DEVELOPMENTAL DYNAMICS 193:359-369 (1992)
Differences in the Histogenesis of EDL and Diaphragm in Rat LING YIPING, DENAH APPELT, ALAN M. KELLY, AND CLARA FRANZINI-ARMSTRONG Departments of Anatomy (C.F.-A.),Biology (L.Y.,D.A.) and Veterinary Pathobiology (A.M.K.), University of Pennsylvania, Philadelphia, Pennsylvania 19104
ABSTRACT We have examined the histogenesis of the diaphragm and extensor digitorum muscle in rat embryos, with the aim of defining differences in developmental patterns that can be related to the functional requirements of these muscles during and after development. Patterns of interactions between myotubes and other cells, and frequency of gap junctions are quite different in the two muscles. In diaphragm, primary myotubes (at day 16 in utero) are closely associated with each other, forming parallel sheets or palisades and communicating by gap junctions. Secondary myotubes have formed by day 18, but are immature, and the frequency of gap junctions is lower. The arrangement in palisades is maintained even after fibers are separated from each other by their individual basal lamina. In EDL primary fibers at day 16 have fewer gap junctions, and the peak in communication occurs afterthe appearanceof secondary myotubes (day 18 and 21). Secondary myotubes are more mature than in diaphragm at day 18. 0 1992 Wiley-Liss, Inc.
Key words: Gap junctions, Muscle development, Rat muscle, Diaphragm, EDL INTRODUCTION Skeletal muscle is progressively assembled from succeeding generations of cells. Early in myogenesis the primordium is established by a small population of primary myotubes. This is then amplified by the addition of variable numbers of secondary myotubes that use the walls of primary myotubes as a scaffold. Subsequently the myofibers in these clusters cells separate and become independent (Kelly and Zacks, 1969; Ontell and Kozeka, 1984; Chiu and Sanes, 1985; Duxson et al., 1989). In the present study we have examined general muscle organization and the incidence of intercellular junctions in the developing diaphragm and EDL of fetal and neonatal rats. We have focused on the incidence of gap junctions because intercellular communication plays an important role at critical moments during cell differentiation and tissue morphogenesis (Potter et al., 1966; Warner, 1973; Spitzer, 1982; Cavaney, 1985) and, in some systems, it is necessary for orderly em0 1992 WILEY-LISS, INC
bryonic development (Warner et al., 1984; Fraser et al., 1987). We have questioned whether muscle histogenesis is stereotyped or whether each muscle has a singular pattern of assembly that may be linked to its function during embryogenesis andlor with its anticipated role a t later stage. From their inception, the functions of diaphragm and EDL differ from each other. The diaphragm must form a coherent barrier between the unfolding thoracic and peritoneal cavities and subsequently withstand mounting trans-diaphragm pressure. In addition, the fetal diaphragm must be differentiated enough to support the abrupt onset of respiration a t parturition, and it prepares by rhythmically contracting in utero (Dawes, 1984). Fetal respiratory movements and the beginning of respiration are thought to play a critical role in differentiation of the tracheobronchial tree. The EDL is not subject to equivalent lateral pressures, and intrauterine and postpartum movements are slow and less active. We find that EDL and diaphragm follow the same general rules of development: At least two waves of myotube formation, transient cell adhesion and communication, followed by a separation of independent fibers. However, there are clear differences, which are noticeable from very early stages, before innervation and/or hormonal influences occur, in the patterns of development of the two muscles. MATERIALS AND METHODS Electron Microscopy Rat embryos a t 16,18,21,and 22 days in utero (E1622, plug day = 0) were obtained following C02 euthanasia of the dam. Pups at 0 and 5 days after birth were similarly euthanised. At E l 6 entire hind limbs were fixed, a t E l 8 and later the EDL was partially dissected at the distal end, pinned away from the leg and fixed for about one hour, then completely dissected. Diaphragms were dissected with a rim of rib cage and pinned for the initial fixation. The muscles were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer, con-
Received September 12, 1991; accepted April 13, 1992. Address reprint requestsicorrespondence to Dr. Clara FranziniArmstrong, Department of Anatomy, University of Pennsylvania, Philadelphia, PA 19104-6058. Dr. Ling Yiping’s current address is Laboratory of Electron Microscopy, Shangai Medical University, Shangai 200032 China.
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taining 5 mM sucrose and 0.1% tannic acid. Fixation was at room temperature for approximately two hours, then the tissues were stored for 1-15 days in the fixative at 4°C. For thin sectioning the muscles were postfixed in 2% OsO, in 0.1 M cacodylate buffer and 0.05 M sucrose for 30 min (or alternatively, in 2% OsO,, 0.8% K,Fe(CN)6), en block stained in 2% uranyl acetate in 70% EtOH for 30 min, dehydrated, and embedded in Spurr. One set of diaphragms, from E16, E18, and E21 in utero were fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer and 0.013% CaC1, overnight at room temperature. Post-fixation was as above. Thin sections were stained in uranyl acetate and lead. For freeze-fracture muscles were fixed in glutaraldehyde, as above, but without tannic acid. The fixed muscles were infiltrated in up to 30% glycerol, frozen in Freon 12 and fractured and replicated in a Balzer’s 400. Platinum shadow was at 45”. Sections and replicas were photographed in JEOL JEM 1005 and Philips 410 microscopes. Photographs for quantitative analysis were taken as follows: a) Cross sections of the entire muscle (for EDL), or of approximately 1mm wide segments (for the diaphragm) were mounted on bare 400 mesh grids. b) Freeze fracture replicas were obtained from longitudinally oriented muscles, and all available replica pieces were mounted on bare grids. All visible areas of replicas and thin sections were photographed at a magnification of 3,000 x . Each grid square measured approximately 2,000 pm, and was covered by a set of overlapping micrographs.
RESULTS General Description At E l 6 the primordia of both diaphragm and EDL are composed of primary myotubes only. The myotubes in the diaphragm are more developed: they have a larger diameter and more myofibrils. In EDL primary myotubes are circular in profile (triangles, Fig. la), and are closely, but randomly intercalated with a number of mononucleated cells (myoblasts, presumptive myoblasts, and fibroblasts). In the diaphragm the situation is different: primary myotubes (triangles, Fig. 2a) form a tight arrangement in long rows (palisades), mostly relegating the mononucleated cells at the periphery of the rows. Proximity between primary myotubes is more prevalent than in EDL, and the cells are so packed that they take a polygonal profile. Mesenchymal cells with polyribosome-studded cytoplasm insert themselves in the spaces between the myotubes at the periphery of the palisades. At E l 8 primary myotubes have moved apart in both muscles, and secondary ones are intercalated in between. Again however, the pattern is different in the two muscles. In the EDL (Fig. lb) each circular profile of cross-sectioned primary myotube (M) has its surrounding group of secondary myotubes (m) and mononucleated cells, forming fairly well defined individual clusters; in the diaphragm (Fig. 2b) primary and sec-
ondary myotubes form tight palisades, where one or two secondary myotubes andlor mononucleated cells squeeze between the primary ones. The fibers maintain polygonal profiles, and face each other over long distances. Fibroblasts tend to take a position peripheral to the developing clusters of cells a t E l 8 and later. Bundles of collagen fibers are more evident in diaphragm than in EDL at early stages of development. The basal lamina, initially very delicate, surrounds clusters of primary and secondary myotubes and the muscle fibers derived from them. At early stages (e.g., day 18, Fib. lb) the cluster boundaries in EDL are not clearly defined because the basal lamina is hardly visible and mononucleate cells contact more than one cluster. At later stages, the muscle fibers forming clusters gradually separate from each other. In EDL separation of all fibers is not completed until several days after birth (Ontell, 1979). In diaphragm the separation is almost complete at birth, but the fibers remain polygonal and tightly clustered, although a t that point they are mostly separated from each other by a layer of basal lamina. Thus, in addition to having a different packing, diaphragm matures earlier, and EDL follows. Innervation also occurs earlier in the upper body: Intercostal muscles are innervated a t day 14, and axons are present in diaphragm at that age (Dennis, 1981; Dennis et al., 1981). In EDL axons are present at day 15, and primary myotubes are innervated at day 16 (Harris, 1981).
Cell to Cell Contacts All cells form contact^" with each other (Fig. 3a, between arrows, and Fig. 3b). For the purpose of our description, we define “contact” as an area of cell surface which closely approximates the surface of another cell. Along contact areas the cell profiles lie at a distance of 10-20 nm from each other and the basal lamina is excluded (Fig. 3a,b, between short arrows). This allows opportunity for the formation of numerous focal junctions of the adhering type, or spot adhesions, distinguished by intracellular densities and proximity of the adhering membranes (Fig. 3a,b, small arrowheads). Gap junctions, in which the adjacent cell membranes are quite straight and parallel to each other across a narrow gap, are less frequent (Fig. 4a). The pattern of cell to cell contact changes with age and shows some differences between EDL and diaphragm. This is clearly demonstrated by the rough quantitation given below. Quantitation of Cell Contacts Cross sections were cut across the approximate middle of the muscle mass. For purposes of quantitation, cell contacts were divided into four categories: extra short contacts, at which the cells interact over a very short distance (less than 0.4 pm); short contacts, which occupy a cell boundary length of 0.4-3.3 pm; medium contacts with a length of 3.4-10 p m ;and long contacts, with lengths above those.
