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Appearance of myosin in the chick limb bud
DEVELOPMENTAL
BIOLOGY
28,
138-141 (1872)
Appearance JUDITH Department
of Myosin MEDOFF’
of Biology,
Bmndeis
Accepted
in the Chick Limb Bud AND EDGAR University,
Februnry
ZWILLING~ Waltham,
Massachusetts
3, 1972
Quantitative microcomplement fixation has been used to detect the appearance of myosin in the chick embryonic limb bud. It has been shown that myosin or a myosinlike molecule is present by stage 23, before muscle can be distinguished histologically. INTRODUCTION
There is no information about the developmental stage at which chick embryonic limb bud mesoderm cells begin to synthesize myosin. In Holtzer’s work (1961) with somites, there was no reaction to fluorescein-labeled antimyosin in either presumptive somite myoblasts or cells outside the muscle-forming area of somites. Specific staining by antimyosin occurred only in differentiating myoblasts (Holtzer et al., 1957). These observations lend support to a notion in developmental literature of a strict correspondence between biosynthesis and tissue histogenesis, i.e., differentiating cells can synthesize terminal products when they have acquired recognizable histological differentiation, and not before (Holtzer, 1961). It has been shown, however, that limb buds synthesize chondroitin sulfate long before acquiring the cytological features of cartilage (Searls, 1965b; Medoff, 1967). Also, a sulfated mucopolysaccharide with the same electrophoretic mobility as chondroitin sulfate was detected in somites of stage 16-17 chick embryos (FrancoBrowder et al., 1963) and a number of enzymes have been cartilage-related identified in somites (Lash, 1968) and limb buds (Medoff, 1967) before histological evidence of chondrogenesis. 1Present address: Biology Department, Washington University, St. Louis, Missouri 63130. 2 Aided by Grant HD-06345 from the Institute of Child Health and Human Development of the National Institutes of Health, U. S. Public Health Service.
This report presents evidence that stage 23-24 (Hamburger and Hamilton, 1951) limbs synthesize myosin or a myosinlike molecule before myoblasts can be detected in the buds. MATERIALS
0 1972 by Academic
Press, Inc.
METHODS
The presence of myosin in early embryonic chick limbs was determined by complement (C’) fixation. Limb buds were dissected from the chick at the appropriate stage, pooled, and homogenized in the cold in 0.15 M NaCl. The extract was sonicated for 1.5 min. After centrifugation at 105,000 g for 1 hr, the antigen preparation was ready for use. Control tissue was prepared in a similar manner. A rabbit antiserum to monkey myosin and a rabbit antiserum to chicken myosin was given to us by Dr. Bela Nagy of the Retina Foundation (Boston, Massachusetts) and Dr. Robert Cahn of the University of Washington (Seattle). The antisera had been tested for specificity and reacted well with purified myosin. The antisera were heat inactivated at 60°C for 20 min and diluted 1:250 before use. Quantitative microcomplement fixation was performed according to the method of Wasserman and Levine (1961). RESULTS
The C’ fixation curves obtained with the two different antimyosin serums and extracts prepared from the leg muscles of 14-day chick embryos are shown on Fig. 1. Chicken myosin fixed C’ with both antimonkey myosin and antichicken myosin 138
Copyright
AND
Myosin
MEDOFF AND ZWILLING
r
too 60 .-x ‘,
60
.* IA.
20 I
I
I
I
0.001
001
0.1
1.0
mg Protein
(Day
14 Muscle
Extract)
100 -
FIG. 1. C’ fixation with two different antimyosin serums and extracts from the leg muscles of 14-day chick embryos.
