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Development of a mammalian series-fibered muscle.

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THE ANATOMICAL RECORD PART A 278A:571–578 (2004)
Development of a Mammalian SeriesFibered Muscle
ANGELIKA C. PAUL,1 PHILIP W. SHEARD,2 AND MARILYN J. DUXSON1*
1
Department of Anatomy and Structural Biology, University of Otago,
Dunedin, New Zealand
2
Department of Physiology School of Medical Sciences, University of Otago,
Dunedin, New Zealand
ABSTRACT
This study examines the processes by which multiply innervated, serially fibered mammalian muscles are constructed during development. We previously reported that primary
myotubes of such a muscle, the guinea pig sternomastoid muscle, span from tendon to tendon
and are innervated at each of the muscle’s four innervation zones. Secondary myotubes form
later, in association with each point of innervation (Duxson and Sheard, Dev. Dyn., 1995;
204:391– 405). We now describe the further growth and development of the muscle. Secondary myotubes initially insert onto and grow along the primary myotube. However, as they
reach a critical length, they encounter other secondary myotubes growing from serially
adjacent innervation zones and may transfer their attachment(s) to these serially positioned
secondary myotubes. Other secondary myotubes maintain attachment at one or both ends to
their primary myotube. Thus, an interconnected network of primary and secondary myotubes
is formed. Patterns of reactivity for cell adhesion molecules suggest that early attachment
points between myotubes are the embryonic precursors of adult myomyonal junctions, characterized by the expression of ␣7B␤1 integrin. Finally, the results show that secondary
myotubes positioned near a tendon are generally longer than those lying in the mid belly of
the muscle, and we suggest that the environment surrounding the tendinous zone may
somehow stimulate myotube growth. Anat Rec Part A 278A:571–578, 2004.
©
2004 Wiley-Liss, Inc.
Key words: skeletal muscle; development; multiple innervation; secondary
myotubes; intrafascicularly terminating fibers; alpha 7 integrins;
myomyonal junction
The architecture of many larger mammalian skeletal
muscles differs from the standard textbook view in that
they have multiple endplate bands along the length of
their fascicles, the majority of individual fibers are shorter
than the fascicles, and fibers terminate intrafascicularly
(Loeb et al., 1987; Paul, 2001). Intrafascicular fiber terminations (IFTs) are characterized by elaborate folding of
the sarcolemma and high expression levels of cell-matrix
adhesion molecules, in particular the ␣7␤1 integrins and
dystrophin (Young et al., 2000; Paul et al., 2002). From the
terminal sarcolemma of the IFT, fibers of the extracellular
matrix extend to contact other fibers terminating in a
complementary manner to form myomyonal junctions.
These junctions may involve entire terminating fibers, or
multiple complementary branches of neighboring muscle
fibers (Torigoe and Nakamura, 1987; Young et al., 2000),
and link muscle fibers within each fascicle into a complex
network, quite unlike anything seen in smaller muscles.
We have previously characterized aspects of the development, functional anatomy, and physiology of such muscles
©
2004 WILEY-LISS, INC.
(Duxson and Sheard, 1995; Sheard et al., 1999; Young et
al., 2000; Paul, 2001; Paul et al., 2002). Here we ask how
the interlinking network of short fibers and myomyonal
junctions that characterizes these muscles is formed during development.
Angelika C. Paul’s present address is Department of Cellular,
Molecular, and Developmental Biology, University of Colorado,
Boulder, CO 80309.
*Correspondence to: Marilyn J. Duxson, Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, P.O. Box 913, Dunedin 9001, New Zealand. Fax:
64-3-479-7254. E-mail: marilyn.duxson@stonebow.otago.ac.nz
Received 23 April 2003; Accepted July 11 2003
DOI 10.1002/ar.a.20020
572
PAUL ET AL.
Fig. 1. Comparison of development of a singly innervated muscle
(left) with that of a multiply innervated, series-fibered muscle (right). The
positions of innervation zones are indicated by arrows in each case.
