It is well established that a rise in circulating thyroid hormone during the second half of chick embryo development significantly influences muscle weight gain and bone growth. We studied thyroid influence on differentiation in slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of embryos rendered hypothyroid by hypophysectomy or administration of an anti-thyroid drug. The expression of native myosins and myosin light chains (MLCs) was studied by electrophoretic analysis, and the myosin heavy chain (MHC) was characterized by immunohistochemistry. The first effects of hypothyroid status were observed at day 21 of embryonic development (stage 46 according to Hamburger and Hamilton). Analysis of myosin isoform expression in PLD muscles of hypothyroid embryos showed persistence of slow migrating native myosins and slow MLCs as well as inhibition of neonatal fast MHC expression, indicating retarded differentiaton of this muscle. In ALD muscle, hypothyroidism maintained fast embryonic MHC and induced noticeable amounts of fast MLCs, thus delaying slow muscle differentiation. Our results suggest that thyroid hormones play a role in modulating the appearance of neonatal fast MHC and the disappearance of isomyosins transiently present during embryogenesis. However, T3 supplemental treatment would seem to compensate in part for the effects of hypothyroidism induced by hypophysectomy, suggesting that thyroid hormone might interfere with other factors also accounting for the observed effects.

Vertebrate skeletal muscle fibres can be classified into two main types, fast type II and slow type I, respectively characteristic of rapidly and slowly contracting muscles. These dynamic properties relate essentially to myosin enzymatic features. Myosin expression during skeletal muscle development is modulated by different epigenetic factors, including thyroid status. In rats, hypothyroidism induces a decrease and hyperthyroidism an increase in the number of fast fibres. These effects are associated with changes in energy metabolism enzymes, myosin adenosine triphosphatase activity and myosin light chain (MLC) (Ianuzzo et al., 1980, 1984; Fitts et al., 1980; Johnson et al., 1980; Nicol and Johnston, 1981; Nwoye et al., 1982) and heavy chain (MHC) composition (Izumo et al., 1986).

In mammalian muscle, MHC is found in several isoforms, e.g., embryonic, neonatal and adult fast type II isomyosins, depending on development stage and muscle type (Whalen et al., 1981; Lyons et al., 1983; Butler-Browne and Whalen, 1984). Many experiments have clearly demonstrated that the transition from neonatal to adult isomyosin is closely correlated with the increase in thyroid hormone plasma concentration during postnatal development (Gambke et al., 1983; Butler-Browne et al., 1984, 1987; Whalen et al.,1985; d’Albis et al., 1987). Similarly, the transition from larval to adult isomyosins in urodelean amphibians is dependent on thyroid hormone concentration (Chanoine et al., 1987).

Peptide mapping techniques and specific monoclonal antibodies enabled three fast MHC isoforms to be characterized in fast chicken muscle. As in mammals, these are referred to as embryonic, neonatal and adult MHCs (Bader et al., 1982; Bandman et al., 1982; Winkelmann et al., 1983; Bandman, 1985; Cerny and Bandman, 1987). In avian fast skeletal muscle development, Hofmann and co-workers (1988) and Van Horn and Crow (1989) identified an early and late isoform of embryonic fast MHCs, and a third isoform was found by Lagrutta et al. (1989). Sweeney et al. (1989) demonstrated that the initiation of skeletal myogenesis is also characterized by the expression of a ventricular MHC stored in all developing muscle cells. Two isoforms (SM1 and SM2) identified within slow type I MHC showed proportional changes during development (Matsuda et al., 1982; Gardahaut et al., 1988).

Isoform switching has also been reported in MLCs. In mammalian development, fast skeletal muscle accumulates a fast embryonic MLC (Whalen et al., 1978) which is progressively replaced by an adult fast isoform (MLC1F), whereas a fast isoform (MLC3F) is only synthesized in significant amounts after birth. Slow muscles, which pass through a development stage in which fast MLCs are predominant, begin to accumulate significant amounts of slow MLCs at birth (for review see Barton and Buckingham, 1985). Although no embryonic MLC has been evidenced in birds, the disappearance of slow MLC isoforms and the activation of MLC3F in developing fast muscle as well as the disappearance of fast MLCs in developing slow muscle have been considered as markers for muscle maturation in avian embryo (Rubinstein et al., 1977; Bandman et al., 1982; Crow et al., 1983; Gardahaut et al., 1985).