5 2
so
-
60
-
2 -cl c
40-
20
-
001
01
IO mg
IO
Protein
FIG. 2. C’ fixation with rabbit antichicken and extracts from early limb buds.
myosin
to about the same extent. Although the chicken myosin-antimonkey myosin probably represents a cross-reaction, we cannot estimate the extent of this cross-reaction since we did not titrate the antiserum with monkey myosin. Nevertheless, at appropriate dilutions of each antiserum, the leg muscle extracts behave serologically essentially the same. There was no C’ fixation with antigen prepared from limb buds of stage 20 through 22 embryos. C’ fixation with undiluted extracts of stage 23, however, was observed, suggesting that there may be some myosin or a myosinlike molecule present in the stage 23 bud. Figure 2 presents the C’ fixation data obtained with the stage 23 preparation and three different preparations of stage 24 limb buds. Al-
in Chick Limb Bud
139
though these data were obtained with rabbit antichicken myosin, these same extracts also fixed C’ in a qualitatively similar manner with rabbit antimonkey myosin. None of these extracts fixed C’ with a rabbit-anti-poly U, which served as control for serologic specificity. Extracts from stage 23 limb buds and three extracts of stage 24 limb buds contained myosin or a myosinlike molecule, as judged from the C’-fixing activity. Myosin serologic activity accumulated rapidly in late stage 24-early stage 25 limb buds, and extracts contained some 50 times more activity than the two early stage 24 extracts. Other embryonic tissues were examined for the presence of myosin. The regions to be tested were extracted in the same manner as the limb buds and assayed. The heads of stage 20 embryos and the neural retina of T-day chicks were negative. Extracts of brain from 15 and 20-day chick embryos fixed C’ with both antisera in a similar manner. This tissue appeared to contain myosin or a myosinlike molecule. This puzzling result caused concern regarding the specificity of both antibodies until Puszkin et al. (1968) reported the isolation of a protein with characteristics similar to those of actomyosin from both rat and cat brain. It may be that chick embryonic brain contains a similar actomyosin-like protein accounting for the positive reaction with myosin antisera. DISCUSSION
The results presented in this paper suggest that myosin or a myosinlike molecule is present in primitive mesoderm cells of the chick embryonic limb bud before morphological evidence of muscle differentiation. We are aware that the crude extracts were prepared at salt concentrations which ordinarily precipitate adult myosin. However, the fixation of complement is inhibited by excess electrolyte, and we chose to prepare our extracts at the optimal salt concentration for fixation, 0.15 M
140
DEVELOPMENTAL BIOLOGY
NaCl. Our results indicate that some myosin or myosinlike material was present in the supernatant, but owing to its limited solubility at 0.15 M NaCl, large amounts of extract had to be used in order to obtain complement fixation. Determination of the specificity of the antiserum is critical in the validation of immunoembryological studies of myosin. Such studies are complicated by the problems encountered in obtaining antigenitally homogeneous preparations of myosin. It has been reported that chick myosin purified by two different methods remained heterogeneous and antisera produced against such myosin were contaminated with heterologous antibodies (Finck, 1965). We feel confident that our antisera do contain antibodies against myosin, but we recognize the possibility that they may be heterogeneous and the reaction may have measured a myosinlike molecule which does not become associated with muscle. The accumulation of serologic activity in older limb buds concomitant with the appearance of myoblasts and myocytes and the fact that extracts prepared from other embryonic areas at the same stages do not react with the antisera suggest that we are measuring myosin or an antigen which is part of the myosin molecule. Actin or actinlike filaments have recently been reported in homogenates of developing chick leg muscle as early as stage 23 (Hitchcock, 1971), which supports our notion that early limb buds contain low levels of muscle specific protein prior to morphological evidence of myogenesis. The possible presence of myosin in stage 23 limb buds, the fact that pieces of mesoderm from both the chondrogenic and myogenic area were equivalent in their ability to form cartilage (Zwilling, 1966), and the report of uniform sulfate fixation in the mesoderm of early limb buds followed by some incorporation of label in the prospective muscle-forming region at later stages (Searls, 1965a), all raise an important question regarding mecha-