Primary myotube formation is similar in both muscle types (top), but
primary myotubes of multiply innervated muscles are each multiply
innervated. Secondary myotubes are initiated in association with all
innervation zones, so that multiply innervated muscles have multiple foci
of secondary myotube formation (middle). As secondary myotubes
grow, they will all extend to the tendons in the singly innervated muscle.
In contrast, most secondary myotubes will form intrafascicularly terminating fibers in the multiply innervated muscle (bottom).
The cellular pattern of embryogenesis of multiply innervated, serially fibered muscles shows many features in
common with development of simple muscles with a single
zone of innervation, but also some crucial differences (Fig.
1). In both muscle types, the muscle is initially formed by
a generation of primary myotubes that extend the entire
muscle length, define the fascicular architecture, and become innervated by the motor nerve (Fig. 1, top). However,
in the multiply innervated muscles, each primary myotube receives innervation at multiple sites spread evenly
along the muscle length, rather than at a unique centrally
located site as in the classical small muscle (Duxson et al.,
1989; Duxson and Sheard, 1995). At the next stage of
development, secondary myotubes form exclusively in association with sites of innervation on the primary myotubes in both muscle types (Fig. 1, middle). In the singly
innervated muscle, this means that new secondaries are
associated only with the single centrally located innervation zone of the primary myotube. However, in the multiply innervated muscle, sites of secondary myotube initiation are staggered along most of the length of the fetal
muscle, corresponding to the multiple innervation sites on
the primary myotubes (Duxson et al., 1989; Duxson and
Sheard, 1995). Finally, as secondary myotubes elongate,
differences again arise. In the singly innervated muscle,
all secondary myotubes will extend fully to the tendon and
insert directly onto it, whereas secondary myotubes in the
multiply innervated muscle will terminate within the
muscle belly at one or both ends, forming the characteristic series-fibered arrangement of these muscles (Fig. 1,
bottom). The question then is, how does this staggered
array of short fibers form into a coherent network that can
effectively transmit force through the muscle?
We hypothesized that in the developing multiply innervated muscle, secondary myotubes might make overlapping contacts with each other, with some or all of these
sites of myotube-to-myotube contact being maintained to
form the myomyonal junctions seen in adult serially fibered muscles. It is well known that developing singly
innervated muscles form transient junctional complexes
between primary and secondary myotubes characterized
by interfolding of the sarcolemmae and expression of a
range of cell-cell adhesion molecules (Duxson and Usson,
1989; Rosen et al., 1992; Fredette et al., 1993; Kaufmann
et al., 1999), but these are lost during maturation. Here,
we investigate whether similar sites of myotube interaction occur in developing multiply innervated muscles and
whether these involve secondary-to-secondary myotube
contacts as well as primary-to-secondary contacts. We also
compare the adhesion molecules expressed at sites of developmental myotube contact with those expressed at mature myomyonal junctions. Continuity of these expression
patterns would be consistent with a developmental relationship between the two sites.
MATERIALS AND METHODS
Tissues for Immunohistochemistry
Guinea pig sternomastoid muscles were collected from
2– 4 animals at each of embryonic day 37 (E37), E47,
postnatal day 1 (PN1), and from mature adults. This age
range covers the period from formation to maturity of
secondary myotubes in this muscle (Duxson and Sheard,
1995). Dated pregnancies were the result of immediate
postpartum matings between stably coupled pairs. Euthanasia of adults and neonates was by overdose of sodium
pentobarbitone; of embryos, by cold anesthesia followed by
decapitation, in accord with protocols approved by the
University of Otago Committee on the Ethical Use of
Animals in Research.
Transmission Electron Microscopy (TEM)
A semiserial section electron microscopic (EM) analysis
was made of a single E37 muscle. The embryo was deeply
anesthetized on ice, then the head fixed in place to maintain the sternomastoid muscles at resting length. Fixation
was by cardiac perfusion with 1% glutaraldehyde, 1%
paraformaldehyde, and 0.05 M glucose in a 0.1 M phosphate buffer (pH 7.3). After perfusion, muscles were excised and immerse-fixed in the same solution for a further
2 hr, postfixed in 2% osmium tetroxide, stained en bloc in
2% aqueous uranyl acetate for 45 min, then dehydrated in
alcohols and propylene oxide before embedding in TAAB
epoxy resin.
