growth and metamorphosis of the rectus abdominis muscle in rana pipiens

JOURNAL OF MORPHOLOGY 182:317-337 (1984)

Growth and Metamorphosis of the Rectus Abdominis Muscle in Rana pipiens

KATHRYN LYNCH Department of Anatomy, Uniformed Seruices University of the Health Sciences, Bethesda, Maryland 20814

ABSTRACT Cross sections through the middle segment of the anuran rectus abdominis muscle were analyzed morphometrically at nine stages of development, from early larval life through full maturity. The numbers, sizes, and relative distributions of twitch and slow muscle fibers, newly differentiated fibers, degenerating fibers, and satellite cells were determined at each stage. The data indicate that the muscle increases slowly in size and fiber content during early larval life. New fibers appear to form primarily along the medial margin of the muscle. During mid-larval stages, when thyroid hormone levels are rising, new fibers form throughout the medial portion of the muscle. At a slightly later stage, fibers in the lateral region of the muscle begin to degenerate. Structurally normal presynaptic elements are present on both degenerating fibers and the empty basal laminae of fibers that had been removed by phagocytes. Both fiber formation and fiber loss slow during mid- metamorphic climax, at the time when thyroid hormone levels reach a peak in anurans and begin to decline. Degenerating fibers appear within the body of the muscle at the end of metamorphosis. By the end of the second postmetamorphic month, neither degenerating nor newly differentiated fibers are present. The muscle continues to grow through adult life primarily by fiber hypertrophy.

During amphibian metamorphosis a number of muscles degenerate, others develop de novo and still others undergo extensive remodeling as the larval body acquires its adult form. Degeneration of the anuran tail musculature and development of the limb have been described morphologically (Watanabe and Sasaki, '74; Muntz, '75). Only recently, however, has a detailed microscopic study been made of metamorphic changes in a set of muscles that is functional in both larval and adult amphibians. Alley and Cameron ('83, '84) have found that metamorphic remodeling of the anuran jaw apparatus en- tails loss of the larval muscle fibers and their simultaneous replacement by a population of new fibers that form the adult muscles.

The rectus abdominis, a segmented muscle in the ventral abdominal wall, is another muscle that is present in anurans from late embryonic stages through maturity. It differs from the jaw muscles in that it is composed in the adult animal of both twitch and slow muscle fibers (Page, '65; Forrester and

Schmidt, '70). Preliminary studies were un- dertaken in Rana pipiens to determine when slow fibers appear during development and what changes this mixed muscle undergoes at metamorphosis. Initial results revealed regional variations in the structure of the larval rectus abdominis and in its response to metamorphic stimuli (Lynch, '83; Lynch and Harris, '84).

The morphometric study reported here was designed to study the regional variations in greater detail in a representative segment of the rectus abdominis. The sizes and distributions of twitch and slow fibers and the distributions of degenerating and newly differentiated fibers were characterized at several critical stages of development. The distribution of satellite cells was also stud- ied, since satellite cells function as the stem cells of muscle (Church, '69; Snow, '83; Alley and Cameron, '84) and the source of myonuclei for growing fibers (Moss and Leblond, '71). The results of the study indicate that the larval rectus abdominis grows by fiber

@I 1984 ALAN R. LISS, INC.

318 K. LYNCH

hypertrophy and hyperplasia. Just prior to the metamorphic climax, fiber formation accelerates, but only in the medial region of the muscle. In the lateral region, fibers abruptly begin to degenerate. By the middle of the metamorphic climax, the lateral degeneration has removed a significant portion of the muscle. Both degeneration and fiber formation continue at reduced rates through the metamorphic climax and the first postmetamorphic weeks. After the first two postmetamorphic months, the muscle grows primarily by fiber hypertrophy.

MATERIALS AND METHODS

Specimens representing embryonic, larval, and early (1 to 3 weeks) postmetamorphic stages were grown in the laboratory from fertilized R. pipiens eggs obtained from Car- olina Biological Supply. The embryos and larvae were kept in distilled water a t 21 to 23°C. Larvae were fed goldfish food and baby brine shrimp. Older juvenile frogs (8 to 9 weeks postmetamorphosis) were purchased from the Michigan Amphibian Facility. Ma- ture adults (winter frogs, 6 to 9 cm snout to vent) were brought from Hazen Farms in Vermont.

The entire ventral abdominal wall was removed from pithed frogs and tadpoles and pinned out on Sylgard in standard frog Ring- er’s solution (111 mM NaC1, 2 mM KC1, 1.8 mM CaC12, 5 mM N-2-hydroxyethylpipera- zine-N’-2-ethanesulfonic acid (HEPES), 3 mM dextrose). The tissue was fixed by immersion in 3% glutaraldehyde in Ringer’s, rinsed in 90 mM cacodylate buffer, postfixed in os- mium-ferrocyanide (Karnovsky, ’71) a t 4”C, dehydrated in methanol, and embedded in Araldite. The embedded preparations were split first along the midline, between the two muscles. One side was then split trans- versely across the middle of the muscle, through the third most rostra1 of the six segments that comprise the rectus abdominis. One-micrometer sections were cut across the entire width of the segment, from its medial to lateral margin. The sections were stained with toluidine blue or paraphenylenedia- mine (modified from Korneliussen, ’72). The thick sections were mapped by projecting them onto tracing paper at 1 6 5 0 ~ or 3 3 0 0 ~ from a Nikon Biophot microscope, using a system of mirrors devised by Lawshe Instru- ments, Rockville, MD.

Ultrathin sections were cut immediately adjacent to the mapped thick sections, stain-

ed with uranyl acetate and lead citrate, and examined in a Philips 400 electron microscope. From the thin sections, muscle fiber types and the positions of myonuclei and satellite cell nuclei were noted on the maps. Muscle fiber cross-sectional area was measured from the maps on a Hewlett-Packard 9874A digitizer and the data were tabulated with a Hewlett-Packard desk-top computer.

Muscle fibers were categorized as slow or twitch, or an immature or degenerating form of either. Slow and twitch fibers were distin- guished on the basis of their Z-line and M- band structure (Page, ’65; Peachey and Hux- ley, ’62). Fiber cross-sections containing lat- ticed Z-lines or an M-band or both were classified as twitch fibers. Those possessing no M-band lattice and an amorphous or punc- tate Z-line were classified as slow fibers. Small fibers with poorly developed myofibrils and/or an abundance of free ribosomes in their sarcoplasm were classified as “new” or immature. Degenerating fibers were frag- mented. If not totally enclosed within phagocytic cells, the fragments were usually separated by lamellar processes of phagocytes.

All cells whose cross-sectional profiles contained no myofibrils and which lay inside the basal lamina of a muscle fiber were classified as satellite cells. Only those whose nuclei were included in the section were counted. The satellite cells were very closely apposed to the muscle fibers (< 20 nm between plasmalemmae in spots). When the basal lamina was absent or poorly developed, therefore, the close juxtaposition of the plasmalemma of a nonmuscle cell to a muscle fiber was the criterion for its classification as a satellite cell. Other investigators have differentiated subcategories of cells in close apposition to muscle fibers (myoblasts, “true” myosatel- lite cells, undifferentiated cells, and fibro- blastlike cells), on the basis of nuclear/ cytoplasmic ratios, the degree of condensa- tion of chromatin, and the concentration of rough ER and free ribosomes (e.g., Kelly and Zacks, ’69; Kikuchi and Ashmore, ’76; On- tell, ’77). Largely because cells fell along a continuum with respect to each of these cri- teria, no attempt was made in the present study to distinguish subdivisions within the population of nonmuscle profiles included within the muscle basal lamina.

Muscles were analyzed from larvae in developmental stages XI (the last stage of premetamorphosis; Etkin, ’68), XVII and XVIII

DEVELOPMENT OF ANURAN RECTUS ABDOMINIS 3 19

(prometamorphic stages), XXI, XXIV, XXV (metamorphic stages), and from frogs 1 to 3 weeks postmetamorphosis (WPM), 8 to 9 WPM, and fully mature. Analysis was restricted in all cases to the third most rostral of the six muscle segments.

In order to detect differences between the medial and lateral sides and the dorsal and ventral halves of the segment, the data were accumulated separately from different regions of the muscle cross-sections. This was accomplished by dividing each muscle map into eight equal compartments or “octants.” The map was first divided into four compartments of equal width medial, centromedial, centrolateral, and lateral. Each of these quarters was then divided into ventral and dorsal halves, resulting in octants: ventro- m e d i a l o , ventro-centromedial (VCM), ven- trocentrolateral (VCL), ventrolateral (VL), dorsomedial (DM), dorsocentromedial (DCM), dorso-centrolateral (DCL), and dorsolateral (DL).

In the larger muscles, it was not feasible to classify and measure every muscle fiber. In these muscles, only the central third or half of an octant was analyzed and the data obtained were extrapolated to obtain values for the entire octant. In addition, it was not possible to obtain thin sections of the entire width of the mature frog muscles. The larger fiber sizes in the mature muscles, however, made it possible to classify the fibers a t the light microscopic level. Partial thin sections were used to check the accuracy of the analysis.

RESULTS Gross structure of the rectus abdominis

The rectus abdominis first appears in St. 21 (Shumway, ’40) embryos as a paired, segmented, fan-shaped muscle continuous caudally with the myotomal musculature. Each of the six segments comprising the muscle is a transverse row of muscle cells whose long axes run rostrocaudally. The segments are linked in series by transverse tendinous intersections. During embryonic and most of larval life, nerve trunks enter the muscle laterally, pass dorsal and parallel to the tendinous intersections, and give off branches rostrally and caudally. Through larval stages I to XIX (Taylor and Kollros, ’46), the muscle retains its fan shape (Fig. 1A) and is adherent to the overlying skin, especially a t the tendinous intersections. The developing pu- bic bone splits the sixth segment during early

larval life, thereby separating the rectus abdominis from the myotomes. During the metamorphic climax (St. XX-XXV), the pectoral muscle develops ventral and lateral to the three most rostral segments. At this time, the muscle narrows rostrally and acquires the well-defined contours that characterize the postmetamorphic muscle (Fig. 1B). The connective tissue around the muscle thickens and the adhesion between skin and muscle loosens. The shape of the adult muscle differs slightly with the gender of the animal. It is narrower in males than in females and pos- sesses a strip of white extracellular material of undetermined nature along the insertion of the lateral abdominal muscles in males.

Structure of the third most rostral segment The third segment of the rectus abdominis

was selected for morphometric analysis on the supposition that qualitative developmental changes would be best represented in this central segment. The more caudal segments of the muscle do not narrow during metamorphosis to the extent that the rostral segments do. Nevertheless, cursory examination of thick sections of caudal segments disclosed that these segments are qualita- tively similar to the mid-segment in their structure and the changes they undergo during development.

Dimensions The mean length of the mid-muscle seg-

ment increased gradually during larval life (Table 1). At the end of metamorphosis, it was 2.1 _+ .31 mm long. It increased to approximately 5 mm in the adult frogs. On the other hand, the width of the muscle (distance between medial and lateral edges) increased from 2.2 f .51 mm at St. XI to a maximum of 3.4 + .35 mm at St. XVII (Table 1; Fig. 2). Beginning at St. XVIII, fibers degenerated in the lateral half of the muscle (Fig. Z), and the muscle narrowed to 1.5 If: .12 mm. It did not increase in width again until metamorphosis was complete.

Premetamorphosis (St. I-XI) is a period of variable length during which the larvae ap- proach their maximal size and their limbs develop through the foot paddle stage. At St. XI, the maximum thickness of the rectus abdominis (distance between ventral and dorsal surfaces) varied from muscle to muscle, ranging from 45 to 125 pm. The St. XI muscles were never more than five fibers in depth and in some regions were as little as one

320 K. LYNCH

A

U 1 mm

Fig. 1. Rectus abdominis muscles from Rana pipiens. A. A muscle from a St. XI larva. The right side of the specimen that was drawn consisted of only five transverse rows of fibers, whereas the left side had the usual six segments. The segments are separated by tendinous intersections (asterisks). The tendinous intersection between the fifth and sixth segments is irregular and often difficult to discern after the earliest larval stages. The more rostra1 segments are composed of bundles and sheets of fibers which may anastomose or span more than one segment. The oblique abdominal muscles are

indicated laterally. B. A muscle from a frog 2 weeks after metamorphosis. An asterisk marks one of the tendinous intersections that divide the muscle into six com- pact segments. The dashed line indicates the line of insertion of the lateral abdominal muscles (not drawn). It also corresponds to the lateral margin of the upper three segments of the rectus abdominis. The pectoral muscle (PI slightly overlaps the lateral edge of the upper three segments. Muscles in A and B were drawn at the same magnification.

TABLE 1. Dimensions of the third segment of the rectus abdominis muscle of Rana pipiens

Number Maximum of Length Width thickness

muscles (mm) (mm) (mm)

Larval stages Premetamorphic

XI 6 1.2 i 0.37* 2.2 ? 0.51 0.08 k 0.029 Prometamorphic

XVII 3 1.3 k 0.25 3.4 k 0.35 0.12 k 0.004 XVIII 3 1.5 k 0.00 3.1 + 0.59 0.10 k 0.019

Metamorphic XXI XXIV xxv

3 1.7 k 0.40 2.1 f 0.46 0.11 i 0.028 3 1.9 * 0.29 1.5 + 0.12 0.09 i 0.006 3 2.1 k 0.31 1.6 i 0.23 0.08 k 0.010

Frogs 1-3 WPM' 6 2.1 i 0.26 1.6 k 0.18 0.07 i 0.048

Adult 6 5.0 i 1.30 7.1 f 1.40 0.29 i 0.062 8-9 WPM 6 2.7 f 0.41 2.6 f 0.29 0.09 * 0.019

*Mean i standard deviation. ' WPM, weeks postmetamorphosis.

DEVELOPMENT OF ANURAN RECTUS ABDOMINIS

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321

METAMORPHOSIS

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A * * / ( . _ - - - / I L - - \ r - . _ _ < - -

POSTMETAMORPHOSIS

* - _--- - _- 1 *

I _ _ _ _ _ -_ L n

ST XXlV

ST XXV

1-3 WPM

- 1.Ornrn Ventral * Degenerating fibers

Fig. 2. A diagram of changes in the cross-sectional dimensions of the third segment of the rectus abdominis. Profiles are tracings of muscle cross-sections representative of each stage analyzed. The muscle widens during premetamorphosis and early prometamorphosis. At St. XVIII, degenerating fibers (asterisks) appear in the lateral half. With the loss of lateral fibers, the muscle narrows. This process was particularly apparent at St.

XXI; profiles of two of the three St. XXI muscles analyzed are shown to illustrate the variability at this stage. The lateral edge contains degenerating fibers through the end of metamorphosis. A second wave of degeneration, of fibers in the body of the muscle, occurs at the end of metamorphosis and in the first postmetamorphic weeks. The muscle enlarges during adult life, primarily by fiber hypertrophy.

322 K. LYNCH

DEVELOPMENT OF ANURAN RECTUS ABDOMINIS 323

fiber thick (< 15 pm). Some of them consisted of fiber bundles separated by gaps in which no fibers were present.

Premetamophosis is followed by prometamorphosis (St. XII-XIX), a period of approximately 3 weeks during which the limbs achieve their adult form. The maximum thickness of the muscle increased through prometamorphosis and subsequently diminished during the metamorphic climax (St. XX-XXV), a period which lasts approximately 2 weeks and is characterized by emergence of the forelimbs, tail resorption, and other dramatic alterations in the larvae. The maximum thickness of the muscle did not increase significantly during the first 2 months after metamorphosis, but its width (Table 1) and the number of fibers in it (Table 2) increased. The adult muscles were longer, thicker, and wider than those from 8-9-WPM frogs (Table l), but had only 23% more fibers (Table 2).

Distribution of fibers of different types and sizes-general patterns

Several structural patterns were common to muscles from a number of stages. One of these was the distribution of slow fibers. Slow fibers were present in all of the muscles analyzed. As a class, they were smaller than the twitch fibers (Table 2) and less variable in size. (The ratio of the standard deviation of their cross-sectional area to the mean cross- sectional area averaged 0.47, whereas the ratio for twitch fibers was 1.03). In all of the muscles, the slow fibers were more numerous in ventral and medial regions of the muscle than in dorsal and lateral regions.

The range of twitch fiber sizes was broad (1 to 70 pm in diameter in larval muscles), and varied from muscle to muscle in any experi-

Fig. 3. The central region of a St. XXII muscle. In almost all of the muscles analyzed, the ventral surface was easily distinguishable from the dorsal surface because of its relatively high concentration of small fibers, both slow (arrowheads) and twitch (arrows). ~ 5 1 0 .

Fig. 4. The medial margin of a premetamorphic (St. XI) rectus abdominis muscle. Through St. XXN, the larval muscle tapers to a sharp medial edge composed of very small fibers. ~ 7 4 0 .

Fig. 5. The lateral edge of the same muscle shown in Figure 4, at the same magnification. Except at St. XXV, fibers in the lateral half of the muscle are consistently larger than those in the medial half. The lateral half of premetamorphic muscles contains twitch fibers with a wide range of diameters and no slow fibers. X740.

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324 K . LYNCH

mental group. (The greatest variation in the range of twitch fiber diameter was in the 1- 3-WPM muscles, where the range in one muscle was 2 to 18 pm and in another, 2 to 65 pm). In 35 of 39 muscles examined, however, twitch fibers in the ventral half of the muscle were smaller than those in the dorsal half (Table 3; Fig. 3). In all of the larval muscles except those from St. XXV larvae, twitch fibers in the medial half of the muscle were smaller than those in the lateral half (Table 3; Figs. 4, 51, and the medial margin of the muscle tapered to a sharp edge composed of very small fibers. Newly differentiated fibers and satellite cells were frequently present among these small fibers (Fig. 6). The pattern of small medial fibers and a sharp medial edge was not apparent in most of the muscles from St. XXV larvae and 1-3-WPM frogs, or in muscles from 8-9-WPM frogs (Fig. 7). Twitch fibers in the medial half of the adult muscles, however, were again consistently smaller than those in the lateral half, although the adult muscles lacked a sharp medial edge of small fibers.

Degenerating fibers were present at a number of stages. They first appeared as a consistent feature in the St. XVIII muscles, where they were scattered through the lateral half of the muscle (Fig. 2). Thereafter, through the end of metamorphosis, a few degenerating fibers were present at the lateral edge of almost every muscle. Degenerating fibers were virtually absent from the medial half of the muscle until the final stage of metamorphosis (St. XXV), when they appeared within the bodies of all of the muscles examined. They were present also in a third of the muscles from 1-3-WPM frogs, but were absent from 8-9-WPM and adult muscles.

Distribution of fibers-individual stages The earliest larval stage analyzed was St.

XI, the last stage of premetamorphosis. St. XI muscles contained both twitch and slow fibers. The slow fibers were restricted to the medial half of the muscle (Fig. 8A).

By St. XVII, a prometamorphic stage, the larval muscles had reached their maximal width (Table 1). The number of fibers was approximately twice that of St. XI muscles (Table 2). Slow fibers were present in small numbers in octants VL and VCL in one of the three muscles examined, but were concentrated in the medial octants, especially VM, in all of the muscles (Fig. 8B). Small “new” fibers were far more numerous at St. XVII than at St. XI. They constituted a large

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Fig. 6. The medial edge of a prometamorphic (St. X W muscle. In premetamorphic, prometamorphic, and early metamorphic muscles, the most medial profiles include satellite cells (closed arrows) and small, poorly differentiated fibers (open arrows). x 960.

to 9 weeks postmetamorphosis. The fibers forming the medial edge are similar in size to those elsewhere in the muscle. Fibers in this section stained differentially with toluidine blue. Most of the small, pale fibers along the ventral surface (arrowheads) are slow fibers. Twitch fibers, on the other hand, exhibit a range of staining intensities. X280. Fig. 7. The medial margin of a muscle from a frog 8

326 K. LYNCH

FIBERS PER OCTANT

7 "

Q Tmcn RIEI NEW MIIS SLOW M R S

OM DCM DCl DL VM VCM VCL VL OCTANT

Fig. 8. The distribution of fibers among octants of the third most rostra1 segment of the rectus abdominis muscle during pre- and prometamorphic stages. DCL, dorso- centrolateral; DCM, dorso-centromedial; DL, dorsolateral; DM, dorsomedial; N, number of muscles (each from a different animal); VCL, ventro-centrolateral; VCM, ventrocentromedial; VL, ventrolateral; VM, ventromedial. The octants in each muscle were equal in width (medial to lateral distance), but varied in thickness (dorsal to ventral distance). Column heights indicate the mean number of fibers per muscle cross-section; T-bars indicate standard deviations. The number of degenerating fibers per octant is shown as a numeral above each

Fig. 9. A satellite cell (asterisk) closely apposed to the large fiber that lies below it and enclosed in its thin basal lamina. A small, poorly differentiated fiber (open arrow) is also contained within the wispy basal lamina. The closed arrow indicates a profile that presumably belongs to a satellite cell. Such ribosome-laden profiles were frequently encountered in regions that had a high concentration of developing fibers. x 19,300.

Fig. LO. A cluster of three closely apposed profiles. At spots, the apposed plasmalemmae are less than 20 nm

column. A. Stage XI, the last stage of premetamorphosis. Fibers are smaller and more numerous ventrally. Slow fibers are concentrated medially. New (i.e., small, poorly differentiated) fibers are restricted to the medial margin. B. Stage XVII, a prometamorphic stage. The ventral half of the muscle, especially the ventromedial quarter, contains more and smaller fibers than other regions. Most of the slow fibers and small new fibers are concentrated medially. C. Stage XVIII, a prometamorphic stage. The distribution resembles that in St. XVII muscles, except that degenerating fibers (indicated by numbers above the columns) are present in the lateral half of the muscle.

apart. Such clusters of two or more profiles were sometimes enclosed in a common basal lamina, but the basal lamina was often absent or very scanty (as it is here). Clusters were seen at all larval stages examined and in muscles from 1-3-WPM frogs. They were numerous in muscle regions with a high proportion of new fibers and often included satellite cells and immature fibers. (The arrow points to a process that may belong to either.) The more mature fibers were of the same type or, as here, a combination of slow (asterisk) and twitch fibers. x 15,400.

328 K. LYNCH


proportion of the fiber profiles in the medial half of the muscle but were excluded from the lateral half. Single new fibers were frequently closely apposed (within 20-60 nm) to larger, well-developed fibers and enclosed within the basal laminae of the larger fibers when a basal lamina was present (Fig. 9). Their profiles were often in close apposition also to cytoplasmic processes that contained no myofibrils and presumably belonged to satellite cells. Although the profiles of newly differentiated cells were called “fibers,” at least some of them may have represented mononuclear cells. Myoblasts containing my- ofilaments have been reported as a normal stage in the development of Xenopus myoto- ma1 muscle fibers (Kielbbwna, ’66; Muntz, ’75).

Fiber “clusters” were encountered occa- sionally in St. XI muscles. These were composed of two or more small, closely apposed fibers, enclosed in a common basal lamina when a basal lamina was present (Fig. 10). Such clusters were consistently present in muscles from St. XVII and later stages, through the first postmetamorphic weeks. Immature fibers and satellite cells were frequently present in such clusters, in combination with one or more small but well- differentiated fibers. The well-differentiated fibers in any single cluster were sometimes of the same type, either twitch or slow, and sometimes a mixture of both types.

The St. XVIII muscles (Fig. 8C) resembled St. XVII muscles in all respects except that degenerating fibers, empty basal laminae and macrophages were scattered through their lateral halves. Several of the empty basal laminae were postsynaptic to structurally intact presynaptic elements (Fig. 11).

Stages XX through XXV span the metamorphic climax. At St. XXI, the width of the

Fig. 11. Presynaptic elements (closed arrow) on an empty muscle basal lamina (open arrows). Profiles such as this were seen in St. XVIII, St. XXV, and 1-3-WPM muscles. The presynaptic half of the neuromuscular junction is structurally normal. As in the majority of larval neuromuscular junctions, more than one vesicle- filled axonal profile is present, covered by Schwann cell processes. The profile (asterisk) adjacent to the synaptic basal lamina is presumed to be a remnant of the postsynaptic cell (see Fig. 15). x 13,500.

Fig. 12. A portion of the lateral edge of a metamorphic muscle. ARer St. XVIII, the lateral edge consistently contains signs of fiber degeneration: phagocytes (closed arrows), empty muscle basal laminae (open arrows), and degenerating fibers (not included in this field). ~6,200.

third segment of the rectus abdominis was 3/5 of its maximum width (Table 1). The mean number of fibers, however, did not differ significantly from St. XVII and XVm, the average fiber size was simply smaller CTables 1,2). A small number of degenerating fibers, macrophages, and empty basal laminae were present laterally in all of the St. XXT muscles (Fig. 12). New fibers and clusters of small fibers in common basal laminae were present in all octants, with the smallest proportion in the lateral quarter (VL and DL; Fig. 13A).

During the penultimate stage of metamorphosis, St. XXIV, the muscle was less than half its St. XVII width (Table 1). New fibers, while still present throughout the muscle, were reduced in number (Fig. 13B). Degen- erating fibers and empty basal laminae were still present at the lateral edge of the muscle.

New fibers were fewer in St. XXV muscles (Fig. 13C) than in muscles from St. XXIV larvae. The number of degenerating fibers was significantly higher, however. Degener- ating fiber profiles and empty basal laminae were present in small numbers, scattered through all but the most medial extent of the muscles. Presynaptic elements were occa- sionally present on empty basal laminae.

Like St. XXV muscles, muscles from 1-3- WPM frogs contained empty basal laminae that sometimes bore presynaptic elements. In the rare cases where a degenerating fiber was the postsynaptic element, phagocytic cells had invariably dissected the postsynaptic region away from the rest of the muscle fiber, leaving it adherent to the synaptic basal lamina (Figs. 14, 15). New fibers, clusters, and degenerating fibers were also present in the 1-3-WPM muscles (Fig. 16A). All of these features were not represented in each of the six muscles examined, however. Two of the six muscles were thin, with low fiber counts (426 and 432) and small fibers (cross- sectional area < 100 pm21 These contained neither new fibers nor degenerating fibers. Two of the muscles were moderately thick, with high fiber counts (607 and 615) and small fibers (< 100 pm2). These had both immature and degenerating fibers. The last two muscles were thick, with moderate numbers of fibers (456 and 592) of robust size (mean twitch fiber size > 200 pm?. They contained new fibers, but no degenerating fibers.

By 8 to 9 weeks postmetamorphosis, the mean fiber number was approximately twice that of the 1-3-WPM muscles (Table 2). Nei- ther immature nor degenerating fibers were

K. LYNCH

FIBERS PER OCTANT

T

330

ml A ST XXI

T m

1w-1 c STXXV T

DM DCM DCL DL V M VCM VCL VL OCTANT

Fig. 13. Fiber distribution in the third segment of rectus abdominis muscles from larvae in metamorphic climax. A. Stage XXI. Slow fibers show a strong prefer- ence for the ventral surface of the muscle. With the loss of the fibers that comprised the lateral half of the pre- and prometamorphic muscles, slow fibers and new fibers extend into the lateral octants. B. Stage XXN. The num-

apparent (Fig. 16B). From this period to full maturity, the width and thickness of the muscle approximately tripled and the length almost doubled (Table 1). This increase in mass was largely due to an increase in fiber size. The mean number of fibers in the muscles of fully mature frogs was only 1.2 times greater than the number in frogs 8-9 WPM, whereas the mean cross-sectional area of twitch fibers was 7.4 times greater, and of slow fibers, 10.7 times greater (Table 2). The slow fibers in muscles from female frogs were smaller than those in male muscles (cross- sectional area 585 pm2 + 49.9 (SE) vs 886 pm2 f 67.6 (SE), p < 0.01). Neither this difference nor any other was detectable between males and females at other developmental stages. There were virtually no immature or degenerating fibers in the adult muscles (Fig. 16C). The lateral octants (VL and DL) had fewer and larger fibers than the

ber of new fibers has diminished. A small number of degenerating fibers are consistently present at the lateral edge of the muscle. C. Stage XXV. The muscles are similar to St. XXW muscles except that degenerating fibers are scattered through all but the most medial octants, and the number of new fibers is further reduced.

medial ones and a much smaller proportion of slow fibers. As in all the preceding stages, the slow fibers were concentrated ventrally and medially.

Fig. 14. A degenerating fiber bearing a neuromuscular junction (open arrow). Such profiles were seen in St. XVIII and 1-3-WPM muscles. Phagocyte processes (closed arrows) extend between fragments of the degenerating fiber. ~9,900.

Fig. 15. Another section through the neuromuscular junction shown in Figure 14. The presynaptic elements are structurally similar to those on normal muscle B- bers. (Each synaptic vesicle contains a dense central spot, an unusual feature that was present in all of the synaptic vesicles in the muscle from which this section was cut.) Active zones (arrowheads) are present, although their relationship to postjunctional folds is difficult to assess. A phagocyte process (open arrow) separates the postjunctional region (closed arrows) from the rest of the fiber, leaving it adherent to the synaptic basal lamina. ~21,700.

332 K. LYNCH

FIBERS PER OCTANT

160

120

VI p g 40

4 E y1 m

X

DM DCL DL VM VCM VCL VL DCTANT

Fig. 16. Fiber distribution in the third segment of rectus abdominis muscles from frogs. A. 1-3 WPM. The fiber distribution resembles that in St. XXV muscles in all respects except that the mean number of fibers is less. Degenerating fibers are present in small numbers in some of the muscles. B. 8-9 WPM. The number of fibers is approximately equal in the four octants that comprise the dorsal half of the muscle and in the four

Satellite cell distribution The population of satellite cells was quan-

tified in several ways (Table 4). Direct count was made of satellite nuclei; that is, nuclei within cell profiles that included no myofibrils and were within the muscle basal lamina. The ratio of these nuclei to muscle fiber profiles and their ratio to the sum of myonuclei and satellite cell nuclei were computed as well. "he last measure proved to be ex- tremely variable both within and between experimental groups and was therefore not included in Table 4.

The absolute number of satellite cells was lowest in the St. XI muscles. These muscles

that comprise the ventral half. No degenerating or poorly differentiated fibers are present. C. Fully mature adults. The lateral octants contain fewer fibers than other octants, a finding not entirely attributable to the larger number of small slow fibers in the rest of the muscle. The muscles contain no new fibers and only an occa- sional degenerating fiber (a total of three in one of the six muscles).

also had the fewest muscle fibers (Table 2). When normalized by the number of muscle fibers, the concentration of satellite cells was within the same range as at most other stages. Both the absolute number of satellite cells and their ratio to muscle fibers were highest in the muscles from stages XVII, XVIII, and XXI larvae. These muscles also had the highest proportion of new muscle fibers. Because new fibers often occupied the same positions as satellite cells (i.e., in close apposition to, and within the basal laminae of, well-differentiated fibers), it is quite likely that, for these stages, the satellite cell category included at least a few myotube profiles. The degree to which this affected the satel-


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lite cell count can be determined only by analysis of serial thin sections, a task that is currently underway.

Examination of the distribution of satellite cells within the muscle field showed no significant disparities between ventral and dorsal halves or between medial and lateral halves except in the pre- and prometamorphic muscles (stages XI, XVII, and XVIII). During these three stages, the absolute number of satellite cells was much lower in the lateral half of the muscle than in the medial half. When the ratio of satellite cells to muscle fibers was computed, the degree of difference between the medial and lateral halves of the muscle lessened but was still statisti- cally significant in the St. XI and St. XVII muscles.

Summary Both the size of the muscle and the number

of muscle fibers in it increase during premetamorphosis (St. I-XI) and early prometamorphosis (see summary Figs. 2,171. During prometamorphosis (St. XII-XIX), the rate of fiber addition accelerates throughout the medial half of the muscle. Late in prometamorphosis, the width of the muscle diminishes due to degeneration of large fibers in its lateral half. As the lateral margin shifts toward the midline, new fibers and slow fibers, which were initially restricted to the medial half of the muscle, spread across a progressively greater proportion of the muscle field. The rate of fiber addition slows during metamorphosis (St. XX-XXV). At the end of metamor-

FIBERS PER MUSCLE ,400-I T

X I X V I X V H XXI XXW XXV 1-3 8-0 AWJLT WPM WPM

STAGE

Fig. 17. A summary graph of the mean number and type distribution of fibers in the third segment of the rectus abdominis at the stages analyzed. Columns indicate the mean fiber number per muscle cross section. T- bars indicate the standard deviation. Numerals above the columns are the number of degenerating fibers per muscle cross-section. See text for discussion.

334 K. LYNCH

phosis, degenerating fibers appear in all but the most medial region of the muscle. The number of fiber profiles per muscle cross section increases during the first two postmetamorphic months. The muscle grows thereafter primarily by fiber hypertrophy.

DISCUSSION

This study describes the structural alterations that occur in the rectus abdominis of R. pipiens as the animal progresses from early larval stages to maturity.

Muscle growth During premetamorphosis, the rectus ab-

dominis increases gradually in size as the tadpole grows. Comparison of the St. XI larval muscles with cross sections of two late embryonic muscles (data not included) showed clearly that the number of muscle fibers increases during premetamorphosis. Several features of the St. XI muscles sug- gest that new fibers are added at the medial margin of the muscle. These features are the taper in thickness a t the medial edge, the small size of the most medial fibers, the pres- ence among them of poorly differentiated fibers, and the large size of the lateral fibers. However, muscles from several larvae that failed to progress through the larval stages a t the normal rate possessed the same features (data not included). The develop- mentally retarded tadpoles were in premetamorphosis, ranging from St. I11 to St. IX and from 15 to 17 months in age, whereas the normally developing premetamorphic (St. XI) larvae that were analyzed were 13.5 to 19.5 weeks old. Further study is therefore required to determine whether premetamorphic expansion of the rectus abdominis occurs primarily by fiber addition at the medial margin.

The growth pattern of larval muscles changes during prometamorphosis. Satellite cells in the jaw muscles become mitotically active at St. XVIII and give rise to a population of small new fibers (Alley and Cameron, '84). Fiber proliferation accelerates in the cutaneous pectoris muscle during this period (Lin- den and Letinsky, '831, and small, newly differentiated fibers are scattered throughout the medial half of the prometamorphic rectus abdominis. The generation of new myofibers in the jaw muscles has been described on the basis of serial sections and tritiated thymi- dine labeling experiments (Alley and Cam- eron,'84). Satellite cells adherent to large "larval" muscle fibers divide. Their progeny

give rise to a population of myoblasts that fuse to form myotubes. To this point, the process resembles that deduced from morphological analysis of developing mammalian and avian muscles (e.g., Church, '69; Kelly and Zacks, '69; Kikuchi and Ashmore, '76; Campion et al., '81). In mammals and birds, the new myotubes apparently split away from the supporting "primary" fibers to become new, independent muscle fibers (Kelly and Zacks, '69; Ontell and Dunn, '78; Campion et al., '81). In the anuran jaw muscles, on the other hand, the supporting fibers degenerate (Alley and Cameron, '83), leaving the new fibers independent by default.

The structural relationships of poorly differentiated fibers in the rectus abdominis are consistent with the description of fiber formation in the anuran jaw muscles, up to the point of larval fiber degeneration. Immature fibers are frequently included within the basal laminae of large, well-differentiated fibers, often with a satellite cell in close apposition as well, (Only satelite cell profiles that contained nuclei were recorded; profiles of non- nucleated satellite cell processes were far more common.) However, the separation of new fibers from their support fibers does not occur by larval fiber degeneration in the rectus abdominis as it does in the jaw muscles. Instead the process seems to entail separation of small fibers from larger ones, in the man- ner deduced from observations of the developing muscles of higher vertebrates (Kelly and Zacks, '69; Ontell and Dunn, '78). Cell proliferation and fusion along the surface of small fibers that have separated from their support fibers may account for the clusters of small fibers within common basal laminae that are especially numerous in the late prometamorphic muscles. By 8-9 WPM, all of the new fibers have apparently achieved inde- pendence; fibers no longer lie in close apposition (within 20-60 nm) nor do they share basal laminae.

The difference in the proliferative response of the two halves of the rectus abdominis during prometamorphosis and early metamorphosis may be due to the differential distribution of satellite cells in the pre- and prometamorphic muscles; satellite cells are infrequent in the lateral half of the muscle be- fore the lateral half is lost. Some of the dispar- ity between the medial and lateral satellite cell concentrations in the prometamorphic muscles may be due to artefactual inclusion in the satellite cell category of new fibers that happened to be sectioned through a region


bearing no myofibrils. This cannot totally account for the difference, however. One of the St. XI muscles had no fibers that could be classified as newly differentiated and several had medial octants that contained no new fibers, yet all of the St. XI muscles exhibited a medial to lateral gradient in satellite cell concentration. Once established, a gradient of mitotically active cells could be self-maintain- ing and the basis for a differential response to systemic growth-promoting agents.

The proliferative response of the satellite cells in the rectus abdominis during prometamorphosis is likely to be a response to the hormonal changes that govern anuran metamorphosis (see Galton, '83, for review). Prior to prometamorphosis, the satellite cells presumably serve as the source of myonuclei for enlarging muscle fibers (Moss and Leblond, '71). (The ratio of myonuclei to cross-sectional fiber area remains relatively constant while the cross-sectional area of the muscle fibers increases.) The accelerated formation of new fibers during prometamorphosis indicates a switch, therefore, in the fate of the satellite cell progeny. Instead of fusing with well-established, growing muscle fibers, they must form myoblasts which fuse with each other rather than with the preexisting population of fibers. It is not possible at this point to determine whether the shift reflects the pres- ence of two subpopulations of satellite cells with different hormonal sensitivities, or simply a hormonally induced alteration in a ho- mogeneous population.

During the first 2 months after metamorphosis, the number of muscle fibers in the middle segment of the rectus abdominis approximately doubles, yet there is only a small number of new fiber profiles in the 1-3-WPM muscles and none in the 8-9-WPM muscles. The difference in fiber number may be due to differences in the stocks from which the frogs came (see Materials and Methods). On the other hand, it may be due to fiber elongation. Profiles of new fibers in the 1-3-WPM muscles may represent myoblasts or myotubes which have not yet extended the full length of the muscle segment. Growth and extension of short fibers to their full adult length would re- sult in more profiles in any single muscle cross section (see also Ontell and Dunn, '78).

Postmetamorphic growth of the redus abdominis is largely by hypertrophy. This is in agreement with the discovery by Speny ('81) that the limb muscles of Rana pipiens grow primarily by hypertrophy rather than hyperplasia.

Fiber degeneration Abruptly at St. XVIII, while new fibers are

proliferating in the medial half of the rectus abdominis, fibers in the lateral half begin to degenerate. This spatial segregation of proliferation and degeneration differs from the situation in the jaw muscles, where new and degenerating fibers intermingle in the muscle bed (Alley and Cameron, '83). There is no obvious cause for the differential response on the part of the two regions of the rectus abdominis. In the jaw muscles, the degenerating fibers are the larval fibers, i.e., those that were formed during premetamorphosis, iden- tifiable by their large size. Large larval fibers form the bulk of the lateral rectus abdominis, but they must also be present in the medial half of the muscle; immature fibers are mixed with large well-developed f i - bers during prometamorphosis and early metamorphosis, yet only the laterally dis- posed larval fibers degenerate.

The lateral degeneration is presumably a response to thyroid hormone, as degeneration in the tail and elsewhere in the meta- morphoric anuran has proved to be (reviewed by Galton, '83). The response in the rectus abdominis, as in the jaw muscles (Alley and Cameron, '831, precedes that in the tail, however. If the changes in concentration of cir- culating thyroid hormone in R. pipiens parallel those measured in R a m catesbiana (Regard et al., ,781, hormone levels are just beginning to rise precipitously at St. XXI. Most of the fibers in the lateral rectus abdominis have degenerated by the end of St. XXI, but the tail is just beginning to shorten (Etkin, '68).

Only during the last stage of metamorphosis (XXV) and the first postmetamorphic weeks, when thyroid hormone levels are de- clining (Regard et al., '78), do profiles of degenerating fibers appear within the body of the rectus abdominis (i,e., the original medial half). This second wave of degeneration may be caused by the poor nutritional state of the animals at this phase of their life cycle. Although the 1-3-WPM frogs were supplied with fruittlies, some of them were thin and their digestive tracts empty at the time of sacrifice. The variability in the general state of the early postmetamorphic frogs was re- flected in variability in the structure of their rectus abdominis muscles (see Results). The most robust of these contained no degenerating fibers. On the other hand, neither did the muscles with the fewest, thinnest fibers. An-

336 K. LYNCH

other indication that the second degenera- tive phase may not be due to starvation is the observation that the degeneration begins abruptly at St. XXV, when the animals appear to be quite healthy. All three of the muscles from St. XXV larvae contained degenerating fibers, even though they differed very little from St. XXIV muscles in other respects and possessed a healthy contingent of small new fibers.

Whatever its cause, the second wave of degeneration raises the possibility that in the rectus abdominis, as in the jaw muscles, fibers formed during early larval life do not survive into the adult animal. Their removal from the jaw muscles is complete by mid- metamorphosis. If they are also completely removed from the rectus abdominis, it must occur in two phases: degeneration of lateral fibers during prometamorphosis and early metamorphosis, and loss of the remainder at the end of metamorphosis. It would be of interest to know if there is indeed selective loss of larval fibers, and, if so, why it occurs at different times in the two halves of the muscle.

The structurally normal presynaptic elements observed on basal laminae from which muscle fibers had been phagocytosed is the first direct evidence that denervation does not precede or play a role in metamorphic fiber degeneration. It is also the first view of naturally occurring unilateral neuromuscular junctions, a synaptic configuration here- tofore seen only in experimental preparations. Marshall, Sanes, McMahan, and their colleagues have produced such struc- tures experimentally in the frog cutaneous pectoris (for review see Sanes, '83). By doing so, they discovered that the synaptic region of the muscle basal lamina contains the in- formation necessary to induce regenerating axons to adhere to it and differentiate into functional presynaptic elements. The results of this study further verify their conclusion.

The fate of the isolated presynaptic elements is unknown. They may eventually re- tract as other branches of their motoneurons innervate new fibers elsewhere in the muscle. This is the case in the jaw muscles; the trigeminal motoneurons that innervate larval fibers in the jaw muscles are repro- grammed at metamorphosis to innervate the new adult myofibers (Alley and Barnes, '83; Barnes and Alley, '83).

Slow fiber distribution The results of this study confirm the report

by Uhrik and Schmidt ('73) that slow fibers are concentrated ventrally and medially

within the rectus abdominis. In addition, the results show that slow fibers are present in larval muscles at least as early as St. XI. They are virtually absent from the lateral half of the St. XI muscles. If fibers are added at the medial edge of premetamorphic muscles, then slow fibers do not appear until late in premetamorphosis. The number of slow fibers per muscle is relatively constant through prometamorphosis and metamorphosis, in spite of the prometamorphic increase in new fibers. The number of twitch fibers also remains relatively constant until after metamorphosis. Twitch fibers are being lost to degeneration in the lateral half of the muscle, however, whereas slow fibers are not. The majority of the fibers that form during late prometamorphosis must therefore differentiate into twitch fibers. This suggests that slow fibers differentiate during a narrow developmental window between late premetamorphosis and the end of prometamorphosis. It is by no means clear yet what causes the differentiation of slow fibers or other muscle fiber types during development (e.g., Hanzli- kova and Schiaffino, '73; Gordon et al., '81; Butler et al., '82; Laing and Lamb, '83; and McLennan, '83).

As the lateral half of the muscle degener- ates during early metamorphosis, the medial half of the original prometamorphic muscle becomes the whole muscle. The medial to lateral gradient of slow fibers is diminished by this shift, but is not obliterated. The ventral to dorsal gradient, on the other hand, intensifies. The factors that dictate this differential distribution of slow fibers are unknown.

CONCLUSIONS

The rectus abdominis muscle undergoes substantial remodeling in the course of anuran metamorphosis. Exposure to hormonal changes which are presumably similar throughout the muscle elicits fiber proliferation in one region of the muscle and fiber degeneration in another. Myogenic cells pro- liferate in the medial portion of the muscle during the period when thyroid hormone levels in the blood are slowly rising. Larval f i - bers begin to degenerate in the lateral half of the muscle during the same period, but at a slightly later stage. Both degeneration and proliferation continue at a slower rate during the metamorphic climax, when thyroid hormone levels are at their peak. Additional fibers are lost from the remainder of the muscle during the last stage of metamorphosis and the first postmetamorphic weeks, a pe-


riod during which hormone levels are ap- proaching their premetamorphic low. Muscle growth is minimal during this period. When it resumes, it takes the form of fiber hypertrophy rather than hyperplasia.

ACKNOWLEDGMENTS

The technical assistance of Mr. C. Darryl Harris and the secretarial service of Mrs. Mary Thomson are gratefully acknowledged.

This work was supported by U.S. Public Health Service Grant NLS 5 R01 NS17417- 03 and by the Uniformed Services University of the Health Sciences. The opinions or asser- tions contained herein are the private ones of the author and are not to be construed a s official or reflecting the views of the DoD or the USUHS. The experiments reported herein were conducted according to the prin- ciples set forth in the Guide for Care and Use of Laboratory Animals, Institute of Labora- tory Animal Resources, National Research Council, DHEW Pub. No. (NIH) 78-23.

LITERATURE CITED

Alley, K.E., and M.D. Barnes (1983) Birth dates of trigeminal motoneurons and metamorphic reorganization of the jaw myoneural system in frogs. J. Comp. Neurol. 218:395-405.

Alley, K.E., and J.A. Cameron (1983) Turnover of an- w a n jaw muscles during metamorphosis. Anat. Rec. 205t7A.

Alley, K.E., and J.A. Cameron (1984) Cellular features of metamorphic myofiber turnover in the leopard frog. Anat. Rec. 208t7A.

Barnes, M.D., and K.E. Alley (1983) Maturation and recycling of trigeminal motoneurons in anuran larvae. J . Comp. Neurol. 218:406-414.

Butler, J., E. Cosmos, and J. Brierley (1982) Differentia- tion of muscle fiber types in aneurogenic brachial muscles of the chick embryo. J. Exp. Zool.224:65-80.

Campion D.R., S.P. Fowler, G.J. Hausman, and J.O. Re- agan (1981) Ultrastructural analysis of skeletal muscle development in the fetal pig. Acta Anat. (Basel) 110:277-284.

Church, J.C.T. (1969) Satellite cells and myogenesis: A study in the fruitbat web. J . Anat. 105:419-438.

Etkin, W. (1968) Hormonal control of amphibian metamorphosis. In W. Etkin and L.I. Gilbert (eds): Meta- morphosis: A Problem in Developmental Biology. New York: Appleton, pp. 313-348.

Forrester, T., and H. Schmidt (1970) An electrophysiolog- ical investigation of the slow muscle fibre system in the frog rectus abdominis muscle. J. Physiol. (Lond.) 207:477-491.

Galton, V.A. (1983) Thyroid hormone action in amphibian metamorphosis. In J.H. Oppenheim and H.H. Sam- uels (eds): Molecular Basis of Thyroid Hormone Action. New York Academic Press, pp. 445-483.

Gordon, T., G. Vrbova, and G. Wilcock (1981) The influ- ence of innervation on differentiating tonic and twitch muscle fibers of the chicken. J. Physiol. (Lond.) 319t261- 269.

Hanzlikova, V., and S. Schiaffno (1973) Studies on the effect of denervation in developing muscle. 111. Diver- sification of mvofibrillar structure and orimn of the heterogeneity ’bf muscle fiber types. Z. Zellforsch. 147:75-85.

Karnovsky, M.J. (1971) Use of ferrocyanide-reduced os- mium tetroxide in electron microscopy. J. Cell Biol. 51 :146A.

Kelly, A.M., and S.I. Zacks (1969) The histogenesis of rat intercostal muscle. J. Cell Biol. 42t135-153.

KieJbowna, L. (1966) Cytological and cytophotometrical studies on myogenesis in Xenopus laeuis (Daudin). Zool. Pol. /1:247-255.

Kikuchi, T., and C.R. Ashmore (1976) Developmental aspects of the innervation of skeletal muscle fibers in the chick embryo. Cell Tissue Res. 171:233-251.

Korneliussen, H. (1972) Identification of muscle fiber types in “semithin” sections stained with p-phenylene- diamine. Histochemie 32:95-98.

Laing, N.G., and A.H. Lamb (1983) The distribution of muscle fibre types in chick embryo wings transplanted to the pelvic region is normal. J. Embryol. Exp. Mor- phol. 78:67-82.

Linden, D.C., and M.S. Letinsky (1983) Correlated nerve and muscle differentiation in the bullfrog cutaneous pectoris. In A.D. Grinnell and W.J. Moody, Jr. (eds) The Physiology of Excitable Cells. New York: Alan R. Liss, pp. 423-433.

Lynch, K. (1983) Structure of the rectus abdominis muscle in frog larvae and postmetamorphic juveniles. Anat. Rec. 205t115A.

Lynch, K.J., and C.D. Harris (1984) Metamorphosis of the rectus abdominis muscle in Rana pipiens. Anat. Rec. 208t106A.

McLennan, I.S. (1983) The development of the pattern of innervation in chicken hindlimb muscles: Evidence for specification of nerve-muscle connections. Dev. Biol. 97:229-238.

Moss, F.P., and C.P. Leblond (1971) Satellite cells as the source of nuclei in muscles of growing rats. Anat. Rec. 170:421-436.

Muntz, L. (1975) Myogenesis in the trunk and leg during development of the tadpole of Xenopus Zaeuis (Daudin 1802). J. Embryol. Exp. Morphol. 33t757-774.

Ontell, M. (1977) Neonatal muscle: An electron microscopic study. Anat. Rec. 189t669-690.

Ontell, M., and R.F. Dunn (1978) Neonatal muscle growth A quantitative study. Am. J. Anat. 152:539- 556.

Page, S.G. (1965) A comparison of the fine structure of frog slow and twitch muscle fibers. J . Cell Biol. 26t477- 497.

Peachey, L.D., and A.F. Huxley (1962) Structural identification of twitch and slow striated muscle fibers of the frog. J. Cell Biol. 13:177-180.

Regard, E., A. Taurog, and T. Nakashima (1978) Plasma thyroxine and triiodothyronine levels in spontaneous- ly metamorphosing R a m catesbiana tadpoles and in adult anuran amphibia. Endocrinology 102t674-684.

Sanes, J.R. (1983) Roles of extracellular matrix in neural development. Annu. Rev. Physiol. 45.581-600.

Shumway, W. (1940) Stages in the normal development of Rana pippiens. 1. External form. Anat. Rec. 78:139- 147.

Snow, M.H. (1983) A quantitative ultrastructural analysis of satellite cells in denervated fast and slow muscles of the mouse. Anat. Rec. 207.593-604.

Sperry, D.G. (1981) Fiber type composition and postmetamorphic growth of anuran hindlimb muscles. J. Mor- phol. 170:321-345.

Taylor, A.C., and J.J. Kollros (1946) Stages in the normal development of Rana pipiens larvae. Anat. Rec. 94:7- 23.

Uhrik, B., and H. Schmidt (1973) Distribution of slow muscle fibres in the frog rectus abdominis muscle. Pflugers Arch. 34Ot361-366.

Watanabe, K., and F. Sasaki (1974) Ultrastructural changes in the tail muscles of anuran tadpoles during metamorphosis. Cell Tissue Res. 155t321-336.

growth and metamorphosis of the rectus abdominis muscle in rana pipiens

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