GAP JUNCTIONS IN HISTOGENESIS
Fig. 1. a: Cross section of the EDL at E16. Primary myotubes (triangles) have circular profiles and are separated by numerous cells, mostly myoblasts, with which they make multiple "contacts" (see text). There are also some contacts between myotubes. Outlines of most cell are complex and the contacts tend to be short. x 3700. b: Cross section of EDL at
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E18. Primary myotubes (M) are mostly separated from each other, and secondary myotubes (m) are closely associated with their periphery, forming extensive contacts. Numerous cells are still present between the myotubes and form short contact with them, including fibroblasts (f). x 3700.
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Fig. 2. a: Primary myotubes in diaphragm at E l 6 are more closely clustered than in EDL. Triangles mark a set of closely apposed primary myotubes which form a palisade by making extensive contacts with each other, to the exclusion of other cells. x 2800. b: Diaphragm at E18. Myoblasts and secondary myotubes (m) are present between primary
Note however, that the arrangement remains much more myotubes (M). tight than in the EDL. Contacts tend to be long. Primary and secondary fibers in diaphragm at 16 and 18 days of gestation are more developed than those in EDL at the same age. f: fibroblast. x 2800.
GAP JUNCTIONS IN HISTOGENESIS
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Fig. 3. a: Contacts between two primary myotubes (M) and a cell with rough ER, which might be a fibroblast (F) intercalated between them. Most contacts are "short" in this image (between arrows). Small arrowheads point to focal adhering junctions within areas of contact. EDL, El 6 .
x 18,000. b: Extensive, "long" contact between a primary (bottom) and secondary (top) myotube in EDL, E21. Small arrowheads point to focal adhering junctions. x 18,000.
Data collection involved 3 steps: 1) cell profiles were identified as: a) profiles containing myofibrils (myotubes, M, Table 1); b) profiles not containing myofibrils (nmf, Table 1).The latter category includes a variety of cells, see below. 2) The number of contacts between the first 50 complete myotubes profiles and their immediate neighbours were counted and categorized. 3) From these data we calculated (Table 1): the relative frequencies of contacts between adjacent myotubes (M-M) and of contact between myotubes and
non-myofibrillar cell profiles (M-nmf ); the average number of contact for each myotube profile; the relative frequencies of contacts of various lengths; and the average length of myotube profile occupied by contacts. The latter was estimated by multiplying the number of contacts in the four categories by their average length (0.20, 1.85, 6.70, and 13.3 Km). This of course is not a precise morphometric approach, and the numbers obtained are not exact. However, the separation of junctional lengths into categories is very useful
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Fig. 4. a: Gap junction between primary (left) and secondary (right) myotubes in EDL, E21. x 36,000. b: One large and two small gap junctions (arrows) on the surface of a myotube in diaphragm, E18. Junctions like these are often found on long stretches of the myotube surface indicating that the association between primary and secondary myotubes extends over relatively long distances. Semicircles indicate sites of pe-
ripheral couplings. x 42,000. c: Gap junction (lower left) and peripheral couplings (semicircles) on a myotube in the diaphragm, E18. Asterisks indicate opening of caveolae and/or T tubules. x 19,000, d: Gap junction on a myotube in diaphragm, E16. This is most likely to be a junction between two primary myotubes. x 62,000.
in giving a semiquantitative assessment of trends that are visible to the eye. The myofibrillar (M) and non-myofibrillar (nmf) cell categories lump profiles of different cells at different ages (Fig. 5a-f). At E l 6 all myofibrillar cells are primary myotubes; the non-myofibrillar cells between them are mostly myoblasts and a few fibroblasts. Both
types of nmf cells make brief but numerous contacts with the myotubes and are lumped together, because the distinction between them is not always very clear (Figs. l a , 2a, 3a, 5a and b). At El8 myofibrillar profiles are from primary and secondary myotubes; most nonmyofibrillar profiles are from young developing myotubes, which are closely associated to the surface of the
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TABLE 1. Contacts Between Developing Myotubes and Other Cells in EDL and Diaphragm
Muscle 16 DD EDL 18 DD EDL 21 DD EDL 22 DD EDL 16 DD Diaph 18 DD Diaph 21 DD Diaph
Contacts" M-M 23 44 44 33 50 16 63
total) M-nmf
(% of
Number of
contacts/ myotube
77 56 56 67 50 84 37
Xsmall
Frequency of contacts % of total
8.4 k 3.0 68 4.4 2 2.3 58 2.1 k 1.4 27 1.1 2 0.7 5 5.6 k 1.7 32 3.6 f 1.9 23 2.3 k 1.1 "M-M contacts between cell profiles containing myofibrils (myotubes);M-nmf
Small 26 24 25 26 21 29 4
Medi-
um
Long
6 17 42 62 45 43 78
6 7 2 5 18
Total length of contacts/ myotube (pm) 8.9 4.9 7.6 ? 4.1 8.4 ? 6.9 6.4 k 5.3 16.4 k 7.7 16.5 2 9.7 16.6 ? 7.6
*
contacts between myotubes and cell profiles containing no myofibrils. These include myoblasts, presumptive myoblasts, fibroblasts and some secondary myotubes which happened to be sectioned at a level devoid of myofibrils.
myotubes and either have not yet developed myofibrils, or are cut near the ends (Figs. lb, 2b, 5c,d; Ontell, 1979). These developing myotubes, although apparently devoid of myofibrils, are distinguishable from myoblasts and fibroblasts, because they are more closely apposed to the myotube surface, they tend to form longer contacts and often push into the outline of the older myotubes (Fig. 3b). Myoblasts and fibroblasts are at the periphery of the clusters and palisades of myotubes. At E21 (Figs. 5e,f) and later, most non-myofibrillar cells forming junctions with myotubes are either developing myotubes or future satellite cells. Both types of cells form relatively long contacts with the more developed myotubes. The quantitation of cell contacts shown in Table 1 indicates some interesting common trends and some differences between EDL and diaphragm. Common trends are: a) The number of contacts per myotube decreases with age. Student's T test indicates that within each muscle the difference between each time point and the preceeding one is significant at a level better b) The length of contacts increases with than P < age: The relative frequency of extra short contacts declines and that of medium and long ones increases. This is probably directly related to the decrease in population of myoblasts. c) As a result of these opposite trends, the roughly estimated total length of contact per myotube remains approximately constant with age. d) In both muscles, primary myotubes make contacts with each other before secondary myotubes appear. The apparent discrepancy between rat EDL, in which contacts exist between primary myotubes, and mouse EDL, in which primary myotubes are completely separated from each other (Ontell et al., 1988>,may be due to a small difference in the developmental stage a t which the muscles were examined. There are four important differences between EDL and diaphragm (see diagram in Fig. 5): a) In EDL myotubes tend to form shorter contacts. The difference is clearly visible if one compares Figure l a and l b with Figure 2a and 2b, and it is a reflection of the different geometries of the two muscles, which was noted in the
general description. The average total length of contact per myotube is longer in diaphragm than in EDL a t all ages. The difference is statistically significant a t all ages (P < b) At E l 6 the diaphragm has a higher incidence of contacts between primary myotubes than the EDL. This is visible in Figures l a and 2a, and it also a reflection of the more compact arrangement of primary myotubes in the diaphragm at this age. c) EDL, on the other hand, has a higher incidence of myotube-to-myotube contacts than diaphragm at E18. This is due to a higher incidence of developing secondary myotubes which contain myofibrils in the EDL at that age. d) The trend is apparently reversed a t E21, when diaphragm has a larger incidence of myotube to myotube contacts than the EDL. This has two possible explanations: Either the EDL is less mature, and the nmf profiles belong to a population of developing myotubes, or it is more advanced and the nmf profiles belong to satellite cells. Persistence of contacts between myotubes and other cells in EDL at E22 in utero indicates that as in the mouse (Ontell, 19791, some fibers are still grouped in clusters at this stage. Interestingly the diaphragm also maintains a low density of contacts until a t least the day of birth. These were not counted.