Semiserial sectioning extended over about 3/4 of the
muscle length (about 5 mm) with a group of 10 ultrathin
(90 –100 nm) sections being collected onto Formvar-coated
slot grids every 9 ␮m and subsequently stained with lead
citrate and uranyl acetate. Semiserial photomontages of
five adjacent and easily identified myotube clusters, taken
over the entire extent of the section series, were used to
construct a longitudinal map identifying the extent, origin, and insertion of every myotube within the selected
clusters [some results reported in Duxson and Sheard
(1995)]. Outlines of selected regions of each cluster were
subsequently traced into a 3D reconstruction program,
using a digitizing pad, to produce 3D-rendered illustrations showing relationships between developing myotubes.
DEVELOPMENT OF A SERIES-FIBERED MUSCLE
Immunohistochemistry
Immunohistochemical techniques were used to examine
the presence or absence of selected integrins or their ligands and of cadherins and N-CAM at myomyonal junctions at each developmental stage. Techniques were essentially identical to those described previously (Paul et
al., 2002). The integrin subunits examined were those we
have previously localized to adult myomyonal junctions,
namely, the ␣7A and ␣7B subunits, plus the ␣6 subunit,
which we have not previously investigated. Antibodies
used were rabbit polyclonal anti-␣7A (␣7CDA2) and anti␣7B (␣7CDB1) (Song et al., 1993; Martin et al., 1996) and
a rat monoclonal anti-␣6 integrin (GoH3) (Immunotech,
Marseille, France). A monoclonal antitenascin antibody
(M1-B4) (Chiquet and Fambrough, 1984) was obtained
from the Developmental Studies Hybridoma Bank maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA 52242), under contract NO1HD-7-3263 from the National Institute for Child Health
and Development (NICHD). To look for the presence of
cadherins, we used a mouse monoclonal antibody raised
against ␤-catenin (15B8) kindly gifted by K. Knudson
(Johnson et al., 1993). The anti-N-CAM antibody was a
rabbit polyclonal against rat N-CAM (AO49) from the
laboratory of Urs Rutishauser. The monoclonal mouse
antislow-myosin heavy-chain antibody (NOQ7.1.1A) was
from Dr. Robyn Fitzsimons (Harris et al., 1989).
Sections were observed with an Olympus microscope
and digital fluorescent or bright field images taken with a
Pixera PVC 100C digital camera (Pixera). Two-color images were produced by merging within Adobe Photoshop
5.5.
RESULTS
Our initial aim was to understand the sequence of relationships between new secondary myotubes and their primary and secondary myotube neighbors as they form and
then extend in length within the belly of a multiply innervated muscle.
Transmission Electron Microscopy
We used semiserial EM analysis to visualize the relationships between developing myotubes in the multiply
innervated guinea pig sternomastoid muscle at embryonic
day 37 (E37). This time point falls within the period of
very active formation and growth of new secondary myotubes, so that the muscle contains a complete continuum
of size of secondary myotube, ranging from absolutely new
to almost fully grown.
New myotubes appeared in association with each of the
muscle’s four innervation zones, as previously reported
(Duxson and Sheard, 1995). As secondary myotubes associated with a particular primary myotube elongated, those
initiated at one innervation zone soon overlapped longitudinally with those initiated at adjacent innervation zones.
Three-dimensional reconstruction revealed complex associations between growing secondary myotubes and their
parent primary myotube and, later, between overlapping
or adjacent secondary myotubes. Some examples of these
associations are illustrated in the reconstruction and accompanying electron micrographs of Figure 2. Figure 3
shows a more quantitative analysis in which all the secondary myotubes sectioned and photographed from end to
573
end (37 secondary myotubes in total) are logged in terms
of their length and the nature of their insertion.
