ABSTRACT
Myosin alkali light chain accumulation in developing quail limb musculature has been analysed on immunoblots using a monoclonal antibody which recognizes an epitope common to fast myosin light chain 1 (MLCif) and fast myosin light chain 3 (MLC3f). The limb muscle of early embryos (i.e. up to day 10 in ovo) has a MLC profile similar to that observed in myotubes cultured in vitro-, although MLC)r is abundant, MLC3r cannot be detected. MLC3f is first detected in 11-day embryos. To determine whether this alteration in MLC3f accumulation is nerve or hormone dependent, limb buds with and without neural tube were cultured as grafts on the chorioallantoic membrane of chick hosts. Although differentiated muscle develops in both aneural and innervated grafts, innervated grafts contain approximately three times as much myosin as aneural grafts. More significantly, although aneural grafts reproducibly accumulate normal levels of MLCif, they fail to accumulate detectable levels of MLC3f. In contrast, innervated grafts accumulate both MLCif and MLC3f, suggesting that the presence of neural tube in the graft promotes the maturation, as well as the growth, of muscle tissue. This is the first positive demonstration that innervation is necessary for the accumulation of MLC3f that occurs during normal limb development in vivo.
Introduction
Muscle contractile proteins are the products of multigene families. The number of genes in each family depends upon the organism, but it has been estimated that there are between 7 (Robbins, Freyer, Chisholm & Gilliam, 1982) and 31 distinct myosin heavy chain sequences (Kropp, Gulick & Robbins, 1986) and at least 10 actin-like sequences (Schwartz & Rothblum, 1980) in the chicken genome. Since striated, smooth and cardiac muscles all express different contractile protein isoforms, the expression of some of these family members is obviously tissue specific. Recently, it has been suggested that some isoforms are developmentally regulated within a single tissue. For example, several groups have demonstrated the existence of embryonic and neonatal isoforms of myosin heavy chain which are present for only brief periods during embryonic and early post-hatch development of the chick and which differ from the adult isoforms (Rushbrook & Stracher, 1979; Bandman, Matsuda & Strohman, 1982; Bader, Masaki & Fischman, 1982; Winkelman, Lowey & Press, 1983). Analysis of the mRNA populations of embryonic muscle using cDNA probes to myosin has confirmed this observation (Umeda et al. 1983). Isoform switching during early chick development has also been described for α-actin (Paterson & Eldridge, 1984), tropomyosin (Roy, Sreter & Sarkar, 1979; Montarras, Fiszman & Gros, 1982; Matsuda, Bandman & Strohman, 1983), troponin (Matsuda, Obinata & Shimada, 1981) and myosin light chain subunits (Chi, Fellini & Holtzer, 1975; Rubinstein, Pepe & Holtzer, 1977; Merrifield & Konigsberg, 1986; Crow, Olson & Stockdale, 1983).
In the avian embryo, multiple forms of myosin light chains are coexpressed in the muscles of embryos younger than 10 days, including the adult fast (If, 2f), adult slow (Is, 2s) and embryonic-specific (le) light chains (Stockdale, Raman & Baden, 1981; Takano-Ohmuro et al. 1985). Remarkably, alkali light chain 3 (MLC3f) is not expressed at these early stages and is first detected in developing fast muscle prior to hatching at about the stage at which adult slow and embryonic light chains disappear (Crow et al. 1983; Takano-Ohmuro et al. 1985). In cultured muscle cells, however, the coexpression of fast and slow isoforms persists and MLC3f does not accumulate to detectable levels (Keller & Emerson, 1980). Thus, both the disappearance of slow and embryonic MLC isoforms and the activation of MLC3f synthesis in developing fast muscle have been taken as markers for the maturation of muscle in the avian embryo.
Although the timing of isoform switching seems specific for each contractile protein, it has been suggested that many of these changes may be orchestrated by some common factor in the developing embryo. In light of the well-documented effect of hormones (Johnson et al. 1980; Gambke et al. 1983; Butler-Browne, Herlicoviez & Whalen, 1984; Whalen, Toutant, Butler-Browne & Watkins, 1985; Izumo, Nadal-Ginard, & Mahdavi, 1986) and nerve (Buller, Eccles & Eccles, 1960; Gutmann, 1976; Jolesz & Sreter, 1981) upon adult mammalian muscle, it is possible that either influence may be playing a role in affecting isoform switches. This possibility is supported by the observation that muscle cells cultured in vitro differentiate into embryonic muscle but do not undergo the switches seen in the maturing embryo.
