DEVELOPMENTAL

BIOLOGY

l&16-26

(1992)

Distinct Myogenic Programs of Embryonic and Fetal Mouse Muscle Cells: Expression of the Perinatal Myosin Heavy Chain lsoform in Vitro TIMOTHY *Day

H. SMITH*

Neuromuscular Laboratory, Massachusetts and TProgram in Neuroscience,

AND JEFFREY

BOONE

MILLER*,?

General Hospital, 169 13th Street, Charlestown, Medical School, Boston, Massachusetts

Harvard

Accepted

September

Massachusetts 02115

02129;

9, 1991

Early embryonic and late fetal mouse myogenic cells showed distinct patterns of perinatal myosin heavy chain (MHC) isoform expression upon differentiation in vitro. In cultures of somite or limb muscle cells isolated from Day 9 to Day 12 embryos, differentiated cells that expressed perinatal MHC were rare and perinatal MHC was not detectable by immunoblotting. In cultures of limb muscle cells isolated from Day 13 to Day 18 fetuses, in contrast, the perinatal MHC isoform was easily detected and was expressed in a substantial percentage of myocytes and myotubes. Analyses of clonally derived muscle colonies and cytosine arabinoside-treated fetal muscle cell cultures suggested that different fetal muscle cell nuclei initiated perinatal MHC expression at different times. In both embryonic and fetal cell cultures, the embryonic MHC isoform was expressed by all differentiated cells examined. A small number of myotubes in fetal muscle cell cultures showed a mosaic distribution of MHC isoform accumulation in which the perinatal MHC isoform accumulated in a restricted region of the myotube near particular nuclei, whereas the embryonic MHC isoform accumulated throughout the myotube. Thus, the myogenic program of fetal, but not embryonic, mouse myogenic cells includes expression of the perinatal MHC isoform upon differentiation in culture. 0 1332 Academic Press, Inc.

and Stockdale, 1987; Cossu and Molinaro, 1987). For example, sequential transitions or modulations of MHC isoform expression occur within avian and mammalian myotubes in culture in the absence of innervation (Cerny and Bandman, 1986; Silberstein et aL, 1986; Weydert et aZ., 1987; Miller and Stockdale, 1989). In addition, multiple types of avian and mammalian myoblasts appear sequentially at different stages of muscle development, and the different types of myoblasts form multiple types of myotubes which express distinct MHC isoform phenotypes in vitro (reviewed, Cossu and Molinaro, 1987; Miller and Stockdale, 1987; Miller, 1991). One of the earliest signs of muscle fiber diversification in prenatal rodents is the fiber-specific expression of the perinatal MHC isoform in secondary fibers and a subset of primary fibers (A. J. Harris et aL, 1989; Condon et ak, 1990a). The cellular and molecular processes which restrict perinatal MHC expression to particular fibers are not understood. One possibility is that the diverse types of primary and secondary muscle fibers are formed from different types of myoblasts that are committed to form myotubes with distinct patterns of MHC isoform expression. This possibility is supported by the work of Vivarelli et al, (1988) who showed that myoblasts isolated from early embryonic mouse limbs and somites, before Day 14 in utero (E14), form myotubes in culture which coexpress embryonic and slow

INTRODUCTION Distinct types of fast and slow skeletal muscle fibers, which have different biochemical phenotypes and contraction rates, form during vertebrate muscle development. The contraction rate of a striated muscle cell is largely determined by the ATPase activity of the myosin heavy chain (MHC) isoform or isoforms expressed in the cell (Schwartz et aZ., 1981; Reiser et al., 1985; Sweeney et I& 1988). In the mouse and rat, multiple striated muscle MHC isoforms have been identified, and each MHC isoform is expressed only in certain striated muscle cells and at certain stages of development (Whalen et al., 1981; Izumo et al, 1986; Emerson and Bernstein, 1987; Naruzawa et aL, 1987; Weydert et aL, 1987; Vivarelli et al., 1988; Schiaffino et al., 1988, 1989; A. J. Harris et aL, 1989; Condon et aZ., 1990a). MHC isoform expression is, therefore, both the molecular determinant of and a marker for skeletal muscle fiber diversification during development (Bandman, 1985; Whalen, 1985; Stockdale and Miller, 1987). Though MHC isoform expression is regulated by extrinsic factors such as thyroid hormone levels and the activity pattern of the innervating motor neuron (Jolesz and Sreter, 1981; Pette and Vrbova, 1985; Izumo et ah, 1986); factors intrinsic to myogenic cells also influence the MHC phenotype of muscle fibers (Kelly, 1983; Miller 0012-1606/92 $3.00 Copyright All rights

0 1992 by Academic Press, Inc. of reproduction in any form reserved.

16

SMITH

AND MILLER

Perinati

MHCs, whereas myoblasts isolated from fetal mice, after E15, form myotubes which, in most cases, express only embryonic MHC. In addition, the perinatal MHC isoform is expressed in myotubes formed by some, but not all, mouse muscle cell lines (Silberstein et a& 1986; Weydert et aZ., 1987; Miller, 1990; Cox et aZ., 1991), though it is not clear how expression of perinatal MHC by transformed cell lines compares to expression in primary muscle cell cultures. To determine how expression of the perinatal MHC isoform by untransformed myogenie cells in vitro compares to that of developing myotubes in zliz)o, we have analyzed perinatal MHC expression in myotubes formed from mouse embryonic somite, embryonic limb, and fetal limb myoblasts. Distinct patterns of perinatal MHC expression were found among the myocytes and myotubes formed from embryonic and fetal myoblasts. MATERIALS

