The appearance of muscle protein and myofibrils within the embryonic chick limb-bud

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1 /. Embryol. exp. Morph. Vol. 30, 3, pp , Printed in Great Britain The appearance of muscle protein and myofibrils within the embryonic chick limb-bud By P. V. THOROGOOD 1 From the Department of Zoology, University College of Wales SUMMARY Myotubes are present in the developing hind limb of the embryonic chick at 5 days. An immunofluorescence technique was used to detect actomyosin within the myotubes. The earliest detectable appearance of this muscle protein was at six days of development, at sites located peripherally beneath the flattened dorsal and ventral surface of the limb. These dorsal and ventral loci are interpreted as representing the primordial extensor and flexor muscles. At the ultrastructural level the cytoplasm of the myotubes contains fibrillar components which are apparently aggregating to form myofibrils. A rudimentary banding pattern can be distinguished. INTRODUCTION Attention to tissue differentiation in avian limb-bud development has been focused mainly on chondrogenesis and the emergence of the skeletal pattern. In contrast the phenomenon of myogenesis in the limb has been relatively neglected, in spite of extensive immunofluorescence analysis of avian somite myogenesis both in vitro and in vivo (Holtzer, Marshall & Finck, 1957, reviewed Holtzer, 1970). It has been reported, from work on the mouse embryo, that presumptive limb muscle is first seen as a layer of condensing cells underlying the dorsal and ventral ectoderm and is distinct from the cartilage elements in the central axis of the limb (Milaire 1965). In the avian embryonic limb, Weel (1948) described 'bundles of myoblasts' at 4-\- days and myotubes at 5 days. However, he did not specify at what point such tissue develops within the limb-bud. More recently, Gould, Day & Wolpert (1972) have described the first detectable event in limb myogenesis as the formation of dorsal and ventral myogenic condensations composed of closely packed mesenchyme cells and appearing at 4 days of development - stage 22 (Hamburger & Hamilton, 1951). However, these reports describe the morphological events at the tissue level. It is not known when these myogenic cells within the limb begin to produce the' luxury proteins' (Holtzer, 1970) characteristic of myogenic differentiation. The relationship of actomyosin to the appearance of myogenic cells is important in considerations of tissue differentiation in the embryonic limb. 1 Author's address: The Professorial Clinical Unit, Institute of Orthopaedics, Royal National Orthopaedic Hospital, Stanmore, Middlesex, U.K.

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3 Muscle protein and myofibrils in chick limb-bud 675 This brief communication presents findings from an investigation into the first appearance of muscle protein and myofibrils in the avian limb-bud. Evidence is presented on the earliest detection of actomyosin within the limbbud system by immunofluorescence technique. The appearance of myofibrils within differentiating myotubes is revealed by electron microscopy. These results are subsequently discussed in relation to existing reports of embryonic limb myogenesis. MATERIALS AND METHODS For preliminary histological purpose hind limb bud tissue of the requisite stage was fixed in Bouins fluid, embedded in paraffin wax and sectioned at 6 /mi. The sections were stained by the Mallory triple staining technique. Striated muscle actomyosin was obtained from the thigh musculature of 6- month-old Light Sussex cockerels killed by cervical fracture. The actomyosin extraction, purification and the subsequent production and preparation of the anti-serum against actomyosin, followed the procedure employed by Kemp, Jones & Groschel-Stewart (1971). A non-immunized serum was similarly prepared for control purposes. The conjugation with fluorescein isothiocyanate was executed according to the method described by Rinderknecht (1962). Cryostat sections (6-8 /mi) of hind limbs from Light Sussex chick embryos of stages 26-35, were mounted on pure quartz u.v. slides. The sections were treated with conjugate (of either immunized or non-immunized serum) for 30 min, followed by thorough washing in Dulbecco solution and were finally mounted in 70 % (v/v) glycerol-dulbecco solution. The preparations were examined with a Reichert Zetopan microscope fitted with a dark-field immersion condensor and with u.v. illumination. For electron microscopy, tissue of equivalent stages was fixed by the simultaneous double fixation procedure (Trump & Bulger, 1966). The material was block stained in 5 % uranyl acetate, dehydrated through an ethanol series, FIGURE 1 (A) Longitudinally-sectioned myogenic tissue from the dorsal region of a 5-day hind limb (stage 26). The long, multinucleate myotubes (m.t.) are prominent and between them are uninucleate cells («), presumably mesenchymal or myoblastic in nature. Within the myotube cytoplasm some fibrillar matter can be distinguished (arrows x 1800). (B) Longitudinally-sectioned myogenic tissue from a 6-day hind limb (stage 28). A fluorescence reaction can be seen within the myotubes; at bottom right the myotubes are sectioned obliquely, x 600. (C) Myogenic tissue as in (B) but sectioned transversely, the myotubes are therefore seen end on. x 600. (D) Transversely-sectioned tissue from the same source as (B) and (C). This section has been treated with control conjugate. Note the absence of an immunofluorescence reaction, x 600.