Quantitation of Gap Junctions Gap junctions between myotubes are seen in thin sections (Fig. 4a). In freeze-fracture sizes of gap junctions vary greatly (Fig. 4b,c). We have obtained estimates of the frequency of gap junctions at various stages of differentiation for EDL and diaphragm using the freeze fracture technique (Fig. 4b-d).The first problem was that of differentiating split membranes belonging to myotubes from those of other cells. In most cases, the elongated approximately cylindical shape of the myotubes is sufficient for identification. Smaller fragments of membrane, particularly for younger myotubes, were identified using the following criteria: presence of caveolae (Fig. 412, asterisks); random distribution of intramembranous particles of variable sizes; and presence of junctional
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5a
U
5c
n
5e
5f
Fig. 5. a-f: Diagrams of cell profiles encountered in EDL (a, c, and e) and diaphragm (b, d, and f) at various ages. At E l 6 (a,b) both primordia are composed of primary myotubes (M,), containing myofibrils, and myoblasts (m) and fibroblasts (f), which are non-myofibrillar. Contacts between myotubes are in general longer than contacts between myotubes and non-myofibrillar cells. M,-M, contacts are more numerous in diaphragm. At E l 8 (c,d) most contacts are between primary (M,) and sec-
ondary (M,) myotubes. Fibroblasts have a more peripheral position relative to bundles of myofibers. Note that secondary myotubes are more frequently fibrillar in EDL than in diaphragm at this stage. The diaphragm maintains a "palisade" arrangement of cells. At E 21 contacts are fewer and are either between myotubes or between myotubes and future satellite cells (s).
tetrads at peripheral couplings (Fig. 4b,c, semicircles, see Franzini-Armstrong et al., 1991). Endothelial cells also have caveolae, but their intramembranous particles are far more uniform in size, and tight junctions
are often visible. Fibroblasts and myoblasts have very few, if any, caveolae. All junctions belonging to myotubes were identified in low power micrographs covering the entire area of
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TABLE 2. Rough Estimate of Gap Junction Frequency on Surface of Myotubes at Various Stages of DeveloDment
Muscle 16 DD EDL 18 DD EDL 21 DD EDL 22 DD EDL BIRTH EDL 16 DD DIAPH 18 DD DIAPH 21 DD DIAPH 22 DD DIAPH BIRTH DIAPH
No. =ids 6 9 6 6 3 13 9 18 4 9
No. gap
No. sauares
No. gap iunctions
junctions/ sauare
27 66 77 37 47 127 107 166 34 167
7 49 86 6 5 158 76 20 4 6
0.26 0.74 1.12 0.16 0.11 1.24 0.71 0.12 0.12 0.06
TABLE 3. Frequency of Gap Junctions on Cell Surface of Myotubes at Selected Stages of Development
Total
Total
measured number Number eaD Muscle area (mm2) gap junctions junctions/Gr;12 18 DD EDL 49 1993 2.46 x lop2 21 DD EDL 5.34 x lo-' 86 1619 16 DD DIAPH 5.00 x 158 3164 18DDDIAPH 5.50 x 76 1384 ~~~
each freeze-fracture replica (see Materials and Methods). An initial, rough estimate of gap junction frequency was obtained by counting the gap junctions visible in each opening of the supporting EM grid, and calculating an average (Table 2). The densities thus obtained are not exactly comparable a t different ages, because at the earlier developmental stages there are fewer membrane profiles in each grid square opening and an increasingly larger fraction of the replica surface is occupied by membrane as development progresses. Thus the density a t the younger ages is underestimated raltive to that a t older ages. Despite this limitation, this initial assessment indicated at what ages the frequency is high. There are some interesting differences between EDL and diaphragm regarding the periods a t which peaks in gap junction frequency occur. In EDL gap junctions are relatively scarce a t the primary myotube stage (E16), and more frequent during the period of maximum clustering of primary and secondary fibers (E18-E21). In diaphragm the density of gap junctions is high at the primary myotube stage (E16) and less when secondary myotubes are present. Decline to a low level occurs earlier in diaphragm (between E l 8 and E21) than in EDL (between E21 and E22). Both muscles have a low density of gap junctions on the surface of young muscle fibers a t birth (see also Schmalbruch, 1987), indicating some remaining contacts between the muscle fibers. Comparison of Table 1 and 2 indicates that a high incidence of gap junctions requires the simultaneous presence of a high incidence of contactslmyotube profile and of M-M contacts. This confirms the hypothesis that the gap junctions occur at sites of myotube-to-myotube junction. Further analysis was performed on muscles at the critical stages, by determining the density of gap junction per surface area (Table 3). Gap junctions were counted as above, and surface areas of all myotube surfaces in the replicas were obtained by digital planimetry of the enlarged micrographs. These data allow a more accurate comparison between different stages of the same muscle than those given in Table 2, but they
are still approximate, since the plasmalemma of the myotubes is curved and surface areas were obtained from measurements of their projected images. The density is not different in EDL between E l 8 and E21, but it declines in diaphragm between E l 6 and E18.
DISCUSSION The differences in myotube dispositions, patterns of intercellular contacts, and frequency of gap junctions between developing EDL and diaphragm point to specific differences in their developmental programs that are initiated close to inception of myogenesis. In the middle of the EDL at E16, for example, there are few myotube to myotube contacts, because the myotubes are separated from each other by numerous mononucleated cells, most of which are probably myoblasts. This large population of myoblasts presumably results in the potential for a large population of secondary cells. By contrast, the diaphragm has more myotube to myotube contacts and larger myotubes at this age, indicating a higher proportion of primary cells, and a more mature muscle. Connective tissue also develops more rapidly in the diaphragm, giving it mechanical strength. Knowing that the diaphragm is more mature than the EDL, as indicated by the size of the fibers, their content of myofibrils and of myosin heavy chain (Kelly et al., 19911,we were surprised to find that the EDL at E l 8 has more myotube to myotube contacts and a higher incidence of secondary myotube profiles containing myofibrils than the diaphragm at the same age. This would indicate a more rapid initial maturation of secondary myotubes in EDL. The pattern a t later stages (E21) is more difficult to interpret in this regard, since it may indicate that the EDL is either more or less advanced than the diaphragm (see above, see also Harris et al., 1989). Diversity in the development of EDL and diaphragm are thus not simply related to the fact that the myotubes and muscle fibers in the diaphragm appear to be more mature a t all stages. A related difference exists between the primordium of EDL and soleus in mice: At the same age primary myotubes are differently arranged relative to each other (Ontell and Kozeka, 1984; Ontell et al., 1988). In the development of skeletal muscle there seem to be two, or possibly three, sequential periods of cell communication, during which frequency of gap junction increases, separated by periods of cell independence,
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when junctions decline. In the differentiation of segmental myotomes, unsegmented mesodermal cells communicate with each other, then separate during morphogenetic movements (Hayes, 1975; Blackshaw and Warner, 1976). Myoblasts a t pre-fusion stages are also capable of forming gap junctions, if given the opportunity for close association in vitro (Chow and Young, 1987; Kalderon et al., 1977). At later stages myotubes containing fully differentiated myofibrils are again connected, but become fully distinct functional units in coincidence with innervation and in a manner dependent on activity (Keeter et al., 1975; Dennis, 1981; Dennis et al., 1981; Armstrong et al., 1983). Variations in gap junction frequency during muscle histogenesis are characteristically different in EDL and diaphragm. In EDL the frequency of gap junctions is high during the period of primary to secondary myotube adhesion, as is also seen in thin sections (Kelly and Zacks, 1969; Rubinstein and Kelly, 1981; Hayes, 1975). This arrangement is advantageous because secondary fibers do not extend the full length of the muscle, but insert into the walls of the primary ones (Ontell and Kozeka, 1984; Ontell et al., 1988). Contraction of the latter, without simultaneous activation of the former might be disruptive. The diaphragm, on the other hand, has its highest incidence of gap junctions at the primary myotube stage, and it also maintains a fairly high level during formation of secondary myotubes. This result correlates with the work of Harris (1981), showing that primary myotubes are aggregated into large groups, and with the work of Dennis et al. (19811, showing that focal activation of the diaphragm a t E l 6 in utero induces a wave of activation that spreads across the entire muscle. This is probably essential in the early coordination of respiratory movements, before phrenic innervation is complete. Early development of the diaphragm is reflected in the development of motor neurons and in the innervation of the muscles. Generation and clustering of motor neurons in the thoracic segment of the spinal chord occurs at Ell-El2 and a t E13-El4 respectively. In the lumbar segment the two events are a t E12-El3 and a t E l 4 (Nornes and Das, 1974). Intercostal muscles (and presumably diaphragm) are first innervated at E14-El5 (Kelly and Zachs, 1969; Dennis et al., 1981), leg muscles a day or two later. EDL fibers are extensively coupled a t E18-E21 and uncouple abruptly between E21 and E22. The soleus, which matures more slowly, is still rich in junctions at birth (our unpublished observations, Rash and Staehelin, 1974; Schmalbruch, 1987). Diaphragm has its final decline by E21. The final decline in gap junction frequency is related to the separation between the two generation of myotubes. This in turn might be related to the innervation of the secondary myotubes, but there is not sufficient information on this point. In relating gap junction frequency, and the resultant intercellular communication, with innervation and fiber activity, two proposals have been offered: a) activity of one set of fibers would
be transmitted to the other by means of the gap junctions, and this may be important in the normal development of secondary fibers (Rubinstein and Kelly, 1981); and b) on the other hand, it was shown that activity results in the closing off of junctions (Armstrong et al., 1983). The two possibilities are not mutually exclusive. The time of the last wave of junction formation during myogenesis, which is described here, coincides with the formation of secondary myofibers, and follows innervation, rather than precedes it. This would indicate the necessity of a coupling between primary and secondary fibers a t the time when the former are just innervated. The subsequent abrupt decrease in gap junctions two days later may be the result of activity. Interestingly, although primary and secondary fibers are coupled during development, they do not necessarily follow similar developmental pathways. Many primary cells in the EDL of rat, for example, differentiate into slow type I fibers, whereas all secondary cells become fast type I1 (Rubinstein and Kelly, 1981; Narusawa et al., 1987). Hence, electrical activity does not seem to regulate early muscle specialization. We find that gap junction formation is spatially and temporally related to the formation of adhering junctions between myotubes. The importance of adhering junctions in morphogenesis is clearly established, since they mediate the attachment and interaction between cells which are a necessary step in organogenesis. NCAM, the muscle and nerve specific cell adhesion molecule, is concentrated at areas of myotube to myotube contacts (Covault and Sanes, 1986) roughly during the period of time when we find a high level of intercellular “contacts” and gap junctions, and N-cadherin, the Ca2+-dependent adhesion molecule is present during myogenesis (Knudsen et al., 1990; Knudsen, 1990). While everybody agrees on the occurrence and importance of cell-to-cellinteractions during myogenesis, the specific role of adhering junctions and cell adhesion molecules during myogenesis has not been determined. Clearly, the adhering and gap junctions that we find between myotubes (primary to primary and primary to secondary) and the adhering junctions between fibroblasts and myogenic cells are not related to the process of myoblast fusion. Even though some association was found between gap junction presence and cell fusion (Rash and Staehelin, 1974; Rash an Fambrough, 19731, it is clear that in most cases the two are not causally related (Kalderon et al., 1977; KaYderon and Gilula, 1979, see Cavaney, 1985). What is the specific role of gap and adhering junctions a t specific stages of development? The development-related decrease in junctions is indicative of surface membrane changes, and these in turn may be related to the ability of the muscle to support growth of secondary fibers, by allowing adherence to and interaction with the membrane of the older myotubes. In this manner, junctions contribute to the definition of the number of fibers in the muscle.