AND

METHODS

Cell culture. Embryos were obtained from CD-1 or BALB/c mice (Charles River), and embryo ages were determined according to the stages of Theiler (1989). Cells were prepared for culture by dissecting and mechanically dissociating Embryonic Day 8.5-11.5 somites (with neural tubes included) or by dissecting and enzymatically dissociating whole Ell-El5 limb buds or skinned and deboned muscle from El6 or older limb buds. Limb tissue was dissociated by one or two 15-min incubations at 37°C in 0.05% trypsin, 0.5 mM EDTA with repeated trituration. Cells were cultured at 500020,000 cells/cm2 in a medium consisting of DMEM supplemented with 15 or 20% fetal calf serum, 5% chicken embryo extract, 1 mlM sodium pyruvate, 10 mM Hepes (pH 7.4), 2 mML-glutamine, and 10,000 units/ml penicillin. For clonal analyses, cells were cultured at 2-20 cells/cm2 in a medium consisting of Ham’s F-10 made 1.8 mlM CaCl, and supplemented with 10% horse serum, 5% chicken embryo extract, glutamine, and penicillin (Hauschka et ah, 1979). Medium was used at 0.2 ml/cm2, dishes were gelatin coated, and cultures were incubated in a humidified atmosphere of 95% air, 5% CO2 at 37°C. Fetal calf serum (GIBCO) was selected for its ability to support clonal growth of primary adult human myoblasts (Lev et uZ., 1987) and horse serum (Hazelton) was selected for its ability to support muscle colony formation from Embryonic Day 19 mouse muscle cells (Hauschka et uk, 1979). Identical results were obtained using cells from both strains of mice and both types of culture medium. Cultures were designated by age of donor and duration of incubation. For example, an El3 + 4 culture was prepared with myoblasts from El3 donors and was maintained in culture for 4 days. Myoblasts are consid-

MHC Expression

in

Vitro

17

ered to be myogenic, mononucleated, and mitotic cells that do not express skeletal muscle MHC(s); myocytes are mononucleated cells that express skeletal muscle MHC(s); and myotubes (or muscle fibers) are multinucleated, nonmitotic cells that express skeletal muscle MHC(s). The embryonic period of development is the period of morphogenesis which, for the mouse, lasts until about E13-E14, and the fetal period of development is the period of growth that follows until birth (Theiler, 1989). Mcmoclonul antibodies. The reactivities with mouse MHC isoforms of the mAbs used in these experiments were determined and are shown in Table 1. The preparation and properties of mAbs F47 and F59 have been described (Crow and Stockdale, 1984; Miller et al, 1985; Miller et a& 1989; Miller, 1990). Two additional mAbs, BF-45, which is specific for embryonic MHC, and BF-34, which is specific for perinatal MHC, were gifts of Dr. Stefano Schiaffino (Schiaffino et cd., 1988, 1989). Two mAbs specific for the slow MHC isoform, RllDlO (Khaw et ub, 1984) and NOQ7.5.4.D (Narusawa et al, 1987; A. J. Harris et uL, 1989), were also used. All experiments used either 1:lO or 1:20 dilutions of hybridoma supernatants (F47, F59, and NOQ7.5.4.D), 1:200 dilutions of lyophilized and resuspended ascites (BF-45 and BF-34), or 500 rig/ml of purified antibody (mAb RllDlO). The mAbs did not react with smooth muscle or nonsarcomeric MHCs (Evans et ah, 1988; Miller et al, 1989; and unpublished). Immunocytochemistry and electrophoresis. Cultures were fixed for 5 min in 100% ethanol at room temperature and incubated for 1 hr at room temperature with mAbs diluted in PBS containing 2% horse serum and 2% bovine serum albumin, and mAb binding was visualized with a horseradish peroxidase-linked system using diaminobenzidine as substrate (Vectastain ABC kit, Vector Laboratories) (Miller et ub, 1985). For double immunofluorescence, a modification of the method of Wesse1 and McClay (1986) was used (Miller, 1990). Samples were incubated with the first mAb (e.g., F47 or NOQ754D), followed by sequential incubations with (i) a saturating amount (100 pg/ml) of the monovalent fab fragment of fluorescein-conjugated goat anti-mouse IgG (Cappel Laboratories), (ii) the second mAb, and (iii) 10 pg/ml Texas red-conjugated goat anti-mouse IgG (Vector Laboratories). In some cases when mAb F59 was used as the second antibody, cultures were incubated with the first mAb (i.e., F47, BF-34, BF-45, or NOQ754D), followed by sequential incubations with (i) 5 pg/ml of fluorescein-conjugated goat anti-mouse IgG (H + L specific, Zymed Laboratories), (ii) mAb F59, and (iii) 5 pg/ml Texas red-conjugated goat anti-mouse IgG (H + L, Vector Laboratories). Because mAb F59 reacted

18

DEVELOPMENTAL

BIOLOGY

VOLUME

TABLE REA~TI~ITIES

OF MONOCLONAL

ANTIBODIES

I@,1992

1

WITH MOUSE

MYOSIN

HEAW

CHAIN

ISOFORMS

Reaction of mAb with MHC isoform” Monoclonal antibody

Embryonic

Perinatal

Type IIA

Type IIB

Type 11X

Slow

BF-45 BF-34 NOQ754D RllDlO F47 F59

++ +++

++ +++ +++

+++ +++

+++ +++

+++ +++

++ +++ ++

a MHCs from whole muscles or cultures were subjected to SDS-PAGE in 6% polyacrylamide, 37.5% glycerol gels, transferred to immunoblots, and tested for reaction with each mAb. Individual MHC isoforms were distinguished by electrophoretic mobility and expression patterns in animals of different ages and in different muscles or cell cultures (Schiaffino et al, 1988,1989, Miller, 1990; LaFramboise et al, 1991; see, e.g., Fig. 1). The embryonic MHC was from BC3H-1 cultures and El4 limb muscle; the perinatal MHC was from C&C,, cultures (Silberstein et al, 1986; Miller, 1990) and Postnatal Day 2 (P2) limb muscle; the Type IIA MHC was from adult diaphragm and adult soleus; the Type IIB MHC was from adult quadriceps; the Type 11X was from adult diaphragm; and the slow MHC was from P2 ventricle, adult diaphragm, and adult soleus. Equal amounts of myosin from each sample were analyzed and the intensity of mAb reaction with each sample was compared using the antibody concentrations or hvbridoma sunernatant dilutions stated under Materials and Methods. +++, strong reaction, ++, moderate reaction; +, weak reaction; -, no detectable reaction.

