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Effect of prostaglandin E2 on cyclic AMP levels in limb cells of mouse mutant Brachypodism
DEVELOPMENTAL BIOLOGY 142, 489-492 (1990)
Effect of Prostaglandin E2 on Cyclic AMP Levels in Limb Cells of Mouse Mutant Brachypodism TIMOTHY A. BALLARD* AND WILLIAM A. E L M E R *Department of Biological Sciences, University of North Carolina at Wilmington, Wilmington, North Carolina 28403; and Department of Biology, Emory University, Atlanta, Georgia 30322 Accepted August 29, 1990 Mouse embryo limb cells carrying either the brachypodism (bpH/bpH) mutation or its wild-type ( + / + ) allele were tested for their ability to accumulate cyclic AMP in response to prostaglandin E2 (PGEz) between Embryonic Days E l 2 and El4. M u t a n t cells exhibited a precocious increase in cyclic AMP. In the absence of PGE~ but in the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX), the brachypodism cells accumulated a significantly lower amount of cyclic AMP by Day El4. Limb cells carrying the bpH mutation may provide a useful experimental system to study the PGE2-cyclic AMP-cartilage differentiation interrelationship. © 1999AcademicPress, Inc.
tration in the same buffer used for cell incubations. The final ethanol concentration of the incubations did not exceed 0.4%. Reagents for the assay of cyclic AMP were purchased from Diagnostic Products Corp., while 1methyl-3-isobutylxanthine (MIX) and all other chemicals were purchased from Sigma.
INTRODUCTION
Prostaglandin E~ (PGE2), which acts through a cyclic adenosine 3', 5'-monophosphate (cyclic AMP)-mediated system, has been shown to be involved in limb-bud chondrogenesis (Parker et al., 1981; Ballard and Biddulph, 1983; Gay and Kosher, 1985; Smales and Biddulph, 1985). A cell-surface receptor has been reported to be present in limb mesenchyme (Biddulph et aL, 1988) and inhibition of PGE 2 in limb cells has been shown to prevent chondrogenesis (Chepenik et al., 1984). These data suggest a PGE2-receptor-cyclic A M P - c h o n d r o genic sequence. However, the nature of the interrelationships between these components is not understood. Brachypodism (bpH/bpH), an autosomal recessive trait in mice, is characterized by a delay of and a deficiency in the formation of the limb skeleton (Hewitt and Elmer, 1976; Duke and Elmer, 1977, 1978; Hewitt and Elmer, 1978). Abnormal expression is first recognized at the onset of chondrogenesis (Embryonic Day 12 of gestation) (Landauer, 1952; Konyukhov and Ginter, 1966; Milaire, 1965; Gruneberg and Lee, 1973; Elmer and Selleck, 1975; Elmer, 1976). Pathogenesis has been ascribed to an aberrant processing of the oligosaccharides of cell-surface glycoconjugates (Elmer and Wright, 1983; Elmer et al., 1988). Abnormalities at the cell surface have been correlated with a decrease in cyclic AMP in the bpH limb cells (Elmer et al., 1981). Consequently, it was of interest to examine whether brachypodism cells can recognize PGE 2 and respond to a transmembrane signal by increasing cyclic AMP levels.
Cell Preparation Using the time of vaginal plug appearance as Embryonic Day 0 (Krotoski and Elmer, 1973), embryos of the 11-, 12-, and 14-day post-plug stages were obtained from matings between mice homozygous for either the bpH allele or the normal allele at the same locus. Morphological characterization of the embryos collected was used for precise stage determination (Theiler, 1972). Embryos were removed from the uteri of the mothers in Tyrode's solution and the postaxial hindlimbs collected by dissection. Postaxial hindlimbs were incubated for 4 min at room temperature in calcium-magnesium-free Tyrode's solution (CMF) containing 2.25% t r y p s i n 0.75% pancreatin, pH 8.0. After enzyme treatment, the ectoderm was removed by dissection and the mesoblasts were collected in CMF. After 30 min, the CMF was replaced by 2 ml of a calcium stock solution containing 1.8 mg/ml glucose dissolved in Hanks' solution with 1 m M calcium. Dissociated cells were counted with a Coulter counter (Model ZF) and the cells pelleted by centrifugation. The cell pellet was washed two times with the calcium stock solution, then covered with a total volume of incubation medium equal to 1.5 ml/sample. The cells were dispersed into siliconized flasks on ice.