In their initial stages of growth (length of 1– 400 ␮m;
2– 8 nuclei), young secondary myotubes formed complex
interfolding adhesive junctions with the primary myotube
along most of their length, just as previously seen in small
muscles such as the rat lumbrical (Duxson et al., 1989).
An example of such a very young myotube is cell 8 which
is illustrated in Figure 2 at section level E only. This was
a myotube of 350 ␮m and 7– 8 nuclei and showed complex
interfolding with the primary myotube, similar to that
seen in Figure 2E, along most of its length. In older/longer
secondary myotubes, these interfolded junctional zones
became confined to the cell extremities, and there was a
less intimate relationship with the primary in the central
zone. Cell 7 (Fig. 2D–F) is an example of a more developed
secondary myotube, which extended over 1,100 ␮m along
the axis and formed a complex insertion onto the primary
myotube near both its ends (Fig. 2D and F), while in its
central zone (Fig. 2E) it was in simple apposition to the
primary myotube.
A crucial change occurred once secondary myotubes
reached a length of 1,100 –1,400 ␮m. At this length, a
proportion of them released their insertion onto the parent
primary, at one or both ends, and instead formed a close
interdigitating insertion on a neighboring secondary myotube. One such pair of secondary myotubes is illustrated
in Figure 2. Cell 9 (in blue) is a secondary myotube centered on an innervation point at 1,760 ␮m along the longitudinal axis (top arrow) and extends down along the
primary to 310 ␮m, where it terminates. Cell 3 (in green)
is centered on the innervation zone at 430 ␮m on the axis
and extends up the primary to 1,970 ␮m before terminating. In Figure 2B–F, the relationship between these two
cells can be seen in section at selected levels. In Figure 2B,
cell 9 initially appears as an independent myotube, close
to, but not inserted onto, the primary mytoube. At the next
level (Fig. 2C, 1,600 ␮m), the beginning of cell 3 is seen. It
inserts deeply into cell 9 over about 100 ␮m of axial
length. The two cells then continue side by side, but without any intimate junctional contact (Fig. 2D, 860 ␮m) until
the end of cell 9 is approached (Fig. 2E, 360 ␮m). At this
level, an insertion of secondary to secondary again occurs,
but in a reciprocal direction, so that the terminal portion
of cell 9 inserts cell processes into the substance of cell 3.
Finally, cell 3 continues alone after the termination of cell
9 (Fig. 2F).
The quantitative analysis of secondary myotube insertion (Fig. 3) demonstrates the initial formation and
growth of secondary myotubes on the primary myotube
(squares, lower line), followed by transfer of the insertion
of a proportion of secondaries onto other developing secondaries (diamonds, upper line of plot). The data show
first that all new secondary myotubes arise exclusively on
the surface of the primary myotubes, never purely in
association with an existing secondary myotube. Secondly,
the length at which some secondaries transfer their insertions is significant; 1,100 –1,400 ␮m is the range of separation between adjacent innervation zones in the muscle
at this developmental stage. Thus, it is the length at
which secondary myotubes initiated at neighboring zones
on the same primary myotube will first encounter each
other along the longitudinal axis. It seems to be a matter
of chance whether a particular myotube retains insertion
on the parent primary myotube or encounters a suitable
574
PAUL ET AL.
