Investigative Ophthalmology & Visual Science, Vol. 33, No. 3, March 1992
Copyright © Association for Research in Vision and Ophthalmology
Prenatal Morphogenesis of Primate Extraocular Muscle:
Neuromuscular Junction Formation and
Fiber Type Differentiation
John D. Porfer*t and Robert S. Bakerf
Extraocular muscle represents a distinctive class among mammalian skeletal musculature in exhibiting
the full range of muscle fiber type variability found in vertebrate species. To better understand the basis
for the unique structural/functional diversity of extraocular muscle, the ontogeny of lateral rectus
muscles was studied in Macaca nemestrina fetuses of 62-156 days gestation using light and electron
microscopy. At E62, myotubes and myofibers are evident, but fiber-type differentiation has not yet
occurred and neuromuscular junctions are primitive. By E92, presumptive singly and multiply innervated fiber types could be distinguished on the basis of myofibril delineation. Like other skeletal
muscles, extraocular myogenesis proceeds through at least two generations of myofibers. All primary
and secondary myofibers were generated and were maturing by El 21. The phylogenetically "old"
global multiply innervated fiber type was the first to attain adult form. This was followed by maturation
of global layer singly innervated fiber types, which are developed by El56, except for attainment of
definitive size and mitochondrial content. Orbital layer fiber types, particularly the orbital singly
innervated fiber, are the last to mature. Neuromuscular junction maturation paralleled the changes
observed during fiber-type differentiation. In summary, the sequential development of their constituent
muscle fiber types may reflect the functional pressures the extraocular muscles are exposed to by
maturing visual and visuomotor systems. In particular, ontogenic and phylogenic changes observed in
the orbital singly innervated fiber type may have direct implications for the types, range, and precision
of eye movements used by different species and at different gestational ages. Invest Ophthalmol Vis Sci
33:657-670,1992
The present study examined the prenatal development of fiber types in the extraocular muscles of a
primate. These muscles represent a distinctive class
among mammalian skeletal muscles. Extraocular
musclefibertypes do not easilyfitthe traditional fiber
classification schemes. In addition, these fiber types
include multiply innervated fiber types rare in mammalian skeletal muscle but common in birds and amphibians. The differences between these muscles and
other skeletal muscles may be related to their embryological origin from isolated mesenchymal condensations of neither somite nor branchial arch origin. How-
ever, it is generally believed that the plasticity of a
muscle is dictated by the plasticity of its motoneuron
pool. Thus, the unique fiber type composition of
these muscles (for review, see Spencer and Porter1)
may be, in part, related to the diversity of eye movement systems and the high discharge rates of oculomotor motoneurons.2
Extraocular muscles are responsible for reflexive
eye movements with velocities as low as <l°/sec to
saccadic movements of velocities up to 600°/sec. At
the same time, they maintain precise interocular
alignment to prevent diplopia. Also, oculomotor motoneurons exhibit sustained firing rates of up to 300/
sec and phasic discharges of up to 400/sec. These values are an order of magnitude higher than that seen
for other somatic motoneurons. Given that muscle
metabolic and contractile characteristics are highly
correlated with motoneuron physiological characteristics, the unusual properties of oculomotor motoneurons probably play a role in shaping the definitive
muscle fiber types. The relative contribution of myogenic factors and neurotropic regulation of gene expression infibertype differentiation, however, has yet
to be clearly established for any skeletal muscle.
From the *Department of Anatomy and Neurobiology and the
fDepartment of Ophthalmology, University of Kentucky Medical
Center, Lexington, Kentucky.
Supported by USPHS grant R01 EY05464 from the National
Eye Institute, Bethesda, Maryland, and a grant from the University
of Kentucky Medical Center Research Fund, Lexington, Kentucky.
Submitted for publication: August 9, 1991; accepted October 22,
1991.
Reprint requests: John D. Porter, Dept. of Anatomy and Neurobiology, University of Kentucky Medical Center, 800 Rose Street,
Lexington, KY 40536-0084.
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / March 1992
To better understand the unique features of the extraocular muscles, this study examined their prenatal
development in a primate model to establish the temporal relationship between axon and axon terminal
maturation and fiber type differentiation. Present
findings indicate that the phylogenetically "old"
global multiply innervated fiber type is the first to
develop. This is followed by maturation of global
layer singly innervated fiber types, which are developed by birth except for attainment of definitive size
and mitochondrial content. Orbital layer fiber types,
particularly the orbital singly innervated fiber, are the
last to mature. Ontogenic and phylogenic changes in
the orbital singly innervated fiber type may directly
impact the range and precision of eye movements.