with all differentiated muscle cells, this protocol was sufficient to identify muscle cells that reacted with F59 but did not react with the first mAb tested. Possible binding of mAb F59 to the second IgG binding site of the fluorescein-conjugated antibody thus did not affect the results. Control experiments showed that the observed fluorescence was due to mAb binding to MHC. Nuclei were visualized by blue fluorescent staining with 1 &l!f bisbenzimide (Hoechst 33258). Individual cells were scored for number of nuclei and staining with the mAbs under test. In different experiments, O-7% of the cells examined showed indeterminate staining patterns that were neither clearly positive nor clearly negative with a particular mAb, and these cells were excluded from the analyses shown in Table 2. Staining percentages determined by different observers typically agreed to within 10%. Myosin from cultures or tissues was prepared by cycles of high salt extraction and low salt precipitation (Crow et ah, 1983; Miller et aZ., 1985). MHC isoforms were separated by SDS-PAGE in gels containing 6% polyacrylamide and 37.5% glycerol (Danieli Betto et aL, 1986; Schiaffino et uZ., 1989) and transferred to nitrocellulose (Miller et al, 1985; Miller and Stockdale, 1986a,b). Nitrocellulose transfers were incubated for l-2 hr at room temperature with mAbs diluted in PBS containing 2% nonfat powdered dry milk, and mAb binding was visualized with a horseradish peroxidase-linked system (Vectastain ABC, Vector Laboratories) and a chemiluminescent substrate (ECL kit, Amersham) or with an alkaline phosphatase-linked system (Vectastain AP, Vector Laboratories) and nitroblue tetrazolium/5-bro-

mo-4-chloro-3-indolyl phosphate substrate (Bio-Rad Laboratories). One-dimensional peptide maps of protease-digested MHCs were obtained by modifying previous procedures (Cleveland, 1983; Bandman, 1985; Schafer et d, 1987) for minigels. MHCs were subjected to SDS-PAGE in 5% gels and briefly stained with Coomassie blue, and the regions of the gel containing MHC were excised. The gel pieces were equilibrated in sample buffer, inserted into sample wells of a 15% gel, and partially digested with 250 ng of Staphylococcus uureua V8 protease (Schafer et al, 1987). After electrophoresis, the partial proteolytic fragments were transferred to nitrocellulose and incubated with mAb, and mAb binding was visualized as above. RESULTS

Immunoblot analysis. The perinatal MHC isoform was expressed in differentiated cultures of mouse myogenic cells, but the amount of perinatal MHC expression depended on the age of the myoblast donor. Myogenic cells were prepared from embryonic (El2 or earlier) and fetal (E17-E18) limbs, placed in culture, and incubated for 5 days to allow formation of differentiated cells that expressed MHC. MHCs were prepared from the cultures and from the limb muscles of mice of different ages; the MHC isoforms were separated by electrophoresis in 6% polyacrylamide, 37.5% glycerol gels, and duplicate gels were analyzed by Western blotting with mAbs specific for different isoforms (Table 1). Such analyses (Fig. 1) showed that easily detectable amounts of the perinatal

SMITH

Perinatd

AND MILLER

TABLE 2 MYOSIN

HEAW

Myoblast source

CHAIN ISOFORM EXPRESSION FORMED IN VITRO

Duration of culture (days)

IN MY-S

Monoclonal antibody

Percentage (number examined) of myocytes stained” 4 (84) 99 (103) 100 (150)

El1 somite$ (stage 17/X3)

4

F47 (peri -t II) NOQ754D (slow) BF-45 (emb)

El1 limb (stage 17/18)

4

F47 NOQ754D

4 (135) 93 (112)

El2 limb (stage 20)

4

F47 NOQ754D BF-45

1(125) 99 (142) loo (loo)

El3 limb (stage 21)

F47 F47 NOQ754D BF-34 (peri) F47 NOQ754D

50 83 53 29 94

11 (81)

(106) (59) (88) (91) (119)

El5 limb (stage 23)

4 6

F47 F47 NOQ754D

64 (92) 75 (86) 77 (127)

El7 limb (stage 25)

4 6

F47 F47 BF-34 NOQ754D BF-45

8.3 (96) 95 (100) 96 (152) 63 64 100 (125)

F47 F47 NOQ754D

47 (59) 95 (109) 55 (89)

El8 limb (stage 26)

4 6

o Myocyte MHC expression was examined by fluorescence using isoform-specific mAbs, mAb F59, and Hoechst 33258. Mononucleated myocytes which stained with mAb F59 were observed for staining with the indicated mAbs. Zero to seven percent of the myocytes showed staining that was neither clearly positive nor clearly negative, and these cells were excluded from the analysis. Staining of multinucleated myotubes is described in the text. b Somite, but not limb, cultures also contained neurons. c Stages are according to Theiler (1989).