VOLUME 28, 1972
nisms of differentiation. There is as yet little evidence in support of the so-called exclusivity of differentiated states; instead each cell type is probably characterized by more than one synthetic pathway (Grobstein, 1966). A model of differentiation emerges from these observations. An embryonic cell may contain specific enzymes and metabolic intermediates or even complex macromolecular products in small amounts before terminal differentiation. Differentiation may then be accomplished by a gradual stabilization and amplification of certain metabolic patterns and repression of others. Rigorous evidence is lacking about synthetic activities of individual limb mesoderm cells. The appearance of myosin or a myosinlike molecule at stage 23-24 may, in fact, coincide with the appearance of a new population of cells which have had no chondroitin sulfate synthesizing properties (or activity). However, there are models for restriction of previously general synthetic activities to particular tissues (i.e., and repression in others). Gulonolactone oxidase is widely distributed in early chick blastoderms, but the enzyme disappears from all tissues except the kidney during the course of development of the embryo (Fabro and Rinaldini, 1965). Other examples may be cited. The fact is that muscle appears in tissue areas which initially fix sulfate (Searls, 1965a) and are capable of differentiating into cartilage (Zwilling, 1966). There is no evidence that cells migrate into these areas. It seems likely, then, that myosin-synthesizing cells (myoblasts) are derived from the same mesoderm which forms the other tissue types of the limb. There is at least the likelihood that cells which first engage in low level chondroitin sulfate synthesis may then synthesize myosin or give rise to progeny which do this. SUMMARY
Microcomplement fixation suggests the presence of myosin or a myosinlike molecule in the limb buds of stage 23-24 chick
MEDOFF AND ZWILLINC
embryos before histological evidence of muscle differentiation. Stage 24-25 buds exhibited about 50 times more myosin serologic activity than the limbs of embryos 1 day younger. The for his ecution aration
authors wish to thank Dr. Lawrence Levine invaluable assistance in the planning and exof the experimental work as well as the prepof the manuscript. REFERENCES
FABRO, S. P., and RINALDINI, L. M. (1965). Loss of ascorbic acid synthesis in embryonic development. Deuelop. Biol. 11, 468-488. FINCK, H. (1965). Immunochemical studies of myosin. I. Effects of different methods of preparation on the immunochemical properties of chicken skeletal muscle myosin. Biochim. Biophys. Acta 111, 20% 220. FRANCO-BROWDER,S., DERYDT, J., and DORFMAN, A. (1963). The identification of a sulfated mucopolysaccharide in chick embryos, stages 11-23. Proc. Nat. Acad. Sci. U.S.A. 49, 643-647. GROBSTEIN, C. (1966). What we do not know about differentiation. Amer. Zoo!. 6,89-95. HAMBURGER, V., and HAMILTON, H. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. HITCHCOCK, S. E. (1971). Detection of actin filaments in homogenates of developing m’uscle using heavy meromyosin. Develop. Biol. 25, 492-501.
Myosin in Chick Limb Bud
141
HOLTZER, H. (1961). Aspects of chondrogenesis and myogenesis. In “Synthesis of Molecular and Cellular Structure,” Growth Symp. No. 19 (D. Rudnick, ed.), pp. 35-87. Ronald Press, New York. HOLTZER, H., MARSHALL, J., and FINCK, H. (1957). An analysis of myogenesis by the use of fluorescent antimyosin. J. Biophys. Biochem. Cytol. 3, 705723. LASH, J. W. (1968). Semitic mesenchyme and its response to cartilage induction. In “Epithelial-Mesenchymal Interactions” (R. Fleischmajer and R. E. Billingham, eds.), pp. 165-172. Williams and Wilkins, Baltimore, Maryland. MEDOFF, J. (1967). Enzymatic events during cartilage differentiation in the chick embryonic limb bud. Develop. Biol. 16, 118-143. PUSZKIN, S., BERL, S., PUSZKIN, E., and CLARKE, D. D. (1968). Actomyosinlike protein isolated from mammalian brain. Science 161, 170-171. SEARLS, R. L. (1965a). An autoradiographic study of the uptake of Sz5-sulfate during the differentiation of limb bud cartilage. Deuelop. Biol. 11, 155168. SEARLS, R. L. (1965b). Isolation of mucopolysaccharide from the precartilaginous embryonic chick limb bud. Proc. Sot. Exp. Biol. Med. 118, 1172m 1176. WASSERMAN, E., and LEVINE, L. (1961). Quantitative micro-complement fixation and its use in the study of antigenic structure by specific antigen-antibody inhibition. J. Immunol. 87,290-295. ZWILLING, E. (1966). Cartilage formation from socalled myogenic tissue of chick embryo limb buds. Ann. Med. Exp. Biol. Fenn. 44, 134-139.
