EMBRYOGENESIS OF EXTRAOCULAR MUSCLE John D. Porter, Ph.D.

Lexington. Kentucky

I. INTRODUCTION Postnatal maturation of vision includes a well

documented critical period, during which time the formation of retinogeniculostriate synaptic connectivity is activity dependent (Hubel and Wiese 1965). Congenital strabismus and amblyopia produce direct and permanent deficits in visual function. However, the potential role of developmental muscular and/or neuromuscular deficits in the etiology of misalignment of the eyes, and in the subsequent disruption of the ocular dominance columns of the striate cortex, has received little attention. Although the structuralffunctional properties of the final common pathway for eye movement systems clearly are unique, there have been few descriptive studies and virtually no experimental studies that have addressed extraocular muscle development. Given the unique muscle fiber type composition of adult extraocular muscle, mechanisms regulating their morphogenesis are likely to differ from those that regulate the differentiation of other skeletal muscles. In this review, we: (a) consider the early events in extraocular myogenesis, (b) discuss pre- and postnatal shaping of the extraocular muscle fiber types, and (c) review the genetic and epigenetic mechanisms that regulate the development of the extraocular muscle phenotype. Based upon the unique extraocular muscle phenotype, and the extended postnatal period during which intrinsic and extrinsic factors may influence the expression of muscle fiber characteristics, we propose that there is a postnatal critical period for eye muscle development that is not unlike that of the visual sensory system. II. EARLY DEVELOPMENTAL EVENTS

Two theories exist as to the early events in the ontogenesis of the extraocular muscles. One holds that the anlagen of each muscle condenses from one of three distinct precursors, separately and at distinct tinies (Gilbert 1947, 1957). The altemative theory(Sevel, 1981, 1986) is that the extraocular muscles develop concurrently from a single mesenchymal condensation that subsequently divides into separate superior and inferior mesodermal complexes. Individual extraocular muscles may receive contributions from both mesodermal complexes (medial and lateral recti) or may arise from only one or the other complex (remainder of the oculorotatory muscles plus the levator palpebrae superioris). During organogenesis, the developing brainstem also is segmented into regions known as rhombomeres that give rise to the cranial nerves (Lumdsen and Keynes, 1989). Each of the oculomotor nerves arises from particular rhombomeres, consistent with the segmental nature of the

cranial nerves. A caudal to rostral internuclear gradient for the genesis of oculomotor motoneurons has been described in rats (Altman and Bayer, 1980, 1981). The majority of motoneurons in abducens, trochlear, and oculomotor nuclei are postrnitotic by the time the eye muscles are forming. Recent studies suggest that aggregates of myoblasts may be contacted by oculomotor nerves prior to migration and carry their innervation with them into the developing orbit (Wahl et al., 1994). Whether innervation first occurs in the orbit or while myoblasts are still adjacent to the neural tube, the close proximity of the anlagen of the extraocular muscles may actually facilitate development of anomalous innervation of eye muscles. The classic clinical example of this relationship is Duane's retraction syndrome, wherein the congenital absence of the abducens nerve results in the "inappropriate" innervation of the lateral rectus by the oculomotor nerve. A similar outcome has been seen in a transgenic mouse model in which the oculomotor and trochlear nuclei are absent and the abducens nerve may sprout to innervate incorrect muscles (J.D. Porter, R.S. Baker, and AP. McMahon, unpublished).

The prior notion that the extraocular muscles originate from the neural crest is only partially correct. Recent studies have established that the myoblasts that form the extraocular muscles arise from cranial mesoderm, while it is the orbital connective tissues that originate from neural crest (Couly et al., 1992; Noden, 199 1). A rostral, unpaired mesodermal condensation (the prechordal plate) and contributions from some of the seven paired condensations of paraxial mesoderm (the somitomeres; these are loosely analogous to somites) form the eye muscles. There are conflicting opinions as to whether prechordal mesoderm contributes directly to the formation of extraocular muscle (Couly et al., 1992), or if cells originating from this site first seed the somitomeres, which secondarily give rise to myoblasts that populate the orbit (Wachtler et al., 1984). The precursor cells migrate together in compact aggregates to form condensations around the developing eye. It is the number and fate of these orbital condensations that have led to the controversy detailed above. Once muscle precursor cells are in the primitive orbit, the primary myotube alignment pattern apparently is specified by cell-cell interactions with the neural crest-derived connective tissue, as the proper spatial orientation is obtained even if the myoblasts are from mesoderm that was transplanted to the head from an inappropriate source (Noden, 1986), or if the oculomotor and trochlear nerves are absent (J.D. Porter, R.S. Baker, and A.P. McMahon, unpublished).