Preliminary results have been presented in abstract
form elsewhere.3
Materials and Methods
Tissue was obtained from timed pregnant monkeys
(Macaca nemestrina) at the breeding colony at the
Regional Primate Research Center, University of
Washington, Seattle, WA. All procedures involving
animals adhered to the ARVO Resolution on the Use
of Animals in Research. Monkey gestational ages
were accurate to within a maximum of ±5 d. Fetuses
were delivered by Caesarean section at 62, 92, 121,
135, and 156 days of gestation (embryonic days are
designated as E, postnatal days as P; term in this species is approximately El66) and were anesthetized
with Nembutal intraperitoneally. Two fetuses were
male, two were female, and one was undetermined.
Most animals were perfused with Ringer's/dextrose
solution containing 2% heparin, followed by fixative
containing 2% paraformaldehyde and 2.5% glutaraldehyde in phosphate buffer. Muscles from the E62day case werefixedby immersion. Following fixation,
muscles were shipped to Kentucky for further processing. Lateral rectus muscles were processed for light
and electron microscopy by methods described in detail previously.45
Briefly, muscles were washed in cold phosphate
buffer and cut transversely at 500 ixm using a SmithMcllwain tissue sectioner (Brinkman, Westbury,
NY). Muscle sections then were postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer, stained en
bloc with 0.05% uranyl acetate in maleate buffer, dehydrated in a graded series of methanols, followed by
propylene oxide, and embedded in epoxy resin. Semithin sections were cut with an ultramicrotome,
stained with 0.1 % paraphenylenediamine, and examined with phase contrast optics. Ultrathin sections
containing representative regions of each muscle were
cut, picked up on 135 mesh honeycomb grids, and
Vol. 33
stained with uranyl acetate and lead citrate. Sections
were examined and photographed by using a Hitachi
H-7000 electron microscope. For comparison, adult
extraocular muscles were available from other studies
in this laboratory.5
Results
Muscle Fiber Morphology in Adult Extraocular
Muscle
Adult monkey lateral rectus muscles exhibit six distinct fiber types that do not conform to any of the
routine muscle classification schemes.1-4 Muscles contain a c-shaped orbital layer that lies away from the
eye and a global layer found in relation to the eye and
optic nerve. The extraocular musclefiber-typeclassification of Spencer and Porter1 is used in the present
report. The distinction between singly and multiply
innervated fiber types was based upon the longitudinal separation of neuromuscular junctions on individual fibers. The orbital layer contains one singly
(SIF) and one multiply (MIF) innervated fiber type.
The orbital SIF (Fig. ID) exhibits properties typical of
fast twitch, fatigue-resistant fibers and is characterized
by extensive mitochondrial aggregates and a surrounding capillary bed. Orbital MIFs (Fig. ID) are
smaller in diameter, present fewer mitochondria and
distributed neuromuscular junctions, and exhibit
myofibril characteristics that vary along the length of
individual fibers.6 The orbital MIF resembles the
avian (twitch) type with distributed neuromuscular
junctions. The global layer contains three SIF types
and an additional MIF type distinct from that of the
orbital layer (Fig. 2D). Global SIFs are distinguished
largely according to number, size, and distribution of
mitochondria. All global SIFs are fast twitch (among
the extraocular muscles, slow twitch fibers are found
only in the levator palpebrae superioris7), and include
global red, global intermediate, and global white SIFs.
Because of the presence of the extensive mitochondrial aggregates, the global red SIF closely resembles
the orbital SIF. Global MIFs resemble amphibian
tonic (nontwitch) fibers with their distributed neuromuscular junctions and poorly delineated myofibrils.
The specific details of adult neuromuscular junction
morphology are described in detail elsewhere.1
Early Developmental Events
At the earliest time studied (E62), monkey lateral
rectus muscles did not yet exhibit the layered organization that characterizes the adult but instead contained
primitive myofibers and myotubes scattered among a
loosely organized connective tissue matrix and
sparsely distributed, thin-walled vascular elements
(Fig. 3A). Activate fibroblasts were abundant. None
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Fig. 1. Electron photomicrographs of orbital layer of monkey lateral rectus muscles at E92 (A), E121 (B), E156 (C), and adult (D). At E92
(A), fibers are small in diameter, show little evidence of subtype formation, and the layer contains only scattered vascular elements (v).