GAP JUNCTIONS IN HISTOGENESIS
ACKNOWLEDGMENTS We thank Nosta Glaser for technical help and for the illustrations. Supported by grants from NIH (HL15835 to the Pennsylvania Muscle Institute) and from the Muscular Dystrophy Association. REFERENCES Armstrong, D.L., Turin, L., and Warner, A.E. (1983) Muscle activity and the loss of electrical coupling between striated muscle cells in Xenopus embryos. J . Neurosci. 3:1414-1421. Blackshaw, S.E. and Warner, A.E. (1976) Low resistance junctions between mesodermal cells during development in trunk muscles. J. Physiol. 255:209-230. Cavaney, S. (1985) The role of gap junctions in development. Annu. Rev. Physiol. 47:319-335. Chiu, A.Y. and Sanes, J.R. (1985) Differentiation of basal lamina in synaptic and extrasynaptic portions of embryonic rat muscle. Dev. Biol. 103:456-467. Chow, I. and Young, S.H. (1987) Opening of single gap junction channels during formation of electrical coupling between embryonic muscle cells. Dev. Biol. 122:332-337. Covault, J. and Sanes, J . (1986) Distribution of N-CAM in synaptic and extrasynaptic portions of developing and adult skeletal muscle. J. Cell Biol. 102:716-730. Dawes, G.S. (1984) The central control of fetal breathing and skeletal muscle movements. J . Physiol. 356:l-18. Dennis, M.J. (1981) Development of the neuromuscular junction: Inductive interaction between cells. Annu. Rev. Neurosci. 4:43-68. Dennis, M.J., Ziskind-Conhaim, L., and Harris, A.J. (1981) Development of neuromuscular junctions in rat embryos. Dev. Biol. 81: 266-279. Duxson, M.J., Usson, Y., and Harris, A.J. (1989) The origin of secondary myotubes in mammalian skeletal muscles: Ultrastructural studies. Development 107343-750. Franzini-Armstrong, C., Pincon-Raymond, M., and Rieger, F (1991) Muscle fibers from dysgenic mouse in vivo lack a surface component of peripheral couplings. Dev. Biol. 146:364-376. Fraser, S.E., Green, C.R., Bode, H.R., and Gilula, N.B. (1987) Selective disruption of gap junctional communication interferes with a patterning process in Hydra. Science 237:49-55. Harris, A.J. (1981) Embryonic growth and innervation of rat skeletal muscles. I. Neural regulation of muscle fiber number. Philos. Trans. R. SOC.Lond. B 293:257-277. Harris, A.J., Duxson, M.J., Fitzsimons, R.B., and Rieger, F. (1989) Myonuclear birthdates distinguish the origins of primary and secondary myotubes in embryonic mammalian muscle. Development 107:771-784. Hayes, B.P. (1975) The distribution of intercellular junctions in the developing myotomes of the clawed toad. Anat. Embryol. 147:345354.
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Kalderon, N., Epstein, M.L., and Gilula, N.B. (1977) Cell-to-cell communication and myogenesis. J . Cell Biol. 75788-806. Kalderon, N. and Gilula, N.B. (1979) Membrane events involved in myoblast fusion. J . Cell Biol. 81:411-425. Keeter, J.S., Pappas, G.D., and Model, P.G. (1975) Inter- and intramyotomal gap junctions in the axolotl embryo. Dev. Biol. 4521-33. Kelly, A.M. and Zacks, S.I. (1969) The histogenesis of rat intercostal muscle. J . Cell Biol. 42:135-153. Kelly, A.M., Rosser, B.W.C., Hoffman, R., Panettieri, A,, Schiaffino, S., Rubinstein, N.A., and Nemeth, P.M. (1991) Metabolic and contractile protein expression in developing rat diaphragm muscle. J . Neurosci. 11:1231-1242. Knudsen, K.A. (1990) Cell adhesion molecules in myogenesis. Curr. Biology 2:902-906. Knudsen, K., Myers, L., and McElwee, S. (1990) A role for the Ca2+dependent adhesion molecule, N-cadherin, in myoblast interaction during myogenesis. Exp. Cell Res. 188:175-184. Narusawa, M., Fitzsimons, R., Izumo, S., Nadal-Ginard, B., Rubinstein, N.A., and Kelly, A.M. (1987) Slow myosin in developing rat skeletal muscle. J. Cell Biol. 104:447-459. Nornes, H.D. and Das, G.D. (1974) Temporal pattern of neurogenesis in spinal chord of rat. I. An autoradiographic study. Brain Res. 73:121-138. Ontell, M. (1979) The source of “new” muscle fibers in neonatal muscle. In: “Muscle Regeneration,” Mauro, A. (ed.). New York Raven Press, pp 137-146. Ontell, M. and Kozeka, K. (1984) The organogenesis of murine striated muscle: A cytoarchitectural study. Am. J. Anat. 171:133-148. Ontell, M., Bourke, D., and Hughes, D. (1988) Cytoarchitecture of the fetal murine soleus muscle. Am. J . Anat. 171:133-148 Potter, D.D., Furshpan, E.J., and Lennox, E. (1966) Connections between cells of the developing squid as revealed by electrophysiological methods. Proc. Natl. Acad. Sci. U S A . 55328-336. Rash, J.E. and Fambrough, D. (1973) Ultrastructural and electrophysiological correlates of cell coupling and cytoplasmic fusion during myogenesis in vitro. Dev. Biol. 30:168-186. Rash, J.E. and Staehelin, L.A. (1974) Freeze-cleave demonstration of gap junctions between skeletal myogenic cells in vivo. Dev. Biol. 36:455-461. Rubinstein, N.A. and Kelly, A.M. (1981) Development of muscle fibers specialization in the rat hind limb. J. Cell Biol. 90:128-144. Schmalbruch, H. (1987) Skeletal muscle fibers of newborn rats are coupled by gap junctions. Dev. Biol. 91:485-490. Spitzer, N. (1982) Voltage and stage dependent uncoupling of RohonBeard neurones during embryonic development in Xenopus tadpoles. J . Physiol. 235:267-286. Warner, A.E. (1973) The electrical properties of the ectoderm during induction and early development of the nervous system. J . Physiol. 235:267-286. Warner, A.E., Guthrie, S.C., and Gilula, N.B. (1984)Antibodies to gap junctions protein selectively disrupts junctional communication in early amphibian embryo. Nature 311:127-131.
