Inhibition of prostaglandin synthesis reduces cyclic AMP levels and inhibits chondrogenesis in cultured chick limb mesenchyme.

The present study investigated effects of inhibiting the synthesis of prostaglandins (PGs) on cyclic AMP concentrations and chondrogenesis in cultured chick limb mesenchyme. Indomethacin produced concentration-dependent inhibition of both PGE(2) synthesis and chondrogenesis over a concentration range of 50--200 microM. Half maximal inhibition of PGE(2) was achieved with 50 microM concentrations of the drug which also produced visibly reduced amounts of cartilage matrix in cell cultures as evaluated by Alcian green staining on day 6 of culture. The inhibitory effects of indomethacin on chondrogenesis were largely reversed by addition of 1 mM dibutyryl cAMP, indicating that cells could still respond to cyclic AMP stimulation. Endogenous levels of cyclic AMP, which increased by 6 fold during the six days of culture in control cells, did not increase significantly from dissociated cells at the time of plating (day 0) in indomethacin- treated cultures. The results indicate that inhibition of the prechondrogenic rise in PGE(2) concentrations in limb mesenchyme prevents the increase in cyclic AMP levels which occur during this same period resulting in inhibition of chondrogenesis. The data provide further support for the hypothesis that PGE(2), through its effects on the adenylate cyclase-cAMP system, plays an important role in the differentiation of cartilage.

In vitro production of monoclonal antibodies to cultured embryonic chick limb mesenchyme.

A simple, rapid protocol for the in vitro production of monoclonal antibodies (MAbs) that recognize native antigens in cultured chick limb mesenchyme during chondrogenic differentiation is described. Murine lymphocytes were stimulated by direct exposure to methanol-fixed micromass cultures of limb mesenchyme derived from the distal tip of stage 25 chick limb buds. Initial immunohistochemical characterization of two antibodies (DIDI and DIIA5) produced by this method showed preferential localization of reactivity with antigens in developing cartilage nodules during chondrogenesis in cultured chick limb mesenchyme. This study demonstrates the utility of in vitro immunization of lymphocytes for the production of MAbs to native antigens expressed by differentiating embryonic limb cells in culture. Immunohistochemical data provided by DIDI and DIIA5 suggest that antigens bearing these epitopes may be important in early morphogenetic events during limb skeletal development.

Selective and transient expression of a native chondroitin sulfate epitope in Deiters' cells, pillar cells, and the developing tectorial membrane.

The tectorial membrane (TM) is an acellular connective tissue overlying the sensory hair cells of the organ of Corti. Association of the tectorial membrane with the stereocilia of the sensory hair cells is necessary for proper auditory function. During development, the mature tectorial membrane is thought to arise by fusion of a "major" and "minor" tectorial membrane (Lim, Hear Res 1986;22:117-146). Several proteins and glycoconjugates have been detected in the developing TM; however, the specific molecules which mediate fusion of the two components of the TM have not been identified. In the present study, a novel monoclonal antibody (TC2) that recognizes a native epitope on glycosaminoglycans enriched in chondroitin-4-sulfate revealed a transient and restricted expression in the developing gerbil TM. The localization patterns suggest that Deiters' and pillar cells secrete a TC2-positive matrix prior to birth that later becomes incorporated into the marginal band and superior layer (cover net) of the TM. The developmental timecourse and patterns of TC2 reactivity suggest that this molecule may play a critical role in the fusion of the minor TM with the major TM.

Dynamic expression of a native chondroitin sulfate epitope reveals microheterogeneity of extracellular matrix organization in the embryonic chick heart.

TC2 is a novel monoclonal antibody produced by in vitro immunization of splenocytes with a peanut agglutinin-positive fraction from extracts of prechondrogenic micromass cultures of chick limb mesenchyme. ELISA results demonstrated TC2 reactivity with a native epitope on a glycosaminoglycan (GAG) enriched in chondroitin-4-sulfate and with multiple intact proteoglycans, but not with other GAGs tested. TC2 immunohistochemical reactivity was abolished by pretreatment of sections with chondroitinase AC or preadsorption with chondroitin-4-sulfate GAG. Strong TC2 localization occurred throughout the developing heart at stage 9. As looping ensued, a graded reactivity was observed from lowest in the atrium to highest in the conotruncus that correlated well with versican localization. The superior atrioventricular cushion stained preferentially with TC2 as compared to the inferior cushion at stages 16-18. At these later stages TC2 patterns did not agree completely with anti-versican reactivity. By stage 23 there was a marked reduction in TC2 localization in the heart, however, strong reactivity remained at certain sites, including the conotruncus and in subcompartments of both atrioventricular cushions. A heterogeneous distribution of other native chondroitin sulfate glycosaminoglycan epitopes recognized by monoclonal antibodies d1C4 and CS-56 was observed as well. The distribution of the TC2 epitope usually did not overlap with d1C4 or CS-56 localization at the stages examined. Overall, the spatiotemporal characteristics of TC2 reactivity in the developing chick heart appear to correlate with subdomains of the endocardial cushions as well as with trabecular and atrial septal formation.

The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation.