The biological significance of developmental appearance of MHC isoforms is not fully understood. However, it is clear that adult MHC isoforms are able to induce distinct contractile properties of fibre types (Wagner and Giniger, 1981). At the post-natal stage the switch from a mixture of SM1 and SM2 isomyosins to a predominance of SM2 in ALD muscle is correlated with the maximal velocity of shortening of the fibres (Reiser et al., 1988). Recently, it has been proposed that the different fast MHCs play distinct structural roles during fibrillogenesis (Bandman and Bennett, 1988). Therefore, one of the major effects of myosin isoform switching might be to influence the contractile performance of skeletal muscles.

In chickens, circulating thyroid hormone concentrations rise substantially during the second half of embryonic life. At the end of embryonic development, thyroxine (T4) plasma concentration is about 6 times as high as at day 11, and serum 3,5,3’-triiodothyronine (T3) increases at least threefold between day 17 and 19 (for reviews see Thommes, 1987; Decuypere and Kühn, 1988). Although the role of thyroid hormone has not been studied in this species, its effects on mammalian muscle suggest that increased levels during chick embryogenesis influence muscle differentiation. Accordingly, we induced hypothyroidism by hypophysectomy or administration of an anti-thyroid drug to determine the effect on myosin isozyme transitions in slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of chick embryo. Electrophoretic analysis was used to study native myosin and MLC composition and an immunocytochemical approach to characterize MHCs.

The present work suggests that thyroid hormones play a role in slowing down expression of isomyosins transiently present during chick embryogenesis.

(1) Animals and treatments

Experiments were performed on outbred Rhode Island chickens. Embryo age was determined according to Hamburger and Hamilton stages (H. and H., 1951) and in days of incubation. Control and experimental embryos were killed at various times between day 10 and 21 of development (stages 36 and 46 of H. and H.) and PLD and ALD muscles were removed.

Hypothyroidism

Experiment 1. In one series, hypothyroidism was induced after hypophysectomy by surgical decapitation according to the technique of Fugo (1940). In chick embryos at stage 10 of H. and H. (10 somites), a slit was made through the mid-mesencephalic region, and the brain portion located anteriorly to the slit was removed. The embryos appeared to be devoid of prosencephalic derivatives since the eyes, upper beak, hypothalamus and hypophysis were lacking. They were shown to be hypothyroid (for review see Thommes, 1987; Fig. 1 adapted from Thommes, 1987), and their thyroids upon removal at day 21 (stage 46 of H. and H.) resembled those observed in control embryos at day 11–12 (stage 37 of H. and H.), being composed of small follicles, generally empty but occasionally containing colloid, separated by well-developed sinusoids (Fig. 2).

Fig. 1.

Thyroxine concentrations (ng/ml) measured in plasma of chick embryos between day 9.5 and 19.5 (adapted from Thommes, 1987).

Fig. 1.

Thyroxine concentrations (ng/ml) measured in plasma of chick embryos between day 9.5 and 19.5 (adapted from Thommes, 1987).

Fig. 2.

Sections of thyroid glands from (A) control, (B) hypophysectomized and (C) thiourea-injected chick embryos observed at day 21 of incubation (stage 46 of H. and H.) (×100). Thyroid controls (A) are composed of follicles accumulating large amounts of colloids. In thyroid of hypophysectomized embryos (B), follicles are separated by well-developed sinusoids and appear to be empty or to contain a droplet of colloid. In thiourea-injected embryos (C), heightened follicular cells have columnar features (marked reduction of the follicular lumina and colloid content compared to control glands).

Fig. 2.

Sections of thyroid glands from (A) control, (B) hypophysectomized and (C) thiourea-injected chick embryos observed at day 21 of incubation (stage 46 of H. and H.) (×100). Thyroid controls (A) are composed of follicles accumulating large amounts of colloids. In thyroid of hypophysectomized embryos (B), follicles are separated by well-developed sinusoids and appear to be empty or to contain a droplet of colloid. In thiourea-injected embryos (C), heightened follicular cells have columnar features (marked reduction of the follicular lumina and colloid content compared to control glands).

Experiment 2. In a second series, hypothyroidism was induced by thiourea treatment. Thiourea (2 mg/100 g estimated body weight dissolved in 100 μl of Ringer’s solution; King and King, 1978; McNabb et al., 1984) was pipetted onto the chorioallantoic membrane at day 7, 11, 15 and 19 of incubation (stages 31, 37, 41 and 45 of H. and H.). Thyroid of treated embryos clearly demonstrated hypothyroid status since follicular cells had become columnar and follicular lumina and colloid were markedly reduced and even absent (Fig. 2).