MATERIALS AND METHODS
Materials
Incubations and Assay of Cyclic A M P
Prostaglandin E2 (Sigma Chemical Co.) was dissolved in absolute ethanol and diluted to the desired concen-
Postaxial hindlimb cells, representing 100-300 t~g protein/sample, were incubated at 37°C in an oscilla489
0012-1606/90 $3.00 Copyright © 1990 by Academic Press, Inc. A]| rights of reproduction in any form reserved.
490
DEVELOPMENTALBIOLOGY
tory water bath in 1.5 ml of a buffer, pH 7.5, containing Hanks' solution and at a final concentration of I m M MgC12, 1 mM CaC12, 1.8 mg/ml glucose, and 20 m M T r i s HC1 (Ballard and Biddulph, 1983). In some experiments, the phosphodiesterase inhibitor MIX (Zenser et al., 1977) was added to the incubation medium to obtain a final concentration of 2 mM. Previous studies have shown this concentration to be maximally effective in elevating cyclic AMP (Ballard and Biddulph, 1983, 1984). Prostaglandin E 2 (at a final concentration of 30 ttg/ml) or the ethanol control solution was added after a 5-min preincubation period. The control solution consisted of ethanol and 20 m M Tris buffer, pH 9.0. Cell viability, as measured by the trypan blue exclusion method, was consistently higher than 95%. Incubations were terminated 5 min after exposure to the test agents by the addition of cold 5% trichloroacetic acid (TCA). Tissue extracts were prepared as previously described (Ballard and Biddulph, 1983, 1984). Cyclic AMP was assayed by the method of Gilman (1970), as modified by Tovey et al., (1974). Protein within the TCA pellets was measured by the method of Lowry et al., (1951). RESULTS AND DISCUSSION
Cells of both genotypes incubated in the ethanol control solution exhibited no significant increase in cyclic AMP between Embryonic Days E l l and El4 (Fig. 1A). However, when MIX was added to the ethanol control solution, the cyclic AMP level in the wild-type cells was significantly higher than the bp H value at Day El4 (Fig. 2A). Since inhibition of phosphodiesterase provides a relative measure of adenylate cyclase activity (Ballard and Biddulph, 1983; Ballard and Biddulph, 1984), these data suggest that a reduction in adenylate cyclase activity in brachypodism cells accompanies the cartilage matrix deficiency expressed in the mutant between Days E l l and El4 (Elmer, 1976). These data also corroborate a previous immunofluorescent study which suggested a decrease in the endogenous level of cyclic AMP in bpH limb cells (Elmer et al., 1981). Ballard and Biddulph (1983), using embryonic chick limb mesenchyme between H a m b u r g e r - H a m i l t o n (1951) stages 20-32, have shown that 30 #g/ml of PGE 2 over a period of 5 min can cause a net fivefold increase in cyclic AMP accumulation. In the present study, a smaller (approximately twofold), but still significant, net increase in cyclic AMP in the presence of PGE2 was also observed between Days E l l (stage 18-19) (Theiler, 1972) and El4 (stage 22) in the wild-type mouse cells (Fig. 1B). This increase was not detectable, however, until after Day El2. The smaller increase in mouse cell responsiveness was probably due to: (1) the use of the postaxial region of the mouse limb rather than the en-
VOLUME142, 1990
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DAYS OF GESTATION FIG. 1. Cyclic AMP accumulation in mouse embryo limb mesenchyme cells in the absence of MIX after treatment with ethanol (A) or PGE2 (B). (O) Represents + / + cells and (O) represents bpHcells. Each point is the mean of nine values obtained from three individual experiments. Vertical bars represent SEM. *Value differing significantly (P < 0.05) from + / + cells at the same stage.