The heart defect (hdf) mouse is a recessive lethal that arose from a transgene insertional mutation on chromosome 13. Embryos homozygous for the transgene die in utero by embryonic day 10.5 postcoitus and exhibit specific defects along the anterior-posterior cardiac axis. The future right ventricle and conus/truncus of the single heart tube fail to form and the endocardial cushions in the atrioventricular and conus/truncus regions are absent. Because the hdf mouse mutation provided the opportunity to identify a gene required for endocardial cushion formation and for specification or maintenance of the anterior most segments of the heart, we initiated studies to further characterize the phenotype, clone the insertion site, and identify the gene disrupted. Chromosome mapping studies first identified the gene, Cspg2 (versican), as a candidate hdf gene. In addition, an antibody recognizing a glycosaminoglycan epitope on versican was found to be positive by immunohistochemistry in the extracellular matrix of normal wild-type embryonic hearts, but absent in homozygous hearts. Expression analysis of the Cspg2 gene showed that the 6/8, 6/9, and 7/9 Cspg2 exon boundaries were present in mRNA of normal wild-type embryonic hearts but absent in the homozygous mutant embryos. DNA sequence flanking the transgene was used to isolate from a normal mouse library overlapping genomic DNA segments that span the transgene insertion site. The contiguous genomic DNA segment was found to contain exon 7 of the Cspg2 in a position 3' to the transgene insertion site. These four separate lines of evidence support the hypothesis that Cspg2 is the gene disrupted by the transgene insertion in the hdf mouse line. The findings of this study and our previous studies of the hdf insertional mutant mouse have shown that normal expression of the Cspg2 gene is required for the successful development of the endocardial cushion swellings and the embryonic heart segments that give rise to the right ventricle and conus/truncus in the outlet of the looped heart.

Production of a monoclonal antibody by in vitro immunization that recognizes a native chondroitin sulfate epitope in the embryonic chick limb and heart.

We report the production of a monoclonal antibody (d1C4) by in vitro immunization that has immunoreactivity with a native chondroitin sulfate epitope in embryonic chick limb and heart. Murine lymphocytes were stimulated by direct exposure to unfixed, unsolubilized precartilage mesenchymal aggregates in high-density micromass culture derived from Stage 22-23 chick limb buds. Specificity of d1C4 reactivity was demonstrated by sensitivity of immunohistochemical staining to pretreatment with chondroitinase ABC or AC, preferential immunoreactivity with chondroitin-6-sulfate glycosaminoglycan (CS-C GAG) in ELISA, and competition of immunohistochemical staining with CS-C GAG. Immunohistochemical analysis of the expression of the d1C4 epitope revealed a striking localization of immunoreactivity in the extracellular matrix (ECM) of precartilage aggregates of chick limb mesenchyme in high-density micromass culture by 16 hr and the prechondrogenic limb core at Stage 23 in vivo. Immunoreactivity in both cultured limb mesenchyme and the embryonic limb continued through differentiation of prechondrogenic condensations into cartilage tissue. In the developing chick heart, d1C4 staining was found throughout the ECM of atrioventricular cushion tissue by Stage 25, but was localized to mesenchyme adjacent to the myocardium in the outflow tract cushions. There was an abrupt demarcation between d1C4-reactive intracardiac mesenchyme and unreactive extracardiac mesenchyme of the dorsal mesocardium in the Stage 22 embryo. This study demonstrates the efficacy of in vitro immunization of lymphocytes for the production of MAbs to native ECM constituents, such as CS-GAGs. Immunohistochemical data utilizing d1C4 suggest that CS-GAGs bearing this epitope may be important in early morphogenetic events leading to cartilage differentiation in the limb and valvuloseptal morphogenesis in the heart.

Transformation of cardiac endothelium into cushion mesenchyme is dependent on ES/130: temporal, spatial, and functional studies in the early chick embryo.

ES/130 is a novel 130-kDa protein that has been linked previously to the transformation of endocardial endothelium into cushion mesenchyme. In the present study we report the localization of protein and mRNA for ES/130 in stages 7-plus through 20 chick embryos and present functional data related to a potential mechanism for ES/130. The temporal and spatial regulation of ES/130 expression suggests that this epithelial-to-mesenchymal transformation is a result of homogenetic induction. Functional studies indicate that myocardially derived ES/130 elicits expression of this protein by target AV endothelial cells, which is linked to a signal transduction cascade. The localization of ES/130 to other sites of inductive interactions (e.g., limb bud ectoderm, gut, and notochord) implies that this protein may have a more widespread importance to embryogenesis beyond its involvement in cardiac cushion tissue formation.

Comparative effects of cytosine arabinoside and a prostaglandin E2 antagonist, AH6809, on chondrogenesis in serum-free cultures of chick limb mesenchyme.