Since thyroid hormone is required for central and peripheral nervous system development, the effects of hypothyroidism observed in chick embryos could be due to some deleterious effect on nerve development. The innervation pattern of ALD and PLD muscles from hypophysectomised and thiourea-injected embryos was unchanged compared to control muscles, as revealed by our analysis of junctional acetylcholine receptor cluster distribution on longitudinal sections of fibres labelled with anti-fast- or anti-slow-myosin antibody (data not shown). These observations are in agreement with results of denervation experiments suggesting a direct action of thyroid hormone on muscle tissue (Nwoye et al., 1982; Gambke et al., 1983; Russell et al., 1988).

Interruption of the hypothyroid state

Experiment 3. In a third series, hypophysectomized embryos received four injections (1.2 μg/100 g body weight) on day 7, 11, 15 and 19 of development (stages 31, 37, 41 and 45 of H. and H.) of 3,5,3’-triiodothyronine sodium salt (T3) (King and Delfiner, 1974) dissolved in a total volume of 100 μl of Ringer’s solution containing 0.01 N NaOH.

Control embryos

Controls consisted either of embryos observed at day 21 (stage 46 of H. and H.), which had received vehicle injection solutions (Ringer’s or NaOH ) on day 7, 11, 15 and 19 of incubation (stages 31, 37, 41, and 45 of H. and H.) or noninjected embryos observed at day 10, 14, 16, 18 and 21 of development (stages 36, 40, 42, 44 and 46 of H. and H.). The number of the embryos examined is given in Table 1.

Table 1.

Number and development stages of embryos

Number and development stages of embryos
Number and development stages of embryos

(2) Biochemical analysis

Pyrophosphate gel electrophoresis of native myosin

Samples were prepared from 75 to 150 mg (wet weight) of muscle. Crude myosin extracts were done at 0°C according to the method of d’Albis et al. (1986). Briefly, muscles were cut into small fragments and washed with 5 volumes 20 mM NaCl, 5 mM sodium phosphate, and 1 mM EGTA, pH 6.5. Myosin was extracted in 3 volumes of buffer containing 100 mM Na4P2O7, 5 mM EGTA, and 1 mM dithiothreitol, pH 8.5. After 30 minutes of gentle agitation, the mixture was centrifuged at 10,000 g and the myosin-containing supernatant diluted twice with glycerol for storage at 20°C. Nondenaturing pyrophosphate gel electrophoresis was carried out as described by Hoh et al. (1976) and d’Albis et al. (1979). Cylindrical gels (60×5 mm) were prepared with 4% polyacrylamide, 20 mM sodium pyrophosphate and 10% glycerol, pH 8.5. Running buffer was 20 mM sodium pyrophosphate (pH 8.5) with 10% glycerol, 0.01% 2-mercaptoethanol and 2 mM MgCl2. Gels were loaded with 4-8 μg myosin. Electrophoresis was performed at a constant voltage (80 V) for 19 hours at 4°C. Gels were stained for proteins, and the relative amount of each isomyosin was esti-mated by scanning the gels with a photometric recorder equipped with an automatic integrator.

Two-dimensional gel electrophoresis of MLC

Native myosin samples were diluted with 9.5 M urea, 1.6 % ampholines (LKB), pH 5-8, 0.4% ampholines, pH 3.5–10, 2% Nonidet-P40 and 5% 2-mercaptoethanol. Two-dimensional electrophoresis was performed according to O’Farrell (1975). For the first dimension, 4% acrylamide-bisacrylamide cylindrical gels containing 2% ampholines (1.6%, pH 5–8, 0.4%, pH 3.5–10) were used. For the second dimension, cylindrical gels were applied to SDS-polyacrylamide slab gels containing 15% acrylamide and 0.1% bisacrylamide. The SDS slab gels were stained with Coomassie blue R-250, and the relative amounts of the different MLCs were quantified using a densitometer equipped with an integrator (Gardahaut et al., 1985). Statistical comparisons of percentages of native isomyosins and MLCs in control and experimental embryo muscle were performed using Student’s t-test.

(3) Immunocytochemical analysis

Distribution of the different MHCs was analyzed using specific monoclonal antibodies (mAbs) as summarized in Table 2 (Bandman, 1985; Cerny and Bandman, 1987; Bandman and Bennett, 1988; Bandman et al., 1990). Tissues were frozen in isopentane cooled in liquid nitrogen. Cryostat-cut sections (6 μm thick) were collected on gelatin-coated slides and air-dried for 1 hour at room temperature. Transverse serial sections were incubated overnight at 4°C with the different mAbs (EB165, 2E9, AB8, HV11, NA2) diluted 1/5000 in PBS. Sections were washed three times for 10 minutes in PBS and then incubated with a fluorescein isothiocyanate-labelled goat anti-mouse IgG1 Ab (Southern Biotechnology Associates, Inc.) diluted 1/100 in PBS for 1 hour at room temperature. After washing in PBS, sections were mounted in glycerol-PBS and coverslipped.

