DEVELOPMENTAL

BIOLOGY

144,47-53 (1991)

Gap Junctional Communication

during Limb Cartilage Differentiation

CAROLINEN. D. COELHOAND ROBERTA. KOSHER Department

of Anatomy, University

of Connecticut Health Center, Farmington, Accepted November

Connecticut

06032

23, 1990

The onset of cartilage differentiation in the developing limb bud is characterized by a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely apposed to one another prior to initiating cartilage matrix deposition. During this condensation process intimate cell-cell interactions occur which are necessary to trigger chondrogenic differentiation. In the present study, we demonstrate that extensive cell-cell communication via gap junctions as assayed by the intercellular transfer of lucifer yellow dye occurs during condensation and the onset of overt chondrogenesis in high density micromass cultures prepared from the homogeneous population of chondrogenic precursor cells comprising the distal subridge region of stage 25 embryonic chick wing buds. Furthermore, in heterogeneous micromass cultures prepared from the mesodermal cells of whole stage 23/24 limb buds, extensive gap junctional communication is limited to differentiating cartilage cells, while the nonchondrogenic cells of the cultures that are differentiating into the connective tissue lineage exhibit little or no intercellular communication via gap junctions. These results provide a strong incentive for considering and further investigating the possible involvement of cell-cell o 1%~ Academic press. 1nc. communication via gap junctions in the regulation of limb cartilage differentiation.

minal heparin-binding domain of fibronectin impair the formation of prechondrogenic aggregates in vitro (Frenz et ab, 1989). This latter observation suggests that an adhesive interaction between fibronectin and heparin-like cell surface molecules may be involved in condensation formation, particularly since heparinase treatment also impairs prechondrogenic aggregate formation in vitro (Frenz et ah, 1989). A transient increase in the expression of other matrix macromolecules, including type I collagen (Dessau et al., 1980), tenascin (Mackie et ab, 1987), and a large mesenchymal chondroitin sulfate proteoglycan called PG-M (Kimata et ah, 1986), also occurs during condensation, suggesting that these molecules may play an as yet undefined role in this critical event. The nature of the cell-cell interactions that occur during condensation which trigger cartilage differentiation is not known. Ultrastructural studies have revealed the presence of gap junctions between prechondrogenic mesenchymal cells in the condensing central core of the developing limb bud (Kelley and Fallon, 19’78, 1983; Zimmermann et ah, 1982; Zimmermann, 1984). Since gap junctions play an important role in mediating cell-cell communication during development and differentiation (Loewenstein, 1968; Gilula, 1980; Caveney, 1985), this observation suggests the possibility that cell-cell communication via gap junctions may be involved in promoting limb chondrogenesis during the condensation process. However, it had not previously been directly demonstrated whether or not gap junctional communication actually occurs during chondrogenesis. Therefore, in the

INTRODUCTION

The onset of cartilage differentiation in the developing limb is characterized by a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely juxtaposed to one another prior to initiating cartilage matrix deposition. During this condensation process, critical cell-cell and/or cell-matrix interactions occur that are necessary to trigger chondrogenic differentiation (see Kosher, 1983; Solursh, 1983 for reviews). Condensation is accompanied by the initiation of the expression of the gene for the protein core of cartilage-specific sulfated proteoglycan (Kosher et al., 1986a; Mallein-Gerin et al., 1988) and by a dramatic increase in the expression of the gene for cartilage-characteristic type II collagen (Kosher et al., 198613;Nah et aZ.,1988). The onset of the critical condensation phase of chondrogenesis may be initiated, at least in part, by a progressive decline in the accumulation of extracellular hyaluronate (Kosher et ah, 1981; Knudson and Toole, 1985; Kulyk and Kosher, 1987). The adhesive glycoprotein, fibronectin, may be involved in regulating the onset of condensation and chondrogenesis by promoting prechondrogenic aggregate formation during the process. A striking transient increase in fibronectin gene expression occurs during condensation and at the onset of chondrogenesis in viva and in vitro (Kulyk et ab, 1989); large amounts of fibronectin are present along the surfaces of the closely apposed cells during the process (Dessau et al, 1980; Kosher et aZ.,1982; Tomasek et al, 1982); and monoclonal antibodies against the amino ter47