Fig. 2. A cluster of developing myotubes from E37 guinea pig sternomastoid muscle. A: Reconstruction of part of a primary myotube
(red/orange) and some of its associated secondary myotubes (all other
colors) from semiserial (10 ␮m interval) electron micrographs. The longitudinal axis is shown in ␮m at left. Solid arrows at left indicate the
levels of two innervation zones that lie within the region of reconstruction. Of particular interest are cell 9 (blue, centered on the innervation
zone at 1,760 ␮m and terminating at 310 ␮m) and cell 3 (green, centered
on the innervation zone at 430 ␮m and terminating at 1,700 ␮m). These
two well-grown secondaries form complementary insertions onto each
other, close to their respective points of termination. Cell 4 (gray) is a
large secondary myotube that extends throughout the reconstructed
region. Note that some myotubes (in particular myotubes 7 and 8, visible
in D–F) were removed from the reconstruction for clarity. A–F: Electron
micrographs from selected levels of the reconstruction showing details
of some myotube-myotube relationships. Note particularly the interdigitating insertions between myotubes 3 and 9 close to their respective
terminations at section levels C and E. Myotube 7 is a small myotube
(1,100 ␮m long) seen first at level D and terminating shortly after level F;
it inserts on the surface of the primary myotube. Myotube 8 (350 ␮m in
length) is seen at section level E only. Myotubes referred to in the text
have been artificially outlined to allow resolution of the sarcolemmae at
this low magnification. Cell numbers correspond to those in A.
DEVELOPMENT OF A SERIES-FIBERED MUSCLE
other secondary myotube and transfers its insertion. In
the present study, about half of the secondary myotubes
over 2,400 ␮m in length continued to use the primary
myotube as their main substrate for growth and as their
sole insertion.
Figure 3 also shows that very few secondary myotubes
were found within the length range of 1,400 –2,100 ␮m.
Possible explanations for this gap in the length distribution of secondary myotubes are considered in the discussion.
In summary, this TEM study shows that developing
secondary myotubes in this series-fibered muscle first
grow on and maintain insertion on the primary myotube.
As they reach a length where they encounter other extending secondaries, a proportion transfer their insertion at
one or both ends to these contemporaries. Others retain
all insertion on the primary myotube. A proportion of both
types will finally extend into the tendon zone, and insert
at one end on the tendon, and at the other end on a
primary or secondary myotube. This process results in
formation of an interconnected network of primary and
secondary myotubes, with complex interdigitations between myotubes taking the place of myomyonal junctions
in the adult.
Immunohistochemistry
We next examined the cell adhesion molecules expressed at the interfaces between developing myotubes to
see how these related to those expressed at adult myomyonal junctions. In the adult guinea pig sternomastoid muscle, we have previously reported strong immunoreactivity
for ␣7B integrin and weak immunoreactivity for ␣7A integrin at myomyonal junctions of adult guinea pig sternomastoid muscle, but found no evidence for localization of
N-CAM or cadherins (Paul et al., 2002).
The developmental stages examined immunohistochemically were embryonic day 37 (E37), i.e., early secondary myotube generation (corresponding to the EM
study above); E47, i.e., late secondary myotube development; postnatal day 1 (PN1), i.e., maturing muscle fibers
with few myotubes remaining; and adult. In the fetal
material, a slow-myosin heavy-chain antibody (antislowMyHC, NOQ7.1.1A) was in some instances used to identify myotube type, as only primary myotubes react positively to the antibody in fetal sternomastoid muscle
(Duxson and Sheard, 1995).
Integrin Expression
At E37, the anti-␣7B integrin antibody localized to the
cytoplasm of myotubes, and at a modest level to selected
interfaces between E37 myotubes. The arrow in Figure 4B
indicates one reactive secondary-secondary myotube interface. At E47, irregular but intense staining was more
frequently present at myotube interfaces, particularly
those between adjacent secondary myotubes (arrow, Fig.
4E), while cytoplasmic expression had faded. Generalized
labeling of the sarcolemma of both slow and fast fibers was
present by P1 (Fig. 4G and H), and there were localized
regions of more extensive sarcolemmal staining at myotube interfaces (arrow in Fig. 4G), reminiscent of the pattern seen at adult myomyonal junctions viewed in crosssection. Blood vessels were also labeled by the anti-␣7B
integrin antibody: the bright dots surrounding the muscle
fibers in Figure 4G–I are capillaries. In adult sternomas-
575
toid, the anti-␣7B integrin antibody localized to the entire
sarcolemma of all muscle fibers, with localized enhancement at sites of myomyonal junctions (e.g., arrow in Fig.