BIOLOGY
28,
138-141 (1872)
Appearance JUDITH Department
of Myosin MEDOFF’
of Biology,
Bmndeis
Accepted
in the Chick Limb Bud AND EDGAR University,
Februnry
ZWILLING~ Waltham,
Massachusetts
3, 1972
Quantitative microcomplement fixation has been used to detect the appearance of myosin in the chick embryonic limb bud. It has been shown that myosin or a myosinlike molecule is present by stage 23, before muscle can be distinguished histologically. INTRODUCTION
There is no information about the developmental stage at which chick embryonic limb bud mesoderm cells begin to synthesize myosin. In Holtzer’s work (1961) with somites, there was no reaction to fluorescein-labeled antimyosin in either presumptive somite myoblasts or cells outside the muscle-forming area of somites. Specific staining by antimyosin occurred only in differentiating myoblasts (Holtzer et al., 1957). These observations lend support to a notion in developmental literature of a strict correspondence between biosynthesis and tissue histogenesis, i.e., differentiating cells can synthesize terminal products when they have acquired recognizable histological differentiation, and not before (Holtzer, 1961). It has been shown, however, that limb buds synthesize chondroitin sulfate long before acquiring the cytological features of cartilage (Searls, 1965b; Medoff, 1967). Also, a sulfated mucopolysaccharide with the same electrophoretic mobility as chondroitin sulfate was detected in somites of stage 16-17 chick embryos (FrancoBrowder et al., 1963) and a number of enzymes have been cartilage-related identified in somites (Lash, 1968) and limb buds (Medoff, 1967) before histological evidence of chondrogenesis. 1Present address: Biology Department, Washington University, St. Louis, Missouri 63130. 2 Aided by Grant HD-06345 from the Institute of Child Health and Human Development of the National Institutes of Health, U. S. Public Health Service.
This report presents evidence that stage 23-24 (Hamburger and Hamilton, 1951) limbs synthesize myosin or a myosinlike molecule before myoblasts can be detected in the buds. MATERIALS
0 1972 by Academic
Press, Inc.
METHODS
The presence of myosin in early embryonic chick limbs was determined by complement (C’) fixation. Limb buds were dissected from the chick at the appropriate stage, pooled, and homogenized in the cold in 0.15 M NaCl. The extract was sonicated for 1.5 min. After centrifugation at 105,000 g for 1 hr, the antigen preparation was ready for use. Control tissue was prepared in a similar manner. A rabbit antiserum to monkey myosin and a rabbit antiserum to chicken myosin was given to us by Dr. Bela Nagy of the Retina Foundation (Boston, Massachusetts) and Dr. Robert Cahn of the University of Washington (Seattle). The antisera had been tested for specificity and reacted well with purified myosin. The antisera were heat inactivated at 60°C for 20 min and diluted 1:250 before use. Quantitative microcomplement fixation was performed according to the method of Wasserman and Levine (1961). RESULTS
The C’ fixation curves obtained with the two different antimyosin serums and extracts prepared from the leg muscles of 14-day chick embryos are shown on Fig. 1. Chicken myosin fixed C’ with both antimonkey myosin and antichicken myosin 138
Copyright
AND
Myosin
MEDOFF AND ZWILLING
r
too 60 .-x ‘,
60
.* IA.
20 I
I
I
I
0.001
001
0.1
1.0
mg Protein
(Day
14 Muscle
Extract)
100 -
FIG. 1. C’ fixation with two different antimyosin serums and extracts from the leg muscles of 14-day chick embryos.