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FIG. 2. Fluorescence (A) and corresponding bright light (a) photomicrographs demonstrating the intercellular transfer of lucifer yellow dye via gap junctions in a Day 9 micromass culture established from the distal subridge mesenchymal cells of stage 25 wing buds. Note that the extent of lucifer yellow transfer is less than it is during the onset of overt chondrogenesis at Days 2 and 3 of culture (Figs. lC, lc, lD, and Id).

present investigation we studied gap junctional communication during the progressive chondrogenic differentiation that limb mesenchymal cells undergo in high density micromass culture. We demonstrated that extensive gap junctional communication, as assayed by the intercellular transfer of lucifer yellow dye, does indeed occur during condensation and at the onset of limb cartilage differentiation in vitro. Furthermore, extensive cell-cell communication via gap junctions appears to be limited to differentiating cartilage cells in vitro since nonchondrogenic cells differentiating into the connective tissue lineage exhibit little or no gap junctional communication. These results suggest that cellcell communication via gap junctions may be involved in regulating limb cartilage differentiation. MATERIALS

AND

METHODS

Preparation of cultures. Wing buds were removed from stage 23, 24, and 25 (Hamburger and Hamilton, 1951) embryos of White Leghorn chicks. Distal wing bud tips (subridge regions) were cut away from the stage 25 limb buds as previously described, with the size of the excised subridge regions being 0.3-0.4 mm from the distal apex of the tissue to the proximal cut edge (Kosher et

al., 1979a). Ectoderm was removed from the tissues, and micromass cultures were prepared from the distal subridge mesenchymal cells of the stage 25 wing buds and from the mesodermal cells of whole stage 23/24 wing buds as previously described (Gay and Kosher, 1984). Standard high density micromass cultures were prepared by spotting 2 X lo5 cells in 10 ~1 of medium onto the surface of tissue culture dishes (Ahrens et aZ.,197’7; Gay and Kosher, 1984). Analysis of gap junctional cowmunicatim. Gap junctional communication was assayed by the scrape-loading/dye transfer technique of El-Fouly et al (1987). At various times during culture corresponding to different phases of chondrogenesis, cells were washed with phosphate-buffered saline (PBS), supplied with a 0.05% solution of the gap junction-permeable dye, lucifer yellow (Sigma), and the gap junction-impermeable dye, rhodamine dextran (Molecular Probes, Inc.), in PBS (El-Fouly et al., 1987), and scrapes were made through the diameter of the micromass cultures with a fine scalpel blade. Transfer of lucifer yellow dye from the incised cells to contiguous cells via gap junctions was allowed to proceed for 2 min, after which the cultures were washed with PBS, fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, mounted under cover-

FIG. 1. Fluorescence photomicrographs demonstrating the intercellular transfer of lucifer yellow dye via gap junctions from scrape-loaded cells to contiguous cells at various times during the progressive chondrogenic differentiation of the distal subridge mesenchymal cells of stage 25 wing buds in micromass culture. (A-D) and (a-d) represent two independent experiments illustrating the extent of gap junctional communication at 3 hr (A, a), 24 hr (B, b), 48 hr (C, c), and 72 hr (D, d). At 3 hr, which is before the onset of condensation and overt differentiation, little transfer of lucifer yellow dye from mesenchymal cells at the scraped edges to adjacent cells is observed. Extensive transfer of lucifer yellow from scrape-loaded cells to contiguous cells is observed during condensation at 24 hr (B, b) and continues during the subsequent several days of culture in which cartilage matrix synthesis proceeds (C, c, D, and d). No intercellular transfer of the gap junction-impermeable dye, rhodamine dextran, occurs during any period of culture. At least 10 different cultures at each time point were examined, and results similar to those illustrated were observed in each experiment. x230.

