 × 10⁻⁸ M ) inhibited chondrogenesis judged by the accumulation of Alcian blue bound to the extracellular matrix and depressed PKC activity in cytosolic fraction. When cells were grown for 3 days in control medium after 3 days' treatment with PMA, chondrogenesis resumed and PKC activity recovered to normal values. PKC activity in cultures plated at low density (2 × 10⁶ cells/ml) where chondrogenesis is reduced was as low as that in 1 day cultured cells plated at high density (2 × 10⁷ cells/ml) or that in PMA treated cells. On the other hand, staurosporine promoted chondrogenesis without affecting PKC activity. Furthermore, reversal of PMA's inhibitory effect on chondrogenesis by staurosporine was not accompanied by recovery of PKC activity. These data indicate that increases in PKC activity is closely related to chondrogenesis and that PMA inhibits chondrogenesis by depressing PKC. However, staurosporine's enhancing effect on chondrogenesis is not related to PKC activity.     

Separation of the myogenic and chondrogenic progenitor cells of undifferentiated limb mesenchyme

Undifferentiated limb bud mesenchyme consists of at least two separate, possibly predetermined, populations of progenitor cells, one derived from somitic mesoderm that gives rise exclusively to skeletal muscle and one derived from somatopleural mesoderm that gives rise to the cartilage and connective tissue of the limb. In the present study, we demonstrate that the inherent migratory capacity of myogenic precursor cells can be used to physically separate the myogenic and chondrogenic progenitor cells of the undifferentiated limb mesenchyme at the earliest stages of limb development. When the undifferentiated mesenchyme of stage 18/19 chick embryo wing buds or from the distal subridge region of stage 22 wing buds is placed intact upon the surface of fibronectin (FN)-coated petri dishes, a large population of cells emigrates out of the explants onto the FN substrates and differentiates into an extensive interlacing network of bipolar spindle-shaped myoblasts and multinucleated myotubes that stain with monoclonal antibody against muscle-specific fast myosin light chain. In contrast, the cells of the explants that remain in place and do not migrate away undergo extensive cartilage differentiation. Significantly, there is no emigration of myogenic cells out of explants of stage 25 distal subridge mesenchyme, which lacks myogenic progenitor cells. Myogenic precursor cells stream out of mesenchyme explants in one or occasionally two discrete locations, suggesting they are spatially segregated in discrete regions of tissue at the time of its explantation. There are subtle overall differences in the morphologies of the myogenic cells that form in stage 18/19 and stage 22 distal subridge mesenchyme explants. Finally, groups of nonmyogenic nonfibroblastic cells which are fusiform-shaped and oriented in distinct parallel arrays characteristically are found along the periphery of stage 18/19 wing mesenchyme explants. Our observations provide support for the concept that undifferentiated limb mesenchyme consists of independent subpopulations of committed precursor cells and provides a system for studying the early determinative and regulatory events involved in myogenesis or chondrogenesis.     

Effect of phorbol and bryostatin I on chondrogenic expression of chick limb bud, in vitro

The ability of phorbol esters to promote tumor formation and alter cell differentiation has been attributed to its action on a number of critical cellular functions, in particular, on protein phosphorylation, through the activation of protein C kinase. The present paper describes the effects of PMA (phorbol 12-myristate 13 acetate) on in vitro chondrogenesis in non-passaged, embryonic limb bud cells, relative to the effects of Bryostatin I. This compound also activates C kinase and binds competitively to the phorbol ester receptor, yet does not affect cell differentiation. Levels of PMA as low as 10(-7) M markedly reduced cartilage formation in 4-day cultures, as indicated by nodule count and Alcian blue staining for chondroitin sulfate. Coadministration of Bryostatin I at equimolar concentration prevented the PMA inhibitory effect on chondrocytic expression. This confirms other findings that phorbol activation of C kinase cannot exclusively account for the activity of phorbol on cell expression, i.e., that other pathway(s) must also be involved. Altering the time of PMA exposure demonstrated that PMA inhibited chondrocyte phenotypic expression, rather than cell commitment: early (0-48 h) exposure to PMA (during chondrocytic commitment in vitro) had little inhibitory effect on the staining index, whereas, exposure from 49-96 h (presumably post-commitment) and 0-96 h had moderate and strong inhibitory effects, respectively, on cartilage synthesis. Further research on the phorbol/Bryostatin I interaction should add to our knowledge of the control processes involved in tumor promotion and cell differentiation.     