tire limb bud; and (2) the incomplete formation of the skeletal element by Day El4 in the mouse compared to that by stages 30-32 in the chick. The postaxial region was used because the cartilage deficiency in the mutant is most severe in this region (Krotoski and Elmer, 1973). When bpH cells were exposed to PGE 2 in the absence of MIX, the level of cyclic AMP increased precociously and significantly on Day El2 compared to that in the wildtype (Fig. 1B). A similar response was observed when MIX was present in the incubation medium (Fig. 2B). By Day El4, however, both genotypes exhibited similar
BRIEF NOTES
have begun to express the chondrogenic phenotype (Biddulph et al., 1984; Gay and Kosher, 1985). Interestingly, compared to wild-type, a larger population of bpH postaxial cells continue to express undifferentiated characteristics between Days E l 2 and El4 (Hewitt and Elmer, 1978). Consequently, the increased responsiveness of the mutant cells could be explained as a continued expression of PGE2 receptors over this developmental time period.
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DAYS OF GESTATION FIG. 2. Cyclic AMP accumulation in mouse embryo limb mesenchyme cells in the presence of 2 m M M I X after treatment with ethanol (A) or PGE 2 (B). (©) Represents + / + cells and (e) represents bpg cells. Each point is the mean of nine values obtained from three individual experiments. Vertical bars represent SEM. *Value differing significantly (P < 0.05) from + / + cells at the same stage.
concentrations of cyclic AMP under both incubation conditions (Figs. 1 and 2), even though the values were lower in the absence of MIX. A more revealing picture emerged when differences (A) between matched hormone- and ethanol-treated incubations were compared (Fig. 3). The data indicate that mutant cells at Days E12 and E l 4 were significantly more responsive to PGE 2 than were the wild-type cells. It has been shown in studies using chick limb cells that undifferentiated, prechondrogenic limb mesenchyme cells are maximally responsive to PGE 2 when compared to older cells that
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DAYS OF GESTATION FZG. 3. Effects of PGE2 on cyclic AMP accumulation in + / + and bpH mouse limb mesenchyme cells in the absence (A) or presence (B) of MIX. (©) Represents + / + cells and (O) represents bpH cells. Each point is the mean of nine values obtained from three individual experiments and is the difference (A) from an equal number of ethaaol control samples. Vertical bars represent the SE of the difference. *Values differing significantly (P < 0.05) from + / + cells at the same stage.
492
DEVELOPMENTALBIOLOGY
The incomplete expression of the cartilage phenotype in the mutant appears to be related to the inability of the limb cells to accumulate endogenous cyclic AMP (Fig. 2A) and the precocious responsiveness to PGE 2 at a prechondrogenic stage (Figs. 1B and 2B). The data suggest a defective regulation in the activities of both adenylate cyclase and phosphodiesterase, compared to these activities in wild-type cells from the same stages. From the results of this study, it seems that brachypodism limb cells may serve as a very useful experimental system to study the developmental regulation of PGE 2 receptors and the interrelationships between PGE2, cyclic AMP, and cartilage differentiation. REFERENCES
BALLARD,T. A., and BIDDULPH,D. M. (1983). Effects of prostaglandins on cyclic AMP levels in isolated cells from developing chick limbs. Prostaglandius 25, 474-480. BALLARD, T. A., and BIDDULPH, D. M. (1984). The morphology and hormonal responsiveness of developing skeletal elements in chick limb buds. Amer. J. Anat. 169, 221-236. BIDDULPH, D. M., SAWYER,L. M., and SMALES,W. P. (1984). Chondrogenesis of chick limb mesenchyme in vitro: Effects of prostaglandins on cyclic AMP. Exp. Cell Res. 153, 270-274. BIDDULPH,D. M., DOZIER,M. M., JULIAN,N. C., and SAWYER,L. M. (1988). Inhibition of chondrogenesis by retinoic acid in limb mesenchymal cells in vitro: Effects on PGE2 and cyclic AMP concentration. Cell Differ. Dev. 25, 65-76. CHEPENIK, K. P., HO, W. C., WAITE, B. M., and PARKER, C. P. (1984). Arachidonate metabolism during chondrogenesis in vitro. Calcif. Tissue Int. 36, 175-181. DUKE, J., and ELMER, W. A. (1977). Effect of the brachypod mutation on cell adhesion and chondrogenesis in aggregates of mouse limb mesenchyme. J. EmbryoL Exp. Morphol. 