Secondary generation myofibers are seen in association with the larger primary ones. With the exception of increased vascularization, little
change is evident in this layer by E121 (B). No associations of primary and secondary myofibers are evident at this stage. While orbital layer
fiber types continue to be underdeveloped just before birth (E156, C), this layer is recognizable by means of the higher mitochondrial content
of orbital singly innervated fibers and the increased microvascular network (c). Orbital multiply innervated fibers (M) are small and poorly
developed. In the adult (D), the orbital singly innervated fiber type is characterized by extensive mitochondrial aggregates and dense capillary
bed (c). At neuromuscular junctions, axons (a) encircle individual fibers. Multiply innervated fibers (M) are small and contain fewer, small
mitochondria and poorly delineated myofibrils. Original magnifications X 2250 (A, B, C), X 1500 (D).
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^^^ra^^
Fig. 2. Electron photomicrographs of global layer of monkey lateral rectus muscles at E92 (A) E121 (B), E156 (C), and adult (D). E92
monkey (A) exhibits early appearance of global multiply innervated fiber type (M), distinguished on the basis of myofibril size. Vasculature is
sparse, and scattered, small-diameter unmyelinated axons (a) are present. By E121 (B), the darker matrix of global multiply innervated fibers
(M) easily distinguishes them from singly innervated types. Fibers are larger in diameter and vascular bed development is higher than in the
orbital layer at this same stage. Axons are largely unmyelinated, although some scattered myelinated axons are present (a). Resolution of global
singly innervated fiber subtypes is possible by E156 (C), although their mitochondrial content is not yet that of the adult. Myelinated axons (a)
are distributed throughout the muscle. In the adult (D), singly innervatedfibertypes may be distinguished on the basis of the number, size, and
distribution of mitochondria. Capillaries (c) ring the global red singly innervated fiber type. Original magnifications X2250 (A, B, C),
X15OO(D).
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Fig. 3. Electron photomicrographs of early developmental events in monkey lateral rectus muscles. Two putative primary generation
myotubes are coupled (between arrows) in E62 monkey (A). Similar coupling is apparent (B) between putative primary (at right) and
secondary (at left) generation myofibers. Well developed A and I bands are evident in the primary generation cell. Note that the same basal
lamina surrounds both cells. High-magnification micrograph (C) of E62 myofiber indicates both the early sarcomeric organization of myofilaments and tnadic contacts between t-tubules (arrow) and sarcoplasmic reticulum. Myofibrils are poorly delineated at this stage. Comparison
with myofibrillar organization in an adult global singly innervated fiber (D) is illustrated (arrow indicates a triad). Original magnifications
X9000 (A), X22.5OO (B), X31,500 (C), X31,000 (D).
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / March 1992
of the six extraocular myofiber types could be resolved at this gestational stage. Individual myofibers
)r myofiber clusters were surrounded by a wispy basal
amina and few collagen fibrils were present in the
nterstitial space. Tight clusters of myofibers, with individual fibers often linked by gap junctions, were a
prominent feature at the earliest gestational ages studied (Fig. 3 A). The majority of muscle cells were multinucleate myotubes or myofibers, containing slightly
infolded euchromatic nuclei frequently central in location.
In the E92 monkey, the tight cell clusters were a less
obvious feature, but, when present, they typically
contained early and late differentiating myofibers,
with the smaller and more primitive secondary myofibers often sending cytoplasmic projections into presumptive primary generation myofibers (Fig. 3B).
This contrasts with the E62 case, at which time the
myofibers that made up individual clusters were at
similar developmental stages, and all were likely primary generation cells. Myonuclei contained more heterochromatin and prominent nucleoli. At this stage, it
was not yet possible to clearly distinguish the characteristic muscle layer organization. It was possible only
to identify those general regions of the muscle primordium that ultimately would give rise to orbital and
global layers. Connective tissue elements remained
limited, as much of the intercellular space was filled
with a loosely organized matrix, with collagen bundles particularly evident in relation to neurovascular
elements. Only by El35 to El56 was the interstitial
connective tissue elaborated to the point that it resembled adult form. At E92, the microvascular network
remained relatively sparse, with even the largest vessels consisting of an endothelium and a maximum of
1-2 layers of smooth muscle.
At E62, the basic sarcomeric organization of actin
and myosin filaments, consisting of hexagonal arrangement of actin around each myosin filament, was
already in place (Fig. 3C). The interlocking of actin
filaments at the Z-line did not yet appear to be complete. The early myofibrils were loosely defined, often
surrounded by clear cytoplasmic areas that presumably allowed for their further expansion in size. Likewise, while the extent of internal membrane system
development was slight, clear triadic contacts between elements of the t-tubule system and sarcoplasmic reticulum were evident in some fibers (compare
Fig. 3C with the adult morphology shown in Fig. 3D).