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slips in 10% glycerin in PBS, and examined by fluorescence microscopy. RESULTS

In our initial studies we examined gap junctional communication during the progressive chondrogenic differentiation that the distal subridge mesenchymal cells of stage 25 wing buds undergo in high density micromass culture. Stage 25 subridge mesenchymal cells comprise a relatively homogeneous population of undifferentiated chondrogenic precursor cells, which uniformly progress through the phases of chondrogenesis in micromass culture, and form a virtually uniform sheet of cartilage with little, if any, nonchondrogenic tissue detectable (Gay and Kosher, 1984). In these cultures, widespread prechondrogenic condensations of cells are formed by the end of the first day of culture, after which there is a uniform and progressive accumulation of cartilage matrix by the cells (Gay and Kosher, 1984). At 3 hr after the initiation of culture, which is before overt morphological indications of differentiation and the onset of condensation, little transfer of lucifer yellow dye from scrape-loaded mesenchymal cells to contiguous cells is detectable, indicating that only limited gap junctional communication is occurring at this time (Figs. 1A and la). At 24 hr, at which time the widespread prechondrogenic condensations of cells which characterize the onset of chondrogenesis are present, extensive transfer of lucifer yellow dye via gap junctions from scrape-loaded cells to contiguous cells is observed (Figs. 1B and lb). Extensive transfer of lucifer yellow dye via gap junctions continues through the subsequent several days of culture, duringwhich the synthesis of cartilage matrix proceeds (Figs. lC, lc, lD, and Id). Intercellular transfer of lucifer yellow is less at Day 9 of culture, at which time the cells are spatially segregated from one another by an extensive cartilage matrix (Fig. 2) than it is during the onset of chondrogenesis at Days 2 and 3 (Figs. lC, lc, lD, and Id). No intercellular transfer of the gap junction-impermeable dye, rhodamine dextran, from scrape-loaded cells to contiguous cells was observed during any period of culture. These observations indicate that extensive cell-cell communication via gap junctions occurs during condensation and at the onset of overt chondrogenesis in vitro, at which time crucial cell-cell interactions are occurring which are necessary to trigger cartilage differentiation. We next examined gap junctional communication in heterogeneous micromass cultures prepared from the

51

and Chondrogenesis

cells comprising whole stage 23/24 wing buds, in which the differentiation of chondrogenic and nonchondrogenie cell types takes place. At stage 23/24, cells in the proximal central core of the limb bud initiate chondrogenie differentiation, while cells in the proximal preand postaxial periphery of the limb bud initiate differentiation into primarily nonchondrogenic connective tissue cell types. Therefore, when cells from whole stage 23/24 limb buds are subjected to micromass culture, differentiating cartilage nodules that are separated from one another by nonchondrogenic fibroblastic-like tissue form in discrete regions of the cultures (Gay and Kosher, 1984). As shown in Fig. 3, extensive transfer of lucifer yellow dye from scrape-loaded cells to contiguous cells occurs in the differentiating cartilage nodules of the cultures, whereas the intervening nonchondrogenic cells in the culture exhibit little or no intercellular transfer of the dye. DISCUSSION

The results of the present study indicate that intercellular communication via gap junctions occurs during condensation and at the initiation of limb chondrogenesis, at which time critical cell-cell interactions are occurring that are necessary to trigger cartilage differentiation. Furthermore, extensive cell-cell communication appears to be limited to differentiating cartilage cells in vitro since nonchondrogenic cells differentiating into the connective tissue lineage exhibit little or no gap junctional communication. These observations provide a strong incentive for considering the possibility that cell-cell communication via gap junctions may be involved in regulating limb cartilage differentiation. It is of particular interest that although extensive gap junctional communication occurs among differentiating cartilage cells in micromass cultures derived from whole stage 23/24 wing buds, the intervening nonchondrogenic fibroblastic-like cells of the cultures exhibit little or no intercellular transfer of lucifer yellow dye. It is difficult to conclusively eliminate the possibility that the lack of lucifer yellow dye transfer among the nonchondrogenic cells is due to the selective binding or sequestration of the dye by cytoplasmic components in these fibroblastic-like cells. However, our observation interfaces well with the suggestion of Kelley and Fallon (1983) that the ultrastructural organization of gap junctions in the chondrogenic central core of the developing limb bud is consistent with their functional coupling,