Table 2.

Specificity of monoclonal antibodies for chicken muscle myosin heavy chain isoforms

Specificity of monoclonal antibodies for chicken muscle myosin heavy chain isoforms
Specificity of monoclonal antibodies for chicken muscle myosin heavy chain isoforms

(1) Development of myosin isoforms in fast PLD and slow ALD muscles

Immunocytochemical study was carried out by day 10 (stage 36 of H. and H.) when the common primordium begins to cleave into ALD and PLD muscles (Grim, 1971). The portion giving rise to ALD and PLD muscles initially reacted with HV11 mAb. At this stage, reactivity was distributed throughout these muscles, but by day 14 (stage 40 of H. and H.) it was reduced and then finally eliminated at the end of embryogenesis. Between day 10 and 14 (stages 36 and 40 of H. and H.), immunoreactivity with EB165 mAb was also detected in both fast PLD and slow ALD muscles. Strong immunoreactivity continued throughout embryonic life in all fast PLD muscle fibres but disappeared in almost all slow ALD fibres after day 14 (stage 40 of H. and H.). At day 10 (stage 36 of H. and H.), ALD and PLD muscles were reactive with NA2 mAb which labelled a higher proportion of ALD than PLD fibres. Slow ALD continued to be immunostained at all stages of embryonic development, while almost all PLD fibres no longer reacted with this mAb after day 14 (stage 40 of H. and. H.). No immunoreactivity with AB8 and 2E9 mAbs was observed in ALD and PLD muscles between day 10 and 20 (stages 36 and 45 of H. and H.). Immunoreactivity with 2E9 mAb was evidenced only in PLD muscle at hatching time (stage 46 of H. and H.). Thus, at this stage all ALD myofibres were stained with anti-slow MHC NA2 mAb and all PLD myofibres with anti-fast MHC EB165 mAb; some rare fibres were always immunostained with EB165 mAb in ALD and with NA2 mAb in PLD (Fig. 3).

Fig. 3.

MHC expression in ALD and PLD muscles during embryonic chick development. Vertical lines: (A) observation at day 10 (stage 36 of H. and H; ×500), (B) at day 14 (stage 40; ×180), (C) at day 21 (stage 46; ×180). Each vertical line represents immunocytochemical staining of transverse serial sections with mAbs: (I) HV11, (II) EB165, (III) NA2, (IV) 2E9, (V) AB8. At day 10 (A) in both ALD and PLD muscles, many fibres reacted with mAb HV11 (ventricular cardiac MHC; line I), EB165 (embryonic/adult fast MHC; line II) and NA2 (slow MHCs; line III). At this stage, no immunoreactivity was observed with mAb 2E9 (neonatal fast MHC; line IV) and AB8 (adult fast MHC; line V). At day 14 (B), the immunoreactivity level with HV11 mAb had declined considerably in PLD and ALD fibres, and the proportion of fibres immunostained with NA2 mAb appeared stronger in ALD than in PLD. At hatching time (C), staining with HV11 mAb was maintained in rare fibres of ALD muscle alone. NA2 mAb reactivity had totally disappeared in PLD fibres, whereas almost all ALD fibres were positive. 2E9 mAb reacted in a high percentage of PLD fibres alone. No immunoreaction with AB8 mAb could still be observed in ALD and PLD muscles.

Fig. 3.

MHC expression in ALD and PLD muscles during embryonic chick development. Vertical lines: (A) observation at day 10 (stage 36 of H. and H; ×500), (B) at day 14 (stage 40; ×180), (C) at day 21 (stage 46; ×180). Each vertical line represents immunocytochemical staining of transverse serial sections with mAbs: (I) HV11, (II) EB165, (III) NA2, (IV) 2E9, (V) AB8. At day 10 (A) in both ALD and PLD muscles, many fibres reacted with mAb HV11 (ventricular cardiac MHC; line I), EB165 (embryonic/adult fast MHC; line II) and NA2 (slow MHCs; line III). At this stage, no immunoreactivity was observed with mAb 2E9 (neonatal fast MHC; line IV) and AB8 (adult fast MHC; line V). At day 14 (B), the immunoreactivity level with HV11 mAb had declined considerably in PLD and ALD fibres, and the proportion of fibres immunostained with NA2 mAb appeared stronger in ALD than in PLD. At hatching time (C), staining with HV11 mAb was maintained in rare fibres of ALD muscle alone. NA2 mAb reactivity had totally disappeared in PLD fibres, whereas almost all ALD fibres were positive. 2E9 mAb reacted in a high percentage of PLD fibres alone. No immunoreaction with AB8 mAb could still be observed in ALD and PLD muscles.