MHC isoform were expressed in El7 + 5 cultures (lane 3) as well as in the El8 (lane 4) and Postnatal Day 5 (P5, lane 5) limb samples included for comparison, whereas little or no perinatal MHC was detectable in El1 + 5 (lane 1) or El2 + 5 (lane 2) cultures or adult quadriceps muscle (lane 6). The perinatal MHC isoform was identified by (i) electrophoretic mobility (M,) intermediate between that of the embryonic and slow MHCs; (ii) reaction with the perinatal MHC-specific mAb BF-34, as well as with mAbs F47 and F59; and (iii) lack of reaction with the embryonic MHC-specific mAb BF-45. Thus, this immunoblot analysis, and similar analyses not

19

MHC Expression in Vitro

shown, showed that fetal (E15-El@, but not embryonic (E10.5-E12), mouse limb myoblasts formed differentiated cells that expressed the perinatal MHC isoform. The immunoblot analyses with mAbs BF-45 and RllDlO (Fig. 1) showed that the slow MHC isoform and the embryonic MHC isoform were also expressed in El1 + 5, El2 + 5, and El7 + 5 cultures. The slow MHC isoform was identified based on its rapid M, (identical to that of the slow MHC from the adult soleus muscle, not shown) and its reaction with slow MHC-specific mAbs RllDlO and NOQ754D (Khaw et aL, 1984; Narusawa et aL, 1987). The embryonic MHC isoform was identified based on its less rapid M, and its reaction with the embryonic MHC-specific mAb BF-45 (Schiaffino et al, 1989). The results with slow and embryonic MHC expression in embryonic muscle cell cultures confirmed those seen previously (Vivarelli et al, 1988). The MHCs expressed in differentiated cultures of embryonic limb muscle cells thus appeared to be the embryonic and slow MHC isoforms, whereas the MHCs expressed in cultures of fetal limb muscle cells were the embryonic, slow, and perinatal MHC isoforms.

F59hll) BF-45(emb)

----w

BF-34 (peril RllDlO(slow)

-

-

_

FIG. 1. Embryonic and fetal mouse myogenic cells expressed distinct patterns of MHC isoforms upon differentiation in culture. As indicated, MHCs were prepared from El1 + 5, El2 + 5, and El7 + 5 cultures; El8 mouse limb muscles (El8 limb); Postnatal Day 5 mouse hindlimb (P5 limb); and adult quadriceps muscle (Ad Quad). MHCs were analyzed by SDS-PAGE in 6% polyacrylamide, 37.5% glycerol gels and by immunoblotting with mAbs F59 (reacts with all MHCs), BF-45 (embryonic MHC), BF-34 (perinatal MHC), and RllDlO (slow MHC). Embryonic MHC (slowest mobility) and slow MHC (fastest mobility) were the predominant MHCs expressed in the El1 + 5 and 12 + 5 embryonic cell cultures, whereas the embryonic and perinatal (intermediate mobility) MHCs were the predominant MHCs expressed in the El7 + 5 fetal cell cultures. Embryonic, perinatal, and slow MHCs were coexpressed in the El8 limb; perinatal and a small amount of slow MHC were coexpressed in the P5 limb; and adult fast MHC (mostly Type IIB) was expressed in the adult quadriceps. In some cases, samples analyzed on different blots were juxtaposed for presentation. Detection of mAb binding was by horseradish peroxidase with diaminobenzidine substrate for mAbs F59, BF-45, and RllDlO and by alkaline phosphatase with NBT/BCIP substrate for mAb BF-34.

20

DEVELOPMENTAL BIOLOGY

mAb BF-34

mAb F47

FIG. 2. Comparison of perinatal MHC expressed in mouse muscle and fetal muscle cell cultures. One-dimensional immunopeptide mapping was used to compare MHCs from El8 mouse limb muscles (limb) and El8 + 10 limb cell cultures (culture). Partially proteolyzed fragments of the MHCs were analyzed by SDS-PAGE in 15% gels and immunoblotting with mAb BF-34 or mAb F47. Antibody binding was detected using an alkaline phosphatase-linked system and NBT/BCIP substrate (mAb BF-34) or a horseradish peroxidase-linked system and a chemiluminescent substrate (mAb F47). Perinatal MHCs from the El8 limb and differentiated cultures produced similar patterns of immunoreactive peptides. Minor low-molecular-weight bands identical to those seen in the BF-34 culture samples could also be seen in the limb MHCs when blots were visualized by chemiluminescence. Arrowheads point out one minor band that consistently appeared in F47 maps of culture, but not limb, MHCs (see text).

One-dimensional immunopeptide mapping with mAbs BF-34 and F47 was used to further compare the structure of the perinatal MHC expressed in the cultures with that of the perinatal MHC expressed in the animal. MHCs were isolated from El8 limb muscle and El8 + 10 muscle cell cultures. Partial proteolytic fragments of the MHCs were prepared, separated by SDS-PAGE in 15% gels, and tested for reactivity with mAb BF-34 or mAb F47. MHCs from the El8 limb muscle and the El8 + 10 cultures produced identical patterns of MHC fragments that reacted with the perinatal MHC-specific mAb BF-34 (Fig. 2). When mAb F4’7 was used, the major bands of the immunopeptide maps were always identical, but one or two additional minor bands were sometimes seen in the El8 + 10 culture samples that were not seen in the El8 limb samples (Fig. 2). These bands could have been due to differences in the amount of sample loaded, to the expression of additional F47-reactive MHCs (e.g., Types IIA, IIB, or 11X) in the cultured myotubes that were not expressed in the El8 limb, or to differences in structure-perhaps due to different posttranslational modifications. The immunopeptide mapping results suggested that the perinatal MHC expressed in the animal was very similar, perhaps identi-