An active Golgi apparatus was present in many fibers.
By E92, myofibrils were conspicuously larger, with
little or no surrounding areas of vacant cytoplasm,
and were better delineated by elements of the internal
membrane system. Mitochondria, with well developed cristae, were evident as early as E62.
Vol. 33
Fiber Type Differentiation
Myofibers in 62-day fetal extraocular muscles were
homogeneous in appearance, with individual fibers
exhibiting similar mitochondrial content and degree
of myofibril delineation. Initial expression of any of
the distinctive characteristics of extraocular muscle
fiber types was first apparent at E92 (Figs. 1A and
2A). Singly and multiply innervated types then could
be distinguished as myofibrils were well delineated by
internal membrane systems in the former and remained large and poorly delineated in the later. The
lack of divergence in mitochondrial content at this
time precluded identification of the distinctive SIF
subtypes.
By E121, primary and secondary generation myofibers could no longer be distinguished and the resolution of SIFs from MIFs was clear for both extraocular
muscle layers. In particular, global MIF morphology
began to approach the degree of maturation of myofibrils and internal membrane systems evident in the
adult (Fig. 2B). At this time, thisfibertype also exhibited the higher electron density of myofibrils that characterizes the adult. The global MIF then was the first
type to attain definitive form and, at this stage, was
disproportionately large compared to the singly innervated types. The occurrence of significant elaboration in internal membrane systems and in the number, size, and distribution of mitochondria marked
the early stages of appearance of SIF subtypes.
Throughout the time frame of study, however, orbital
fiber types lagged behind those of the global layer in
obtaining adult characteristics. Concurrent with fiber
type specialization, and likely related to increased metabolic demands, the microvascular network matured
to a level that was considerably more extensive than at
earner stages. With increased fiber size, intercellular
space was markedly reduced.
Changes in oxidative capacity were observed in
SIFs by El35. In particular, the increased mitochondrial content of orbital SIFs allowed better differentiation of extraocular muscle layers at this stage. Mitochondria were largely distributed singly. Only occasionally were they seen to demonstrate the clustering
that characterizes this fiber type in the adult. Taken
together, observations at El35 suggested that the orbital layer continued to lag behind global layer fiber
types in size and elaboration of internal ultrastructure.
The basis for functional and metabolic differences
between the orbital and global layers were clearly evident by El56 (Figs. 1C and 2C). While all fiber types
were qualitatively smaller than in the adult, global
layer intermediate and pale SIFs now were easily distinguished as a result of variations in mitochondrial
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content that presumably translate into fatigability differences. Orbital MIFs remained small and poorly developed at this stage, only to mature rapidly in the
early postnatal period.8 At E156, the global red and its
counterpart orbital SIF types remained immature in
terms of obtaining definitive mitochondrial content.
Continued elaboration of vascular elements resulted
in an extensive capillary network in association with
orbital SIFs. Commensurate increases in the mitochondrial content of this fiber type led to the initiation of formation of the central and subsarcolemmal
clusters that characterize these fiber types. These two
highly fatigue-resistant fiber types were, however, the
last to attain adult form, reaching their definitive anatomical profile in the first few postnatal months.8
Neuromuscular Junction Maturation
The early nerve bundles seen in the E62 animal
contained small (approximately 1-3 ^m diameter)
axons, but, surprisingly, there were few growth cones
observed at this or any later stage (Fig. 4B). The majority of axons were clustered into discrete bundles
surrounded by the processes of presumptive Schwann
cells. (High polyribosome and modest rough endoplasmic reticulum content and the presence of a basal
lamina distinguished Schwann cells.) Axons contain
substantial membranous material, presumably
smooth endoplasmic reticulum, and relatively few
neurotubules and neurofilaments. The individual
axons were not discretely isolated, but typically were
in direct contact with one another. Very few presumptive terminals were observed in direct anatomical contact with myofibers. Rather, most "terminals"
were at distances > 100 nm from the sarcolemma with
synaptic clefts of irregular width (Fig. 4A). Occasionally, Schwann cells and myofibers were attached by
junctional complexes (Fig. 4A). Synaptic vesicles
were clear and spherical, with a few dense core vesicles, and were sparsely distributed in preterminal and
terminal axons. Thickening of the sarcolemma immediately adjacent to nerve terminals represented the
only postjunctional change observed at this time.
By E92, most axons were isolated from one another
by Schwann cell processes, and nerve bundles were
well defined by connective tissue elements (Fig. 4D).