The time pattern of native myosin and MLC expression was previously determined in developing PLD and ALD muscles (Gardahaut et al., 1985, 1988, 1990). In PLD, only one fast native myosin co-migrating with the adult fast component FM3 was expressed up to day 12 (stage 38 of H. and H.). Subsequently, two additional fast isoforms with the same mobility as adult FM2 and FM1 successively appeared. At this time, slow native components migrating at SM1 and SM2 positions were detected in trace amounts in this muscle. At day 12 (stage 38 of H. and H.), slow and fast MLC subunits were co-expressed, and then at day 16 (stage 42 of H. and H.) the accumulation of slow MLCs markedly decreased (3% of total MLCs). ALD muscle at day 11 (stage 37 of H. and H.) contained two slow native isomyosins (SM1 and SM2), the former being predominant at all embryonic stages. Trace amounts of fast native isomyosins were detected at this time. Whereas ALD muscle co-accumulated fast and slow MLCs at day 12 (stage 38 of H. and H.), fast MLCs were progressively replaced by slow MLCs between day 12 and 18 (stages 38 and 44 of H. and H.), with fast MLCs decreasing from 40 to 20 % of total MLCs.

(2) Effects of hypothyroidism on myosin isoform expression

The effects of hypothyroid status induced after hypophysectomy or thiourea treatment were first observed at day 21 of incubation (stage 46 of H. and H.), with no change occurring before this developmental stage. Our studies were carried out simultaneously on native isomyosin, MLC and MHC subunit compositions in PLD and ALD muscles of hypothyroid embryos.

Fast-twitch PLD muscle

Native isomyosins

At day 21 (stage 46 of H. and H.), in both control and experimental PLD muscles, electrophoretic analysis showed three prominent bands migrating at the positions of adult fast isoforms FM3, FM2 and FM1. PLD muscles of hypophysectomized and thiourea-injected embryos exhibited two additional bands not detected in controls which had mobilities similar to slow-type isomyosins SM2 and SM1 (Figs 4A, 5A). In these muscles, slow isomyosins reached 30% of total myosin, whereas no slow isomyosin was detected in PLD muscles of hypophysectomized T3-treated embryos (Figs 4A, 5A).

Fig. 4.

Myosin expression in PLD muscle from day 21 control and experimental embryos (stage 46 of H. and H.). The vertical lines represent (A) nondenaturing gel electrophoresis of myosin; (B) two-dimensional gel electrophoresis of myosin light chains (MLC); (C) immunocytochemical detection of neonatal MHC (mAb 2E9) (×350). The horizontal lines represent (I) control embryo, (II) hypophysectomized embryo, (III) thiourea-injected embryo and (IV) hypophysectomized-T3-treated embryo. SM1 and SM2 = slow native isomyosins; S1, S2 = slow MLCs; FM1, FM2 = fast native isomyosins; F1, F2, F3 = fast MLCs.

Fig. 4.

Myosin expression in PLD muscle from day 21 control and experimental embryos (stage 46 of H. and H.). The vertical lines represent (A) nondenaturing gel electrophoresis of myosin; (B) two-dimensional gel electrophoresis of myosin light chains (MLC); (C) immunocytochemical detection of neonatal MHC (mAb 2E9) (×350). The horizontal lines represent (I) control embryo, (II) hypophysectomized embryo, (III) thiourea-injected embryo and (IV) hypophysectomized-T3-treated embryo. SM1 and SM2 = slow native isomyosins; S1, S2 = slow MLCs; FM1, FM2 = fast native isomyosins; F1, F2, F3 = fast MLCs.

Fig. 5.

Accumulation of myosin isoforms in PLD muscle from control and experimental embryos at day 21 (stage 46 of H. and H.): (A) native isomyosins, (B) MLC isoforms. Empty bars represent slow components, dotted bars fast components.

Fig. 5.

Accumulation of myosin isoforms in PLD muscle from control and experimental embryos at day 21 (stage 46 of H. and H.): (A) native isomyosins, (B) MLC isoforms. Empty bars represent slow components, dotted bars fast components.