VOLUME 149, 1992

cal, to the MHC isoform that reacted with the perinatal MHC-specific mAb BF-34 and was expressed in cultures of fetal mouse muscle cells. Immunocytochemical analysis of embryonic muscle cell cultures. Double immunofluorescence analysis was used to analyze MHC isoform expression patterns in individual myocytes and myotubes. These analyses confirmed the different perinatal MHC expression patterns of embryonic and fetal cultures that were seen by immunoblotting and in addition revealed heterogeneity of MHC expression patterns among the differentiated cells within single cultures. Muscle cells were prepared from somites or limbs of donors of different ages, cultured, and analyzed by double immunofluorescence as described under Materials and Methods. The results are presented in Table 2, Fig. 3, and the text. Consistent with the immunoblot analysis, very few of the differentiated cells formed from embryonic somite and limb muscle cells expressed the perinatal MHC isoform. When myogenic cells were prepared from E8.5El1 somites or Ell-El2 limbs, the MHC-expressing cells formed in the cultures after 4 to 7 days of incubation were almost entirely mononucleated myocytes; fewer than 5% contained two to four nuclei (Fig. 3) (Vivarelli et al, 1988). Somite, but not limb, cell cultures contained many neurons which originated from neural tube cells included in the dissections and formed very long, highly fasiculated neurites. Fewer than 5% of the myocytes in embryonic (El2 or earlier donor) muscle cell cultures stained positively with mAb F47 or perinatal MHC-specific mAb BF-34, indicating that the perinatal MHC isoform was expressed in only a small subset of the differentiated cells (Table 2, Fig. 3). The small percentage of perinatal MHC-expressing cells did not increase when culture duration was lengthened. In contrast, 93-99% of the myocytes formed from El2 or younger myoblasts stained with the slow MHC-specific mAb NOQ754D, and 100% of the myocytes (identified by staining with mAb F59) also stained with the embryonic MHC-specific mAb BF-45. When E12.5 + 5 cultures were doubly stained with mAbs F47 and NOQ754D, the small percentage of myocytes that stained with F47 and thus expressed perinatal MHC also stained with NOQ754D and thus also expressed slow MHC (n = 103). Within the detection limits of the double immunofluorescence analysis, therefore, differentiated cells formed from embryonic somite or limb muscle cells appeared to be of one predominant type and two minor types: (i) greater than 90% of the myocytes expressed both the embryonic and the slow MHC isoform, much as seen previously (Vivarelli et al, 1988); (ii) l-4% of the myocytes expressed embryonic, perinatal, and slow MHC;

SMITHANDMILLER

Perinatal

F59

I FIG. 3. Double immunofluorescence analysis of MHC isoform expression patterns in cultures of embryonic and fetal mouse muscle cells. As indicated, cells from embryonic (E12) and fetal (A, E18; B, E17) limbs were cultured, and the cultures were analyzed after 5 days of incubation by double immunofluorescence with mAbs F47 and F59 (A) or with mAbs NOQ754D (NOQ) and F59 (B). Binding of mAb F59 was detected with a Texas red-conjugated secondary antibody and binding of mAbs F47 and NOQ754D was detected with fluoresceinconjugated secondary antibodies (see Materials and Methods). Note that mAbs F47 and NOQ754D did not stain all of the myocytes and myotubes that stained with mAb F59. Cells that stained with mAb F47 or failed to stain with NOQ754D were rare in El2 muscle cell cultures. See Table 2 for quantitative analyses. Bar represents 20 pm.

and (iii) l-7% of the myocytes expressed only the embryonic MHC isoform. Immunocytochemical analysis of fetal muscle cell cultures. Diverse patterns of perinatal MHC isoform expression were found among the myocytes and the myotubes formed from the fetal limb muscle cells of El3 or older donors. To determine if myoblast fusion and myotube formation affected MHC expression, perinatal MHC isoform expression was separately analyzed in

MHC Expression in Vitro

21

mononucleated myocytes, which are formed from a single myoblast, and in multinucleated myotubes containing four or more nuclei. In El3 cultures, myocytes were the predominant MHC-expressing cells and myotubes were rare, whereas, in El&El8 cultures, myocytes usually amounted to 30-50% of the MHC-expressing cells and myotubes were abundant. Consistent with the immunoblot results, which suggested that the embryonic MHC isoform was abundant in fetal muscle cell cultures (Fig. 1), every myocyte and myotube examined in fetal muscle cell cultures stained with the embryonic MHC-specific mAb BF-45 (Table 2 and not shown) and thus appeared to express the embryonic MHC isoform. A substantial portion of the myocytes and myotubes in fetal muscle cell cultures also stained with mAb NOQ754D and thus appeared to express slow MHC (Table 2). The percentage of myocytes and myotubes that reacted with NOQ754D decreased as the age of the donor increased. In every case examined, mAb NOQ754D stained a smaller percentage of the differentiated cells in cultures of El3 or older donor cells than in cultures of El2 or younger donors. Similarly, Vivarelli et al. (1988) found that slow MHC is expressed by all MHC-expressing cells in somitic and embryonic muscle cell cultures and in a proportion of the myotubes in fetal muscle cell cultures. Perinatal MHC was expressed in a much larger percentage of the myocytes formed in fetal cell cultures (defined here as from El3 or older donors) than in embryonic cell cultures (El2 or younger donors). For example, about one-third of the myocytes that formed from El3 limb cells and reacted with mAb F59 also reacted with mAbs F47 or BF-34 and thus expressed perinatal MHC (Table 2). In contrast, less than 5% of the myocytes formed from El2 donor cells expressed perinatal MHC. The proportion of myocytes that expressed perinatal MHC tended to increase as the age of the donor increased from El3 to El8 or the duration of culture was lengthened from 3 to 6 days, but these trends were not always seen (Table 2). When comparing cultures prepared on different days from the same age donor, the percentage of perinatal MHC-expressing cells that was observed varied, probably because differences in initial culture densities affected the rate of differentiation. Myocytes that did and did not express perinatal MHC were often located adjacent to each other in these highdensity cultures (Fig. 3). Perinatal MHC appeared to be expressed in a higher percentage of myotubes than myocytes in cultures of fetal muscle cells. Initial observations suggested that perinatal MHC was expressed by nearly all myotubes (four or more nuclei) in El8 muscle cell cultures, even in those cases when a significant percentage of the myo-