5 2
so
-
60
-
2 -cl c
40-
20
-
001
01
IO mg
IO
Protein
FIG. 2. C’ fixation with rabbit antichicken and extracts from early limb buds.
myosin
to about the same extent. Although the chicken myosin-antimonkey myosin probably represents a cross-reaction, we cannot estimate the extent of this cross-reaction since we did not titrate the antiserum with monkey myosin. Nevertheless, at appropriate dilutions of each antiserum, the leg muscle extracts behave serologically essentially the same. There was no C’ fixation with antigen prepared from limb buds of stage 20 through 22 embryos. C’ fixation with undiluted extracts of stage 23, however, was observed, suggesting that there may be some myosin or a myosinlike molecule present in the stage 23 bud. Figure 2 presents the C’ fixation data obtained with the stage 23 preparation and three different preparations of stage 24 limb buds. Al-
in Chick Limb Bud
139
though these data were obtained with rabbit antichicken myosin, these same extracts also fixed C’ in a qualitatively similar manner with rabbit antimonkey myosin. None of these extracts fixed C’ with a rabbit-anti-poly U, which served as control for serologic specificity. Extracts from stage 23 limb buds and three extracts of stage 24 limb buds contained myosin or a myosinlike molecule, as judged from the C’-fixing activity. Myosin serologic activity accumulated rapidly in late stage 24-early stage 25 limb buds, and extracts contained some 50 times more activity than the two early stage 24 extracts. Other embryonic tissues were examined for the presence of myosin. The regions to be tested were extracted in the same manner as the limb buds and assayed. The heads of stage 20 embryos and the neural retina of T-day chicks were negative. Extracts of brain from 15 and 20-day chick embryos fixed C’ with both antisera in a similar manner. This tissue appeared to contain myosin or a myosinlike molecule. This puzzling result caused concern regarding the specificity of both antibodies until Puszkin et al. (1968) reported the isolation of a protein with characteristics similar to those of actomyosin from both rat and cat brain. It may be that chick embryonic brain contains a similar actomyosin-like protein accounting for the positive reaction with myosin antisera. DISCUSSION
The results presented in this paper suggest that myosin or a myosinlike molecule is present in primitive mesoderm cells of the chick embryonic limb bud before morphological evidence of muscle differentiation. We are aware that the crude extracts were prepared at salt concentrations which ordinarily precipitate adult myosin. However, the fixation of complement is inhibited by excess electrolyte, and we chose to prepare our extracts at the optimal salt concentration for fixation, 0.15 M
140
DEVELOPMENTAL BIOLOGY
NaCl. Our results indicate that some myosin or myosinlike material was present in the supernatant, but owing to its limited solubility at 0.15 M NaCl, large amounts of extract had to be used in order to obtain complement fixation. Determination of the specificity of the antiserum is critical in the validation of immunoembryological studies of myosin. Such studies are complicated by the problems encountered in obtaining antigenitally homogeneous preparations of myosin. It has been reported that chick myosin purified by two different methods remained heterogeneous and antisera produced against such myosin were contaminated with heterologous antibodies (Finck, 1965). We feel confident that our antisera do contain antibodies against myosin, but we recognize the possibility that they may be heterogeneous and the reaction may have measured a myosinlike molecule which does not become associated with muscle. The accumulation of serologic activity in older limb buds concomitant with the appearance of myoblasts and myocytes and the fact that extracts prepared from other embryonic areas at the same stages do not react with the antisera suggest that we are measuring myosin or an antigen which is part of the myosin molecule. Actin or actinlike filaments have recently been reported in homogenates of developing chick leg muscle as early as stage 23 (Hitchcock, 1971), which supports our notion that early limb buds contain low levels of muscle specific protein prior to morphological evidence of myogenesis. The possible presence of myosin in stage 23 limb buds, the fact that pieces of mesoderm from both the chondrogenic and myogenic area were equivalent in their ability to form cartilage (Zwilling, 1966), and the report of uniform sulfate fixation in the mesoderm of early limb buds followed by some incorporation of label in the prospective muscle-forming region at later stages (Searls, 1965a), all raise an important question regarding mecha-