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III. EXTRAOCULAR MYOGENESIS After muscle precursor cells migrate into the orbit,

!ll)'Ogenesis follows the same general stages that have been described for other skeletal muscles (Kelly, 1983; Seve!, 1981) Six distinct myogenically defmed cell lineages have been identified in extraocular muscle primordia (Lucas et a!., 1991), and may correspond to the six adult muscle fiber t)pes. Close parallels can be drawn between the development of human and macaque monkey extraocular muscles; thus we have studied subhuman primates in detail. As in other skeletal muscles, the extraocular muscles are generated from at least two waves of myogenesis that form primary and secondary generation myofibers (Porter and Baker, 1992). In the monkey, myoblasts fuse to form primary myotubes prior to embryonic day 62, while secondary myotubes appear between embryonic days 62-92 (term in the monkey is about 165 days). As in other skeletal muscles, the secondary myotubes form in close association with primary myotubes. While muscle fibers still are homogeneous and relatively undifferentiated, early neuromuscular contacts are observed (by embryonic day 62; Porter and Baker, 1992). By embryonic day 92, cytological differences between singly innervated and multiply innervated fiber types can be distinguished. All primary and secondary generation fibers are generated and maturing by embryonic day 121. As a general rule, the phylogenetically "old" global multiply innervated fibers are the first to form, while fibers in the orbital layer mature last. Based upon evidence from ultrastructural and myosin expression studies, it appears that the extraocularm multiply innervated fiber types derive from primary myoblasts and singly innervated fiber t) pes from secondaries. IV. FillER TYPE MATURATION

Despite the considerable prenatal development that occurs in extraocular muscle, there is significant postnatal maturation of these muscles (Hanson et a!. , 1980; Spencer and Po[ter, 1988), even in the primate (Porter and Baker, 1992). Like the other skeletomotor systems, the oculomotor system exhibits overproduction of motoneurons and competition for synaptic sites, resulting in the activity dependent adjustment of motoneuron number to target size (Sohal, 1977: Sohal et a!., 1991; Sohal and Weidman, 1978). In !:)pica! skeletal muscle, multiple axons contact a muscle fiber at a single site, but, through competitive interactions, all but one axon is eliminated. A percentage of motoneurons die during this perinatal process. Similarly, the singly innervated fibers in extraocular muscle most likelv exhibit this same transient and focal multiple innervatio of muscle fibers. However, the rules that govern neuromuscular junction formation and stabilization must be quite different for multiply innervated fibers. Instead of neuromuscular junction formation at one site that precludes the formation of junctions at other sites on the same fiber, multiply innervated

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fibers must allow synaptogenesis at sites that are distributed along their length. The mechanisms responsible for the formation of either focal or distributed neuromuscular junctions in extraocular muscle have not yet been addressed.

By birth, the six fiber !:)pes are recognizable in monkey extraocular muscles (Porter and Baker, 1992). Beyond the postnatal increase in size of all fiber types, muscle maturation occurring after birth in the monkey largely involves increases in the mitochondrial content of the orbital singly innervated fiber type (J.D. Porter, RS. Baker, and RF. Spencer, unpublished). This postnatal shaping of fiber characteristics occurs in the human as well and likely is critical for appropriate function of eye movement systems. During the postnatal period the defmitive muscle characteristics are established. This period roughly corresponds with the period of visual system maturation, and eye muscle characteristics are subject to aberrant development and/or developmental delays. We believe that this period of time represents a critical period for eye muscle development (approximately the first 3-6 months after birth in primates), during which time the eye muscles acquire the structural/functional characteristics demanded by binocular vision, but are more susceptible to insult than in the adult.