22

DEVELOPMENTAL

BIOLOGY

cytes did not express perinatal MHC (e.g., El8 + 4 culture in Table 2). For further analysis, El8 limb muscle cells were cultured at relatively low density (-500 cell/ cm’) and sister cultures were observed by double immunofluorescence with mAbs F47 and F59 after different durations of incubation. These cultures differentiated more slowly than the higher density cultures. After ‘7 days of culture, 89% of the myotubes (n = 98) and 37% of the myocytes (n = 210) that stained with mAb F59 also stained with mAb F47. After 10 days of culture, 95% of the myotubes (n = 124) and 54% of the myocytes (n = 268) stained with mAb F47. To further analyze myogenic cell heterogeneity in fetal muscle cell cultures, MHC isoform expression patterns were analyzed in clonally derived muscle colonies and cytosine arabinoside-(AraC) treated cultures. For the clonal analysis, El8 limb cells were cultured at clonal density (see Methods) for 7-10 days to allow muscle colonies to form. The MHC isoform expression patterns among the myocytes and myotubes within each colony were then determined by double immunofluorescence with mAbs F47 and F59. The results showed that most muscle colonies were composed of a heterogeneous population of MHC-expressing cells, some of which expressed perinatal MHC and some of which did not (Fig. 4). In one experiment, for example, El8 limb cells were cultured at clonal density and incubated for 7 days until myotube formation had begun, and 58 muscle colonies were examined. Forty five of these colonies (77.6%) were found to contain a mixture of F47-positive and F47-negative myocytes and myotubes; 12 colonies (20.7%) contained only F47-positive muscle cells; and 1 colony (1.7%), composed of only 6 myocytes, contained only F47-negative muscle cells. At the time of examination, colonies consisted of 5-50 myocytes and small myotubes and many undifferentiated myoblasts. The percentage of the MHC-expressing cells that expressed perinatal MHC within the colonies varied. In some colonies only one or two cells expressed perinatal MHC, whereas in other colonies only one or two cells failed to express perinatal MHC. Within muscle colonies (Fig. 4), as in high-density cultures (Fig. 1, Table 2), heterogeneity of perinatal MHC expression was found mostly among myocytes, whereas almost all myotubes expressed perinatal MHC. A clonal analysis of embryonic myogenic cells was not performed because culture conditions under which embryonic cells form muscle colonies have not been determined (cf. Vivarelli et aZ., 1988). In the second set of experiments, nearly confluent El8 limb cell cultures were either left untreated or treated with 1 pg/ml AraC. AraC efficiently killed dividing cells and stopped the formation of new myocytes and myotubes (cf. Miller and Stockdale, 1989). Perinatal MHC

VOLUME

149.1992

F59

F47

FIG. 4. MHC isoform expression patterns in myocytes and myotubes within a fetal muscle colony. Cells from fetal (EN) limbs were cultured at clonal density and the differentiated cells within a single muscle colony that formed after 7 days of incubation were analyzed by double immunofluorescence with mAbs F47 and F59 as indicated. Binding of mAb F59 was detected with a Texas red-conjugated secondary antibody and binding of mAb F47 was detected with a fluoresceinconjugated secondary antibody (see Materials and Methods). In the portion of the muscle colony shown here, two myocytes failed to stain with mAb F4’7 (arrows), whereas other myocytes and two myotubes did stain with mAb F47. Bar represents 25 pm.

expression patterns were analyzed by double immunofluorescence in the untreated and AraC-treated cultures after different durations of culture. In each such experiment, the percentage of cells that expressed perinatal MHC (i) was higher in AraC-treated than in untreated cultures and (ii) increased as culture duration lengthened in both untreated and treated cultures. For example, in one high-density El8 + 6 culture which was treated with AraC from Day 1.5 to Day 4,100% (n = 72) of the myotubes and 93% (n = 151) of the myocytes expressed perinatal MHC, whereas in the untreated parallel culture, 99% (n = 79) of the myotubes and only 69% (n = 182) of the myocytes expressed perinatal MHC. In an additional set of lower density El8 cultures, the percentage of F47-positive myocytes was 54% (n = 273) in El8 + 5 cultures, at which time AraC was added to half

SMITH AND MILLER

Perinatal

of the parallel cultures. Two days later, El8 + 7, the percentage of F47-positive myocytes had increased to 79% (n = 298) in AraC-treated cultures but had remained at 54% (n = 284) in untreated cultures, and 4 days later, El8 + 9, the percentage of F47-positive myocytes had further increased to 93% (n = 300) in the AraC-treated cultures and had increased to 82% (n = 357) in the untreated cultures. The percentages of myocytes and myotubes formed in these cultures did not appear to be affected by AraC, as 30-50% of the differentiated cells were myocytes in all cases. Longer experiments were not performed, because the total number of MHC-expressing cells decreased after Day 9 in both treated and untreated cultures, apparently due to the limited lifetime of the cells in culture (cf. Miller and Stockdale, 1989). Because AraC-treated cultures contained a higher percentage of cells that expressed perinatal MHC than did the untreated cultures, it appeared that older myocytes and myotubes were more likely to express perinatal MHC than newly formed myocytes and myotubes. Within individual myotubes in fetal muscle cell cultures, the staining due to mAb F47 was in most cases coincident with the staining due to mAb F59, suggesting that the perinatal MHC isoform was distributed throughout the MHC-containing regions of the myotube (Fig. 5A). In a small number of myotubes amounting to less than 1% of the total, however, staining with mAb F47 was restricted to a much smaller region of the myotube than was staining with mAb F59 (Figs. 5B and 5C). In such myotubes, staining with mAb F47 appeared to be restricted to the vicinity of only a subset of the nuclei in the myotube. In Fig. 5C, for example, staining with mAb F47 was largely restricted to the region surrounding only one of the five nuclei in the myotube. Thus, in addition to the predominant type of myotube that expressed perinatal MHC throughout the cell, and the less abundant type of myotube that did not express any detectable perinatal MHC, there was a small population of mosaic myotubes in which perinatal MHC accumulated in only a portion of the MHC-containing regions of the myotube. DISCUSSION