42, 209-217. DUKE, J., and ELMER, W. A. (1978). Cell adhesion and chondrogenesis in brachypod mouse limb mesenchyme: Fragment fusion studies. J. Embryol. Exp. MorphoL 48, 161-168. ELMER, W. A., and SELLECK, D. K. (1975). In vitro chondrogenesis of limb mesoderm from normal and brachypod mouse embryos. J. EmbryoL Exp. Morphol. 33, 371-386. ELMER, W. A. (1976). Morphological and biochemical modification of cartilage differentiation in brachypod and other micromelic mouse embryos. In "M~chanismes De La Rudimentation Des Organes Chez Les Emhryons De Vertebras" (A. Raynaud, Ed.), CNRS N ° 266, pp. 235-242. ELMER, W. A., SMITH,i . A., and EDE, D. A. (1981). Immunohistochemical localization of cyclic AMP during normal and abnormal chick and mouse limb development. Teratology 24, 215-223. ELMER, W. A., and WRIGHT, J. T. (1983). Changes in plasma membrane proteins and glycoproteins during normal and brachypod mouse
VOLUME142, 1990
limb development. In "Limb Development and Regeneration, Part A" (J. F. Fallon and A. I. Caplan, Eds.), pp. 355-364. A. R. Liss, New York. ELMER, W. A., PENNYPACKER, M. F., KNUDSEN, T. B., and KWASIGROCH, T. E. (1988). Alterations in cell surface galactosyltransferasP activity during limb chondrogenesis in brachypod mutant mouse embryos. Teratology 38, 475-484. GAY, S. W., and KOSHER,R. A. (1985). Prostaglandin synthesis during the course of limb cartilage differentiation in vitro. J. EmbryoL Exp. Morphol. 89, 367-382. GmMAN, A. G. (1970). A protein binding assay for adenosine 3', 5'-cyclic monophosphate. Proc. NatL AcacL Sc~ USA 67, 305-312. GRUNEBERG,H., and LEE, A. J. (1973). The anatomy and development of brachypodism in the mouse. J. EmbryoL Exp. Morphol. 30, 119141. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. MorphoL 88, 49-91. HEWITT, A. T., and ELMER, W. A. (1976). Reactivity of normal and brachypod mouse limb mesenchymal cells with conA. Nature (London) 264, 177-178. HEWITT, A. T., and ELMER, W. A. (1978). Developmental modulation of lectin-binding sites on the surface membranes of normal and brachypod mouse limb mesenchymal cells. D~erentiation 10, 31-38. KONYUKHOV, B. V., and GINTER, E. (1966). A study of the brachypodism-H gene on development of the long bones of the hind limb in the mouse. Folia Biol. 12, 199-206. KROTOSKI,D. M., and ELMER, W. A. (1973). Alkaline phosphatase activity in fetal hind limbs of the mouse mutation brachypodism. Teratology 7, 99-106. LANDAUER,W. (1952). Brachypodism: A recessive mutation of housemice. J. HerecL 43, 293-298. LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin-phenol reagent. J. BioL Che~ 193, 265-275. MILAIRE, J. (1965). Etude morphog~n~tique de trois malformations congenitales de l'autopode chez la souri (syndactylisme-brachypodisme-hemim~liae dominante) par des m~thodes cytochimiques. Aca~ R. Me& Belg. Mere. 16, 1-120, PARKER, C. L., BIDDULPH,D. U., and BALLARD,T. A. (1981). Development of the cyclic AMP response to parathyroid hormone and prostaglandin E 2 in the embryonic chick limb. Calci~. Tissue Int. 33, 641648. SMALES, W. P., and BIDDULPH, D. M. (1985). Limb development in chick embryos: Cyclic AMP-dependent protein kinase activity, cyclic AMP, and prostaglandin concentrations during cytodifferentiation and morphogenesis. J. Cell PhysioL 122, 259-265. THEILER, K. (1972). "The House Mouse." Springer-Verlag, New York. TOVEY, K. C., OLDHAM,K. G., and WHELAN, J. A. M. (1974). A simple direct assay for cyclic AMP in plasma and other biological samples using an improved competitive binding technique. Clin. Chim. Acta 56, 221-230. ZENSER, T. V., CRAVEN,P. A., DERUBERTIS,F. R., and DAVIS, B: B. (1977). Differential inhibition of cyclic AMP and cyclic GMP hydrolysis in rat renal cortex. Arch. Biochem~ Biophys. 178, 598-606.