In order to examine the role of hormonal and nervous influences upon the activation of MLC3f synthesis in the avian embryo, we have used the approach of grafting both aneural and innervated limb buds onto the chorioallantoic membrane (CAM) of chick embryo hosts and then analysing the developmental potential of these grafts using a monoclonal antibody that can detect the developmental switch in myosin fast alkali light chains. The technique of CAM grafting was first used to study the developmental capacity of early limb buds by Murray & Huxley (1925), who observed that such grafts could attain ‘a form which is extraordinarily close to the normal’. Murray (1926) concluded that limb buds from 3- to 5-day-old chick embryos are ‘self-differentiating’ and that morphogenesis of the limb is independent of innervation and function. These observations were subsequently confirmed and extended by others (Hunt, 1932; Eastlick, 1943; Bradley, 1970; Kenny-Mobbs & Hall, 1983). While these latter studies have been concerned with the ability of aneural and innervated limb buds to produce differentiated muscle when grafted onto the CAM, this study is largely concerned with the ability of these grafts to develop mature muscle, i.e. muscle that expresses the adult-specific contractile protein isoform pattern. Using low ionic strength precipitation of myosin and immunoblot analysis, we have been able to demonstrate that the inclusion of neural tube in the grafted limb bud influences not only the growth but also the expression of MLC3f in muscle tissue of the developing graft.
Materials and methods
Fertile quail eggs (Coturnix Coturnix japonica) were obtained from our own breeding stock and used to obtain donor limb buds. Chicken eggs (Gallus domesticus, Hubbard x Hubbard strain) obtained from Heatwole Hatchery, Harrisonberg, Virginia were used as hosts. Both chick and quail embryos were incubated in a forced draft, humidified incubator at 37·6°C and staged according to the criteria of Hamburger & Hamilton (1951) and Sato, Hoshino, Mizuma & Nishida (1971), respectively.
Chorioallantoic grafting procedure
Grafting of limb buds from 3-day quail embryos onto the CAM of 7-day chick hosts was carried out as described by Hamburger (1942). This choice of experimental protocol was largely dictated by technical considerations. In choosing a donor limb bud, we wanted initially to obtain buds that had not yet become innervated. Since nerve fibres first penetrate the limb bud of the chick at Hamilton and Hamburger (H.H.) stage 23 (approximately day 4 of incubation) (Fouvet, 1973), 3-day-old (H.H. stage 20) chicks were the oldest embryos from which we could reproducibly obtain truly ‘aneural’ limb buds. Since the CAM of the chick embryo is not sufficiently developed to receive grafts before day 7 of development and because the blood vessels of the CAM that support the graft begin to degenerate on day 19 (Hall, 1978), limb buds from 3-day-old chick embryos could only be incubated as grafts to a total chronological age of 15 days. This approach would be ineffective, since it has been shown that MLC3f accumulation cannot be detected in the chick embryo until day 16 of development. To circumvent this difficulty, we decided to obtain limb buds from 3-day quail rather than chick donors. Since quail embryos develop faster (16 versus 21 days to hatching) and activate MLC3f earlier than the chick (day 11 versus day 16), these aneural limb buds were grafted onto a day-7 chick host and incubated to a chronological age of 15 days. It has been known for some time that quail somitic muscle precursor cells transplanted into chick hosts develop into cytologically normal muscle (Christ, Jacob & Jacob, 1977). Thus, even if the grafts were somewhat retarded in their growth (as is often the case) activation of MLC3f in the graft would most likely be detected, should it occur.
Quail embryos were isolated sterilely, placed in Howard’s saline (Howard, 1953) and dissected free of all extra-embryonic membranes using electrolytically sharpened tungsten needles (Dossel, 1958). Aneural limb buds were dissected to contain the somatopleure lateral to the somites at the level of the limb. Innervated limb buds were excised to include both somite and neural tube (see Fig. 3). After puncturing the shell above the air space, a 3 mm square window was cut into the chick egg, the shell membrane removed and the graft placed onto the chorioallantoic membrane (using a Spemann pipette) at a site where two blood vessels bifurcated. The shell was replaced on the window, sealed with paraffin and the hosts were incubated (without rolling) for 12 days. The grafts were removed, freed of any adherent membrane, weighed, photographed and extracted for electrophoresis and immunoblot analysis.