Early embryonic and late fetal mouse myogenic cells showed distinct patterns of perinatal MHC expression upon differentiation in vitro. In cultures of fetal muscle cells from El3 or older mice, the perinatal MHC isoform amounted to a large proportion of the total MHC and was expressed in a large percentage of myocytes and myotubes. In embryonic somite or limb muscle cell cultures, in contrast, very few differentiated cells ex-

MHC Expression in Vitro

23

A

B

C

FIG. 5. MHC isoform expression patterns in myotubes formed from fetal mouse muscle cells. Cells from fetal (E18) limbs were cultured and the differentiated cells that formed after 5 days of incubation were analyzed by double immunofluorescence with mAbs F4’7 and F59 as indicated. Binding of mAb F59 was detected with a Texas red-conjugated secondary antibody and binding of mAb F47 was detected with a fluorescein-conjugated secondary antibody (see Materials and Methods). In most myotubes, identical staining patterns were observed with mAbs F47 and F59 (A). In a small number of myotubes, mAb F47 stained a smaller region of the myotube than mAb F59 (B, C). Note also in A and B that some myocytes and myotubes reacted with mAb F59 but did not react with mAb F47. Bar represents 15 pm.

pressed perinatal MHC. These patterns of perinatal MHC expression suggest that embryonic and fetal myoblasts are different types of myogenic cells which express distinct myogenic programs upon differentiation. These results are consistent with and extend previous work on myoblast heterogeneity in the mouse (Hauschka et CZL,1979; Cossu and Molinaro, 1987; Vivarelli et aZ., 1988). The previous studies demonstrated that early and late mouse myoblasts differ in ability to form clonal muscle colonies, responses to phorbol esters, and expression of several proteins. In addition, Vivarelli et al. (1988) showed that the differentiated cells formed by early myoblasts in culture coexpress embryonic and

24

DEVELOPMENTALBIOLOGY

slow MHCs, whereas those formed by late myoblasts mostly express only embryonic MHC. Perinatal MHC expression was not found previously, perhaps due to use of different antibodies and electrophoresis systems, mouse strains, or culture conditions. The pattern of perinatal MHC expression in culture is thus an additional characteristic that distinguishes between the myogenic programs of embryonic and fetal muscle cells. MHC expression in embryonic cell cultures resembled the initial MHC expression pattern of primary myotubes. Slow and embryonic MHCs were coexpressed in cultures of early embryonic muscle cells and are also coexpressed in primary myotubes soon after they form in the developing animal (Vivarelli et al., 1988; Condon et al, 1990a). Similarly, coexpression of the perinatal and embryonic MHCs in cultures of late fetal muscle cells resembled the initial MHC expression pattern in secondary myotubes. Secondary muscle fibers in the rat leg coexpress embryonic and perinatal MHCs as soon as the fibers begin to form on about E&E19 (Condon et al, 1990a). Perinatal MHC expression is also seen in a subset of primary muscle fibers in the rat leg beginning on E16,1-3 days after the primary fibers form. Restriction of perinatal MHC expression to secondary fibers and a subset of primary fibers is not initially affected by denervation (Condon et aZ., 1990b). Perinatal MHC expression in culture also did not appear to depend on neurons, because perinatal MHC was expressed in aneural fetal muscle cell cultures, and coculture of somitic cells with neurons did not increase perinatal MHC expression above the low level seen in embryonic limb muscle cell cultures. The present results are consistent with the possibility that primary muscle fibers may initially be formed from early embryonic myoblasts and that secondary fibers may initially be formed from late fetal myoblasts. When cultured under identical conditions, embryonic and fetal myoblasts had intrinsically different abilities to express perinatal MHC upon differentiation. The molecular basis of this difference is unknown but could arise if embryonic and fetal myogenic cells express distinct patterns of the regulators of perinatal MHC gene transcription, intracellular signaling pathways, or receptors for extracellular signals, or if embryonic cells require a different culture environment to express perinatal MHC. In the animal, embryonic myoblasts and the myotubes they form might be influenced by neurons, fusion with fetal myoblasts (Allen et aZ., 1979), and other extracellular signals (Kelly, 1983; Miller, 1991). In culture, however, perinatal MHC expression was not dependent on nerves, and fusion was also not required because most mononucleated myocytes formed from fetal myoblasts did express perinatal MHC. Because embry-