Occasional bundles of small diameter, presumably
preterminal axons still lacked the separation from
each other. Axons exhibited the adult complement of
organelles, including organized bundles of neurofilaments with interspersed neurotubules. Myelination of
some axons was encountered only near point of entry
of nerve into the muscle. Even then, only a very thin
layer of myelin was present. Elongated neuromuscu-
663
lar contact zones, usually with overlying Schwann
cells, were characteristic of junctions associated with
presumptive SIFs at this developmental stage (Fig.
4C). Synaptic contacts with putative MIFs were
smaller and did not occupy such an extensive portion
of the fiber surface. Neuromuscular junctions were
seen in association with presumptive primary generation myofibers only, as terminals were not observed
on the small, primitive cells that were tightly associated with the primary myofibers. Terminals were
filled with spherical, clear synaptic vesicles and small
mitochondria. The beginnings of postjunctional specializations, including aggregations of mitochondria
and highly infolded myonuclei, were observed occasionally at junctions associated with SIFs. Postjunctional folding of the sarcolemma was not noted at this
or any other prenatal stage.
At E121, the terminals on global SIFs had become
more discrete, as the elongated nerve/muscle appositions seen at E92 were reduced to focal contacts. At
this point, however, presynaptic elements associated
with singly innervated fibers still lay on the fiber surface and were not yet embedded in sarcolemmal depressions. In keeping with the degree of maturation of
the global MIF structural characteristics, the neuromuscular junctions associated with this fiber closely
resembled those of the adult (Fig. 5). Compared to the
E92 case, myelin sheaths of intramuscular axons were
thicker and were no longer restricted to the nerve
entry zone, but extended farther into the substance of
the muscle.
Further changes in neuromuscular contacts and, in
particular, postsynaptic elements, characterized El 35
monkey extraocular muscles (Figs. 6A and 6B). While
neuromuscular contacts continued to become more
focal in appearance (opposed to the elongated contacts of E92), stacking of preterminal synaptic elements upon one another was first evident in some
junctions at this stage. Some of the neuromuscular
junctions associated with global SIF types showed
embedding of the presynaptic element in deep depressions of the sarcolemma. The distinctive spiraling of
nerve terminals around orbital SIFs seen in the adult
also became apparent by this stage.19 Massive aggregations of highly infolded myonuclei were noted at
the junctional region of singly innervated fiber types
(Fig. 6 A). Postjunctional accumulations of mitochondria also became evident in some SIFs at this stage.
El56 day neuromuscular junctions were structurally mature in virtually all respects, except for elaboration of postjunctional folds in global SIF types
(Fig. 6C). Such folding typifies the neuromuscular
junctions of adult global intermediate and pale SIF
types (Fig. 6D). This feature develops within the first
three postnatal months.8
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / March 1992
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Fig. 4. Electron photomicrographs of early neuromuscular junctions (A, C) and intramuscular nerves (B, D). At E62 (A), developing
synaptic terminals (s) closely approach developing primary myotubes. Scattered synaptic vesicles are evident in putative terminals. Schwann
cells (Sch) are associated with developing neural elements, and, occasionally, form junctional complexes with muscle (arrow). At this stage,
intramuscular axons are small and unmyelinated (B). Schwann cell processes (arrows) surround bundles of axons, but do not enclose
individual axons. An early myotube, characterized by central nuclei (n), exhibits a closer synaptic contact (s) and postsynaptic sarcolemmal
thickening at E92 (C). By this stage, individual axons (a) are surrounded by Schwann cell processes, and nerve bundles are enclosed by collagen
andfibroblastprocesses (D). X2O,7OO (A), X 16,500 (B), X 16,000 (C), X4600 (D).
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Fig. 5. Low- (A) and high- (B) power electron photomicrographs of a neuromuscuiar junction associated with a global multiply innervated
fiber at EI21. Both fiber and synaptic terminal (s) closely resemble that of adult extraocular muscle. X6000 (A), XI 2,000 (B).
Discussion
The extraocular muscles arise from maxillomandibular and premandibular condensations of mesoderm that appear within the orbit. The maxillomandibular condensation gives rise to muscles innervated
by the abducens and trochlear nerves, while the premandibular condensation forms those muscles innervated by the oculomotor nerve.10 At the cellular level,
the generation of the extraocular muscles follows the
same general stages described for other skeletal muscles: mesenchymal cell, early myoblast, myoblast, fusion of myoblasts, myotube, and mature myofiber.'l
Whether germinative cells are pluripotential, subject
to neural or environmental influence, or exist as distinct myoblast lines already destined to form singly or
multiply innervatedfibertypes has not yet been determined for extraocular muscle. Distinct myoblast lines
have been described in skeletal muscles of birds.12 The
present study has established the sequential maturation offibertypes in lateral rectus muscles of the monkey from the myotube/early myofiber stage to birth.