Modification of the cell cycle of limb bud mesenchyme during in vitro cartilage differentiation

A consistent chondrogenesis takes place in micro high-density cultures derived from limb mesenchymal cells of chick embryos of stages 23-24. Flow-cytometric measurements of DNA content showed that cells in the phase of G1 or G0 made up 51% of the dispersed cell suspensions. The proportion of these cells increased to 71% by the onset of cartilage differentiation in day-2 cultures. This ratio was 84% when the voluminous matrix formation began on the 4th day of culturing. Thereafter, it increased to 90% by the 6th day, and to 93% by the 14th day. The results suggest that cartilage differentiates from G0 mesenchymal cells of the limb. In our measurements, however, the G0 phase includes all non-proliferative cell population which have identical DNA content with G1 cells. Therefore, the G0 phase contains also an increasing number of chondroblasts and chondrocytes as the chondrogenesis proceeds.     

In vitro linoleic acid activation of protein kinase C

The importance of membrane fluidity in the activation of protein kinase C (PKC) was examined using the membrane fluidizer, linoleic acid, in a well-defined model membrane system. Biochemical and biophysical properties of the system were monitored. Linoleic acid activated PKC to a level of 50% of that observed for diacylglycerol. In contrast, linoleic acid did not directly interact with the phorbol ester binding site as did diacylglycerol. This was determined by the lack of involvement of the ionizable group of the fatty acid with activity and the enhancement of phorbol ester binding by linoleic acid and its ester analogs. The membrane fluidity of this model membrane system in the presence of linoleic acid was increased as determined by fluorescence polarization. This increased the availability of phospholipids, thus, explaining the linoleic acid-induced enhancement of phorbol ester binding. The PKC conformation as determined from intrinsic tryptophan fluorescence spectra was different for lipid mixtures containing linoleic acid or diacylglycerol correlating with the difference in biochemical activation properties. This study provides evidence that membrane fluidization is not the predominant function of the lipid activator in PKC activation, but may play a role in obtaining the preferred membrane state for maximal activation.     

In vitro histogenic capacities of limb mesenchyme from various stage mouse embryos

Mesenchyme cells isolated from mouse embryo forelimb buds (stages 15 through 21) and placed in high-density micromass cultures are compared with respect to their in vitro histogenic capacities. Particular emphasis is placed on changes in in vitro chondrogenic capacity. Stage 15 mouse limb cultures form numerous aggregates which uniformly fail to differentiate into cartilage nodules. On the other hand, cartilage nodules are observed in cultures prepared from all subsequent stage limbs, although there is a linear decrease in the size of nodules between stage 16–17 and middle-late stage 21 cultures. This decrease correlates with simultaneous decreases in both the proportion of aggregating cells and the extent of dibutyryl cyclic AMP-stimulated cartilage formation. At the same time, observations indicate that the proportions of nonaggregating and nonchondrogenic mesenchyme, myogenic cells, and, perhaps, fibrogenic mesenchyme are increasing. The only exceptions to these patterns are observed in cultures from middle-late stage 21 limbs, when cartilage differentiation in situ is already extensive. Unlike earlier stage cultures, which form nearly identical numbers of aggregates and nodules, middle-late stage 21 cultures form variable numbers of aggregates, only a few of which differentiate into cartilage nodules. Middle-late stage 21 cultures also contain unexpectedly low numbers of myogenic cells/unit area of culture. Based on changes in the in vitro histogenic capacities, it is concluded that concurrent with a progression of morphogenic events in the limb, there is a progression of changes in the relative proportions of cell subpopulations. Both the existence of the different subpopulations and the changes in their relative proportions can be detected in vitro. Furthermore, it is concluded that cartilage formation in the limbs of both mouse and chick embryos probably occurs according to very similar developmental programs.     