Effect of Prostaglandin E2 on Cyclic AMP Levels in Limb Cells of Mouse Mutant Brachypodism TIMOTHY A. BALLARD* AND WILLIAM A. E L M E R *Department of Biological Sciences, University of North Carolina at Wilmington, Wilmington, North Carolina 28403; and Department of Biology, Emory University, Atlanta, Georgia 30322 Accepted August 29, 1990 Mouse embryo limb cells carrying either the brachypodism (bpH/bpH) mutation or its wild-type ( + / + ) allele were tested for their ability to accumulate cyclic AMP in response to prostaglandin E2 (PGEz) between Embryonic Days E l 2 and El4. M u t a n t cells exhibited a precocious increase in cyclic AMP. In the absence of PGE~ but in the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX), the brachypodism cells accumulated a significantly lower amount of cyclic AMP by Day El4. Limb cells carrying the bpH mutation may provide a useful experimental system to study the PGE2-cyclic AMP-cartilage differentiation interrelationship. © 1999AcademicPress, Inc.
tration in the same buffer used for cell incubations. The final ethanol concentration of the incubations did not exceed 0.4%. Reagents for the assay of cyclic AMP were purchased from Diagnostic Products Corp., while 1methyl-3-isobutylxanthine (MIX) and all other chemicals were purchased from Sigma.
INTRODUCTION
Prostaglandin E~ (PGE2), which acts through a cyclic adenosine 3', 5'-monophosphate (cyclic AMP)-mediated system, has been shown to be involved in limb-bud chondrogenesis (Parker et al., 1981; Ballard and Biddulph, 1983; Gay and Kosher, 1985; Smales and Biddulph, 1985). A cell-surface receptor has been reported to be present in limb mesenchyme (Biddulph et aL, 1988) and inhibition of PGE 2 in limb cells has been shown to prevent chondrogenesis (Chepenik et al., 1984). These data suggest a PGE2-receptor-cyclic A M P - c h o n d r o genic sequence. However, the nature of the interrelationships between these components is not understood. Brachypodism (bpH/bpH), an autosomal recessive trait in mice, is characterized by a delay of and a deficiency in the formation of the limb skeleton (Hewitt and Elmer, 1976; Duke and Elmer, 1977, 1978; Hewitt and Elmer, 1978). Abnormal expression is first recognized at the onset of chondrogenesis (Embryonic Day 12 of gestation) (Landauer, 1952; Konyukhov and Ginter, 1966; Milaire, 1965; Gruneberg and Lee, 1973; Elmer and Selleck, 1975; Elmer, 1976). Pathogenesis has been ascribed to an aberrant processing of the oligosaccharides of cell-surface glycoconjugates (Elmer and Wright, 1983; Elmer et al., 1988). Abnormalities at the cell surface have been correlated with a decrease in cyclic AMP in the bpH limb cells (Elmer et al., 1981). Consequently, it was of interest to examine whether brachypodism cells can recognize PGE 2 and respond to a transmembrane signal by increasing cyclic AMP levels.