Like other skeletal muscles, the extraocular muscles
are generated asynchronously from at least two waves
of myogenesis responsible for formation of primary
and secondary fibers. This report is concerned with
the maturation of adult eye muscle fiber types from
the ultrastructurally homogeneous population of cells
present at E62. Global layer fiber types, beginning
with the global MIF, matured earlier than fiber types
in the orbital layer. The potential correlations between the sequence of events in morphogenesis of the
extraocular muscles and maturation of visuomotor
systems are discussed below.
At the earliest stage examined (E62), the extraocular muscles consisted of a homogeneous population of
myotubes and myofibers. The lack of resolution of
ultrastructurally defined fiber types at this stage does
not, however, exclude the possibility of early divergence in cell lineage, as individual cells already may
be committed at the level of gene expression. Tight
coupling between primary myotubes was characteristic of E62 monkey extraocular muscles. The presence
of myofiber linkage by way of gap junctions is typical
of developing skeletal muscle and is hypothesized to
provide for spread of activation between innervated
and noninnervated myotubes. Like other skeletal
muscles, second generation myofibers in extraocular
muscle appear to form only through associations with
primary generation fibers (secondary fibers are generated in monkey lateral rectus muscles between E62
and E92). At E92, multiple secondary myotubes are
coupled to individual primary myotubes, all contained within the same basal lamina. Given the ab-
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Fig. 6. Electron photomicrographs of neuromuscular junctions associated with singly innervated fiber types at E135 (A, B), E156 (C), and
adult (D) stages of development. El 35 terminals (A, B) are characterized by dense aggregations of terminal boutons (s) and myonuclei (n).
Many nerve terminals are wellfilledwith synaptic vesicles, and postjunctional accumulations of mitochondria have become evident (B). A
nerve terminal associated with a global white singly innervated fiber is mature at E156, except for the formation of subjunctional folding in the
sarcolemma (C), Subjunctional folds are clearly evident (arrows) in this samefibertype in the adult (D), X6900 (A), X11,500 (B), X16,000 (C),
XI 1,500 (D).
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sence of neuromuscular contacts on secondary generation myotubes, the presence of such coupling suggests
that neural activation, via gap junction contacts with
the primary myotube, may be important to subsequent maturation of the cell. Such secondary generationfibersdepend greatly upon innervation and muscle contraction, as loss of either halts their formation.13 The present report's findings in the monkey
suggest that extensive formation of new myofibers
continues until between E92 and E121. Thus, the loss
of the fiber-fiber coupling by E121 may reflect the
status of maturation of motor innervation and onset
of independent function in all developing myofibers.
For most skeletal muscles, the anatomical and contractile properties of slow twitch (type I) fibers developfirst.Relating extraocular muscles to other skeletal muscles, Lennerstrand and coworkers1415 indicate
that, structurally and functionally, cat extraocular
muscle slow-fiber systems develop earlier than fast
systems. These data are consistent with present observations in that the global MIF precedes all other types
in attainment of adult characteristics. However, the
slow fiber systems in extraocular muscle, which are
unusual in presentation of multiple innervation, are
neither the structural nor functional equivalent of typical skeletal slow twitchfibers.The early development
of a slow, tonically contracting fiber is difficult to interpret, particularly because the functional role of this
fiber type in the adult is not well understood. However, because this fiber type is a component of the
principal sensory receptor found in extraocular muscle,1617 it is plausible that early maturation of the
global MIF allows proprioceptive information to be
used in visual system development.18"21
Global SIFs mature rapidly in the late prenatal period. Although their mitochondrial content does not
attain adult levels until after birth, ultrastructural
characteristics were such that the three global SIF
types could be discerned by El56. The observation
that orbital layer fiber types mature last is consistent
with the hypothesized role of orbital SIF in oculomotor control.1-22 The exceptionally high mitochondrial
content of orbital SIFs is consistent with elevated fatigue resistance, and thus is consistent with a sustained level of activity in eye position maintenance.
Indeed, motor units in the orbital layer are recruited
early during initiation of an eye movement.23"25 As
oculomotor systems mature, the increased fatigue resistance of this fiber type presumably is an adaptive
response to the development of disjunctive eye movements and fusion, which are particularly important to
primates. Consistent with this view, across mammalian species, the orbital SIF exhibits its highest mitochondrial content in the monkey and man.