FIG. 3. Intercellular transfer of lucifer yellow dye via gap junctions in 24 hr (A), 48 hr (B), and 72 hr (C, D) heterogeneous micromass cultures prepared from the cells comprising whole stage 23124 wing buds. As illustrated in the bright light photomicrographs shown in (E), differentiating cartilage nodules (*) separated from one another by nonchondrogenic libroblastic-like tissue (arrows) form in discrete regions of the cultures. Note that extensive transfer of lucifer yellow dye from scrape-loaded cells to contiguous cells occurs in the differentiating cartilage nodules (*), whereas the intervening nonchondrogenic cells (arrows) exhibit little or no intercellular transfer of the dye. At least seven different cultures were examined at each time point, and results similar to those illustrated were observed in each experiment. X218.

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DEVELOPMENTALBIOLOGY

whereas the structural appearance of gap junctions in the nonchondrogenic pre- and postaxial mesoderm suggests that they may be functionally uncoupled. The differentiation of nonchondrogenic connective tissue cell types in the proximal pre- and postaxial periphery of the developing chick limb bud is influenced by the dorsal/ventral ectoderm of the limb (Kosher et aZ., 1979a; Solursh et aZ.,1981a). It appears that dorsal/ventral limb ectoderm exerts an anti-chondrogenic effect on mesenchymal cells subjacent to it, in that it inhibits their chondrogenic differentiation and allows or directs their differentiation into the nonchondrogenic connective tissue lineage (Kosher et aZ., 1979a; Solursh et aL, 1981b). The results of the present study suggest the possibility that dorsal/ventral limb ectoderm may exert its anti-chondrogenic effect, at least in part, by preventing gap junctional communication among mesenchymal cells under its influence. Cyclic AMP (CAMP) is an intercellular molecule that has been implicated in the regulation of limb cartilage differentiation. A transient increase in cellular CAMP content occurs during the crucial cellular condensation phase of chondrogenesis (Solursh et aZ., 1979; Elmer et aZ.,1981; Ho et al., 1982; Biddulph et aZ.,1984, 1988), and agents which elevate cellular CAMP levels stimulate in vitro limb chondrogenesis (Kosher et aZ., 1979b, 1986; Kosher and Savage, 1980; Solursh et al., 1981a). Recently, we have demonstrated that the ability of CAMP to stimulate limb chondrogenesis is dependent on cell density (Rodgers et ak, 1989). Cyclic AMP is a potent stimulator of the chondrogenic differentiation of limb mesenchymal cells cultured at high densities which allow close cellular juxtapositions and interactions, but fails to promote the chondrogenesis of mesenchyma1 cells cultured at subconfluent densities which preclude intimate cell-cell associations and interactions (Rodgers et aL, 1989). On the basis of this observation, we suggested that CAMP might regulate chondrogenesis by promoting gap junction formation and/or gap junctional communication during condensation or that gap junctional communication at the onset of chondrogenesis might facilitate the direct transfer of CAMP itself from cell to cell (Rodgers et ah, 1989). It should be noted that CAMP is indeed an important regulator of gap junctional intercellular communication in a number of systems (Flagg-Newton et al, 1981; Enomoto et al, 1984; Azarnia and Russell, 1985; De Maziere and Scheurermann, 1985; Saez et aZ., 1986), and we are currently investigating its possible involvement in the gap junctional communication that is occurring at the onset of limb cartilage differentiation. This research was supported by NIH Grants HD22896 and HD22610 to R.A.K.

VOLUME144, I991 REFERENCES

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Gap junctional communication during limb cartilage differentiation.

The onset of cartilage differentiation in the developing limb bud is characterized by a transient cellular condensation process in which prechondrogen...
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