Cell Preparation Using the time of vaginal plug appearance as Embryonic Day 0 (Krotoski and Elmer, 1973), embryos of the 11-, 12-, and 14-day post-plug stages were obtained from matings between mice homozygous for either the bpH allele or the normal allele at the same locus. Morphological characterization of the embryos collected was used for precise stage determination (Theiler, 1972). Embryos were removed from the uteri of the mothers in Tyrode's solution and the postaxial hindlimbs collected by dissection. Postaxial hindlimbs were incubated for 4 min at room temperature in calcium-magnesium-free Tyrode's solution (CMF) containing 2.25% t r y p s i n 0.75% pancreatin, pH 8.0. After enzyme treatment, the ectoderm was removed by dissection and the mesoblasts were collected in CMF. After 30 min, the CMF was replaced by 2 ml of a calcium stock solution containing 1.8 mg/ml glucose dissolved in Hanks' solution with 1 m M calcium. Dissociated cells were counted with a Coulter counter (Model ZF) and the cells pelleted by centrifugation. The cell pellet was washed two times with the calcium stock solution, then covered with a total volume of incubation medium equal to 1.5 ml/sample. The cells were dispersed into siliconized flasks on ice.
MATERIALS AND METHODS
Materials
Incubations and Assay of Cyclic A M P
Prostaglandin E2 (Sigma Chemical Co.) was dissolved in absolute ethanol and diluted to the desired concen-
Postaxial hindlimb cells, representing 100-300 t~g protein/sample, were incubated at 37°C in an oscilla489
0012-1606/90 $3.00 Copyright © 1990 by Academic Press, Inc. A]| rights of reproduction in any form reserved.
490
DEVELOPMENTALBIOLOGY
tory water bath in 1.5 ml of a buffer, pH 7.5, containing Hanks' solution and at a final concentration of I m M MgC12, 1 mM CaC12, 1.8 mg/ml glucose, and 20 m M T r i s HC1 (Ballard and Biddulph, 1983). In some experiments, the phosphodiesterase inhibitor MIX (Zenser et al., 1977) was added to the incubation medium to obtain a final concentration of 2 mM. Previous studies have shown this concentration to be maximally effective in elevating cyclic AMP (Ballard and Biddulph, 1983, 1984). Prostaglandin E 2 (at a final concentration of 30 ttg/ml) or the ethanol control solution was added after a 5-min preincubation period. The control solution consisted of ethanol and 20 m M Tris buffer, pH 9.0. Cell viability, as measured by the trypan blue exclusion method, was consistently higher than 95%. Incubations were terminated 5 min after exposure to the test agents by the addition of cold 5% trichloroacetic acid (TCA). Tissue extracts were prepared as previously described (Ballard and Biddulph, 1983, 1984). Cyclic AMP was assayed by the method of Gilman (1970), as modified by Tovey et al., (1974). Protein within the TCA pellets was measured by the method of Lowry et al., (1951). RESULTS AND DISCUSSION
Cells of both genotypes incubated in the ethanol control solution exhibited no significant increase in cyclic AMP between Embryonic Days E l l and El4 (Fig. 1A). However, when MIX was added to the ethanol control solution, the cyclic AMP level in the wild-type cells was significantly higher than the bp H value at Day El4 (Fig. 2A). Since inhibition of phosphodiesterase provides a relative measure of adenylate cyclase activity (Ballard and Biddulph, 1983; Ballard and Biddulph, 1984), these data suggest that a reduction in adenylate cyclase activity in brachypodism cells accompanies the cartilage matrix deficiency expressed in the mutant between Days E l l and El4 (Elmer, 1976). These data also corroborate a previous immunofluorescent study which suggested a decrease in the endogenous level of cyclic AMP in bpH limb cells (Elmer et al., 1981). Ballard and Biddulph (1983), using embryonic chick limb mesenchyme between H a m b u r g e r - H a m i l t o n (1951) stages 20-32, have shown that 30 #g/ml of PGE 2 over a period of 5 min can cause a net fivefold increase in cyclic AMP accumulation. In the present study, a smaller (approximately twofold), but still significant, net increase in cyclic AMP in the presence of PGE2 was also observed between Days E l l (stage 18-19) (Theiler, 1972) and El4 (stage 22) in the wild-type mouse cells (Fig. 1B). This increase was not detectable, however, until after Day El2. The smaller increase in mouse cell responsiveness was probably due to: (1) the use of the postaxial region of the mouse limb rather than the en-
VOLUME142, 1990
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DAYS OF GESTATION FIG. 1. Cyclic AMP accumulation in mouse embryo limb mesenchyme cells in the absence of MIX after treatment with ethanol (A) or PGE2 (B). (O) Represents + / + cells and (O) represents bpHcells. Each point is the mean of nine values obtained from three individual experiments. Vertical bars represent SEM. *Value differing significantly (P < 0.05) from + / + cells at the same stage.