The present study was designed to compare effects of an established inhibitor of cell proliferation and growth, cytosine arabinoside (Ara C), with that of a prostaglandin E2 (PGE2) antagonist, AH6809, on chondrogenesis in cultured mesenchyme derived from stage 25 chick limb buds. Continuous treatment of cell cultures with 10(-4) M AH6809 prevented completely the twofold increases in DNA content of control cultures which occurred between Day 1 and Day 5 of culture and also produced 90% inhibition of chondrogenesis occurring in control cultures during this same period. Treatment of cells with Ara C (0.1-0.5 microgram/ml) produced equivalent inhibition of DNA content during the same time period; however, chondrogenesis, as evaluated on Day 5 of cell culture, remained at approximately 90% of control cultures. These results indicate that the inhibitory effect of PGE2 receptor blockade on cell growth in these cultures cannot account for the potent inhibitory effects observed on differentiation of cartilage and provide further evidence in support of the notion that PGE2 plays an important initiating role in the process of chondrocyte differentiation within limb mesenchyme.

Effects of a putative prostaglandin E2 antagonist, AH6809, on chondrogenesis in serum-free cultures of chick limb mesenchyme.

In the present study, we have examined the effects of a putative antagonist of prostaglandin E2 (PGE2), AH6809, on chondrogenesis in serum-free cultures of mesenchyme from distal tips of stage 25 chick limb buds in order to test the hypothesis that endogenous PGE2, through receptor-linked adenylate cyclase (AC), initiates differentiation of cartilage in limb mesenchyme. Daily addition of 10(-4) M concentrations of AH6809 produced marked inhibition of chondrogenesis over a 5-day period of cell culture as evaluated by Alcian green binding to cartilage matrix components. Inhibition of chondrogenesis by this compound was further shown to be reversible and treatment of cells with the antagonist limited to periods when chondrocytes had differentiated and were actively secreting cartilage-specific matrix components had little effect. Preincubation of control cells in 10(-4) M concentrations of AH6809 inhibited PGE2-induced activation of AC by greater than 80% without significant (P greater than .05) inhibition of basal activity by the antagonist. Responses to parathyroid hormone, which increased AC activity by 7-fold, and forskolin which increased AC activity by 23-fold in control cells, were also uninhibited by preincubation in AH6809. The results demonstrate that blockade of PGE2-AC linked receptors in prechondrogenic limb mesenchyme inhibits chondrogenesis supporting the hypothesis that endogenous PGE2 concentrations in undifferentiated limb mesenchyme play an initiating role in the differentiation of cartilage.

Development of PTH-responsive adenylate cyclase activity during chondrogenesis in cultured mesenchyme from chick limb buds.

The present study investigated the development of parathyroid hormone (PTH)-responsive adenylate cyclase (AC) activity in chondrogenic cells differentiating from chick limb mesenchyme in culture. Mesenchyme from stage 25 chick embryos was removed from the distal tip (0.3 mm) of limb buds and cultured for a 6 day period in high density micromass cultures. Under these conditions, initial appearance of cartilage matrix and chondroblasts occurred on day 3 of culture and rapidly progressed over the next 3 days to produce, by day 6, a highly confluent and homogeneous layer of cartilage matrix and chondrocytes. Cells initially dissociated from limb mesenchyme on day 0 were essentially unresponsive to PTH, but development of AC-coupled, PTH receptors occurred rapidly during the initial 24 hours of culture. Based on data from dose-response experiments, prechondrogenic cells on day 1 of culture had synthesized their full complement of these receptors relative to fully differentiated chondrocytes in cultures at day 6. Inhibition of chondrocyte differentiation by retinoic acid did not significantly affect the initial development of AC-coupled, PTH receptors but it almost completely prevented synthesis of cartilage matrix. The results indicate that development of AC-coupled PTH receptors during chondrogenesis precedes, by at least 48 hours, overt differentiation of chondrocytes and the accumulation of cartilage-specific extracellular matrix and appears to represent one of the earliest reported events in chondrocyte differentiation. The lack of effect of retinoids on development of these receptors indicates that the inhibitory effects of retinoids on differentiating cartilage are at least somewhat specific for genes regulating synthesis of extracellular matrix molecules.

Responsiveness of adenylate cyclase to PGE2 and forskolin in isolated cells from micromass cultures of chick limb mesenchyme during chondrogenesis.

Exogenous PGE2 stimulation of adenylate cyclase (AC) in intact and enzymatically dissociated micromass cultures of mesenchymal cells derived from the distal tip of stage 25 chick limb buds was examined over a six day period of culture. Responsiveness to PGE2 was measured in both dissociated and intact cell layers in an effort to determine if an inhibitory interaction occurred between PGE2 receptors and the extracellular matrix synthesized by differentiating chondrocytes. PGE2 responsiveness was maximal in both dissociated and intact prechondrogenic mesenchyme after 24 hours in culture and declined significantly as chondrocyte differentiation occurred on days 3 and 6. Equivalent activation of AC activity by PGE2 at each time point examined was noted in both cell groups. In contrast to the decreased responsiveness of differentiating chondrocytes to PGE2, stimulation of AC by forskolin resulted in increased levels of activity in differentiating chondrocytes of both cell groups between days 3-6. The results of the present study demonstrate that the decline in PGE2 responsiveness of differentiating chondrocytes most likely involves specific changes in the PGE2 receptor complex and not in either the interaction of the receptor with extracellular matrix components or a reduction in the available pool of AC present.