MLCs

Figs 4B and 5B show the electrophoretic pattern of MLCs and their relative proportions in control and experimental PLD muscles at day 21 (stage 46 of H. and H.). In all cases, muscle extracts revealed a predominance of fast MLCs (LC1F, LC2F, LC3F), with few slow MLCs (LC1S, LC2S). However, the proportions of slow isoforms in hypophysectomized and thiourea-injected embryos were respectively 2 and 4 times as high as those observed in control muscles (P < 0.05). No significant differences were detected between PLD muscles of hypophysectomized and hypophysectomized-T3-treated embryos. The MLC pattern of muscles was not fully correlated with the native isomyosin profile. Indeed, PLD muscles of control and hypophysectomized-T3-treated embryos contained slow MLCs, whereas no slow native myosin was detected by pyrophosphate gel electrophoresis. These findings may reflect the differences in the detection limits of these two electrophoretic methods. Moreover, slow MLCs, in conjunction with fast MHCs, can constitute hybrid myosin molecules, as has been reported for embryonic PLD muscle (Rubinstein et al., 1977; Takano-Ohmuro et al., 1982). It is thus likely that variations in native isomyosin composition are insufficient to produce a change in fast myosin mobility on nondenaturing gels.

MHCs

At day 21 (stage 46 of H. and H.), there was no PLD fibre reaction with HV11 mAb in hypophysectomized, thiourea-injected and hypophysectomized-T3-treated embryos. Whereas every muscle fibre was strongly immunostained with EB165 mAb, only some rare fibres reacted with NA2 mAb. No immunoreactivity was observed with AB8 mAb. The immunoreactivity of experimental PLD muscles with HV11, EB165, NA2 and AB8 mAbs was similar to that described for control muscles. However, unlike control PLD muscles, which contained a substantial percentage of fibres labelled with 2E9 mAb, PLD muscles of hypophysectomized or thiourea-injected embryos exhibited only a few weakly labelled fibres (Fig. 4C I,II,III). This situation was not modified in hypophysectomized embryos by T3-replacement treatment (Fig. 4 C IV).

Slow-tonic ALD muscle

Native isomyosins

In control ALD muscle at day 21 (stage 46 of H and H.), the isomyosin pattern revealed two bands with mobilities similar to those of adult SM2 and SM1 (faster migrating isomyosin SM1 being more predominant). ALD muscle of hypophysectomized or thiourea-injected embryos exhibited two additional bands in the region corresponding to adult fast native myosins, FM3 being the strongest fast band (13% in PLD of hypophysectomized embryos, 18% in PLD of thiourea-injected embryos). Following T3 administration, fast myosin components were virtually eliminated from ALD muscle of hypophysectomized embryos (Figs 6A, 7A).

Fig. 6.

Myosin expression in ALD muscle from control and experimental embryos at day 21 (stage 46 of H. and H.). The vertical lines represent (A) nondenaturing gel electrophoresis of myosin; (B) two-dimensional gel electrophoresis of myosin light chains (MLC); (C) immunocytochemical staining of fibres with antibody EB165 specific for fast embryonic/adult MHC. The horizontal lines represent (I) control embryo, (II) hypophysectomized embryo, (III) thiourea-injected embryo and (IV) hypophysectomized-T3-treated embryo. SM1 and SM2 = slow native isomyosins; S1, S2 = slow MLCs; FM2, FM3 = fast native isomyosins; F1, F2 = fast MLCs.

Fig. 6.

Myosin expression in ALD muscle from control and experimental embryos at day 21 (stage 46 of H. and H.). The vertical lines represent (A) nondenaturing gel electrophoresis of myosin; (B) two-dimensional gel electrophoresis of myosin light chains (MLC); (C) immunocytochemical staining of fibres with antibody EB165 specific for fast embryonic/adult MHC. The horizontal lines represent (I) control embryo, (II) hypophysectomized embryo, (III) thiourea-injected embryo and (IV) hypophysectomized-T3-treated embryo. SM1 and SM2 = slow native isomyosins; S1, S2 = slow MLCs; FM2, FM3 = fast native isomyosins; F1, F2 = fast MLCs.

Fig. 7.

Accumulation of myosin isoforms in ALD muscle from control and experimental embryos at day 21 (stage 46 of H. and H.): (A) native isomyosins, (B) MLC isoforms. Empty bars represent slow components, dotted bars fast components.

Fig. 7.

Accumulation of myosin isoforms in ALD muscle from control and experimental embryos at day 21 (stage 46 of H. and H.): (A) native isomyosins, (B) MLC isoforms. Empty bars represent slow components, dotted bars fast components.