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5 Muscle protein and myofibrils in chick limb-bud 677 cleared in propylene oxide and embedded in Taab resin. Ulthrathin sections were cut at nm on an LKB 4800 A Ultratome I and double stained with 5 % uranyl acetate and lead citrate (Reynolds, 1963). The sections were examined in an AET EM 6B electron microscope at 60 kv. RESULTS The first appearance of well-defined multinuclear myotubes, as distinct from the earlier condensations of uninucleate myogenic mesenchyme cells, is at 5 days - stage 26. Long 'tubular' cells can be found dorsally and ventrally within the limb; the long axis of these cells runs parallel to the proximo-distal axis of the limb (Fig. 1A). These cells typically possess a number of nuclei: up to ten within a single myotube were observed. Uninucleate mesenchyme cells (myoblasts?) are present between the myotubes. However, the earliest stage at which an immunofluorescence reaction occurred within sectioned limb-bud tissue was at 6 days - stage 28. At stage 27 and earlier no reaction could be detected (Figs. 1B, C). In transverse section this activity was localized in two positions; peripherally beneath the dorsal ectoderm and beneath the ventral ectoderm. These two loci, although subjacent to the ectoderm, were not contiguous with it. In longitudinal section the fluorescence activity was localized in long strands similar in size and scale to the myotubes (Fig. 1 B). In the later stages (29-35) these two simple blocks of immunofluorescence activity were replaced by a complex pattern of muscle blocks as the functional muscular system of the limb emerges. At the ultrastructural level at stage 28, concomitant with the detectable appearance of actomyosin, the cytoplasm of the myotube contains fibrillar material (Fig. 2 A). At higher magnifications it is apparent that two categories of fibre exist: thick and thin fibres can be distinguished measuring approximately 14 and 7 nm respectively. These two types of fibre are aggregated in a parallel fashion which is interpreted by the author as the first indication of a myofibrillar FIGURE 2 (A) Myogenic tissue taken from a dorsal position in a stage 28 hind-limb bud. Three closely associated myogenic cells (1-3) are situated in the centre of the plate and in the cytoplasm of the middle cell (2) some fibrillar components (/) can be distinguished. Polyribosomes (r). Intercellular space (/). x (B) The parallel arrangement of the fibrils can be clearly seen in the cytoplasm of this myogenic cell. Two categories of fibril exist-thick (large arrows) and thin (small arrows). Plasmalemma (p). Intercellular space (/). Mitochondrion (///). x (C) The organization of the myofibrils is more advanced than in the previous plate. Denser regions occur at regular distances along the myofibrils and these are probably the emerging Z-discs (z). Small endoplasmic reticulum profiles (arrows) are associated with these dense regions, x