Differences in the Histogenesis of EDL and Diaphragm in Rat LING YIPING, DENAH APPELT, ALAN M. KELLY, AND CLARA FRANZINI-ARMSTRONG Departments of Anatomy (C.F.-A.),Biology (L.Y.,D.A.) and Veterinary Pathobiology (A.M.K.), University of Pennsylvania, Philadelphia, Pennsylvania 19104
ABSTRACT We have examined the histogenesis of the diaphragm and extensor digitorum muscle in rat embryos, with the aim of defining differences in developmental patterns that can be related to the functional requirements of these muscles during and after development. Patterns of interactions between myotubes and other cells, and frequency of gap junctions are quite different in the two muscles. In diaphragm, primary myotubes (at day 16 in utero) are closely associated with each other, forming parallel sheets or palisades and communicating by gap junctions. Secondary myotubes have formed by day 18, but are immature, and the frequency of gap junctions is lower. The arrangement in palisades is maintained even after fibers are separated from each other by their individual basal lamina. In EDL primary fibers at day 16 have fewer gap junctions, and the peak in communication occurs afterthe appearanceof secondary myotubes (day 18 and 21). Secondary myotubes are more mature than in diaphragm at day 18. 0 1992 Wiley-Liss, Inc.
Key words: Gap junctions, Muscle development, Rat muscle, Diaphragm, EDL INTRODUCTION Skeletal muscle is progressively assembled from succeeding generations of cells. Early in myogenesis the primordium is established by a small population of primary myotubes. This is then amplified by the addition of variable numbers of secondary myotubes that use the walls of primary myotubes as a scaffold. Subsequently the myofibers in these clusters cells separate and become independent (Kelly and Zacks, 1969; Ontell and Kozeka, 1984; Chiu and Sanes, 1985; Duxson et al., 1989). In the present study we have examined general muscle organization and the incidence of intercellular junctions in the developing diaphragm and EDL of fetal and neonatal rats. We have focused on the incidence of gap junctions because intercellular communication plays an important role at critical moments during cell differentiation and tissue morphogenesis (Potter et al., 1966; Warner, 1973; Spitzer, 1982; Cavaney, 1985) and, in some systems, it is necessary for orderly em0 1992 WILEY-LISS, INC
bryonic development (Warner et al., 1984; Fraser et al., 1987). We have questioned whether muscle histogenesis is stereotyped or whether each muscle has a singular pattern of assembly that may be linked to its function during embryogenesis andlor with its anticipated role a t later stage. From their inception, the functions of diaphragm and EDL differ from each other. The diaphragm must form a coherent barrier between the unfolding thoracic and peritoneal cavities and subsequently withstand mounting trans-diaphragm pressure. In addition, the fetal diaphragm must be differentiated enough to support the abrupt onset of respiration a t parturition, and it prepares by rhythmically contracting in utero (Dawes, 1984). Fetal respiratory movements and the beginning of respiration are thought to play a critical role in differentiation of the tracheobronchial tree. The EDL is not subject to equivalent lateral pressures, and intrauterine and postpartum movements are slow and less active. We find that EDL and diaphragm follow the same general rules of development: At least two waves of myotube formation, transient cell adhesion and communication, followed by a separation of independent fibers. However, there are clear differences, which are noticeable from very early stages, before innervation and/or hormonal influences occur, in the patterns of development of the two muscles. MATERIALS AND METHODS Electron Microscopy Rat embryos a t 16,18,21,and 22 days in utero (E1622, plug day = 0) were obtained following C02 euthanasia of the dam. Pups at 0 and 5 days after birth were similarly euthanised. At E l 6 entire hind limbs were fixed, a t E l 8 and later the EDL was partially dissected at the distal end, pinned away from the leg and fixed for about one hour, then completely dissected. Diaphragms were dissected with a rim of rib cage and pinned for the initial fixation. The muscles were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer, con-
Received September 12, 1991; accepted April 13, 1992. Address reprint requestsicorrespondence to Dr. Clara FranziniArmstrong, Department of Anatomy, University of Pennsylvania, Philadelphia, PA 19104-6058. Dr. Ling Yiping’s current address is Laboratory of Electron Microscopy, Shangai Medical University, Shangai 200032 China.
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taining 5 mM sucrose and 0.1% tannic acid. Fixation was at room temperature for approximately two hours, then the tissues were stored for 1-15 days in the fixative at 4°C. For thin sectioning the muscles were postfixed in 2% OsO, in 0.1 M cacodylate buffer and 0.05 M sucrose for 30 min (or alternatively, in 2% OsO,, 0.8% K,Fe(CN)6), en block stained in 2% uranyl acetate in 70% EtOH for 30 min, dehydrated, and embedded in Spurr. One set of diaphragms, from E16, E18, and E21 in utero were fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer and 0.013% CaC1, overnight at room temperature. Post-fixation was as above. Thin sections were stained in uranyl acetate and lead. For freeze-fracture muscles were fixed in glutaraldehyde, as above, but without tannic acid. The fixed muscles were infiltrated in up to 30% glycerol, frozen in Freon 12 and fractured and replicated in a Balzer’s 400. Platinum shadow was at 45”. Sections and replicas were photographed in JEOL JEM 1005 and Philips 410 microscopes. Photographs for quantitative analysis were taken as follows: a) Cross sections of the entire muscle (for EDL), or of approximately 1mm wide segments (for the diaphragm) were mounted on bare 400 mesh grids. b) Freeze fracture replicas were obtained from longitudinally oriented muscles, and all available replica pieces were mounted on bare grids. All visible areas of replicas and thin sections were photographed at a magnification of 3,000 x . Each grid square measured approximately 2,000 pm, and was covered by a set of overlapping micrographs.
RESULTS General Description At E l 6 the primordia of both diaphragm and EDL are composed of primary myotubes only. The myotubes in the diaphragm are more developed: they have a larger diameter and more myofibrils. In EDL primary myotubes are circular in profile (triangles, Fig. la), and are closely, but randomly intercalated with a number of mononucleated cells (myoblasts, presumptive myoblasts, and fibroblasts). In the diaphragm the situation is different: primary myotubes (triangles, Fig. 2a) form a tight arrangement in long rows (palisades), mostly relegating the mononucleated cells at the periphery of the rows. Proximity between primary myotubes is more prevalent than in EDL, and the cells are so packed that they take a polygonal profile. Mesenchymal cells with polyribosome-studded cytoplasm insert themselves in the spaces between the myotubes at the periphery of the palisades. At E l 8 primary myotubes have moved apart in both muscles, and secondary ones are intercalated in between. Again however, the pattern is different in the two muscles. In the EDL (Fig. lb) each circular profile of cross-sectioned primary myotube (M) has its surrounding group of secondary myotubes (m) and mononucleated cells, forming fairly well defined individual clusters; in the diaphragm (Fig. 2b) primary and sec-
ondary myotubes form tight palisades, where one or two secondary myotubes andlor mononucleated cells squeeze between the primary ones. The fibers maintain polygonal profiles, and face each other over long distances. Fibroblasts tend to take a position peripheral to the developing clusters of cells a t E l 8 and later. Bundles of collagen fibers are more evident in diaphragm than in EDL at early stages of development. The basal lamina, initially very delicate, surrounds clusters of primary and secondary myotubes and the muscle fibers derived from them. At early stages (e.g., day 18, Fib. lb) the cluster boundaries in EDL are not clearly defined because the basal lamina is hardly visible and mononucleate cells contact more than one cluster. At later stages, the muscle fibers forming clusters gradually separate from each other. In EDL separation of all fibers is not completed until several days after birth (Ontell, 1979). In diaphragm the separation is almost complete at birth, but the fibers remain polygonal and tightly clustered, although a t that point they are mostly separated from each other by a layer of basal lamina. Thus, in addition to having a different packing, diaphragm matures earlier, and EDL follows. Innervation also occurs earlier in the upper body: Intercostal muscles are innervated a t day 14, and axons are present in diaphragm at that age (Dennis, 1981; Dennis et al., 1981). In EDL axons are present at day 15, and primary myotubes are innervated at day 16 (Harris, 1981).
Cell to Cell Contacts All cells form contact^" with each other (Fig. 3a, between arrows, and Fig. 3b). For the purpose of our description, we define “contact” as an area of cell surface which closely approximates the surface of another cell. Along contact areas the cell profiles lie at a distance of 10-20 nm from each other and the basal lamina is excluded (Fig. 3a,b, between short arrows). This allows opportunity for the formation of numerous focal junctions of the adhering type, or spot adhesions, distinguished by intracellular densities and proximity of the adhering membranes (Fig. 3a,b, small arrowheads). Gap junctions, in which the adjacent cell membranes are quite straight and parallel to each other across a narrow gap, are less frequent (Fig. 4a). The pattern of cell to cell contact changes with age and shows some differences between EDL and diaphragm. This is clearly demonstrated by the rough quantitation given below. Quantitation of Cell Contacts Cross sections were cut across the approximate middle of the muscle mass. For purposes of quantitation, cell contacts were divided into four categories: extra short contacts, at which the cells interact over a very short distance (less than 0.4 pm); short contacts, which occupy a cell boundary length of 0.4-3.3 pm; medium contacts with a length of 3.4-10 p m ;and long contacts, with lengths above those.