4I), as we have previously reported. Thus, the pattern of
immunolocalization of the ␣7B integrin subunit suggests a
continuity in identity between early myotube interfaces
and adult myomyonal junctions.
In developing sternomastoid muscle, we saw no sign of
reactivity with the anti-␣7A integrin antibody at myotube
interfaces. Embryonic day 37 myotubes showed some cytoplasmic localization but this faded by E47, with localization to myomyonal junctions visible only in the adult
[embryonic data not shown; see Paul et al. (2002) for
adult].
Finally, we examined expression of the ␣6 integrin subunit, as its presence has been reported during formation of
myotubes (Bronner-Fraser et al., 1992), and of the ␣9
integrin subunit, which is characteristically expressed at
sites of tension transmission such as mature tendons and
tendon primordia. We screened for ␣9 integrin using an
antibody to its only known extracellular ligand, tenascin
(Wang et al., 1995; Yokosaki et al., 1998). No evidence was
found for localization of either ␣6 integrin or tenascin at
intrafascicular fiber terminations at any time, although
tenascin always localized to fiber terminations at the tendon (results not shown).
Cell-Cell Adhesion
No cell-cell adhesion molecules have been found at adult
myomyonal junctions (Paul et al., 2002), but their presence is widely reported at myotube surfaces and interfaces
during development of singly innervated muscles. We
wondered if they would be present at sites of primarysecondary and secondary-secondary myotube interaction
in the embryonic series-fibered muscle.
We screened for cadherins using an antibody against
their most widespread intracellular ligand, ␤-catenin.
Myotube type was here identified by size and position
rather than with the antislow-MyHC antibody due to
problems with antibody cross-reactivity. During early secondary myogenesis (E37, Fig. 5A and B), ␤-catenin was
localized over most myotube surfaces but enhanced at
both primary-secondary (arrowhead in Fig. 6B) and secondary-secondary (arrow) myotube interfaces, supporting
the previously reported role of cadherins in early myotube
adhesion. At E47 (Fig. 5C and D), it was hard to identify
primary myotubes based on their size, but ␤-catenin expression was much reduced at all myotube interfaces (the
arrow in Fig. 5C and D indicates one myotube interface
with slight immunoreactivity) and was absent by P1 and
at adult myomyonal junctions (not shown). The strong
expression visible in Figure 5D was associated with the
developing capillary bed, rather than myotubes.
Immunoreactivity to the N-CAM antibody showed an
initial pattern of expression similar to ␤-catenin, being
prominently expressed at virtually all myotube interfaces
as well as on the free surfaces of myotubes at E37 (Fig. 6A
and B). However, by E47, staining at many myotube interfaces was specifically reduced (e.g., arrow in Fig. 6D).
With further maturation, N-CAM expression on myotubes
was generally downregulated and largely restricted to
satellite cells at P1 and later (Fig. 6E and F), consistent
with previous reports of N-CAM expression patterns in
developing muscle (Fredette et al., 1993). N-CAM reactiv-
576
PAUL ET AL.
ity was never seen at interfaces between muscle fibers at
adult myomyonal junctions.
DISCUSSION
Fig. 3. A plot to show the nature of insertion of secondary myotubes
of various lengths. The plot includes all secondary myotubes traced from
end to end in the serial section study. The bottom line (squares) shows
secondary myotubes that insert entirely on the primary myotube. The top
line (diamonds) shows secondary myotubes that insert on other secondaries, at one or both ends. Secondary myotubes that extend into the
tendon region are marked with an asterisk.
Fig. 4. Localization of ␣7B integrin during development of guinea pig
sternomastoid muscle. Primary myotubes are identified by antislow-MyHC
antibody in red, with ␣7B integrin shown in green. A–C: Embryonic day 37.