MLCs

At day 21 (stage 46 of H. and H.), control ALD muscles accumulated both slow and fast MLCs, with slow isoforms constituting about 80% of total MLCs (Figs 6B, 7B). The absence of fast native isomyosins probably means that fast MLCs detected in control ALD bound to slow MHC, thus contributing to hybrid molecule expression as suggested above for embryonic PLD muscle. Hypophysectomy as well as thiourea treatment resulted in a significant increase (P < 0.05) in fast MLC content compared to control ALD muscles. The MLC pattern in ALD muscles of hypophysectomized embryos treated with T3 closely corresponded to that of control ALD (Figs 6B, 7B).

MHCs

At day 21 (stage 46 of H. and H.), ALD of hypophysectomized (injected or not with T3) and thiourea-injected embryos showed no immunoreactivity with HV11 mAb. In these experimental embryos, ALD muscle cells were intensely stained with NA2 mAb but were unreactive with 2E9 and AB8 mAbs. As for control muscle, the immunocytochemical profile in experimental muscle revealed by HV11, NA2, 2E9 and AB8 mAbs was unchanged. In contrast to ALD control muscle fibres, which were not labeled with EB165 mAb, most ALD fibres in thiourea-injected or hypophysectomized embryos strongly reacted with this mAb (Fig. 6C I,II,III). In T3-treated hypophysectomized embryos, fibre reactivity with EB165 mAb was similar to that observed after hypophysectomy (Fig. 6C IV).

Our study in chick embryo indicates that a low level of circulating hormones (T3, T4) experimentally induced by hypophysectomy or thiourea treatment is responsible for changes in developmental myosin isoform expression. The effects of experimental hypothyroidism were evidenced from day 21 (stage 46 of H. and H.) onwards.

With regard to developmental analysis of myosin isoforms in control embryos, hypophysectomy and anti-thyroid drug maintained the expression of slow migrating native isomyosins and slow MLCs in PLD muscle until the end of embryonic development. At that time, maintenance of fast native isomyosins and persistence of noticeable amounts of fast MLCs in ALD muscle of hypophysectomized or thiourea-treated embryos were also noted.

In our study, the pattern of MHC expression was initially similar in developing slow ALD and fast PLD muscles. At the beginning of embryonic development, both of these muscles expressed the same ventricular embryonic fast and slow isoforms. The content in ventricular and embryonic fast MHCs was gradually reduced and finally totally eliminated in slow ALD. The same process occurred with ventricular and slow MHCs in fast PLD. In PLD muscles, neonatal MHCs began to be accumulated around day 21 (stage 46 of H. and H.). Following hypophysectomy or thiourea treatment, embryonic fast MHC was maintained in all PLD fibres and no transition from embryonic to neonatal MHC occurred. With respect to these data, detection of fast native myosins (FM3, FM2, FM1) probably indicated the presence of embryonic fast MHC in experimental PLD muscle. In PLD muscle of hypothyroid embryos, the absence of immunoreactivity with anti-slow MHC mAb, when slow native myosin was detected, implies that the latter contained MHCs of embryonic type. Considering the accumulation of slow embryonic MHCs and slow MLCs as well as the inhibition of neonatal MHC expression in PLD muscle from hypophysectomized or thiourea-treated embryos, it is likely that hypothyroidism retards PLD muscle differentiation. Whereas fast embryonic MHC disappeared around day 16 in normal development of ALD muscle, it was still present on the day of hatching in all ALD muscle fibres from hypophysectomized or thiourea-treated embryos. Thus, the fast migrating native myosins detected in these hypothyroid embryo muscles may be considered to be embryonic fast MHC isoforms. In comparison with myosin isoform expression in control ALD muscle at the identical development stage, the persistence of fast embryonic MHC and the presence of noticeable amounts of fast MLCs in ALD from hypophysectomized or thioureatreated embryos indicate that the differentiation of slow muscle was delayed.