VOLUME 28, 1972
nisms of differentiation. There is as yet little evidence in support of the so-called exclusivity of differentiated states; instead each cell type is probably characterized by more than one synthetic pathway (Grobstein, 1966). A model of differentiation emerges from these observations. An embryonic cell may contain specific enzymes and metabolic intermediates or even complex macromolecular products in small amounts before terminal differentiation. Differentiation may then be accomplished by a gradual stabilization and amplification of certain metabolic patterns and repression of others. Rigorous evidence is lacking about synthetic activities of individual limb mesoderm cells. The appearance of myosin or a myosinlike molecule at stage 23-24 may, in fact, coincide with the appearance of a new population of cells which have had no chondroitin sulfate synthesizing properties (or activity). However, there are models for restriction of previously general synthetic activities to particular tissues (i.e., and repression in others). Gulonolactone oxidase is widely distributed in early chick blastoderms, but the enzyme disappears from all tissues except the kidney during the course of development of the embryo (Fabro and Rinaldini, 1965). Other examples may be cited. The fact is that muscle appears in tissue areas which initially fix sulfate (Searls, 1965a) and are capable of differentiating into cartilage (Zwilling, 1966). There is no evidence that cells migrate into these areas. It seems likely, then, that myosin-synthesizing cells (myoblasts) are derived from the same mesoderm which forms the other tissue types of the limb. There is at least the likelihood that cells which first engage in low level chondroitin sulfate synthesis may then synthesize myosin or give rise to progeny which do this. SUMMARY
Microcomplement fixation suggests the presence of myosin or a myosinlike molecule in the limb buds of stage 23-24 chick
MEDOFF AND ZWILLINC
embryos before histological evidence of muscle differentiation. Stage 24-25 buds exhibited about 50 times more myosin serologic activity than the limbs of embryos 1 day younger. The for his ecution aration
authors wish to thank Dr. Lawrence Levine invaluable assistance in the planning and exof the experimental work as well as the prepof the manuscript. REFERENCES
FABRO, S. P., and RINALDINI, L. M. (1965). Loss of ascorbic acid synthesis in embryonic development. Deuelop. Biol. 11, 468-488. FINCK, H. (1965). Immunochemical studies of myosin. I. Effects of different methods of preparation on the immunochemical properties of chicken skeletal muscle myosin. Biochim. Biophys. Acta 111, 20% 220. FRANCO-BROWDER,S., DERYDT, J., and DORFMAN, A. (1963). The identification of a sulfated mucopolysaccharide in chick embryos, stages 11-23. Proc. Nat. Acad. Sci. U.S.A. 49, 643-647. GROBSTEIN, C. (1966). What we do not know about differentiation. Amer. Zoo!. 6,89-95. HAMBURGER, V., and HAMILTON, H. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. HITCHCOCK, S. E. (1971). Detection of actin filaments in homogenates of developing m’uscle using heavy meromyosin. Develop. Biol. 25, 492-501.
Myosin in Chick Limb Bud
141
HOLTZER, H. (1961). Aspects of chondrogenesis and myogenesis. In “Synthesis of Molecular and Cellular Structure,” Growth Symp. No. 19 (D. Rudnick, ed.), pp. 35-87. Ronald Press, New York. HOLTZER, H., MARSHALL, J., and FINCK, H. (1957). An analysis of myogenesis by the use of fluorescent antimyosin. J. Biophys. Biochem. Cytol. 3, 705723. LASH, J. W. (1968). Semitic mesenchyme and its response to cartilage induction. In “Epithelial-Mesenchymal Interactions” (R. Fleischmajer and R. E. Billingham, eds.), pp. 165-172. Williams and Wilkins, Baltimore, Maryland. MEDOFF, J. (1967). Enzymatic events during cartilage differentiation in the chick embryonic limb bud. Develop. Biol. 16, 118-143. PUSZKIN, S., BERL, S., PUSZKIN, E., and CLARKE, D. D. (1968). Actomyosinlike protein isolated from mammalian brain. Science 161, 170-171. SEARLS, R. L. (1965a). An autoradiographic study of the uptake of Sz5-sulfate during the differentiation of limb bud cartilage. Deuelop. Biol. 11, 155168. SEARLS, R. L. (1965b). Isolation of mucopolysaccharide from the precartilaginous embryonic chick limb bud. Proc. Sot. Exp. Biol. Med. 118, 1172m 1176. WASSERMAN, E., and LEVINE, L. (1961). Quantitative micro-complement fixation and its use in the study of antigenic structure by specific antigen-antibody inhibition. J. Immunol. 87,290-295. ZWILLING, E. (1966). Cartilage formation from socalled myogenic tissue of chick embryo limb buds. Ann. Med. Exp. Biol. Fenn. 44, 134-139.