GAP JUNCTIONS IN HISTOGENESIS
Fig. 1. a: Cross section of the EDL at E16. Primary myotubes (triangles) have circular profiles and are separated by numerous cells, mostly myoblasts, with which they make multiple "contacts" (see text). There are also some contacts between myotubes. Outlines of most cell are complex and the contacts tend to be short. x 3700. b: Cross section of EDL at
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E18. Primary myotubes (M) are mostly separated from each other, and secondary myotubes (m) are closely associated with their periphery, forming extensive contacts. Numerous cells are still present between the myotubes and form short contact with them, including fibroblasts (f). x 3700.
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Fig. 2. a: Primary myotubes in diaphragm at E l 6 are more closely clustered than in EDL. Triangles mark a set of closely apposed primary myotubes which form a palisade by making extensive contacts with each other, to the exclusion of other cells. x 2800. b: Diaphragm at E18. Myoblasts and secondary myotubes (m) are present between primary
Note however, that the arrangement remains much more myotubes (M). tight than in the EDL. Contacts tend to be long. Primary and secondary fibers in diaphragm at 16 and 18 days of gestation are more developed than those in EDL at the same age. f: fibroblast. x 2800.
GAP JUNCTIONS IN HISTOGENESIS
363
Fig. 3. a: Contacts between two primary myotubes (M) and a cell with rough ER, which might be a fibroblast (F) intercalated between them. Most contacts are "short" in this image (between arrows). Small arrowheads point to focal adhering junctions within areas of contact. EDL, El 6 .
x 18,000. b: Extensive, "long" contact between a primary (bottom) and secondary (top) myotube in EDL, E21. Small arrowheads point to focal adhering junctions. x 18,000.
Data collection involved 3 steps: 1) cell profiles were identified as: a) profiles containing myofibrils (myotubes, M, Table 1); b) profiles not containing myofibrils (nmf, Table 1).The latter category includes a variety of cells, see below. 2) The number of contacts between the first 50 complete myotubes profiles and their immediate neighbours were counted and categorized. 3) From these data we calculated (Table 1): the relative frequencies of contacts between adjacent myotubes (M-M) and of contact between myotubes and
non-myofibrillar cell profiles (M-nmf ); the average number of contact for each myotube profile; the relative frequencies of contacts of various lengths; and the average length of myotube profile occupied by contacts. The latter was estimated by multiplying the number of contacts in the four categories by their average length (0.20, 1.85, 6.70, and 13.3 Km). This of course is not a precise morphometric approach, and the numbers obtained are not exact. However, the separation of junctional lengths into categories is very useful
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Fig. 4. a: Gap junction between primary (left) and secondary (right) myotubes in EDL, E21. x 36,000. b: One large and two small gap junctions (arrows) on the surface of a myotube in diaphragm, E18. Junctions like these are often found on long stretches of the myotube surface indicating that the association between primary and secondary myotubes extends over relatively long distances. Semicircles indicate sites of pe-
ripheral couplings. x 42,000. c: Gap junction (lower left) and peripheral couplings (semicircles) on a myotube in the diaphragm, E18. Asterisks indicate opening of caveolae and/or T tubules. x 19,000, d: Gap junction on a myotube in diaphragm, E16. This is most likely to be a junction between two primary myotubes. x 62,000.
in giving a semiquantitative assessment of trends that are visible to the eye. The myofibrillar (M) and non-myofibrillar (nmf) cell categories lump profiles of different cells at different ages (Fig. 5a-f). At E l 6 all myofibrillar cells are primary myotubes; the non-myofibrillar cells between them are mostly myoblasts and a few fibroblasts. Both
types of nmf cells make brief but numerous contacts with the myotubes and are lumped together, because the distinction between them is not always very clear (Figs. l a , 2a, 3a, 5a and b). At El8 myofibrillar profiles are from primary and secondary myotubes; most nonmyofibrillar profiles are from young developing myotubes, which are closely associated to the surface of the
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TABLE 1. Contacts Between Developing Myotubes and Other Cells in EDL and Diaphragm
Muscle 16 DD EDL 18 DD EDL 21 DD EDL 22 DD EDL 16 DD Diaph 18 DD Diaph 21 DD Diaph
Contacts" M-M 23 44 44 33 50 16 63
total) M-nmf
(% of
Number of
contacts/ myotube
77 56 56 67 50 84 37
Xsmall
Frequency of contacts % of total
8.4 k 3.0 68 4.4 2 2.3 58 2.1 k 1.4 27 1.1 2 0.7 5 5.6 k 1.7 32 3.6 f 1.9 23 2.3 k 1.1 "M-M contacts between cell profiles containing myofibrils (myotubes);M-nmf
Small 26 24 25 26 21 29 4
Medi-
um
Long
6 17 42 62 45 43 78
6 7 2 5 18
Total length of contacts/ myotube (pm) 8.9 4.9 7.6 ? 4.1 8.4 ? 6.9 6.4 k 5.3 16.4 k 7.7 16.5 2 9.7 16.6 ? 7.6
*
contacts between myotubes and cell profiles containing no myofibrils. These include myoblasts, presumptive myoblasts, fibroblasts and some secondary myotubes which happened to be sectioned at a level devoid of myofibrils.
myotubes and either have not yet developed myofibrils, or are cut near the ends (Figs. lb, 2b, 5c,d; Ontell, 1979). These developing myotubes, although apparently devoid of myofibrils, are distinguishable from myoblasts and fibroblasts, because they are more closely apposed to the myotube surface, they tend to form longer contacts and often push into the outline of the older myotubes (Fig. 3b). Myoblasts and fibroblasts are at the periphery of the clusters and palisades of myotubes. At E21 (Figs. 5e,f) and later, most non-myofibrillar cells forming junctions with myotubes are either developing myotubes or future satellite cells. Both types of cells form relatively long contacts with the more developed myotubes. The quantitation of cell contacts shown in Table 1 indicates some interesting common trends and some differences between EDL and diaphragm. Common trends are: a) The number of contacts per myotube decreases with age. Student's T test indicates that within each muscle the difference between each time point and the preceeding one is significant at a level better b) The length of contacts increases with than P < age: The relative frequency of extra short contacts declines and that of medium and long ones increases. This is probably directly related to the decrease in population of myoblasts. c) As a result of these opposite trends, the roughly estimated total length of contact per myotube remains approximately constant with age. d) In both muscles, primary myotubes make contacts with each other before secondary myotubes appear. The apparent discrepancy between rat EDL, in which contacts exist between primary myotubes, and mouse EDL, in which primary myotubes are completely separated from each other (Ontell et al., 1988>,may be due to a small difference in the developmental stage a t which the muscles were examined. There are four important differences between EDL and diaphragm (see diagram in Fig. 5): a) In EDL myotubes tend to form shorter contacts. The difference is clearly visible if one compares Figure l a and l b with Figure 2a and 2b, and it is a reflection of the different geometries of the two muscles, which was noted in the
general description. The average total length of contact per myotube is longer in diaphragm than in EDL a t all ages. The difference is statistically significant a t all ages (P < b) At E l 6 the diaphragm has a higher incidence of contacts between primary myotubes than the EDL. This is visible in Figures l a and 2a, and it also a reflection of the more compact arrangement of primary myotubes in the diaphragm at this age. c) EDL, on the other hand, has a higher incidence of myotube-to-myotube contacts than diaphragm at E18. This is due to a higher incidence of developing secondary myotubes which contain myofibrils in the EDL at that age. d) The trend is apparently reversed a t E21, when diaphragm has a larger incidence of myotube to myotube contacts than the EDL. This has two possible explanations: Either the EDL is less mature, and the nmf profiles belong to a population of developing myotubes, or it is more advanced and the nmf profiles belong to satellite cells. Persistence of contacts between myotubes and other cells in EDL at E22 in utero indicates that as in the mouse (Ontell, 19791, some fibers are still grouped in clusters at this stage. Interestingly the diaphragm also maintains a low density of contacts until a t least the day of birth. These were not counted.