Preparation and analysis of limb myosin
Normal limbs and grafts were homogenized in 10 vol. (w/v) of high salt extraction buffer [0·6M-NaCl, 15 mM-Tris-HCl (pH 7·4) containing 1 mm-PMSF] in a ground-glass homogenizer and extracted for 15 min on ice (Burridge & Bray, 1975). Insoluble material was pelleted by centrifugation at 12 800g for 15 min and the supernatant was made 50 % in glycerol and stored at −20°C. Extracts were either analysed directly or diluted by dialysis to lower the ionic strength of the extract and precipitate out a crude myosin fraction. To obtain this fraction, glycerinated extracts were dialysed against 50 vol. of cold distilled water containing 1 mM-PMSF for 6h at 4°C using Spectaphor 6000Mr cut-off dialysis tubing. Myosin was pelleted at 10000g for 15 min in a Sorvall HS-4 rotor, solubilized in 0·5 % SDS and assayed for protein concentration (Lowry, Rosebrough, Farr & Randall, 1951). Samples to be run on gels were mixed with 5× Laemmli sample buffer and run on 12-5% acrylamide gels as described by Laemmli (1970) and either stained with Coomassie Blue (Fairbanks, Steck & Wallach, 1971) or electrophoretically transferred onto 0·1 μM-nitrocellulose paper (Schleicher & Schuell, PH 79) overnight at 10 watts constant power (Towbin, Staehelin & Gordon, 1979).
Detection of myosin alkali light chains on immunoblots
A monoclonal antibody to adult quail breast muscle myosin was used to detect the presence of MLClf and MLC3f in crude myosin samples transferred to nitrocellulose. The antibody (QBM-2) is specific for fast alkali light chains (Merrifield, Payne & Konigsberg, 1983) and recognizes an epitope common to both MLC|f and MLC3f. Following electrophoretic transfer, the nitrocellulose replica was rinsed with PBS, blocked for 2 h at 37°C with 5 % BSA in PBS and then incubated overnight with shaking at 4 °C with monoclonal antibody QBM-2. The blot was then washed with several changes of PBS (45 min) and incubated with a 1/1000 dilution of peroxidase-conjugated goat anti-mouse IgG (TAGO) in 01% BSA in PBS for 2h at 37°C. Following a final wash, peroxidase activity was localized using o-dianisidíne HC1 in 10mM-Tris-HCl buffer (pH 7·4) containing 0·033 % hydrogen peroxide as a substrate.
Results
Myosin light chains during normal limb development
When total high-salt-soluble extracts of limb buds from different-aged quail embryos are analysed on immunoblots for their alkali light chain content, we observe that limb buds from 9-day embryos accumulate detectable levels of MLC|1f but not MLC3f (Fig. 1B). The presence of MLC1f in this preparation, as well as the presence of myosin heavy chain and other muscle-specific proteins in the stained gel (Fig. 1A), demonstrate that differentiated muscle is certainly present in 9-day limbs, and the absence of MLC3f suggests that the muscle fibres that differentiate first in the developing limb are significantly different from adult fibres. The alkali light chain content of the early muscle fibres in the embryo is, in fact, very similar to that previously described for quail myotubes cultured in vitro (Merrifield et al. 1983). MLC3f is also absent from the limb musculature of day-10 and -11 embryos, and is first detected by immunoblot analysis of total limb extracts in 12day embryos. The MLC3f content increases in older embryos until, by day 16, MLC3f is detectable, although faintly, by Coomassie Blue staining of total protein.
Myosin alkali light chain content of embryonic quail limb muscle as determined by Coomassie Blue staining and immunoblot analysis with monoclonal antibody QBM-2. Total high salt extracts (A,B) or precipitated myosin (C,D) from limbs of 9- to 16-day-old embryos were analysed on 12·5 % polyacrylamide-SDS gels by staining (A,C) and duplicate gels were electrophoretically transferred onto nitrocellulose and probed with antibody QBM-2 (B,D). Although MLCu is detected in both total extracts and myosin precipitates from all ages and in myosin precipitated from cultured myotubes (Mt), MLC3f is first detected in total extracts at day 12 and in precipitated myosin at day 11. MHC, myosin heavy chain; A, actin; MLClf, myosin alkali light chain 1; MLC3f, myosin light chain 3; Mt, myosin precipitated from cultured myotubes. Each lane contains 25 μg of protein.
Myosin alkali light chain content of embryonic quail limb muscle as determined by Coomassie Blue staining and immunoblot analysis with monoclonal antibody QBM-2. Total high salt extracts (A,B) or precipitated myosin (C,D) from limbs of 9- to 16-day-old embryos were analysed on 12·5 % polyacrylamide-SDS gels by staining (A,C) and duplicate gels were electrophoretically transferred onto nitrocellulose and probed with antibody QBM-2 (B,D). Although MLCu is detected in both total extracts and myosin precipitates from all ages and in myosin precipitated from cultured myotubes (Mt), MLC3f is first detected in total extracts at day 12 and in precipitated myosin at day 11. MHC, myosin heavy chain; A, actin; MLClf, myosin alkali light chain 1; MLC3f, myosin light chain 3; Mt, myosin precipitated from cultured myotubes. Each lane contains 25 μg of protein.