tire limb bud; and (2) the incomplete formation of the skeletal element by Day El4 in the mouse compared to that by stages 30-32 in the chick. The postaxial region was used because the cartilage deficiency in the mutant is most severe in this region (Krotoski and Elmer, 1973). When bpH cells were exposed to PGE 2 in the absence of MIX, the level of cyclic AMP increased precociously and significantly on Day El2 compared to that in the wildtype (Fig. 1B). A similar response was observed when MIX was present in the incubation medium (Fig. 2B). By Day El4, however, both genotypes exhibited similar
BRIEF NOTES
have begun to express the chondrogenic phenotype (Biddulph et al., 1984; Gay and Kosher, 1985). Interestingly, compared to wild-type, a larger population of bpH postaxial cells continue to express undifferentiated characteristics between Days E l 2 and El4 (Hewitt and Elmer, 1978). Consequently, the increased responsiveness of the mutant cells could be explained as a continued expression of PGE2 receptors over this developmental time period.
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DAYS OF GESTATION FIG. 2. Cyclic AMP accumulation in mouse embryo limb mesenchyme cells in the presence of 2 m M M I X after treatment with ethanol (A) or PGE 2 (B). (©) Represents + / + cells and (e) represents bpg cells. Each point is the mean of nine values obtained from three individual experiments. Vertical bars represent SEM. *Value differing significantly (P < 0.05) from + / + cells at the same stage.
concentrations of cyclic AMP under both incubation conditions (Figs. 1 and 2), even though the values were lower in the absence of MIX. A more revealing picture emerged when differences (A) between matched hormone- and ethanol-treated incubations were compared (Fig. 3). The data indicate that mutant cells at Days E12 and E l 4 were significantly more responsive to PGE 2 than were the wild-type cells. It has been shown in studies using chick limb cells that undifferentiated, prechondrogenic limb mesenchyme cells are maximally responsive to PGE 2 when compared to older cells that
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DAYS OF GESTATION FZG. 3. Effects of PGE2 on cyclic AMP accumulation in + / + and bpH mouse limb mesenchyme cells in the absence (A) or presence (B) of MIX. (©) Represents + / + cells and (O) represents bpH cells. Each point is the mean of nine values obtained from three individual experiments and is the difference (A) from an equal number of ethaaol control samples. Vertical bars represent the SE of the difference. *Values differing significantly (P < 0.05) from + / + cells at the same stage.
492
DEVELOPMENTALBIOLOGY
The incomplete expression of the cartilage phenotype in the mutant appears to be related to the inability of the limb cells to accumulate endogenous cyclic AMP (Fig. 2A) and the precocious responsiveness to PGE 2 at a prechondrogenic stage (Figs. 1B and 2B). The data suggest a defective regulation in the activities of both adenylate cyclase and phosphodiesterase, compared to these activities in wild-type cells from the same stages. From the results of this study, it seems that brachypodism limb cells may serve as a very useful experimental system to study the developmental regulation of PGE 2 receptors and the interrelationships between PGE2, cyclic AMP, and cartilage differentiation. REFERENCES
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VOLUME142, 1990
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