Few studies have examined the pattern of myosin heavy chains expressed by particular extraocular muscle fiber !)pes during development. Each of the six fiber types contains characteristic myosin heavy chain (MHC) isoforms demonstrable by immWlocytochemistry at the light microscopic level. Different fiber !)pes have distinct spatial and temporal patterns of myosin heavy chain expression in e>.1raocular muscle. We have characterized the transitions in MHC expression in extraocular muscles of rats between the ages of embryonic day (E) 17 and postnatal day (P)45 using monoclonal antibodies directed against various myosin heavy chain isoforms for immunocytochemical and Western analysis (Brueckner et a!., 1994).

Typically, the embryonic MHC protein is transiently expressed early in skeletal muscle development and is repressed subsequently in lieu of neonatal MHC for putative fast fibers and slow MHC for putative slow fibers. This

developmental isoform is uniquely retained in adult extraocular muscle, however, in a fiber type-specific fashion as demonstrated by immunocytochemistry. It is synthesized in all extraocular muscle fibers as early as E 17. Putative fast-contracting, singly-innervated fibers in the global layer downregulate synthesis of the embryonic MHC protein during the second postnatal week (by Pll). This protein is retained Wltil P20 in the global layer multiply-innervated fibers, where it is co-expressed with slow-twitch MHC. After P35, embryonic MHC is strictlv confined proximally and distally in orbital layer fibers, with

. fast MHC expression

in the mid belly region of these fibers. Eye muscle is unique m that the developmental myosin isoforms are not absent

from adult muscle; thus, these myosin heavy chain genesmaynot be completely repressed as they are in other skeletal muscles. This atypical myosin gene expression may be a contributing factor to their unusual capacity to adapt to changing innervation levels and/or disease states. The atypical pattern of spatial and temporal protein expression is likely the result of both genetic and epigenetic factors that will be discussed below. Unique neural and mechanical cues may elicit rapid sarcomeric turnover, resulting in the continuous expression of the embryonic isoform only in the peripheral nuclear domains of orbital layer extraocular muscle fibers.

As in other skeletal muscles, fast MHC is detected prenatally in secondary extraocular muscle fibers exclusively. After birth, there is a dramatic increase in fast MHC expression as the proportion of secondary fibers to primary ftbers rises. These high protein levels are maintained into adulthood. IIA/IIX MHCs represent a subset of these fast isoforms and are unique in the late onset of their expression (postnatal day 15). During the ftrst postnatal week, IIAJIIX MHC expression becomes restricted to orbital layer extraocular muscle fibers in addition to a subset of global layer fibers (red singly innervated fibers). Given the high fatigue resistance of e>.1raocular muscle, it is noteworthy that the expression of IINIIX is so limited that it cannot be detected by Western analysis. Two other fast MHCs were identified by Western blotting and these most likely represent the liB and extraocular muscle-specific MHC isoforms. These gradually increased in abundance from birth until postnatal day 18, after which time an abrupt increase in protein content was observed. On the basis of the limited distribution of JIA/IIX myosin, it is likely that the EOMspecific myosin is a major component, substituting for JIA/liX in a fast-twitch, fatigue resistant role.

Slow MHC is expressed prenatally only in primary fibers. Early in development, the proportion of primary to secondary fibers is high, so there is a higher percentage of primary (slow) fibers. As myogenesis proceeds, however, and the number of fast MHC-containing secondary fibers increases, the proportion of slow-twitch fibers declines and remains at about 20%. Postnatally, slow-twitch MHC is located in orbital and global multiply innervated fibers. Western blotting revealed the existence of two slow MHC proteins in these fibers that gradually increase in abundance postnatally.

Interestingly, many of the specific transitions in the localization of MHC isoforms occur around the time of eyelid opening (at Pl4), suggesting that functional, rather than solely myogenic stage-specific, factors may be critical in determining e>.1raocular muscle phenotype. Clearly, MHC expression is, at least in part, activity-dependent. Experimental perturbations of oculomotor function (e.g., denervation, monocular deprivation) will provtde mstght mto

the role of neural cues in differential myosin expression in extraocular muscle. V. GENETIC REGULATORY FACTORS