In determining the precise developmental stage at which MLC3f can first be detected in the limb, we thought it important to consider other changes taking place in this dynamic structure. Because the mass of differentiated muscle changes dramatically during embryonic limb development relative to other nonmuscle tissues (McLennan, 1983u; Butler, Cosmos & Brierley, 1982), we used a low ionic strength precipitation to isolate crude myosin fractions from the high-salt-soluble extracts of developing limbs. A plot of the amount of precipitated myosin (as a percentage of total protein) versus developmental age thus provides a biochemical representation of how muscle mass changes during limb development (see Fig. 5), and confirms the histological evidence presented by others. Preliminary analysis of limb extract from day-12 embryos showed that precipitation of myosin was complete by 6h and that this precipitation was independent of myosin concentration in the extract over a 10-fold range (Fig. 2). This method is also reproducible since, in three different ionic strength precipitations of extracts from 12-day limbs, a mean value of 22·1% was obtained with a standard deviation of ±1-6. Although the myosin precipitated in this way contains other proteins of the contractile apparatus, the supernatant fraction is essentially devoid of myosin (Fig. 2). Consequently, by analysing a constant amount of precipitated myosin from embryos of different ages, we can compensate for the difference in muscle mass between individual embryos. Using precipitated myosin in our immunoblot analysis, rather than total limb extract, we observe a similar developmental appearance of MLC3f. except that MLC3f is first detected in 11-(rather than 12-) day embryos (Fig. ID). Thus, ionic strength precipitation represents a convenient and reproducible method for concentrating and quantifying myosin prior to gel electrophoresis, and provides for a greater degree of sensitivity in our immunoblot assay for MLC3f.
Precipitation kinetics of myosin during dilution of a high salt extract of limb by dialysis against 50 vol. of cold water containing 1 mM-PMSF. A crude extract from 12-day embryos was diluted 1:1 with glycerol and either dialysed directly (•) or diluted 1:10 with extraction buffer and then dialysed (□). At each time point, triplicate samples were centrifuged to precipitate myosin and the protein content of the pellet was determined using the Lowry assay. In a separate experiment (see inset), the crude extract (E) and the precipitates (P) and supernatants (S) at 1, 3, 6, 12 and 24 h were analysed on SDS gels. Each lane contains 25 μg protein stained with Coomassie Blue; MHC, myosin heavy chain.
Precipitation kinetics of myosin during dilution of a high salt extract of limb by dialysis against 50 vol. of cold water containing 1 mM-PMSF. A crude extract from 12-day embryos was diluted 1:1 with glycerol and either dialysed directly (•) or diluted 1:10 with extraction buffer and then dialysed (□). At each time point, triplicate samples were centrifuged to precipitate myosin and the protein content of the pellet was determined using the Lowry assay. In a separate experiment (see inset), the crude extract (E) and the precipitates (P) and supernatants (S) at 1, 3, 6, 12 and 24 h were analysed on SDS gels. Each lane contains 25 μg protein stained with Coomassie Blue; MHC, myosin heavy chain.
Aneural and innervated limb buds of a 3-day quail embryo before (A) and after (B,C) incubation on the chorioallantoic membrane. The area contained by the solid line in panel A represents a typical dissection of a limb bud which excluded somites and neural tube (i.e. an aneural graft like G27) while the broken line indicates all of the tissue which was typically included in an ‘innervated’ graft like G17. Two representative grafts which resulted from a 12-day incubation on the CAM are shown in panels B (G17) and C (G27). G17 was subsequently shown to contain both MLC3f and MLC3f while G27 contains only MLC1f (Table 1). The bar represents 1 mm in each respective field.
Aneural and innervated limb buds of a 3-day quail embryo before (A) and after (B,C) incubation on the chorioallantoic membrane. The area contained by the solid line in panel A represents a typical dissection of a limb bud which excluded somites and neural tube (i.e. an aneural graft like G27) while the broken line indicates all of the tissue which was typically included in an ‘innervated’ graft like G17. Two representative grafts which resulted from a 12-day incubation on the CAM are shown in panels B (G17) and C (G27). G17 was subsequently shown to contain both MLC3f and MLC3f while G27 contains only MLC1f (Table 1). The bar represents 1 mm in each respective field.