A: The primary myotube of a cluster, identified by its reaction for slowMyHC. B: Diffuse ␣7B cytoplasmic is present in all myotubes with occasional stronger sarcolemmal staining at interfaces between secondary myotubes (arrow). C: Overlay of A and B. D–F: Embryonic day 47. D: AntislowMyHC-positive primary myotubes. E: Cytoplasmic staining for ␣7B is
Multiply innervated, series-fibered muscles have a
physical architecture very different to that of simple muscles. Many previous studies have led to a good descriptive
understanding of how the form of simple muscles develops. An initial, rather sparse, framework of primary myotubes lays out the skeleton of the muscle and provides a
substrate on which a much larger number of secondary
myotubes will grow. The latter are initiated as binucleate
cells near the central innervation zone of the primary
myotubes, then elongate through to the tendon, where
they form attachments to the ECM and separate entirely
from the primary myotube to form independent contractile
units (Duxson and Usson, 1989; Duxson et al., 1989). This
current study shows that more complex muscles use the
reduced, but sarcolemmal staining at myotube interfaces is more widespread (arrows). F: Overlay of D and E. G and H: Postnatal day 1. G: ␣7B
integrin is present on the sarcolemma of all young muscle fibers, with areas
of more extensive staining (arrow) perhaps corresponding to forming myomyonal junctions. H: Overlay of slow-MyHC immunoreaction. I: Adult. ␣7B
is localized on capillaries (bright dots) and all regions of the sarcolemma,
with intense staining (arrow) indicating regions of myomyonal junctions (L).
Scale bars ⫽ 50 ␮m.
DEVELOPMENT OF A SERIES-FIBERED MUSCLE
577
Fig. 5. Localization of ␤-catenin during development (B and D) compared with differential interference contrast (DIC) images (A and C). A
and B: Embryonic day 37. ␤-catenin reactivity is present at primarysecondary (arrowhead) and secondary-secondary (arrow) myotube interfaces. C and D: Embryonic day 47. Strong ␤-catenin reactivity is now
associated mainly with the developing capillary bed, although occasional secondary-secondary interfaces still react weakly (arrows in C and
D). Scale bars ⫽ 50 ␮m.
same basic principles of development, but work several
variations on this basic scheme.
In multiply innervated muscles, primary myotubes
again form a simple scaffold, predicting the muscle form
and attachments. However, secondary myotubes are initiated at many rather than at a single innervation site on
the primary myotubes (Duxson and Sheard, 1995). This
present study shows that secondary myotubes at first
elongate on the surface of the primary myotube, but soon
encounter myotubes growing in the opposite direction
from adjacent innervation zones. At this point, many of
them form connections onto other secondary myotubes
and transfer one or both of their terminal points of insertion away from the primary myotube. Not all secondary
myotubes transfer away from the primary myotube, however. At the stage observed in this serial EM study, about
half of all the longer (⬎ 1,400 ␮m) secondary myotubes
maintained connection with the primary myotube at both
ends, while others inserted at one end on the primary and
at the other end on a secondary myotube. Thus, an interconnecting network of secondary and primary myotubes is
slowly built, in contrast to the progressive production of
independent parallel myotubes in simple muscles.
What is the relation between the sites of insertion of
developing myotubes onto each other and the myomyonal
junctions that connect adult fibers in serially organized
muscles? Our studies of the cell adhesion molecules characterizing these two structures suggest developmental
continuity, and therefore possible identity, between the
two. Adult myomyonal junctions are most easily characterized by their high specific levels of expression of the
␣7B integrin subunit so this is a key marker. Early myotubes (E37) showed significant widespread expression of
cell-cell adhesion molecules (␤-catenin/cadherins and NCAM) over the whole sarcolemma and at myotube inter-
Fig. 6. Localization of N-CAM (B, D, F) with corresponding differential interference contrast (DIC) images (A, C, E). A and B: Embryonic day
37. N-CAM immunoreactivity is ubiquitously present on myotube surfaces. C and D: Embryonic day 47. N-CAM is still widely present on the
sarcolemma, but some myotube interfaces seem to show specifically
reduced immunostaining (arrow). E and F: Postnatal day 1. N-CAM
immunoreactivity restricted to satellite cells (arrow). Scale bars ⫽ 50 ␮m.