Little is known regarding the genetic mechanisms that may be responsible for many of the unique features of the extraocular muscles. Studies of myosin heavy chain protein and gene expression suggest that extraocular muscle has a broader developmental potential than virtually all other skeletal muscles (Amussen et al., 1993; Rushbrook et a!., 1994; Wieczorek et al., 1985). It is now recognized that, when compared to other skeletal muscles, the extraocular muscles exhibit remarkable diversity among fiber types at the transcriptional, translational, and ultrastructural levels. Given their origin from cephalic mesoderm, the heterogeneity of extraocular muscle may reflect the developmental potential of a unique lineage of muscle precursor cells. The disparate origins of mesenchyme that forms orbital tissues (Noden 1991; Northcutt, 1990) makes study of the specificity of early cell-cell interactions difficult. An understanding of these interactions is necessary in order to address the mechanisms that may be responsible for specification of the patterned spatial distribution of fiber types into distinct layers and the unusual phenotype of individual- fiber types in these muscles. Interestingly, modifications of extraocular muscle primordia into heatproducing cells in billfish (Block and Franzini-Armstrong, 1988), and into electric organs in weakly electric fish (Leonard and Willis, 1979), represent extremes in the genetic potential of muscle precursor cells. The developmental mechanisms responsible for this unusual fate have not yet been addressed, but the phenomenon does serve to highlight the unique character of these muscles. VI. EPIGENETIC REGULA TORY FACTORS

The sequential development of extraocular muscle fiber types may reflect, at least in part, the functional pressures coinciding with the maturation of visual and visuomotor systems. During infancy, the postnatal maturation of extraocular muscle parallels the maturation of the retinal circuitry (Abramov et al., 1982) and the establishment of interocular alignment (Braddick and Atkinson, 1983). The most significant change in the postnatal maturation of extraocular muscle, at the structural level, is the increase in mitochondrial content. Thus, there is a strong correlation between the structural basis for fatigue resistance in the eye muscles and the increasing reliance upon precise eye movements. Experimental monocular deprivation paradigms produce reductions in both contraction velocity and fatigue resistance of extraocular muscle, as well as corresponding alterations in muscle morphology (Lennerstrand, 1980). AI tered visual processing presumably would change the patterned activity of oculomotor motoneurons and thereby directly alter the biochemical and morphological properties of some or all of the extraocular muscle fiber types. These

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data suggest that the maximwn potential for fatigue resistance in a muscle fiber may be set genetically, but \\nether the potential is achieved in a particular fiber type is contingent upon the level of demand placed upon the muscle. The extraocular muscles of Siamese cats, a species with abnormal development of retinogeniculate pathways, also are structurallv altered (Lennerstrand, 1980). The impact that monocular-deprivation or strabismus would have upon many other muscle properties, such as myosin isoform expression, has not vet been addressed. While it is likely that the basic motor mhanisms for vestibula-ocular, pursuit, and saccadic eve movements are in place at birth (albeit with properties different from the adult), the sensory functions of spatial localization and retinal slip detection are immature (Aslin and Salapatek, 1975; Finocchio et a!., 1991; Hansen, 1994; Naegele and Held, 1982; Ornitz et a!., 1985; Shea and Aslin, 1990; Shupert and Fuchs, 1988). The postnatal development of sensory input and feedback systems, and the corresponding changes in motor system parameters (gain, time constant, etc.), then parallels and exerts influence upon the fiber type characteristics of extraocular muscle.

Thus. the atypical pattern of myosin expression in extraocular muscle clearly has its origins in development. A key issue is why the developmental myosin heavy chain genes are not completely repressed in extraocular muscle, as they are in most other skeletal muscles. Such differences between extraocular and skeletal muscles may be related more to functional than to embryological factors (see Spencer and Porter, 1988). In other skeletal muscles, establishment of innervation and the physical loading of muscle are, in part responsible for the developmental myosin heavy cham transitions (Caplan et a!., 1983). Extraocular muscles may continue to express embryonic and neonatal isoforrns as a result of their small, unchanging load--the eye. Interestingly, when other skeletal muscles are immobilized in a stretched position they may exhibit re-expression of ernbl}onic myosin as well (McCormick and Schultz, 1994 ). Indeed, the complex pattern of myosin expression suggests that development of the different extraocular muscle fiber types, particularly those of the orbital layer (Jacoby et a!., 1989), is not regulated solely by developmental stagespecific factors, but rather some fiber types may be particularly susceptible to extrinsic factors that modulate their e'-'jlression. Perhaps the retention of embryonic myosin in the orbital singly innervated fiber, but not in its close counterpart in the global layer (the global red singly innenated), is related to differing muscle force dynamics for the two layers. The maintenance of extraocular muscle force levels at a minimum of 10 g (Collins, 1975), even in extreme off-direction gaze positions, is most likely due to the continuous activity of this particular fiber type. This level of sustained force may be responsible for maintenance of the expression of embl}onic myosin in the adult. The plasticity