Since it is impossible to prove a negative, we cannot absolutely rule out the possibility that MLC3f is synthesized in myotubes cultured in vitro and in developing limbs prior to 11 days in ovo. However, we do know that our immunoassay is sufficiently sensitive to detect both alkali light chains in a sample containing as little as 1 μg of myosin precipitated from adult quail muscle (results not shown). Such samples contain 15 ng of each light chain. Since we routinely apply 25 μg of precipitated myosin to each lane of our gels prior to electrophoresis and immunoblot analysis (i.e. 375 ng of each light chain, assuming identical stoichiometry to adult myosin) MLC3f, if it occurs at all before 11 days of incubation, is present at levels lower than 4 % of that found in adult muscle. This is consistent with a study of incorporation of [35S]methionine into the light chains of avian muscle cultures (Keller & Emerson, 1980) in which MLC3f was shown to represent approximately 4 % (±3 %) of total light chain synthesis. Thus, even if MLC3f is present at these very low levels, its contribution to the physiological state of the muscle is of little significance. The question which is a natural consequence of these observations concerns the nature of the signal which is responsible for the activation of MLC3f synthesis and accumulation to physiologically significant levels.
Characteristics of quail limbs grafted onto chorioallantoic membranes of chick hosts
In order to assess whether humoral or neural influences are involved in the expression of MLC3f at day 11, we grafted limb buds from 3-day quail embryos onto the chorioallantoic membrane of 7-day-old chick hosts and allowed them to develop for 12 days prior to analysis for MLC3f content. We reasoned that if humoral influences (like hormones) were the stimulus for MLC3f activation, then aneural grafts would produce mature muscle with normal accumulation of MLC3f. If, on the other hand, innervation were the stimulus, then only the grafts containing neural tube would contain detectable levels of MLC3f.
Of a total of 93 grafts performed. 43 of the chick hosts survived until the end of the graft incubation period (day 19). Of those chick hosts that survived. 23 supported the successful growth and morphological development of grafts. These grafts ranged in weight from 22 to 293 mg but only those grafts that attained a mass greater than 45 mg were extracted for further analysis (Table 1). Although grafts which included neural tube seemed to survive better, their average size and morphological development were very similar to aneural grafts (Fig. 3). Morphologically, the grafts ranged from feathered tissue masses of approximately 2 mm in diameter to normal but stunted limbs of 13–15 mm in length with normal scale, feather and digit formation (Fig. 4). They differed significantly, however, in the amount of myosin precipitated per mass of total protein in the extract. In the 12 aneural grafts analysed, myosin as a percentage of total protein ranged from 7·5 to 18·8 % with a mean of 12·0% (Table 1). In contrast, the myosin content of innervated grafts ranged from 12·6 to 58·8% with a mean value of 31·6 -almost three times the amount obtained in aneural grafts. The range of values for innervated grafts was similar to that observed in limbs of 9- to 16-day embryos (Fig. 5), with one of the grafts (G33) containing a level of myosin comparable to that seen in a normal limb from an embryo of the same age (i.e. 15 days). The myosin content of aneural limb bud grafts was in all cases below the value obtained for a 9-day embryo.
Potential of the CAM grafting procedure. Aneural (G3) and innervated (G33) grafts sometimes attained morphological development comparable to that of normal limbs from a 16-day embryo (L16). Although stunted, G3 contains bone (at arrow), feathers, scales, rudimentary digits and a well-formed ankle joint, G33 was the largest graft obtained and contained the greatest muscle mass (see Table 1). Subsequent immunoblot analysis revealed that the muscle in G3 contained only MLClf whereas G33 contained both MLC1f and MLC3f.
Potential of the CAM grafting procedure. Aneural (G3) and innervated (G33) grafts sometimes attained morphological development comparable to that of normal limbs from a 16-day embryo (L16). Although stunted, G3 contains bone (at arrow), feathers, scales, rudimentary digits and a well-formed ankle joint, G33 was the largest graft obtained and contained the greatest muscle mass (see Table 1). Subsequent immunoblot analysis revealed that the muscle in G3 contained only MLClf whereas G33 contained both MLC1f and MLC3f.
Myosin content of developing limbs and innervated grafts as a measure of the relative mass of muscle. Myosin was precipitated from limb extracts (in triplicate ±S.D.•) and grafts (see Table 1 for characteristics), quantified using the Lowry assay and expressed as a % of the total protein present in the extract. Aneural grafts all contained less myosin (as a relative %) than limbs of 9-day-old embryos and were not plotted on the graph. Innervated grafts contain amounts of myosin comparable to that of normal limbs from 9- to 15-day embryos.