Chondrogenesis in chick limb mesenchyme in vitro derived from distal limb bud tips: changes in cyclic AMP and in prostaglandin responsiveness

Chondrogenesis was monitored in micromass cultures of mesenchymal cells derived from the distal tip of stage-25 chick limb buds over a 6-day period. Alcian green staining and immunofluorescent localization of cartilage-specific proteoglycans revealed the appearance of cartilage matrix by day 3 of cell culture. By day 6, cultures contained a uniform and homogeneous population of fully differentiated chondrocytes throughout the cell layer, with only a narrow rim of nonchondrogenic cells around the extreme periphery of the culture. Synthesis of sulfated glycosaminoglycans also progressively increased between days 3 and 6, being 8-fold higher at day 6 than at day 1 of culture. Both adenylate cyclase (AC) activity and cAMP concentrations increased dramatically during the first 2 days of culture, reaching maximal levels by day 2, which remained elevated and stable throughout the remaining chondrogenic period (days 3-6). Responsiveness of both AC and cAMP concentrations of the cells to PGE2 was maximal by day 1 of culture and was increased over control cells by 12-fold and 8-fold respectively. Both responses, however, were dramatically reduced by day 3, at which time the initiation of cartilage formation was apparent. Responsiveness of cells during the prechondrogenic period to PGE2 was relatively specific in that no effects could be demonstrated with equivalent concentrations of PGF2 alpha or 6-keto-PGF1 alpha, although PGl2 did produce increases in cAMP concentrations of about 50% of those of PGE2. These results indicate that previously reported changes in the cAMP system in heterogeneous cell cultures derived from whole limb buds reflect changes occurring in the chondrogenic cell type and indicate further that peak responsiveness of the cAMP system of these cells to prostaglandins is restricted to prechondrogenic developmental periods.     

Speed of pattern appearance in reaction-diffusion models: Implications in the pattern formation of limb bud mesenchyme cells

It has been postulated that fibroblast growth factor (FGF) treatment of cultured limb bud mesenchyme cells reinforces the lateral inhibitory effect, but the cells also show accelerated pattern appearance. In the present study, we analyze how a small change in a specific parameter affects the speed of pattern appearance in a Turing reaction-diffusion system using linear stability analysis. It is shown that the sign of the change in appearance speed is qualitatively decided if the system is under the diffusion-driven instability condition, and this is confirmed by numerical simulations. Numerical simulations also show that a small change in parameter value induced easily detectable differences in the appearance speed of patterns. Analysis of the Gierer-Meinhardt model revealed that a change in a single parameter can explain two effects of FGF on limb mesenchyme cells—reinforcement of lateral inhibition and earlier appearance of pattern. These qualitative properties and easy detectability make this feature a promising tool to elucidate the underlying mechanisms of biological pattern formationwhere the quantitative parameters are difficult to obtain.     

Position-specific growth of mouse limb bud cells in vitro.

The relationship between cellular position and growth control has been studied in cultures of dissociated fragments of mouse limb bud cells. Using cells derived from various positions along the anterior-posterior axis of the limb bud we have developed culture conditions that optimize growth of positionally isolated cells. Under these conditions limb bud cells display an inherent, position-specific growth response; proliferation of cells derived from anterior and central regions of the limb is enhanced over that of posterior derived cells. Thus, within the total population of limb bud cells the in vitro growth of posterior cells is unique and correlates with the positional activity associated with the zone of polarizing activity. Anterior and posterior cells were cocultured to determine whether interactions between these two groups of positionally distinct cells lead to the stimulation of growth that has been observed in vivo. We observe a slight but consistent position-dependent stimulation of growth that is indicative of a mitogenic signal passing between these positionally disparate cells. Similarities between position-related growth dynamics in vivo and in vitro suggest that positional interactions that are important for limb formation can occur between dissociated cells cultured under standard conditions.     

Glutamate-induced protein phosphorylation in cerebellar granule cells: Role of protein kinase C

Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells.³²P-labelled cells have been stimulated with 100 M glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells.Abbreviations (NMDA) N-methyl-D-aspartate - (PKC) protein kinase C - (EAA) excitatory aminoacids - (GAMSA) -D-glutamylaminomethylsulfonate - (MK801) (+)-10,11-dihydro-5-methyl-5-H-dibenzo-(a,d)-cyclohepten-5,10imine - (TPA) phorbol 12-myristate 13-acetate - (MARCKS) myristoylated alanine-rich C kinase substrate - (GAP-43) growth-associated protein-43 - (SDS) sodium dodecyl sulfate - (PAGE) polyacrylamide gel electrophoresis - (H7) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - (DIV) days in vitro     