Quantitation of Gap Junctions Gap junctions between myotubes are seen in thin sections (Fig. 4a). In freeze-fracture sizes of gap junctions vary greatly (Fig. 4b,c). We have obtained estimates of the frequency of gap junctions at various stages of differentiation for EDL and diaphragm using the freeze fracture technique (Fig. 4b-d).The first problem was that of differentiating split membranes belonging to myotubes from those of other cells. In most cases, the elongated approximately cylindical shape of the myotubes is sufficient for identification. Smaller fragments of membrane, particularly for younger myotubes, were identified using the following criteria: presence of caveolae (Fig. 412, asterisks); random distribution of intramembranous particles of variable sizes; and presence of junctional
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5a
U
5c
n
5e
5f
Fig. 5. a-f: Diagrams of cell profiles encountered in EDL (a, c, and e) and diaphragm (b, d, and f) at various ages. At E l 6 (a,b) both primordia are composed of primary myotubes (M,), containing myofibrils, and myoblasts (m) and fibroblasts (f), which are non-myofibrillar. Contacts between myotubes are in general longer than contacts between myotubes and non-myofibrillar cells. M,-M, contacts are more numerous in diaphragm. At E l 8 (c,d) most contacts are between primary (M,) and sec-
ondary (M,) myotubes. Fibroblasts have a more peripheral position relative to bundles of myofibers. Note that secondary myotubes are more frequently fibrillar in EDL than in diaphragm at this stage. The diaphragm maintains a "palisade" arrangement of cells. At E 21 contacts are fewer and are either between myotubes or between myotubes and future satellite cells (s).
tetrads at peripheral couplings (Fig. 4b,c, semicircles, see Franzini-Armstrong et al., 1991). Endothelial cells also have caveolae, but their intramembranous particles are far more uniform in size, and tight junctions
are often visible. Fibroblasts and myoblasts have very few, if any, caveolae. All junctions belonging to myotubes were identified in low power micrographs covering the entire area of
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GAP JUNCTIONS IN HISTOGENESIS
TABLE 2. Rough Estimate of Gap Junction Frequency on Surface of Myotubes at Various Stages of DeveloDment
Muscle 16 DD EDL 18 DD EDL 21 DD EDL 22 DD EDL BIRTH EDL 16 DD DIAPH 18 DD DIAPH 21 DD DIAPH 22 DD DIAPH BIRTH DIAPH
No. =ids 6 9 6 6 3 13 9 18 4 9
No. gap
No. sauares
No. gap iunctions
junctions/ sauare
27 66 77 37 47 127 107 166 34 167
7 49 86 6 5 158 76 20 4 6
0.26 0.74 1.12 0.16 0.11 1.24 0.71 0.12 0.12 0.06
TABLE 3. Frequency of Gap Junctions on Cell Surface of Myotubes at Selected Stages of Development
Total
Total
measured number Number eaD Muscle area (mm2) gap junctions junctions/Gr;12 18 DD EDL 49 1993 2.46 x lop2 21 DD EDL 5.34 x lo-' 86 1619 16 DD DIAPH 5.00 x 158 3164 18DDDIAPH 5.50 x 76 1384 ~~~
each freeze-fracture replica (see Materials and Methods). An initial, rough estimate of gap junction frequency was obtained by counting the gap junctions visible in each opening of the supporting EM grid, and calculating an average (Table 2). The densities thus obtained are not exactly comparable a t different ages, because at the earlier developmental stages there are fewer membrane profiles in each grid square opening and an increasingly larger fraction of the replica surface is occupied by membrane as development progresses. Thus the density a t the younger ages is underestimated raltive to that a t older ages. Despite this limitation, this initial assessment indicated at what ages the frequency is high. There are some interesting differences between EDL and diaphragm regarding the periods a t which peaks in gap junction frequency occur. In EDL gap junctions are relatively scarce a t the primary myotube stage (E16), and more frequent during the period of maximum clustering of primary and secondary fibers (E18-E21). In diaphragm the density of gap junctions is high at the primary myotube stage (E16) and less when secondary myotubes are present. Decline to a low level occurs earlier in diaphragm (between E l 8 and E21) than in EDL (between E21 and E22). Both muscles have a low density of gap junctions on the surface of young muscle fibers a t birth (see also Schmalbruch, 1987), indicating some remaining contacts between the muscle fibers. Comparison of Table 1 and 2 indicates that a high incidence of gap junctions requires the simultaneous presence of a high incidence of contactslmyotube profile and of M-M contacts. This confirms the hypothesis that the gap junctions occur at sites of myotube-to-myotube junction. Further analysis was performed on muscles at the critical stages, by determining the density of gap junction per surface area (Table 3). Gap junctions were counted as above, and surface areas of all myotube surfaces in the replicas were obtained by digital planimetry of the enlarged micrographs. These data allow a more accurate comparison between different stages of the same muscle than those given in Table 2, but they
are still approximate, since the plasmalemma of the myotubes is curved and surface areas were obtained from measurements of their projected images. The density is not different in EDL between E l 8 and E21, but it declines in diaphragm between E l 6 and E18.
DISCUSSION The differences in myotube dispositions, patterns of intercellular contacts, and frequency of gap junctions between developing EDL and diaphragm point to specific differences in their developmental programs that are initiated close to inception of myogenesis. In the middle of the EDL at E16, for example, there are few myotube to myotube contacts, because the myotubes are separated from each other by numerous mononucleated cells, most of which are probably myoblasts. This large population of myoblasts presumably results in the potential for a large population of secondary cells. By contrast, the diaphragm has more myotube to myotube contacts and larger myotubes at this age, indicating a higher proportion of primary cells, and a more mature muscle. Connective tissue also develops more rapidly in the diaphragm, giving it mechanical strength. Knowing that the diaphragm is more mature than the EDL, as indicated by the size of the fibers, their content of myofibrils and of myosin heavy chain (Kelly et al., 19911,we were surprised to find that the EDL at E l 8 has more myotube to myotube contacts and a higher incidence of secondary myotube profiles containing myofibrils than the diaphragm at the same age. This would indicate a more rapid initial maturation of secondary myotubes in EDL. The pattern a t later stages (E21) is more difficult to interpret in this regard, since it may indicate that the EDL is either more or less advanced than the diaphragm (see above, see also Harris et al., 1989). Diversity in the development of EDL and diaphragm are thus not simply related to the fact that the myotubes and muscle fibers in the diaphragm appear to be more mature a t all stages. A related difference exists between the primordium of EDL and soleus in mice: At the same age primary myotubes are differently arranged relative to each other (Ontell and Kozeka, 1984; Ontell et al., 1988). In the development of skeletal muscle there seem to be two, or possibly three, sequential periods of cell communication, during which frequency of gap junction increases, separated by periods of cell independence,
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when junctions decline. In the differentiation of segmental myotomes, unsegmented mesodermal cells communicate with each other, then separate during morphogenetic movements (Hayes, 1975; Blackshaw and Warner, 1976). Myoblasts a t pre-fusion stages are also capable of forming gap junctions, if given the opportunity for close association in vitro (Chow and Young, 1987; Kalderon et al., 1977). At later stages myotubes containing fully differentiated myofibrils are again connected, but become fully distinct functional units in coincidence with innervation and in a manner dependent on activity (Keeter et al., 1975; Dennis, 1981; Dennis et al., 1981; Armstrong et al., 1983). Variations in gap junction frequency during muscle histogenesis are characteristically different in EDL and diaphragm. In EDL the frequency of gap junctions is high during the period of primary to secondary myotube adhesion, as is also seen in thin sections (Kelly and Zacks, 1969; Rubinstein and Kelly, 1981; Hayes, 1975). This arrangement is advantageous because secondary fibers do not extend the full length of the muscle, but insert into the walls of the primary ones (Ontell and Kozeka, 1984; Ontell et al., 1988). Contraction of the latter, without simultaneous activation of the former might be disruptive. The diaphragm, on the other hand, has its highest incidence of gap junctions at the primary myotube stage, and it also maintains a fairly high level during formation of secondary myotubes. This result correlates with the work of Harris (1981), showing that primary myotubes are aggregated into large groups, and with the work of Dennis et al. (19811, showing that focal activation of the diaphragm a t E l 6 in utero induces a wave of activation that spreads across the entire muscle. This is probably essential in the early coordination of respiratory movements, before phrenic innervation is complete. Early development of the diaphragm is reflected in the development of motor neurons and in the innervation of the muscles. Generation and clustering of motor neurons in the thoracic segment of the spinal chord occurs at Ell-El2 and a t E13-El4 respectively. In the lumbar segment the two events are a t E12-El3 and a t E l 4 (Nornes and Das, 1974). Intercostal muscles (and presumably diaphragm) are first innervated at E14-El5 (Kelly and Zachs, 1969; Dennis et al., 1981), leg muscles a day or two later. EDL fibers are extensively coupled a t E18-E21 and uncouple abruptly between E21 and E22. The soleus, which matures more slowly, is still rich in junctions at birth (our unpublished observations, Rash and Staehelin, 1974; Schmalbruch, 1987). Diaphragm has its final decline by E21. The final decline in gap junction frequency is related to the separation between the two generation of myotubes. This in turn might be related to the innervation of the secondary myotubes, but there is not sufficient information on this point. In relating gap junction frequency, and the resultant intercellular communication, with innervation and fiber activity, two proposals have been offered: a) activity of one set of fibers would
be transmitted to the other by means of the gap junctions, and this may be important in the normal development of secondary fibers (Rubinstein and Kelly, 1981); and b) on the other hand, it was shown that activity results in the closing off of junctions (Armstrong et al., 1983). The two possibilities are not mutually exclusive. The time of the last wave of junction formation during myogenesis, which is described here, coincides with the formation of secondary myofibers, and follows innervation, rather than precedes it. This would indicate the necessity of a coupling between primary and secondary fibers a t the time when the former are just innervated. The subsequent abrupt decrease in gap junctions two days later may be the result of activity. Interestingly, although primary and secondary fibers are coupled during development, they do not necessarily follow similar developmental pathways. Many primary cells in the EDL of rat, for example, differentiate into slow type I fibers, whereas all secondary cells become fast type I1 (Rubinstein and Kelly, 1981; Narusawa et al., 1987). Hence, electrical activity does not seem to regulate early muscle specialization. We find that gap junction formation is spatially and temporally related to the formation of adhering junctions between myotubes. The importance of adhering junctions in morphogenesis is clearly established, since they mediate the attachment and interaction between cells which are a necessary step in organogenesis. NCAM, the muscle and nerve specific cell adhesion molecule, is concentrated at areas of myotube to myotube contacts (Covault and Sanes, 1986) roughly during the period of time when we find a high level of intercellular “contacts” and gap junctions, and N-cadherin, the Ca2+-dependent adhesion molecule is present during myogenesis (Knudsen et al., 1990; Knudsen, 1990). While everybody agrees on the occurrence and importance of cell-to-cellinteractions during myogenesis, the specific role of adhering junctions and cell adhesion molecules during myogenesis has not been determined. Clearly, the adhering and gap junctions that we find between myotubes (primary to primary and primary to secondary) and the adhering junctions between fibroblasts and myogenic cells are not related to the process of myoblast fusion. Even though some association was found between gap junction presence and cell fusion (Rash and Staehelin, 1974; Rash an Fambrough, 19731, it is clear that in most cases the two are not causally related (Kalderon et al., 1977; KaYderon and Gilula, 1979, see Cavaney, 1985). What is the specific role of gap and adhering junctions a t specific stages of development? The development-related decrease in junctions is indicative of surface membrane changes, and these in turn may be related to the ability of the muscle to support growth of secondary fibers, by allowing adherence to and interaction with the membrane of the older myotubes. In this manner, junctions contribute to the definition of the number of fibers in the muscle.
