/. Embryol. exp. Morph. Vol. 30, 3, pp. 673-679, 1973
673
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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P. V. THOROGOOD
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 6month-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.
676
P. V. THOROGOOD
Muscle protein and myofibrils in chick limb-bud
677
cleared in propylene oxide and embedded in Taab resin. Ulthrathin sections
were cut at 60-90 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 9000.
(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 35700.
(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 35700.
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
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).
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{Received 12 March 1973)
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