Myosin content of developing limbs and innervated grafts as a measure of the relative mass of muscle. Myosin was precipitated from limb extracts (in triplicate ±S.D.•) and grafts (see Table 1 for characteristics), quantified using the Lowry assay and expressed as a % of the total protein present in the extract. Aneural grafts all contained less myosin (as a relative %) than limbs of 9-day-old embryos and were not plotted on the graph. Innervated grafts contain amounts of myosin comparable to that of normal limbs from 9- to 15-day embryos.
Although the crude myosin prepared by a single ionic strength precipitation contains some noncontractile proteins, it is nevertheless a good approximation of the amount of muscle protein per mass of total protein. Since all grafts were extracted in the same way, this value is an estimate of the amount of differentiated muscle tissue contained in each graft. Clearly, those grafts that contained neural tube consistently contained more muscle than aneural grafts.
Myosin light chains in aneural and innervated grafts
The most striking difference between aneural and innervated grafts was the myosin light chain 3 content. When a constant amount of myosin from each graft was analysed with QBM-2 following immunoblotting, MLClf was always present in detectable amounts. MLC3f, however, was never detected in the aneural grafts. In this respect, these myosin preparations had the same isoform content as those prepared from cultured myotubes or from the limbs of normal quail embryos sampled prior to 11 days in ovo (Fig. 6). In contrast, most of the innervated grafts contained detectable levels of both MLClf and MLC3f when analysed under the same conditions (Fig. 7; Table 1). This contrast is most apparent when similar amounts of actomyosin from an innervated (G17) and an aneural (G3) graft are analysed on the same immunoblot along with myosin from normal limbs (Fig. 8). MLC3f cannot be detected in the aneural graft extract while the amount of MLC3f in the innervated graft is comparable to that seen in normal limbs from day-11 to -12 embryos. Of the nine innervated grafts analysed for MLC content, only one (G15) did not contain detectable levels of MLC3f (Table 1). However, this particular graft also did not contain MLClf, suggesting that for some reason it was completely devoid of muscle. These results suggest that the quality of the muscle, as well as the quantity, differs in limb buds grafted with and without neural tube. Innervated grafts that contained differentiated muscle always contained detectable levels of MLC3f.
Myosin alkali light chain content of an aneural graft in relation to normal limb muscle. Total limb extracts from limbs of 6-, 9-, II-, 13- and 16-day-old quail embryos and from an aneural graft (G3) were electrophoresed on 12’5 % SDS gels and either stained (A) or immunoblotted for analysis with antibody QBM-2(B) . Al, extract of adult limb muscle; Ab, purified myosin from adult breast muscle; MHC, myosin heavy chain; MLC], myosin light chain 1; MLC3, myosin light chain 3.
Myosin alkali light chain content of an aneural graft in relation to normal limb muscle. Total limb extracts from limbs of 6-, 9-, II-, 13- and 16-day-old quail embryos and from an aneural graft (G3) were electrophoresed on 12’5 % SDS gels and either stained (A) or immunoblotted for analysis with antibody QBM-2(B) . Al, extract of adult limb muscle; Ab, purified myosin from adult breast muscle; MHC, myosin heavy chain; MLC], myosin light chain 1; MLC3, myosin light chain 3.
Myosin alkali light chain content of representative aneural and innervated grafts. Myosin precipitated from innervated grafts (32, 33) and aneural grafts (31, 43, 45, 46) were electrophoresed on 12·5 % SDS gels and stained (A) or immunoblotted (B) for analysis with antibody QBM-2. MLC3f can only be detected in the innervated grafts, in spite of the fact that grafts 45 and 46 were larger than the innervated graft 32 and constant amounts of precipitated myosin (25 μg) from each sample were analysed.
Myosin alkali light chain content of representative aneural and innervated grafts. Myosin precipitated from innervated grafts (32, 33) and aneural grafts (31, 43, 45, 46) were electrophoresed on 12·5 % SDS gels and stained (A) or immunoblotted (B) for analysis with antibody QBM-2. MLC3f can only be detected in the innervated grafts, in spite of the fact that grafts 45 and 46 were larger than the innervated graft 32 and constant amounts of precipitated myosin (25 μg) from each sample were analysed.
Immunoblot analysis of the myosin alkali light chain content of aneural (G3) and innervated (G17) grafts in relation to normal limb muscle. Myosin was precipitated from limb extracts of day-11 and -12 embryos (Lil, L12) as well as from grafts G3 and G17 and compared by immunoblot analysis. 50 μg of precipitated myosin from each graft or normal limb and 10 μg of actomyosin (A) and purified myosin (M) were applied per well. Only the light chain portion of the blot is shown. The amount of MLC3f in G17 is comparable to that seen in day-11 to -12 limbs from developing embryos.