Muscle fiber differentiation and motor unit devel-
667
opment are known to parallel one another.26-27 Indeed, neural influence upon muscle properties apparently can occur at any of several stages. Given the
primitive state of neuromuscular junctions at E62, it
is likely that the early differentiation of basic muscle
structure can occur in the absence of innervation. In
other muscles, the lack of this early activity leads to an
arrest in formation of secondary fibers.13 Likewise,
denervation at birth interferes with the transition
from "slow" to "fast" characteristics that typifies the
development of most skeletal musculature.28 However, later differentiation of the definitive fiber type
composition of these muscles most likely requires not
only innervation, but probably the particular patterned activity imposed by the motor control system
(see below). Based on the complex nature of extraocular muscle myosin expression, which includes the presence of embryonic and neonatal isoforms as well as
cardiac and extraocular muscle specific types,29"31 it is
likely that the role of neurogenic factors in the regulation of gene expression in these muscles is significant.32 Extraocular muscles, however, show evidence
of possessing a high degree of intrinsic plasticity, including the expression of multiple developmental
myosin isoforms in the adult,29 extraordinarily high
resistance to denervation atrophy533 and development despite the absence of the globe in an anophthalmic mouse model.34 Thus, the potential myogenic
origin of some of their unique features cannot be ignored.
Species Comparisons
The early events observed in developing monkey
extraocular muscles closely paralleled those seen in
limited studies of human fetuses.35 Muscles of the E62
monkey closely resembled those of an approximately
E70 human fetus in presenting tight clusters of myotubes/myofibers linked by gap junctions and axon
bundles that were wrapped simply by Schwann processes. Likewise, the tight associations of primary and
secondary myofibers apparent in the E92 monkey
also were seen in E84 and El05 human fetuses. An
E161 human fetus differed from the later stages of
monkey extraocular muscle development. The primary-secondary myofiber associations were present
over a longer time than was observed for the monkey.
Together, these data suggest a longer proliferative
phase in man. Near-term human fetal extraocular
muscles, comparable to the later stages examined in
the present study, have not been previously studied.
The monkey exhibits a greater degree of fiber-type
differentiation during the prenatal period than either
of the experimental animals (cat and rat) in which
extraocular muscle development has been stud-
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Morch 1992
ied.115'36 Moreover, muscle morphogenesis is much
more compressed to within the perinatal and early
postnatal periods in the cat and rat. Later maturation
of extraocular muscle in these species may be related
to timing of eyelid opening (open at birth in monkey,
while open about P14 in cat and rat). Extraocular
muscles of an E62 monkey look much like those of
El8 rat36 in that myofibers are homogeneous, lacking
differentiation of any of the definitive adult fiber
types. The similarities appear to end there. In contrast
to the monkey, development of the orbital layer precedes that of the global layer in the rat. Moreover,
these authors indicate that the global MIF, the first
type to be defined in the monkey, is the last to mature
in the rat.
In contrast, development of the layered organization of cat extraocular muscles closely resembles that
of the monkey,115 with global preceding orbital. The
two species differ in the overall time course of myogenesis (many events that are prenatal in the monkey
occur in the early postnatal period in the cat) and in
the ultimate degree of mitochondrial development in
the orbital SIF (with higher mitochondrial content in
monkey). In summary, species comparisons of extraocular muscle ontogeny suggest that the sequential
development of fiber types is conserved in frontaleyed organisms but may follow a different sequence
in lateral-eyed species. If such developmental differences are consistently observed in comparisons of animals with frontally and laterally placed eyes, a strong
tie can be made between muscle morphogenesis and
requirements for interocular alignment, fusion, and
vergence eye movements.
In general, neuromuscular junction formation appears to follow similar paths in rat,36 monkey (present
findings), and man.35 Events occurring during synaptogenesis did not differ markedly from those previously described in other skeletal musculature.37"39 Initial contacts are small and evolve into much elongated contacts that ultimately are reduced to the form
seen in the adult (eg, P7 rat exhibits the same elongated contacts seen in E92 monkey, although these
later become focal in both species). Similar to the situation in the monkey, postjunctional folds form in the
postnatal period,40 but only in association with particular fiber types.1 The restriction of postjunctional
folds to particular fiber types suggests that their formation is regulated by activity levels. Using an extraocular muscle model, Sohal41 argues that postjunctional specializations may develop in the absence of
innervation. However, while thickenings of the sarcolemma reminiscent of those at neuromuscular junctions are seen in aneural muscles, the synaptic folds
that form in this situation are shallow and few.