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that is implicit in extraocular muscle myosin expression may be particularly important in light of the degree of adaptive regulation of motoneuron firing rates that is seen in theoculomotor control systems. That is, factors such as the load distribution among orbital and global layers, the continuing influence of multiple innervation of some fiber types, and motoneuron discharge rates that are an order of magnitude higher than seen for other skeletal muscles all must play important roles in shaping fiber phenotype.

Recently, in vitro techniques have been used to examine the specificity of neural influences upon developing extraocular muscle (Porter and Hauser, 1993). Motoneuroncontaining slices of either fetal rat spinal cord or midbrain were co-cultured with neonatal rat extraocular muscle. Thigh muscle co-cultured with spinal motoneurons served as a control. During the first few weeks in culture, cells originating from both types of muscle explants developed into myotubes. Myotubes became innervated and contractile and maturation rate was not affected by myotube source. During this time, muscle explant development was not dependeot upon motoneuron source. However, after the third week in vitro, muscle survival became dependent upon the type of motoneurons with which extraocular muscle was cocultured. Eye muscle degenerated when co-cultured with spinal cord motoneurons, but thrived when co-cultured \\ith midbrain motoneurons, many of which are the appropriate oculomotor motoneurons. These results provide evidence that the trophic requirements of eye muscle are different from those of other skeletal muscles, and further suggest that maldevelopment of this specific nerve/muscle interaction may play a role in congenital strabismus and amblyopia. More recent data show that select, identified growth factors can substitute for oculomotor motoneurons in maintaining eye muscle survival in vitro. Along these same lines, Porter and Baker ( 1993) have shown that a monkey species that is prone to development of strabismus (Macaca nemestrima) exhibits developmental features in extraocular muscle that can be interpreted as the result of altered motoneuron discharge rates in this species. Thus, all members of this species may be prone to strabismus, as evidenced by the transient pathology in their extraocular muscles, but only 5% actually develop esotropia (Boothe et al., 1985, 1990; Kiorpes and &xJthe, 1980; Kiorpes et a!., 1985; Quick eta!., 1992).

In addition to the neural influences, skeletal muscle precursor cell proliferation, differentiation, and survival are regulated by soluble grov.th factors, hormones, and cell adhesion molecules (Fiorini et a!., 1991; Joseph-Silverstein eta!., 1989; Olsen, 1992; Sohal and Holt, 1980). Epigenetic factors can act as both positive and negative muscle gro\\th regulators. Although it is likely that multiple peptide growth factors affect extraocular muscle differentiation, the role of gro\\th factors remains largely unexplored in this system.

Basic fibroblast growth factor (FGF) can be localized \\ithin chick extraocular muscle and is developmentally regulated (Joseph-Silverstein et al., 1989). In addition, thyroid hormones strongly influence skeletal muscle development and cause profound hypertrophic effects on extraocular muscles in adults. Nuclear triiodoth)Tonine (T3) receptors are abundant in orbital layer fibers compared to other skeletal muscle types (Schmidt et al., 1992), suggesting that T 3 has a unique functional role in extraocular muscle. VTI. CONCLUSIONS

The extraocular muscles are highly diverse muscles that are well suited to the performance of the \\ide range of tasks that are specified by the multiple eye movement control systems. The phenotype of any skeletal muscle arises from an interaction of myoblast genotype and environmental influences. The heterogeneity and Wlique fiber composition of extraocular muscle most likely are indicative of differences from typical skeletal muscle in both cell lineage and motoneuron discharge properties. Oculomotor motoneurons are characterized by the eye position at which

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