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onic and fetal myoblasts likely coexist in the El3 mouse limb (Table 1) and embryonic myoblasts do not appear, at least in birds, to be precursors of fetal myoblasts (Rutz and Hauschka, 1982; Seed and Hauschka, 1984), the embryonic and fetal myoblasts appear to be distinct myogenic cell types which are committed to expressing distinct myogenic programs as reflected in the pattern of perinatal MHC expression. Though embryonic and fetal myoblasts expressed distinct myogenic programs, the heterogeneity of perinatal MHC expression among the myocytes and myotubes in fetal muscle cell cultures appeared to be due to the modulation of MHC isoform expression rather than to the presence of different types of fetal myoblasts (cf. Miller and Stockdale, 1989). That is, many myocytes and myotubes in fetal cultures did not appear to initially express perinatal MHC, but did appear to initiate synthesis as duration of culture was lengthened. Heterogeneity of perinatal MHC expression in fetal cultures apparently arose due to the initiation of expression at different times in different nuclei. This mechanism was supported by both the clonal analysis, which showed that muscle cells with different perinatal MHC expression patterns could form among the progeny of a single fetal myoblast, and the AraC experiments, which showed that more muscle cells expressed perinatal MHC as culture duration was lengthened. In addition, the small number of mosaic myotubes found here might have arisen due to differential perinatal MHC gene expression in different nuclei and subsequent restricted diffusion of the perinatal MHC protein (Salviati et cd, 1986; Pavlath et ab, 1989; Hall and Ralston, 1989; D. A. Harris et al., 1989). Nuclei with qualitatively or quantitatively different perinatal MHC isoform expression patterns thus appeared to coexist within myotubes in the absence of innervation or other external signals (cf. Harris, D. et aZ., 1989). Both commitment and modulation mechanisms have been found to regulate perinatal MHC expression in mouse muscle cell lines. Cells of the BCSH-1 and BDlO lines, for example, fail to accumulate perinatal MHC protein upon differentiation (Miller, 1990) and thus appear to be committed to expressing a different pattern of MHCs than cells of lines such as C,/7 which markedly increase expression of perinatal MHC mRNA when culture duration is lengthened (Cox et cd, 1991). The different patterns of perinatal MHC expression in cultures of embryonic and fetal mouse muscle cells suggest that myoblast heterogeneity might underlie the initial formation of primary and secondary muscle fibers. Cultures of untransformed embryonic and fetal myogenic cells, as well as muscle cell lines (Miller, 1990), will be useful models for studying the molecular mechanisms of commitment and modulation which lead to the

SMITH AND MILLER

fiber type-specific patterns of perinatai sion seen in the developing animal.

Perinatal

MHC expres-

We thank Dr. Kathleen M. Buckley for excellent advice; Dr. Doug Falls for chicken embryo extract; and Drs. Alan Kelly (University of Pennsylvania), Phillip Nicholl (Massachusetts General Hospital), Stefano Schiaffino (University of Padova), and Frank Stockdale (Stanford University) for gifts of monoclonal antibodies. This work was supported by the NIH, the Cecil B. Day Foundation, and the W. R. Hearst Foundation. Jeffrey B. Miller is an Established Investigator of the American Heart Association. REFERENCES ALLEN, R. E., MERKEL, R. A., and YOUNG, R. B. (1979). Cellular aspects of muscle growth: Myogenic cell proliferation. J. Anim. Sci 49,115 127. BANDMAN, E. R. (1985). Continued expression of neonatal myosin heavy chain in adult dystrophic skeletal muscle. Science 227, 780782. CERNY, L. C., and BANDMAN, E. R. (1986). Contractile activity is required for the expression of neonatal myosin heavy chain in embryonic chick pectoral muscle cultures. J. Cell Biol. 103,2153-2161. CLEVELAND, D. W. (1983). Peptide mapping in one dimension by limited proteolysis of sodium dodeeyl sulfate-solubilized proteins. In “Biomembranes, part J” (S. Fleischer and B. Fleischer, Eds.), Vol 96, pp. 222-229. Academic Press, New York. CONDON, K., SILBERSTEIN, L., BLAU, H. M., and THOMPSON, W. J. (1990a). Development of muscle fiber types in the prenatal rat hindlimb. Dev. BioL 138,256-274. CONDON, K., SILBERSTEIN, L., BLAU, H. M., and THOMPSON, W. J. (1990b). Differentiation of fiber types in aneural musculature of the prenatal rat hindlimb. Dev. Biol. 138,275-295. Cossu, G., and MOLINARO, M. (1987). Cell heterogeneity in the myogenie lineage. Cum: Top. Den Biol 23,185-208. Cox, R. D., WEYDERT, A., BARLOW, D., and BUCKINGHAM, M. (1991). Three linked myosin heavy chain genes clustered within 370 kb of each other show independent transcriptional and posttranscriptional regulation during differentiation of a mouse muscle cell line. Dev. Biol. 143,36-43. CROW,M. T., OLSON, P. S., and STOCKDALE, F. E. (1983). Myosin light chain expression during avian muscle development. J. Cell Bid. 96, 736-744. CROW, M. T., and STOCKDALE, F. E. (1984). Myosin isoforms and the cellular basis of skeletal muscle development. Ex~. Biol Med 9, 165-174. DANIELI BETPO, D., ZERBATO, E., and BET~Q R. (1986). Type 1,2A, and 2B myosin heavy chain electrophoretic analysis of rat muscle fibers. Biockem. Biophys. Res. Commun. 138,981-987. EMERSON, C. P., JR., and BERNSTEIN, S. I. (1987). Molecular genetics of myosin. Annu. Rev. Biochem. 56,695-726. EVANS, D., MILLER, J. B., and STOCKDALE, F. E. (1988). Developmental patterns of expression and coexpression of myosin heavy chains in atria and ventricles of the avian heart. Dev. Biol. 127,376-383. HALL, Z. W., and RALSMN, E. (1989). Nuclear domains in muscle cells. Cell 59, 771-772. HARRIS, A. J., FI~SIMONS, R. B., and MCEWAN, J. C. (1989). Neural control of the sequence of expression of myosin heavy chain isoforms in foetal mammalian muscles. Development 107,751-769. HARRIS, D. A., FALLS, D. L., and FISCHBACH, G. D. (1989). Differential activation of myotube nuclei following exposure to an acetylcholine receptor-inducing factor. Nature 337,173-176. HAUSCHKA, S. D., LINKHART, T. A., CLEGG, C., and MERRILL, G. (1979).

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