faces, while ␣7B integrin was detected only at select interfaces, generally between more mature secondary
myotubes (e.g., Fig. 4C). As development progressed,
␤-catenin expression was generally lost from the sarcolemma and that of N-CAM was specifically downregulated
at myotube interfaces, whereas strong expression of ␣7B
integrin continued to appear at a selection of myotube
interfaces (Fig. 4F). At postnatal day 1 (by which most
myotubes are mature in the guinea pig), select areas of
close apposition between adjacent myotubes/muscle fibers
showed intense reactivity for ␣7B integrin, but none at all
for the cell-cell adhesion molecules tested. This expression
pattern is typical of that seen at adult myomyonal junctions (Paul et al., 2002). Thus, physical attachment between young myotubes in guinea pig sternomastoid muscle is generally facilitated by a dynamically modulated
pattern of expression of cell-cell adhesion molecules, as
previously shown by other authors in simple muscle systems (Knudsen et al., 1990a, 1990b; Fredette et al., 1993;
Irintchev et al., 1994). However, as myotubes in the multiply innervated muscle begin to mature, some of these
sites begin to express the ␣7B␤1D integrin, which binds
myotube to myotube across the developing basal lamina.
Given their relative scarcity, we suggest that the sites
expressing ␣7B␤1D integrin likely correspond to the sites
of more specialized myotube-to-myotube attachment
578
PAUL ET AL.
found near the ends of the developing myotubes, and that
these sites mark the beginnings of the formation of the
permanent myomyonal junctions of adult series-fibered
muscles.
A remaining puzzle is the presence of a discontinuity in
the length-distribution of the growing secondary myotubes. Figure 3 shows that only a single myotube was
found within the length range of 1,400 –2,300 ␮m. We do
not believe this is a sampling error, as the serial section
study would tend to select against the inclusion of complete, very long myotubes, rather than excluding those of
middle length. One possible explanation is that end-to-end
fusion of extending myotubes might sometimes occur. For
example, two myotubes initiated from adjacent innervation zones might each grow to 1,200 ␮m, at which point
they encounter each other and fuse, producing one myotube of 2,400 ␮m. Unfortunately, close analysis does not
support this idea. Of the 16 myotubes above the critical
length of 2,400 ␮m, 10 of them overlap only a single
innervation zone, and so could not possibly have formed by
fusion of two separately initiated myotubes.
An alternative explanation arises from the observation
that nearly 80% (10/13) of the myotubes longer than 2,300
␮m extend into the zone where the tendon is forming,
distal to the last innervation zone (Fig. 3, data points
marked with a star). We suggest that the length of secondary myotubes may be influenced by the region of the
muscle in which they grow. Myotubes that lie partly
within the tendon may be stimulated to grow faster, or for
a more extended period, whereas myotubes with two intrafascicular terminations might slow their growth prematurely. Our own observations of fiber length in adult
guinea pig sternomastoid muscles would support the idea
of a growth-stimulating effect of the tendon zone; in
whole-mount immunohistochemical studies, fibers with
one end in a tendon and the other terminating intrafascicularly were always significantly longer than fibers terminating intrafascicularly at both ends (data not shown).
In summary, this study traces the relationships between developing myotubes in a series-fibered muscle and
shows how they evolve into the physically interconnected
network of adult muscle fibers. A highly dynamic system
is apparent, where early myotube interactions are dominated by cell-cell adhesion, whereas adult fibers are connected by junctions bridging through the extracellular
matrix. Finally, there is evidence suggesting differential
rates of myotube growth in the region of the tendon versus
the mid belly of the muscle.
ACKNOWLEDGMENTS
The authors thank Ms. Judy Rodda and the staff of
EMTech for their capable technical assistance with these
studies. The work was supported in part by grants from
the Health Research Council of New Zealand, Lottery
Health, and the Deans’ Fund of the University of Otago
Medical School.
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