GAP JUNCTIONS IN HISTOGENESIS
ACKNOWLEDGMENTS We thank Nosta Glaser for technical help and for the illustrations. Supported by grants from NIH (HL15835 to the Pennsylvania Muscle Institute) and from the Muscular Dystrophy Association. REFERENCES Armstrong, D.L., Turin, L., and Warner, A.E. (1983) Muscle activity and the loss of electrical coupling between striated muscle cells in Xenopus embryos. J . Neurosci. 3:1414-1421. Blackshaw, S.E. and Warner, A.E. (1976) Low resistance junctions between mesodermal cells during development in trunk muscles. J. Physiol. 255:209-230. Cavaney, S. (1985) The role of gap junctions in development. Annu. Rev. Physiol. 47:319-335. Chiu, A.Y. and Sanes, J.R. (1985) Differentiation of basal lamina in synaptic and extrasynaptic portions of embryonic rat muscle. Dev. Biol. 103:456-467. Chow, I. and Young, S.H. (1987) Opening of single gap junction channels during formation of electrical coupling between embryonic muscle cells. Dev. Biol. 122:332-337. Covault, J. and Sanes, J . (1986) Distribution of N-CAM in synaptic and extrasynaptic portions of developing and adult skeletal muscle. J. Cell Biol. 102:716-730. Dawes, G.S. (1984) The central control of fetal breathing and skeletal muscle movements. J . Physiol. 356:l-18. Dennis, M.J. (1981) Development of the neuromuscular junction: Inductive interaction between cells. Annu. Rev. Neurosci. 4:43-68. Dennis, M.J., Ziskind-Conhaim, L., and Harris, A.J. (1981) Development of neuromuscular junctions in rat embryos. Dev. Biol. 81: 266-279. Duxson, M.J., Usson, Y., and Harris, A.J. (1989) The origin of secondary myotubes in mammalian skeletal muscles: Ultrastructural studies. Development 107343-750. Franzini-Armstrong, C., Pincon-Raymond, M., and Rieger, F (1991) Muscle fibers from dysgenic mouse in vivo lack a surface component of peripheral couplings. Dev. Biol. 146:364-376. Fraser, S.E., Green, C.R., Bode, H.R., and Gilula, N.B. (1987) Selective disruption of gap junctional communication interferes with a patterning process in Hydra. Science 237:49-55. Harris, A.J. (1981) Embryonic growth and innervation of rat skeletal muscles. I. Neural regulation of muscle fiber number. Philos. Trans. R. SOC.Lond. B 293:257-277. Harris, A.J., Duxson, M.J., Fitzsimons, R.B., and Rieger, F. (1989) Myonuclear birthdates distinguish the origins of primary and secondary myotubes in embryonic mammalian muscle. Development 107:771-784. Hayes, B.P. (1975) The distribution of intercellular junctions in the developing myotomes of the clawed toad. Anat. Embryol. 147:345354.
369
Kalderon, N., Epstein, M.L., and Gilula, N.B. (1977) Cell-to-cell communication and myogenesis. J . Cell Biol. 75788-806. Kalderon, N. and Gilula, N.B. (1979) Membrane events involved in myoblast fusion. J . Cell Biol. 81:411-425. Keeter, J.S., Pappas, G.D., and Model, P.G. (1975) Inter- and intramyotomal gap junctions in the axolotl embryo. Dev. Biol. 4521-33. Kelly, A.M. and Zacks, S.I. (1969) The histogenesis of rat intercostal muscle. J . Cell Biol. 42:135-153. Kelly, A.M., Rosser, B.W.C., Hoffman, R., Panettieri, A,, Schiaffino, S., Rubinstein, N.A., and Nemeth, P.M. (1991) Metabolic and contractile protein expression in developing rat diaphragm muscle. J . Neurosci. 11:1231-1242. Knudsen, K.A. (1990) Cell adhesion molecules in myogenesis. Curr. Biology 2:902-906. Knudsen, K., Myers, L., and McElwee, S. (1990) A role for the Ca2+dependent adhesion molecule, N-cadherin, in myoblast interaction during myogenesis. Exp. Cell Res. 188:175-184. Narusawa, M., Fitzsimons, R., Izumo, S., Nadal-Ginard, B., Rubinstein, N.A., and Kelly, A.M. (1987) Slow myosin in developing rat skeletal muscle. J. Cell Biol. 104:447-459. Nornes, H.D. and Das, G.D. (1974) Temporal pattern of neurogenesis in spinal chord of rat. I. An autoradiographic study. Brain Res. 73:121-138. Ontell, M. (1979) The source of “new” muscle fibers in neonatal muscle. In: “Muscle Regeneration,” Mauro, A. (ed.). New York Raven Press, pp 137-146. Ontell, M. and Kozeka, K. (1984) The organogenesis of murine striated muscle: A cytoarchitectural study. Am. J. Anat. 171:133-148. Ontell, M., Bourke, D., and Hughes, D. (1988) Cytoarchitecture of the fetal murine soleus muscle. Am. J . Anat. 171:133-148 Potter, D.D., Furshpan, E.J., and Lennox, E. (1966) Connections between cells of the developing squid as revealed by electrophysiological methods. Proc. Natl. Acad. Sci. U S A . 55328-336. Rash, J.E. and Fambrough, D. (1973) Ultrastructural and electrophysiological correlates of cell coupling and cytoplasmic fusion during myogenesis in vitro. Dev. Biol. 30:168-186. Rash, J.E. and Staehelin, L.A. (1974) Freeze-cleave demonstration of gap junctions between skeletal myogenic cells in vivo. Dev. Biol. 36:455-461. Rubinstein, N.A. and Kelly, A.M. (1981) Development of muscle fibers specialization in the rat hind limb. J. Cell Biol. 90:128-144. Schmalbruch, H. (1987) Skeletal muscle fibers of newborn rats are coupled by gap junctions. Dev. Biol. 91:485-490. Spitzer, N. (1982) Voltage and stage dependent uncoupling of RohonBeard neurones during embryonic development in Xenopus tadpoles. J . Physiol. 235:267-286. Warner, A.E. (1973) The electrical properties of the ectoderm during induction and early development of the nervous system. J . Physiol. 235:267-286. Warner, A.E., Guthrie, S.C., and Gilula, N.B. (1984)Antibodies to gap junctions protein selectively disrupts junctional communication in early amphibian embryo. Nature 311:127-131.