Immunoblot analysis of the myosin alkali light chain content of aneural (G3) and innervated (G17) grafts in relation to normal limb muscle. Myosin was precipitated from limb extracts of day-11 and -12 embryos (Lil, L12) as well as from grafts G3 and G17 and compared by immunoblot analysis. 50 μg of precipitated myosin from each graft or normal limb and 10 μg of actomyosin (A) and purified myosin (M) were applied per well. Only the light chain portion of the blot is shown. The amount of MLC3f in G17 is comparable to that seen in day-11 to -12 limbs from developing embryos.
Discussion
Although it has previously been demonstrated that limb buds from chick embryos older than stage 18 can develop cytologically normal muscle when grafted onto the chorioallantoic membrane (Eastlick, 1932; Kenny-Mobbs, 1985), the present study is the first to use immunological approaches to analyse the contractile protein isoforms of muscle from CAM grafts to assess its degree of maturation. Using a monoclonal antibody that can detect the developmentally regulated accumulation of MLC3f, we have demonstrated that the muscle that forms in aneural limb bud grafts expresses MLClf but not MLC3f, in spite of the fact that it has a chronological age of 15 days. In this respect, it is embryonic-like and has not matured normally. Since these grafts had to be exposed to the blood supply of the normal host in order to survive, this observation suggests that circulating hormones do not play a major role in activating the accumulation of MLC3f. If hormonal influences play a role, it is strictly permissive and not sufficient to allow MLC3f accumulation to reach detectable levels.
Our observation that grafts containing neural tube and cultured on the CAM under similar conditions accumulate both MLC,f and MLC3f strongly suggests that some influence of the neural tube is responsible for the accumulation of MLC3f that occurs normally in developing limb muscle in vivo. At present, the precise nature of this neural influence is unknown. However, experiments by others on cultured chick muscle cells have demonstrated that MLC3f accumulation can be promoted by electrostimulation (Srihari & Pette, 1981) and is positively correlated with a high level of contractile activity (Moss, Micou-Eastwood & Strohman, 1986). Recently, it has been reported that, at least in the case of troponin C, coculture with nerve or culture in the presence of nerve extract results in normal switching of tissue-specific isoforms (Toyota & Shimada, 1983). In addition, denervation experiments on newly hatched chicks have demonstrated that the expression of MLC3f can be repressed by denervating the muscle and rendering it paralysed (Saitoh, Kitani & Obinata, 1983). This latter experiment should be evaluated carefully, however, since denervation is known to promote degeneration and subsequent regeneration of muscle fibres. While it is difficult to quantify the spontaneous contractile activity of the limb grafts described in this study, it is important to note that several of the innervated grafts were observed to twitch spontaneously during removal from the CAM. Aneural grafts, on the other hand, were never observed to twitch. Experiments are currently in progress to determine whether treatment of developing embryos and innervated CAM grafts with curare will affect the normal accumulation of MLC3f in the functionally denervated muscles.
The increased myosin content per gram tissue that we observe in our innervated grafts may be a result of the same trophic influence that is responsible for promoting MLC3f accumulation, or may be promoted independently by the neural tube. Although the trophic effect of nerve on muscle formation in limb bud grafts has been recognized since 1943 (Eastlick, 1943; Bradley, 1970; Kenny-Mobbs & Hall, 1983), the mechanism for this induction has not been elucidated. While the migration of muscle cell precursors from the somite has been implicated in affecting muscle mass in some of the CAM-grafting experiments done by others (Kenny-Mobbs, 1985), this is clearly not the case in our study since limb buds were dissected from stage-20 embryos after the migration of somitic cells had been completed (Chevallier, 1978). In a very-careful study, Kenny-Mobbs (1985) has confimed that myogenesis in H.H. stage 18 and later stage wing buds is not affected by the presence or absence of the somitic tissues.
In related studies, McLennan (19836) has shown that the functional denervation of chick hindlimb muscles with curare inhibits the normal formation of secondary myotubes in developing chick embryos, with a concomitant reduction in muscle mass. Similarly, others have shown a dramatic decrease in the wing muscle cross-sectional area (Butler et al. 1982) and myotube number (Phillips & Bennett, 1984) in denervated chick embryos relative to control birds. Although there is a correlation between these two parameters of growth, it is obvious that the failure of nerve-dependent secondary myotubes to differentiate normally cannot account for all of the deficit in the size of aneural muscles (McLennan, 19836). Since both the myotube number and the muscle crosssectional area actually decrease in denervated embryos after stage 42, it is clear that, as both Hunt (1932) and Eastlick (1943) observed, nerve plays a role in maintaining muscle once it has formed. Thus, the decreased muscle mass that we observe in our aneural grafts may result from the absence of secondary myotube formation, an increased rate of muscle degeneration or perhaps from a combination of these two factors. We have no way, at present, to evaluate the relative importance of hyperplasia and hypertrophy in this trophic effect.