6 678 P. V. THOROGOOD organization (Figs. 2B, C). The characteristic banding pattern is not distinguishable although rudimentary Z discs are discernible and are usually associated with endoplasmic reticulum profiles which possibly represent the beginning of the T-system. Generally there is little endoplasmic reticulum: that which is present is of the smooth type. The cytoplasm of the cells has a granular appearance due to the abundance of free ribosomes and polyribosome clusters. Helical arrangements of ribosomes, characteristic of myofibrillogenesis (Waddington & Perry, 1963), are occasionally seen. The appearance of the myofibrils suggest that they are not yet in a functional state and are still being assembled at this time. DISCUSSION It has been demonstrated that the contractile protein, actomyosin, can be detected for the first time at stage 28 - six days. The localized presence of actomyosin within myotubes is interpreted by the author as indicative of confirmed myogenic differentiation. Such an interpretation is endorsed by the ultrastructural findings, which demonstrate myofibrillar components in the cytoplasm of these syncytial cells. From the findings presented here it appears that detectable amounts of muscle proteins are present soon after the appearance of myotubes. Such a relationship between actomyosin synthesis and myotube genesis is compatible with current knowledge of myogenic cell lineages and differentiation (reviewed Holtzer, 1970). The dorsal and ventral location of such myogenic tissue confirms the findings of previous workers in the murine embryonic limb (Milaire 1965), and in the avian embryonic limb (Gould et al. 1972): that is, muscle blastemae first appear in the peripheral soft tissue between the preskeletal balstema and the dorsal and ventral ectoderm. As Milaire proposed, it is probable that these Anlagen represent primordial flexor and extensor muscle blocks. In the case of limb chondrogenesis the histological event of condensation occurs subsequent to metabolic differentiation indicated by enhanced chondroitin sulphate synthesis (Searls, 1965; Thorogood, 1972). However in myogenesis the first detectable event is the formation of dorsal and ventral myogenic condensations of mesenchyme cells at stage 22 (Gould et ah 1972). This fact, together with the evidence presented here, suggests that histogenesis precedes metabolic differentiation in limb-bud myogenesis. It is possible that actomyosin is present before the time stated in this report, yet not detectable by the immunofluorescence technique employed here. In fact, recently, it has been claimed that myosin or a myosin-like molecule can be extracted from limb-mesenchyme cells as early as stage 24 (Medoff & Zwilling 1972). The present results should not be regarded as conflicting with those published by Medoff, but rather they should be viewed as complementary. The two approaches employ different techniques possessing disparate degrees of sensitivity and precision. Medoff's findings are

7 Muscle protein and myofibrils in chick limb-bud 679 based on the detection of myosin in extracts of whole limbs whereas the present report visualizes the first appearance of muscle protein within the myogenic condensation of the intact limb. The author thanks Dr R. B. Kemp for practical advice during the work, and Dr J. R. Hinchliffe for reading the manuscript. A part of this work is taken from a thesis submitted to the University of Wales for the degree of Ph.D. (1972). REFERENCES GOULD, R. P., DAY, A. & WOLPERT, L. (1972). Mesenchymal condensation and cell contact in the early morphogenesis of the chick limb. Expl Cell Res. 72, HAMBERGER, V. & HAMILTON, H. L. (195.1). A series of normal stages in development of the chick embryo. /. Morph. 88, HOLTZER, M". (1970). Myogenesis. In Cell Differentiation (ed. O. A. Schjeide and J. de Vellis). New York: Van Nostrand Reinhold. HOLTZER, M, MARSHALL, J. M". JR. & FJNCK, H. (1957). An analysis of myogenesis by the use of fluorescent anti-myosin. /. biophys. biochem. Cytol. 3, KEMP, R. B., JONES, B. M. & GROSCHEL-STEWART, U. (1971): Aggregative behaviour of embryonic chick cells in the presence of antibodies directed against actomyosins. /. Cell Sci. 9, MEDOFF, J. & ZWILLING, E. (1972). Appearance of myosin in the chick limb bud. Devi Biol 28, MILAIRE, J. (1965). Aspects of limb morphogenesis in mammals. In Organogenesis (ed. R. L. Denham and H. Ursprung). New York: Holt, Rinehard and Winston. REYNOLDS, E. A. (1963). The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. /. Cell Biol. 17, RINDERKNECHT, H. (1962). Ultra-rapid fluorescent labelling of proteins. Nature, Lond. 193, SEARLS, R. L. (1965). An autoradiographic study of the uptake of S 35 sulphate during the differentiation of limb bud cartilage. Devi Biol. 11, THOROGOOD, P. V. (1972). Patterns of chondrogenesis and myogenesis in the limb buds of normal and talpid 3 chick embryos. Ph.D. Thesis, University College of Wales, Aberystwyth. TRUMP, B. & BULGER, R. E. (1966). New ultrastructural characteristics of cells fixed in a glutaraldehyde-osmium tetroxide mixture. Lab. Invest. 15, WADDINGTON, C. M. & PERRY, M. M. (1963). Helical arrangement of ribosomes in differentiating muscle cells. Expl Cell Res. 30, WEEL, P. B. VAN (1948). Histophysiology of the limb bud of the fowl during its early development. /. Anat. 82, {Received 12 March 1973) 44 E M B 3C

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