Because extraocular muscles contain SIF and MIF
Vol. 33
types, the mechanisms of neuromuscular junction
formation and stabilization must be different from
those that are operative in other skeletal musculature.
During development, initial synaptogenesis upon a
putative skeletal muscle twitch fiber or extraocular
SIF excludes the formation of junctions at other sites
on that samefiber.42Thefiberthen may be transiently
polyneuronally innervated, but the multiple contacts
are restricted to the one synaptic site. The stacking of
axon terminals at individual neuromuscular junctions of El 35 monkeys may represent such transient
multiple innervation of SIFs. In contrast, synaptogenesis must be allowed to occur at numerous sites along
the length of individual MIFs. Several muscle extracellular matrix and cell surface markers (see Sanes43
for review) have been hypothesized to serve as trophic
factors that attract ingrowing axons. The restricted expression of such markers to the neuromuscular junction may be a consequence of the myonuclear aggregations seen in SIFs at El35. Perhaps differences in
the pattern of expression of such markers in extraocular muscles are responsible for the diverse innervation
pattern seen in these muscles. Furthermore, at all
stages examined in the present study, there was a surprising paucity of axonal growth cones, suggesting
that the remodelling of neuromuscular junctions is
minor or that it must occur rapidly enough to have
been missed in the present study. It has been established that skeletal muscle fibers initially are innervated by multiple axons and that all but one retract
during the perinatal period. The timing and duration
of transient multiple innervation of SIFs in the extraocular muscles are unclear. Oculomotor motoneuron
death is observed in the perinatal period,44"47 however, suggesting that the competition for synaptic sites
in extraocular muscle is similar to that seen in other
skeletal muscles.
Functional Considerations
The basic determination of general fiber types (ie,
slow versus fast) is evident early in development as the
definitive ATPase staining pattern in cat extraocular
muscles is observed at birth and the relative proportions offibertypes do not change thereafter.14-15 Mitochondrial pattern and microvascular network, however, were the last features to fully develop, occurring
predominantly in the postnatal period for rat, cat, and
monkey. These features are responsible for determining the aerobic energy production capacity of muscle
fiber and thereby translate directly into the relative
fatigability of the fiber. In the cat, motor unit studies
show that slowfibersystems are mature by 6 wk postnatal, but fast systems continue to develop until the
adult stage.48 These findings are consistent with pres-
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No. 3
EXTRAOCULAR MUSCLE MORPHOGENESIS / Porrer ond Doker
ent data that indicate early maturation of global MIFs
and significant postnatal increases in fiber diameter
and mitochondrial content. Convincing evidence exists that fatigue resistance is shaped by the functional
demands placed upon the muscles by developing visual and oculomotor systems. Hanson and coworkers15 have shown that the differentiation of succinic dehydrogenase (a mitochondrial oxidative enzyme) staining patterns that lead to definitive
extraocular muscle fiber types in cat begins as late as
P28. In addition, when normal visual system maturation is impaired by monocular lid suture in normal
cats or impaired genetically in albino Siamese cats,
the extraocular muscles exhibit slower contraction
times and reduced fatigue resistance.48*49 The anatomical correlate of these functional changes was reduced
fiber diameter and lower capillary density/fiber.14-49
The development of motoneurons subserving eye
movement function also is impaired by monocular lid
suture.50 Interestingly, the morphological changes induced by visual deprivation did not alter the overall
rotational stiffness of the eye in the orbit.51
In summary, extraocular muscles exhibit greater
structural and functional diversity than any other skeletal muscle. This muscle complexity reflects the diversity and plasticity inherent in eye movement control systems. We interpret the pattern of extraocular
muscle development to indicate an adaptation to the
changing demands placed upon these muscles. The
sequential development of the six muscle fiber types
may reflect the functional pressures the extraocular
muscles are exposed to by maturing visual and visuomotor systems. Several questions remain regarding extraocular muscle development. First, whether there is
any difference in the fiber types derived from the primary and secondary generation myoblasts formed in
these muscles is undetermined. Second, the role of
intrinsic muscle properties in generation of the
unique extraocular fiber types and the stage or stages
at which neural influences may play a role in such
determinations remain to be established. Finally, also
unexplored are the positional or neural cues responsible for the formation of the orbital and global muscle
layers in extraocular muscle.
Key words: extraocular muscle, myogenesis, eye movement, neuromuscular, monkey.
Acknowledgments
The authors thank Dr. Anita Hendrickson for providing
the fetal monkey extraocular muscles for these studies. The
technical expertise of Linda Simmerman and comments on
an earlier version of the manuscript by Drs. Philip Bonner
and Robert Spencer also are much appreciated.
669
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