The precise relationship between increased muscle mass and MLC3f accumulation in innervated grafts is not clear. While it is interesting to speculate that both MLC3f accumulation and increased muscle mass may be associated with the appearance of secondary myotubes, there is no evidence at present to support this hypothesis. Experiments are currently under way, however, to examine whether MLC3f accumulates preferentially in secondary myotubes during normal limb development and in innervated limb buds grafted onto the CAM.
Our demonstration that nervous influence plays a role in the relative expression of MLClf and MLC3f is especially important in light of recent evidence that these two proteins are actually transcribed from the same gene (Nabeshima, Fujii-Kuriyama, Muramatsu & Ogata. 1984). Since MLClf and MLC3f mRNAs may be produced by differential splicing and/or the use of different initiation sites of the same gene, our results suggest that nerve-muscle interactions can regulate gene expression at the level of RNA processing. It is not yet known if other developmentally regulated contractile protein isoforms (i.e. α-cardiac and a-skeletal actin; embryonic, neonate and adult myosin heavy chain) are regulated by neural influences in a similar way to MLC3f. Experiments on nerve and hormonal regulation of myosin heavy chain isoform switching in mammals have, to date, been highly contradictory. While there is good evidence that neonatal and adult isoforms of myosin heavy chain are induced in cultured mouse muscle fibres by coculture with embryonic mouse spinal cord (Ecob-Prince, Jenkison, Butler-Browne & Whalen, 1986), the accumulation of adult myosin heavy chains has also been reported in pure cultures of differentiated mouse C2C|2 cells in the absence of nerve (Silberstein, Webster, Travis & Blau, 1986). Evidence from in vivo experiments have also demonstrated that thyroid hormone can have a profound effect on the expression of myosin heavy chain isoforms in embryonic and adult rats (Johnson et al. 1980; Gambke et al. 1983; Butler-Browne, Herlicoviez & Whalen, 1984; Whalen et al. 1985; Izumo et al. 1986). Denervation, on the other hand, does not seem to affect the normal accumulation of adult myosin heavy chain in newborn rats (Butler-Browne et al. 1982). These reports may indicate that different contractile protein multigene families are regulated differently. Alternatively, the role of nervous influences in the process of muscle maturation may be much more important in birds than in mammals.
Our successful use of innervated limb bud grafts to mimic normal nerve-muscle interactions in the intact embryo is entirely consistent with Eastlick’s (1943) earlier demonstration that the normal interneuronal connections within the spinal column are not required for muscle maintenance by a neural tube segment. In coelomic grafts of limb buds containing neural tube, those that became attached loosely to the mesenteries seemed to contain as much striated muscle as the limbs that were grafted to the lateral body wall and were thus innervated by intact trunk or limb nerves. Similarly, Weiss (1950) demonstrated that neural tube transplanted together with an embryonic amphibian limb into the tail fin of a larva established functional connections with the limb and discharged impulses into it in the absence of external stimulation. In addition, we now know that the uncoordinated movements of the chick limb which can be observed from day 5 to ca. 19 days of development in ovo are reflexogenic and thus are independent of the brain (Hamburger & Balaban, 1963). Although the rate of spontaneous movements in the embryo is reduced by isolating the spinal cord from the brain, they do continue at a significant rate (Hamburger, Wenger & Oppenheim, 1966). It is not clear at this point, however, whether normal synaptic transmission and generation of an action potential in the muscle is necessary for muscle maturation or whether some other trophic factor from the nerve is responsible for the maturational events which we have observed in our innervated grafts and which are known to occur during normal development.
ACKNOWLEDGEMENTS
We would like to thank Anita Mentzer for her excellent technical assistance and Dr Michael Payne for generating the QBM-2 antibody used in these studies. This work was supported by grants from the NIH (07083) and the Muscular Dystrophy Association of America (MDA) to IRK. Part of this work was carried out while PAM was a postdoctoral fellow of the Muscular Dystrophy Association of America and was presented during a poster session at the UCLA Symposium on the Molecular Biology of Muscle Development held in Park City, Utah; March 15-22, 1985.