The fact that hypophysectomy induced changes similar to those observed after thiourea treatment suggests that its effects on myosin isoform expression could be due to a thyroid hormone deficiency. The results of our study tend to favor the notion that thyroid hormones play a role in slowing down the expression of isomyosins transiently present during embryogenesis and, especially, that the increase in circulating thyroid hormones during embryonic development of the chick is probably involved in repression of fast embryonic MHC in fast and slow muscles. In mammals the transition from neonatal to adult fast myosin muscle is also highly responsive to thyroid hormone levels (Gambke et al., 1983; Butler-Browne et al., 1984, 1987; Whalen et al., 1985; d’Albis et al., 1987). However, the influence of thyroid hormones on the MHC pattern seems complex. Ventricular cardiac MHC, abundantly expressed at early embryonic stages in both slow ALD and fast PLD muscles, decreased rapidly during the second half of incubation in normal as well as hypophysectomized or thiourea-treated embryos, suggesting that the level of expression of this isoform is unaffected by hypothyroidism. Conversely, in developing skeletal mammalian muscle, a cardiac α-MHC has recently been described with an expression level evidently dependent on that of thyroid hormones (d’Albis et al., 1991), whereas in adults thyroidectomy had no effect on the expression of a cardiac β-MHC gene in skeletal muscle fibres (Izumo et al., 1986; Mahdavi et al., 1987). These data indicate that the expression of cardiac MHC genes in skeletal muscle may or may not be regulated by thyroid hormones.

In our work, hypothyroidism-induced changes in MLC levels would seem to indicate that there is a correlation between the expression of fast embryonic MHCs and fast MLCs in slow ALD muscle and of slow embryonic MHCs and slow MLCs in fast PLD muscle. Furthermore, in the hypothyroid state, the relative proportions of fast MLCs increased in slow muscles and decreased in fast muscles, whereas the relative amount of slow MLCs decreased in slow ALD while increasing in fast PLD. It appears that genes encoding fast and slow myosin isoforms can be differently regulated according to the muscle. Thus, in adult mammals, the same MHC gene may react differently to thyroid hormone depending on the muscle from which it has been isolated (Izumo et al., 1986).

As previously reported for body weight and bone growth (for review see King and May, 1984), the effects of hypothyroidism on myosin isoform expression are apparent during late embryogenesis when T3 and T4 levels are increasing. It is noteworthy that hypophysectomy by surgical decapitation not only alters thyroid hormone level but also pituitary hormone level, consequently affecting hormone production in glands influenced by hypophysis. To determine the influence of hypothyroidism on modifications of isomyosin expression in hypophysectomized embryos, surgically decapitated embryos were treated with T3, the metabolically active hormone for tissues. Only T3 receptors are present in chicken tissues (Bellabarba et al., 1988), and growth hormone (and/or TRH) is required for peripheral deiodination of T4 into T3 (Decuypere and Kühn, 1988). Thus, T3 supplemental treatment would presumably be more efficient than T4 supplemental treatment in controlling hypothyroidism induced by hypophysectomy. However, T3 supplementation appears to compensate only partly for changes in isomyosin expression induced by hypophysectomy. Especially in experimental embryos, ALD muscle fibres still express fast embryonic MHC and no transition from fast embryonic MHC to neonatal MHC occurs in PLD. Studies performed on hereditary dwarf mice lacking anterior pituitary cells capable of producing growth hormone suggest that thyroid hormone has a direct effect on myosin isozyme transition in fast skeletal muscle (Butler-Browne et al., 1987; Russell et al., 1988). Nevertheless, the absence of growth hormone or other hormone production in decapitated embryos could explain why T3 supplementation partially prevents the effects of hypophysectomy. It is well established that T3 acts first through interaction with nuclear receptors (Oppenheimer and Samuels, 1983) and that the hormone-receptor complex interacts with some regulatory sequences able to stimulate transcription of genes coding for numerous proteins involved in development, such as growth hormone (Evans et al., 1982; Spindler et al., 1982; Yaffee and Samuels, 1984). The major effect of growth hormone on tissues is mediated by an increase in cell production of somatomedins, e.g., stimulating the proliferation and differentiation of muscle cells (see for review Florini, 1991). These data suggest that the incomplete effect of T3 supplementation observed in our study might be due to somatomedin deficiency. We have recently determined that IGF-I injected in nanomolar concentrations is capable of accelerating muscle development. In PLD muscle of 18-day-old embryos, synthesis of fast FM1 isomyosin (MLC3F homodimer) was increased by 30% when compared to controls, and expression of neonatal fast MHC was detected; however, this isoform was not expressed at that time in PLD controls. In ALD muscle, IGF-I increased the expression of adult SM2 isomyosin by 15%. In view of these results, we may postulate that T3 and IGF-I applied simultaneously to hypophysectomised embryos are more efficient than T3 alone, in compensating for the effects of hypophysectomy. Experiments are currently in progress to test this hypothesis.

The authors would like to thank Maryvonne Zampieri for technical assistance. This work was supported by the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale and the Association Française contre les Myopathies.

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