In vitro protein kinase C phosphorylation sites of placental lipocortin

Human placental lipocortin is a high-affinity substrate for rat brain protein kinase C in vitro with phosphorylation occurring on serine and threonine residues in a ratio of approximately 2 to 1. Comparison of the ability of various N-terminal-truncated derivatives of lipocortin to serve as phosphorylation substrates, and direct analysis of the N-terminal peptides cleaved from 32P-labeled lipocortin, indicated that threonine-24, serine-27, and serine-28 were the phosphorylation sites. The possibility is discussed that a lysine residue near the carboxy side of the phosphorylation site was involved in lipocortin interaction with the catalytic site of protein kinase C.     

Experimental analysis of the in vivo chondrogenic potential of the interdigital mesenchyme of the chick leg bud subjected to local ectodermal removal

Current in vitro investigations suggest that ectoderm plays a major role in limb morphogenesis by producing a diffusible factor which inhibits the chondrogenesis of the underlying mesenchyme. In the present work we report evidence supporting such an ectodermal role in vivo. Surgical removal of the marginal ectoderm from the third interdigit of chick leg buds at stages 27 to 30 induces the formation of PNA-positive prechondrogenic mesenchymal condensations 15 hr after the operation. The incidence of prechondrogenic condensations achieved 47, 95.2, and 92.8 of the experimental embryos of stages 27, 28, and 29, respectively. This high rate of prechondrogenic aggregate formation contrasted with a lower incidence of ectopic cartilage formation detectable by Alcian blue staining 40 hr after the operation. The sequential analysis of the experimental interdigits by means of peanut lectin labeling suggests that a number of prechondrogenic condensations undergo disaggregation 20 and 30 hr after the operation failing to form fully differentiated cartilages. When ectoderm removal was accompanied by the elimination of a variable amount of interdigital mesenchyme the incidence of prechondrogenic aggregates showed little differences but the formation of fully differentiated cartilages was reduced at a rate proportional to the amount of interdigital mesenchyme removed. From this study it can be concluded that the ectoderm in vivo appears to inhibit the process of aggregation of the mesenchymal cells to form prechondrogenic condensations. Furthermore our results suggest that as observed in vitro (C. P. Cotrill, C. Archer, and L. Wolpert, 1987, Dev. Biol. 122, 503-515) the transformation of prechondrogenic aggregates into fully differentiated cartilage requires the involvement of a critical amount of mesenchymal cells.     

Immunohistochemical study of creatine kinase isozymes in the human upper limb bud

Creatine kinase (CK) is involved in the production of ATP and is composed of two immunologically distinct subunits, B (CK-B) and M (CK-M). In the differentiation of myoblasts, the isozyme of CK changes from CK-B to CK-M. In the present study, the expression of CK subunits was studied immunohistochemically in the upper limb bud of human embryos (Carnegie stages 13-21). It was found that CK-B and CK-M immunoreactive cells appeared at stage 15 and at stage 18, respectively.     

Evidence for a role of protein kinase C in FGF signal transduction in the developing chick limb bud

In developing limbs, numerous signaling molecules have been identified but less is known about the mechanisms by which such signals direct patterning. We have explored signal transduction pathways in the chicken limb bud. A cDNA encoding RACK1, a protein that binds and stabilizes activated protein kinase C (PKC), was isolated in a screen for genes induced by retinoic acid (RA) in the chick wing bud. Fibroblast growth factor (FGF) also induced RACK1 and such induction of RACK1 expression was accompanied by a significant augmentation in the number of active PKC molecules and an elevation of PKC enzymatic activity. This suggests that PKCs mediate signal transduction in the limb bud. Application of chelerythrine, a potent PKC inhibitor, to the presumptive wing region resulted in buds that did not express sonic hedgehog (Shh) and developed into wings that were severely truncated. This observation suggests that the expression of Shh depends on PKCs. Providing ectopic SHH protein, RA or ZPA grafts overcome the effects of blocking PKC with chelerythrine and resulted in a rescue of the wing morphology. Taken together, these findings suggest that the responsiveness of Shh to FGF is mediated, at least in part, by PKCs.     

In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways

Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2(+)-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Km for C3 was 2.6 microM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis.