Differential expression of the Ca2+-binding protein parvalbumin during myogenesis in Xenopus laevis

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BIOLOGY

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(1988)

Differential Expression of the Ca*+ -Binding Protein Parvalbumin during Myogenesis in Xenopus laevis LAWRENCE

M. SCHWARTZ* AND BRIAN K.

KAY?

*Department of Zodogy, Mwrill Science Center, University of Massachusetts, Amherst, Massachusetts and fDepa&nent of Biology, University of North Carolina Chapel Hill, North Carolina 275993280 Accepted April

01003;

19, 1988

We have used immunocytological techniques to examine the developmental expression of the Ca’+-binding protein parvalhumin in Xenopus Levis embryos. Western blot experiments show that at least three different forms of parvalbumin are expressed during embryogenesis; the tadpole tail expresses one form, adult brain expresses another, mylohyoid muscle expresses both, and gastrocnemius and sartorius muscles express these two plus a third form. Parvalbumin (PV) is first detectable by immunofluorescence at stages 24-25 of development, a time when myotomal muscles are differentiating and contractile activity occurs spontaneously in embryos. At metamorphosis, PV is expressed in developing limb muscles. While the majority of skeletal muscle fibers express high levels of PV in both embryos and adults, a second fiber type has no detectable PV. The arrangement of PV-containing fibers is stereotyped in each muscle group examined. Histochemical staining of tadpole muscles indicate that PV-containing fibers correspond to fast-twitch skeletal muscles, whereas those without PV correspond to slow-twitch muscles. During tail resorption at metamorphosis, PV appears to be extruded from dying tail muscle cells and taken up by phagocytic cells. o 1988 Academic press, IX.

INTRODUCTION

Amphibia, in contrast to most other vertebrates, display a range of profoundly different developmental programs for the generation of skeletal muscle. At least three major developmental programs can be identified: embryonically derived muscle which undergoes programmed cell death at metamorphosis, embryonically derived muscle which survives in the adult, and postembryonically derived muscle which forms at metamorphosis. At present, however, little is known about the factors which determine the developmental decisions required for each of these muscle programs. Besides these more gross developmental programs, there are additional differentiative decisions made by muscles. Within most muscles, there are individual fibers which differ regarding their fiber type. Muscle fibers can be categorized on the basis of their morphology, contractile properties, and metabolic capabilities (Carlson and Wilkie, 1974). While the distribution of fiber types has been extensively examined in birds and mammals, much less is known about different fiber types in amphibia. In an attempt to understand how amphibian muscle cells make differentiative developmental decisions, we have begun to examine the temporal and spatial expression of certain proteins involved in muscle physiology. One of these proteins is parvalbumin (PV). PV is a 12,000-Da polypeptide which contains two high-affinity Ca2+-binding domains (Kretsinger, 1980; Heizmann and 441

Berchtold, 1987). Several lines of evidence suggest that PV is intimately involved with relaxation by fasttwitch skeletal muscle fibers. First, PV is predominantly found in fast-twitch fibers, where its concentration in myoplasm can approach 1 mM (Baron et aL, 1975; Celio and Heizmann, 1982; Wnuk et ab, 1982). Second, denervated rat extensor digatorum longus (EDL) muscle displays both a dramatic increase in relaxation time and a corresponding decrease in PV content when compared to control muscles (Heizmann and Berchtold, 1987). Third, fast-twitch muscles from the mouse mutant “arrested development of righting response” (c&r) are very deficient in PV content and undergo an aberrant series of tetanic contracture following the termination of nerve stimulation, unlike normal muscles which cease contractures once motorneuron stimulation ends (Stuhfauth et ab, 1984). In this study, we have examined the temporal and spatial expression of PV during myogenesis in the African clawed frog Xenopus laevis. We have focused on three selected muscles which are representative of the major developmental programs described above. The myotomal muscles form in the tail of the embryo and degenerate at metamorphosis. The mylohyoid muscle, which resides at the floor of the mouth, appears in the embryo and persists in the adult. The third group studied were the muscles of the limbs, which appear de novo with metamorphosis. We found that all of these muscles contained fibers which express PV at some point of 0012-1606/88 $3.00 Copyright All rights

0 1988 by Academic Press, Inc. of reproduction in any form reserved.

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their development. Moreover, the arrangement of the PV-containing fibers is stereotyped and suggests that all muscle groups have a mixture of more than one fiber type. METHODS

Animals Xenopus la&s adults were purchased from Xenopus I (Ann Arbor, MI) and maintained as previously described (Nieuwkoop and Faber, 1967). Eggs were obtained from females injected with human chorionic gonadotropin (Sigma Chemical Co., St. Louis, MO) and fertilized with sperm from testis explants. Embryos were reared at approximately 21°C and were staged according to Nieuwkoop and Faber (1967). Immuno$wnvscent

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Staining

The presence of PV in cells was monitored by indirect immunoflourescence with a polyclonal antibody generated in rabbits against Xenow leg muscle PV (Kay et al, 1987). Tadpoles were fixed from 2 hr to overnight in alcoholic Bouin’s fixative (Humason, 1979) at room temperature. Individual animals were either embedded in paraffin for sectioning or left intact for whole mount processing and immunoflourescent staining (Kay et aL, 1987). Following immunofluorescent staining, intact animals or dissected tissues were then cleared in glycerol with 2.0% n-propyl gallate (Giloh and Sedat, 1982) for examination by epifluorescence with a Nikon Optiphot microscope. Specimens were photographed with Tri-X film at 400 ASA. Rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR) was used to stain the actin filaments in muscle cells (Wulf et aL, 1979). Tissue for phalloidin staining was fixed in 2.0% paraformaldehyde overnight and then treated as above. A mouse monoclonal antibody (JLABO) to rat skeletal actin was the gift of Benjamin Peng and J. Lin (Lin, 1981). This antibody was detected with goat anti-mouse Ig antibodies coupled to FITC (Cappel Biochemical, Inc.). Histochemical Staining The tails from stage 55 tadpoles were frozen in isopentane in liquid nitrogen and sectioned at 8 pm on a cryostat. Sections were air-dried and treated for NADH tetrazolium reductase or myosin ATPase enzymatic activity according to standard protocols (Dubowitz and Brooke, 1973). Western Blot Tissue was isolated from tadpoles or adults, homogenized in gel loading buffer (Laemmli, 1970), boiled, and

fractionated by SDS-polyacrylamide gel electrophoresis in a 12% gel. Proteins were then blotted to nitrocellulose (Towbin et aL, 1979) and reacted with rabbit anti-PV antibodies, and immune complexes were detected with goat anti-rabbit IgG antibodies conjugated to horseradish peroxidase (BioRad, Richmond, CA) and 4-chloro-1-napthol (Sigma Co.). RESULTS

Tissue Distribution

of Parvalbumin

All of the data generated in this study were obtained with a polyclonal antibody generated in rabbits against purified Xenopus leg muscle parvalbumin (Kay et al, 1987). To validate the specificity of the antibody, and to examine the tissue distribution of the recognized antigens, a western blot was performed. The tissues examined were whole tadpole tail extract, adult brain, and mylohyoid, sartorius, and gastrocnemius muscles from adults (Fig. 1). Interestingly, the sizes of the detected proteins differed among tissues. In tadpole muscle, a single protein was resolved with an apparent molecular weight of 12 kDa. On screening tadpole cDNA libraries,

A

B

C

D

E

kD 9768-

25-

18-

14-

FIG. 1. Western blot of various tissues probed with the PV antibody. Lanes (A-E) contain equivalent amounts of protein extracted from stage 55 tadpole tail, adult brain, adult mylohyoid muscle, adult gastrocnemius muscle, and adult sartorius muscle, respectively. The adult tissues were from a malexenqpus. The molecular weights of size standards are labeled on the left in kilodaltons &Da).

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Parvalbmnin Exprwwion during Mzlogenesis

Kay et al. (1987) obtained clones encoding for a single protein with a predicted translation product size of 12 kDa. In adult brain, a single protein of approximately 13.5 kDa was detected. Extracts of mylohyoid and sartorius muscles had three immunoreactive species with apparent molecular weights of 12, 13.5, and 14 kDa, whereas the gastrocnemius muscle had only the 12- and 1GkDa species. These data suggest that up to three related parvalbumin polypeptides can be expressed in these tissues. Polymorphic expression of parvalbumin has been described in other vertebrates, and sequence determination has shown the isoforms to vary in primary sequence (Goodman et aL, 1979). These patterns of expression have been confirmed by a number of independent approaches. First, polyclonal antibodies affinity purified to the bacterially expressed /3-galactosidase-PV fusion polypeptide encoded by a characterized recombinant bacteriophage (Kay et uL, 1987) react with these same sized polypeptides on Western blots (Alexander and Kay, unpublished). Second, Western blots of proteins separated by two-dimensional polyacrylamide gel electrophoresis have demonstrated the existence of three distinct cross-reacting species of similar size and isoelectric point (Schwartz and Kay, in preparation). Third, three different cDNA species encoding parvalbumin proteins have been isolated and sequenced from Xgtll libraries from adult muscle (Alexander and Kay, unpublished). Together, these data suggest that (a) the antibody is highly specific for parvalbumin proteins, and (b) multiple species of parvalbumin are expressed in different adult tissues of xe??Qwus.

Development Appearance of Parvalbumin We next sought to determine both the cellular distribution of PV and the development timing of its expression. The histological technique of indirect immunofluorescence was used to localize PV-reactive antigens in fixed specimens. It has been previously shown by Northern and Western blotting procedures that parvalbumin RNA and protein, respectively, are absent from Xm oocytes and eggs (Kay et cd, 1987). These molecules are first detected in embryos at stage 24 of development. In agreement with the blotting experiments, sections of stage 23 embryos did not display PV immunoreactivity (Fig. 2A). However, about an hour and a half later, at stage 24, PV could be faintly seen in the dorsal region of the myotomal muscles (Fig. 2B). This is an important stage in development for the neuromuscular system, as it is at stage 24 that the myotoma1 muscles are first able to undergo spontaneous, motorneuron-induced contractures (Hamilton, 1969; Blackshaw and Warner, 1976). Staining was more ap-

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parent approximately 1 hr later, when the embryos reach stage 25 (Fig. 2C). During the next 5 hr, the animals progressed to stage 28, and strong PV immunoreactivity was evident in the myotomal muscles (Fig. 2D). At stage 31 of development, 5 hr later, the accumulation of PV in muscle fibers was quite pronounced (Fig. 2E). We have followed development of the myotomal muscles as far as stage 55, a time when the hindlimbs are well formed. Despite the large size of the myotomal muscle fibers, PV immunoreactivity was still quite striking (Fig. 2F). At no stage of development was PV immunoreactivity seen in cardiac or smooth muscles. This result is clearly demonstrated in Fig. 2E, as this section includes regions of the central nervous system, notochord, gill arches, and gut. Interestingly, PV staining was seen in the retina, but none was seen in the brain (for example, note the absence of neuronal staining in Fig. 3B). The lack of staining in the brain may be due to either the absence of PV in larval brain or, more likely, a detection problem due to the sparse arrangement of presumptive PV-containing neurons. In other organisms, PV has been detected in retinal cells and certain brain neurons (Celio, 1985; Endo et al, 1986).

Arrangement of P V Expressing and Nonexpressing Cells in M~otomd Muscles The myotomes of the tail begin in the head cavity and extend toward the tip of the tail. This can be seen in a sagittal section through the anterior region of a stage 41 tadpole (Fig. 3A). The first myotomal segments flank the base of the brain, and then repeat for the length of the animal. When this specimen was examined for PV immunoreactivity, the myotomal muscles were prominently stained (Fig. 3B). On close examination, it appeared that the outermost muscle fiber from each segment showed greatly reduced immunoreactivity. Along the length of the animal the nonreactive (PV-) muscle cells became increasingly more apparent. This is demonstrated in Fig. 3C, which shows a caudal region of a whole mount tail preparation stained with the PV antibody. At the rostra1 end of the animal, the PV+ cells run the entire length of the myotomal segments, whereas at the caudal end they appear truncated. Moreover, these PV+ cells exist as short fat fibers which reside more or less centrally within each segment (Fig. 3D). Peripheral to these cells are long, thin muscle fibers that are PV-. The organization of these two fiber types in the tadpole tail is readily seen following staining with phalloidin (Fig. 3E), a phallotoxin which intercalates into actin filaments (Wulf et aL, 1979). In the phalloidin stained tail strips, the PV+ fibers span the entire myotomal segment in the anterior tail fragment (bottom), and there is a characteristic striped pattern of PV+ fibers in

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FIG. 2. Immunolocalization of PV in different staged Xe-nop~~ embryos. Stage (A) 23, (B) 24, (C) 25, (D) 2’7, (E) 31, and (F) 55 embryos (Nieuwkoop and Faber, 1967) were preserved in Bouin’s fixative, embedded in paraffin, and cross-sectioned in the midportion of the embryo (A-E) or the tadpole tail (F). Rabbit anti-parvalbumin antibodies were detected with goat anti-rabbit IgG antibodies conjugated to FITC. Due to the abundance of yolk in frog embryos, there is a high level of autofluorescence in the sections of stage 23-24 embryos. In (E) some structures are labeled (C, neural tube (CNS); N, notochord; S, somite, G, gill arches; E, endoderm). Bar = 100 pm in (A-E) and 250 pm in (F).

the posterior region of the tail (top) (Fig. 3F). The short, fat PV staining fibers are more heavily stained than the long, thin fibers due to a greater number of myofilaments per cell (Fig. 4B) (Weber, 1964). In addition to their rostral/caudal distribution, PVcontaining fibers also displayed a stereotyped periph-

era1 to axial distribution. Figure 4A shows a phasecontrast photograph of a portion of the myotomal muscles from a stage 55 embryo. The muscle fibers just under the skin of the animal (right side of the section) have a narrow cross-sectional area. In contrast, the more internal fibers are distinctly larger. All of these

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FIG. 3. Organization of muscle fiber types in tadpole tail, Tails from stage 41 embryos were fixed, permeablised, and stained with either phalloidin or the PV antibody. (A) The tail in longitudinal section in phase-contrast, the hindportion of the brain is labeled (Br). (B) The same section stained for PV immunofluorescence. (C) PV-stained whole mount of a strip of fibers teased from the caudal portion of stage 41 tadpole tail. (D) Phase-contrast micrograph of a comparable tail strip; the black dots are melanocytes. (E) Rhodamine-conjugated phalloidin staining of a tail strip. (F) PV immunoflourescence staining of strips of muscle teased away from different regions of the same tail. Bar = 100 pm in (A-C) 50 &cmin (D), 85 pm in (E), and 250 pm in (F).

fibers stained with a mouse monoclonal antibody (JLA20) directed against skeletal muscle actin (Fig. 4B). As suggested by the phalloidin-stained material

above, the pattern of myofibrils differs between the two fiber types; the entire cross-sectional area of the large fibers stains uniformly, while the thin peripheral fibers

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FIG. 4. Fiber type composition of tail muscle. (A) Phase-contrast image of a crosssection through a stage 55 embryo at the anterior end of the tail. (B) The identical section stained with a mouse monoclonal antibody (JLA20) which reacts with skeletal actin. (C) PV immunoreactive fibers within the same section. The arrows in (A-C) highlight a single muscle cell at the outer rind of the tail which reacts with phalloidin, but does not react with PV antibodies. (D) bright-field view of a cryostat sectioned stage 55 tail that has been stained for NADH tetrazolium reductase, an enzyme activity which is typically restricted to slow-twitch muscle fibers. In (D), the histochemical deposits are restricted to the muscle cells at the outer rind of the tail. Bar = 38 pm.

display small bundles of myofibrils. Both fiber types tion, PV content, myofibrillar organization, and NADH are polynucleated at this stage of development, as de- reductase activity. termined by staining with the fluorescent nuclear dye DAPI (data not shown). When this same section was PV Expression in the M~otmnal Muscles stained for PV immunoreactivity, only the larger fibers during Metawmphosis reacted. In a comparable frozen section (Fig. 4D), these As stated in the Introduction, the myotomal muscles peripheral non-PV-staining fibers selectively stained display a very unique developmental program. These for NADH tetrazolium blue reductase, an activity which serves as a marker for slow-twitch fibers (Dubo- embryonically derived muscles undergo a developmenwitz and Brooke 1973). Comparable sections were then tally programmed death at metamorphosis. We were tested for acid-stable myosin ATPase activity at pH 4.3, curious to follow the fate of PV during tail reabsorpwhich is diagnostic for fast-twitch fibers. In this case, tion. Whole embryos were fixed at different stages of tail resorption and permeablized, and the PV-containthe large diameter fibers were more darkly stained than the thin peripheral fibers (data not shown). These ing cells were stained as described under Methods. At stage 61 of development, the tail was essentially full data are in good agreement with the reported distribution of fast- and slow-twitch muscle fibers in Runa tad- length, but did not generate rhythmic beats as in younger animals These animals displayed the normal pole tails (Watanabe et aZ., 1978b). Taken together, these data suggest that there are two fiber types in patterns of PV fibers in the tail as described above for Xbnopus myotomal muscle, which differ in size, loca- stage 55 animals. On closer examination, however, it

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was apparent that PV staining of some cells was greater at the periphery of the cell (Fig. 5B). In many cells, there were “blebs” on the surface of the fiber which stained intensely. These blebs were concentrated in patches and appeared to be extracellular when viewed with phase-contrast optics. At stage 62, about a day later in development, there was much more pronounced degeneration of the myotoma1 musculature. Many small nonmuscle cells in the tail, most notably at the tip, were intensely immunoreactive and may be macrophages phagocytosing PV from the extracellular space (Weber, 1964; Watanabe et aZ., 19’78a).There were no staining muscle cells toward the tip of the tail and the ones in the anterior half were obviously degenerate. Figures 5C and 5D show two cells at different stages of degeneration; the lower cell looks grossly normal, except that it has poorly resolved sarcomeres and displays peripheral PV staining, and the upper cell is extremely deteriorated and consists of apparent macrophages and blebs of material. A comparably staged animal was fixed and sectioned for examination (Figs. 5E and 5F). Here one can see muscle fibers which display irregular cross-sectional profiles and sarcomeres. The PV immunoreactive material is restricted to the periphery of the cells or extruded into the extracellular spaces. By stage 63.5, the tail had regressed to the point that it was much shorter than the body of the animal and was curled upward at the end. Only a few muscle cells could be found in the anterior portion of the tail and they were wrapped around the notochord as a thin rind. Of the few PV reactive cells present, most were loosely connected blebs of material outlining the sarcolemma.

PV Expression in the Mglohgoid Muscle The mylohyoid muscle reflects the second form of developmental programs displayed by amphibian muscle. In this case, the muscle arises during embryogenesis and persists throughout the life span of the animal. The mylohyoid muscle is organized as a trapezoidal sheet of flattened fibers at the base of the floor of the mouth (Fig. 6A), where it is used in both respiration and swallowing (Deuchar, 1975). This whole mount preparation of a stage 43 animal was stained for PV immunoreactivity (Fig. 6B). As was seen in the myotoma1 muscles, the mylohyoid is composed of both PVand PV+ fibers. The fibers at the anterior of the mylohyoid were PV-, while the posterior-most fibers were PV+. This pattern of staining was confirmed in stained sections of tadpole heads, where we found that only a small number of the posterior fibers in the mylohyoid muscle reacted with the antibody (Fig. 6D). Thus, the mylohyoid muscle, like those of the myotome, displays

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two fiber types based on fiber location and PV immunoreactivity. In a separate experiment, whole mount preparations of the mylohyoid muscle were examined from animals at three stages of metamorphosis (stages 61-64). As the animal progressed through development, the mylohyoid muscle increased in size. Interestingly, the disparity between the PV- and PV+ fibers was difficult to resolve in these tissues and uniform PV staining was evident (not shown). At present we do not know the reason for the appearance of PV staining in all fibers of the mylohyoid muscle at this stage, but it may reflect the activation of PV expression in the anterior fibers as the muscle changes to participate in adult-specific behaviors.

PV Expression in the Limb Muscles The final major developmental program displayed in amphibian myogenesis is to generate new muscles late in development which survive for the rest of the animal’s life. This is exemplified by the muscles of the developing limbs which begin to differentiate with the approach of metamorphosis. As demonstrated by the Western blot in Fig. 1, limb muscles contain parvalbumin proteins. To determine if all fibers within all muscle groups express PV, we examined the cellular localization of PV in sectioned material. Figure ?A shows a phase-contrast photograph of a histological section through the knee of a hindlimb from a stage 55 embryo. Developing bone, muscle, and epidermis can be readily discerned. When the same section was examined for PV immunoreactivity (Fig. 7B), the muscles were very clearly revealed. As with the embryonically derived muscle groups examined above, some muscles display both PV+ and PV- cells. This is demonstrated in Figs. 7C and 7D, which show a muscle bundle with predominantly PV+ cells, although a distinct region of PVfibers is also present. DISCUSSION

In embryos, PV expression begins at about stage 24 of development, which is late in the process of myogenesis. This is well after many of the major differentiative events involved in neuromuscular development have occurred. For example, skeletal muscle actin is first expressed at stages 11/12,14 hr before the appearance of PV (Gurdon and Cascio, 1987). By stage 24, the muscles have assembled myofibrils and can generate coordinated behaviors (Hamilton, 1969; Muntz, 1975). Given the small size of PV and its location within the myoplasm, it seems reasonable that this protein would be produced late in myogenesis since it doesn’t need to intercalate in developing myofibrils. As well, since the

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FIG. 5. Immunofluorescence staining for PV in degenerating stage 64 tadpole tails. (A) Phase-contrast image of the myotomal muscles from the middle region of the tail. (B) The same section stained for PV. (C) Phase-contrast image of a strip of muscle from the caudal end of the tail, with two dying PV+ cells, of which the upper one is advanced in its degeneration. (D) Corresponding PV immunofluorescence. The black star-like objects in (A) and (C) are melanocytes, and the black column in (C) is a blood vessel. (E) Phase-contrast view of a cross-section of a caudal portion of the tail. (F) Corresponding PV immunofluorescence staining. Bar = 38 pm in (A) and (B), 19 pm in (C) and (D), and 75 pm in (E) and (F).

role of PV appears to modulate the contractile properties of fast-twitch muscle fibers, its presence presumably isn’t required until the muscles are functional and involved in the generation of behaviors. These hypothe-

ses are supported by the observation that in the developing chick, PV is not expressed in leg muscles until just prior to hatching (Le Peuch et &, 1979). The expression of PV is coincident with, or shortly

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FIG. 6. Immunofluorescence staining in the tadpole mylohyoid muscle. (A) Whole mount preparation of a fixed stage 41 mylohyoid muscle. (B) The same muscle stained with parvalbumin antibodies; there are more immunoreactive fibers in the posterior half than in the anterior half of the mylohyoid. (C) phase-contrast section through a mylohyoid muscle in a stage 41 tadpole head, with the corresponding immunofluorescence staining for parvalbumin in (D) which has been overexposed in the posterior half of the mylohyoid. Bar = 152 pm in (A) and (B) and 38 nm in (C) and (D).

follows, both synaptogenesis (Hayes and Roberts, 1973; Kullberg et al, 1977) and synaptic transmission (Blackshaw and Warner, 1976). It is interesting to speculate that innervation may be required for, or facilitate, PV gene activation. This hypothesis is supported by the observation that PV expression in adult rabbit muscle is under the direct control of fast-twitch motor innervation (Leberer and Pette, 1986). Moreover, PV protein and mRNA levels can be altered in cross-reinnervated rat muscles (Miintener et c& 1987). However, if innervation does play a role in the differentiation of the frog myotomal muscles, it is more complex than simple neural stimulation of the muscles. Embryos raised in the continuous presence of the anesthetic lidocaine, while completely paralyzed, still produced myotomal muscle fibers which were morphologically normal and contained high levels of PV (Schwartz and Kay, unpublished).

The expression of PV in myotomal muscles is restricted to a subset of fibers arrayed in a stereotyped pattern. At the rostra1 end, the majority of the cells contain PV, while at the caudal end two types of cells are found. The transition in pattern is gradual and begins slightly back from the head region. From various data presented under Results, we believe that the PVcontaining cells are fast-twitch in nature and the nonPV-containing cells are slow-twitch. These observations agree well with the histochemical characterization of muscles of Ranu tadpole (Watanabe et al. 197813). Correlated with this arrangement is the observation of Nordlander (1986) that there are two major classes of motorneurons in Xenopus tadpoles which exist in a rostral/caudal gradient. It is interesting to speculate on the functional ramifications of the arrangement of the two types of muscle fibers. Tadpoles display two major locomotory behaviors with their tails: a slow tail beat

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FIG. 7. Immunofluorescence staining of hindlimb from a stage 55 embryo. (A) phase-contrast image through the knee. (B) PV+ cells from the same section. (C) phase-contrast micrograph of a region of a hindlimb with its cartilage. (D) PV staining of the same section. Bar = 190 pm in (A) and (B) 38 pm in (C) and (D).

for “grazing” behavior which involves predominately the tip of the tail, and an escape response which is generated by rapid sweeps of the entire tail (Gradwell, 1971). Thus, the brief bursts of rapid tail movement in escape would utilize the fast-twitch musculature at the head region, while the rhythmic locomotory behavior of feeding would use the slow-twitch fibers at the tail tip. This arrangement is very reminiscent of the fiber type organization seen in fish, where there is a rind of slowtwitch muscles surrounding a core of fast-twitch fibers (Bone, 1978). Many laboratories dissociate myotomal muscles and grow the myoblasts or myotomal cells in culture for experimentation. Historically, all myotomally derived muscle cells have been treated as equivalent. Since these cells represent two distinct fiber types, this assumption may be incorrect. In preparations of dissociated cells, both long, thin cells and short, fat ones can be identified (H. B. Peng, unpublished). Not surprisingly, these two cell types display different physiological properties; for example, these two size classes have

dramatically different resting calcium levels (Peng, Schwartz, and Kay, data not shown). Taken together, these findings suggest that previously reported experiments utilizing myotomal cells in wivo or in vitro may be more complicated to interpret than previously realized. At metamorphosis, the tail musculature undergoes programmed cell death. One major change seen in degenerating muscle cells at the light microscope level, is the extrusion of sarcoplasm in small blebs along the surface of the fibers. These blebs appear first at the tips of the PV immunoreactive cells, and later are seen along the entire surface. During tail reabsorption, macrophages can be found throughout the tail musculature (Weber, 1964; Watanabe et aL, 19’78a), and as shown here, may be involved in phagocytosing the released PV. PV is a cytoplasmic protein and its release from dying muscle cells presumably reflects general loss of sarcoplasm, although there is the possibility that this protein is selectively extruded. The tadpole mylohyoid muscle and the muscles of the developing limb are also composed of both PV+ and PV-

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muscle fibers. In the limb, as with the myotome, PV immunoreactivity is seen late in myogenesis, after the generation of the contractile apparatus. The PV protein can be found in the muscles throughout the life span of the adult. In the mylohyoid muscle of young tadpoles, the anterior half is composed almost exclusively of PVfibers, whereas the posterior half is composed of predominantly PV+ fibers. This arrangement appears to change with metamorphosis, although our sample size is small in this study. It is interesting to speculate that hormonal changes associated with metamorphosis may bring about a change in fiber composition. This hypothesis is supported by the observation that androgen steroids appear to play a role in determining the composition of different fiber types in the Xeno~~ clasp muscles (Rubinstein et al, 1983) and larynx muscles (Sassoon et oL, 1987). As stated in the Introduction, the major purpose of this study is to begin to understand how developing myoblasts in Xenop~ make developmental decisions. Having characterized the normal patterns of PV expression during myogenesis, we are currently using molecular approaches to perturb its expression. Our hope is that by modulating the expression of PV during development, we may gain some insight into the role(s) of this Ca2+-binding protein in muscle differentiation and function. We would very much like to thank Dr. Nadia Malouf and Mr. David Cowan for their help in performing the histochemical staining of fiber types. We also appreciate the photographic assistance of Susan Whittield and the editorial comments of Drs. Nadia Malouf, Page Anderson, and Benjamin Peng. This work was supported by grants from the American Cancer Society (IN 15-29) and the North Carolina Center for Biotechnology and Molecular Biology to L.M.S. and the American Cancer Society (IN 15-28 and CD-263) and University Research Council to B.K.K. REFERENCES BARON,G., DEMAILLE, J., and DUTRUGE,E. (1975). The distribution of parvalbumins in muscle and in other tissues. FEBS kti 56,156-160. BLACKSHAW,S., and WARNER,A. (1976). Onset of acetylcholine sensitivity and endplate activity in developing myotome muscles of Xenopus Nature (Landon) 262,217-218. BONE, Q. (1978). Locomotor muscle. In Fish Physidogy: Locomotion (W. S. Hoar, and D. J. Randall, Eds.), vol. VII, pp. 361-417. New York: Academic Press. CARLSON,F. D., and WILKIE, D. R. (1974). Muscle Physidogy. Prentice-Hall, New Jersey. CELIO,M. R. (I986). Parvalbumin is in most -r-aminobutyric acid-containing neurons of the rat cerebral cortex. Science 231,995-997. CELIO, M. R., and HEIZMANN, C. W. (1982). Calcium-binding protein parvalbumin is associated with fast contracting muscle fibers. Noture (London) 297,504~506.

DEUCHAR, E. M. (1975). “Xenqpus: The South African Clawed Frog” pp. 33-37. Wiley, London.

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