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Differentiation and Morphogenesis of Extraocular Muscles
Differentiation and Morphogenesis of Extraocular Muscles D M Noden, Cornell University, Ithaca, NY, USA ã 2010 Elsevier Ltd. All rights reserved.
Glossary Mesenchyme – Embryonic cells with a fibroblast-like appearance, surrounded by extracellular matrix, lacking tight junctions with their neighbors, and often capable of undergoing extensive migratory movements. These can be of several different embryonic origins, and include cells that will contribute to many lineages. Morphogenesis – It includes those processes that establish the correct locations and three-dimensional organization of tissues and organs. This includes the proper positioning of extraocular muscles around the globe and their attachments to the sclera and orbital skeleton. Myoblasts – Mitotically active cells committed to the skeletal muscle lineage but not yet expressing muscle-specific proteins such as desmin and myosins, which generally are not evident until after myoblasts fuse to form multinucleated myofibers. Myotome – The several regions of each somite that contain progenitors of skeletal muscle progenitors. Neural crest – Mesenchymal cells that are derived from neural fold tissues and that move peripherally along well-delineated pathways to form neurons and glia of the peripheral nervous system and, in the head region, the connective tissues of the midface and branchial regions. Paraxial mesoderm – Early embryonic cells that are located beside and beneath the developing brain and spinal cord, and include the precursors of most skeletal muscles.
Introduction Muscles that move and stabilize the eye have been highly conserved during vertebrate evolution. While a few remarkable adaptations have occurred, such as co-opting of the dorsal (superior) oblique muscle to generate protective heating for the brain in some fishes, these muscles have retained an anatomical organization linking axes of the body and the eye that arose hundreds of millions of years ago. Considering their ancient status, it is logical to assume that the early development of extraocular muscles would
34
similarly be well conserved among different species, and therefore amenable to comparative analyses that supplant the absence of direct examination in mammals, including humans. However, compared to trunk and limb muscles, our understanding of the origins of ocular muscles and the mechanisms that initiate then maintain their development is at best fragmentary. Myogenesis of skeletal muscles is a lengthy process, with several parameters continuing to be function-dependent throughout the life of an animal. Primary myogenesis spans the period during which populations of myoblasts – committed, mitotically active muscle progenitors – arise, emigrate to their sites of differentiation, fuse to form multinucleated innervated myofibers, and establish intimate connections with connective tissues. This population forms a scaffolding, including the delineation of global and orbital domains, within which secondary myogenesis occurs. During secondary myogenesis stages, previously sequestered latent myoblasts are activated to proliferate and differentiate, forming more than 90% of the myofibers present in mature muscles and generating region-specific specialized fiber types that in most species are present before or soon after birth.
Origins of Extraocular Muscles Striated (skeletal) muscles throughout the body arise within paraxial mesoderm, which is located in close apposition to the embryonic brain and spinal cord. Exceptions to this are the avian striated ciliary muscle that is of neural crest origin, and possibly the striated muscles of the esophageal wall; however, both of these are involuntary. Among voluntary muscles, some of the more ventrally located branchial muscles arise from lateral mesoderm that is contiguous with paraxial mesoderm. Many early accounts of head myogenesis placed the embryonic origin of some eye muscles, especially the lateral rectus, in the same category as branchial (pharyngeal) arch muscles, and ascribed both to a lateral mesoderm origin. These claims were based on the sites at which muscle condensations are first grossly evident in the embryo. However, with the advent of mapping methods and assays for early muscle-specific gene expression patterns, separate and distinct sites of origin for all eye muscles within preotic (i.e., located rostral, in front of the developing inner ear) paraxial mesoderm was confirmed (Figure 1). The sites of origin of extraocular muscles parallel the sites of emergence of the three cranial motor nerves that
Differentiation and Morphogenesis of Extraocular Muscles
Prosencephalon
Extraocular Medial rectus Ventral rectus Dorsal rectus
1st arch Mandibular adductors Intermandibular
3rd arch Pharyngeals Stylopharyngeus Hypaxial Laryngeal Glossal Rectus capitis
Mesencephalon
Dorsal oblique
n. V
n. IV
Retractor bulbi Lateral rectus
Metencephalon
Pterygoideus 2nd arch Stapedial Digastricus, facials
n. III
Ventral oblique
Paraxial mesoderm Lateral mesoderm
35
n. VI
n. VII DO n. IX
Myelencephalon n. XII
1st som.
n. X
Epaxial Biventer, splenius complexus (a)
2nd som. 3rd som. (b)
Figure 1 Sites of origin of craniofacial striated muscles in avian embryos. Panel (a) shows, in dorsal view, the locations of each muscle primordia within paraxial and lateral mesoderm. Panel (b) illustrates one mapping method used in avian embryos, wherein a small bolus of replication-incompetent retrovirus injected into early mesoderm and the embryo harvested 2 weeks later and stained for the appropriate reporter gene, in this case a bacterial galactosidase. In this embryo, in which the eye has been removed to reveal underlying tissues, only the dorsal (superior) oblique muscle was labeled.
innervate them. However, in contrast to axial and branchial muscles, sites of myogenesis are not congruent with locations of motor nerve emergence. Indeed, each of these cranial nerves needs to elongate considerably through peripheral tissues before initial contacts with target muscles are established. Some axons, such as those of the abducens nerve, must extend longitudinally beneath the brain to reach their target lateral rectus muscles, while others, such as the oculomotor nerve fibers, project perpendicular to the floor of the brain before decussating to approach their several target muscles. In extant vertebrates, head paraxial mesoderm constitutes a sparse population of mesenchymal cells (Figure 2). This contrasts with the situation in the neck and trunk regions, where paraxial mesoderm first forms somites, which are segmentally arranged, cuboidal aggregates of epithelial cells. As each somite matures, it becomes delineated into distinct myogenic (myotome) and connective
tissue-forming (sclerotome) regions. The most cranial somite is located beside the hindbrain, immediately caudal to the otic vesicle, and paraxial mesoderm rostral to this site fails to form epithelial tissues and lacks overt evidence of segmentation. Head paraxial mesoderm is present adjacent to the prospective eye-forming regions of the rostral neural plate, but is largely displaced caudally as the optic vesicles emerge and expand lateral to the diencephalon. In the midline just in front of the notochord, this population of loose paraxial mesoderm cells is contiguous with a sparse and species-variable population of prechordal mesenchymal cells. Mapping experiments in avian embryos have shown that prechordal mesoderm contributes to the genesis of extraocular muscles innervated by the oculomotor nerve (Figure 3), but it is not known whether this contribution is exclusive of or supplementary to that of paraxial mesoderm. It is not known if the same occurs in mammalian embryos.
36
Differentiation and Morphogenesis of Extraocular Muscles
Neural crest
Optic vesicle
Mesencephalon
Pharynx Lateral mesoderm
Heart tube Neural crest
(a)
Paraxial mesoderm
(b)
Figure 2 Colorized scanning electron micrographs showing the early relations of neural crest (blue) to mesodermal (red) populations in dorsal (a) and transverse (b) views. Small arrows indicate the direction of movement of the neural crest cells.
(a)
(b)
(c)
Figure 3 Contributions of prechordal mesoderm to developing extraocular muscles is shown by labeling their precursors at stage 4–5 (early gastrulation) with DiI, a fluorescent membrane-binding tag (site ‘o’ in (a)), and fixing the embryos over a day later((b), stage 12, ventral view). Labeled cells in (c) are within the eye muscle-forming region of paraxial mesoderm.
Determination of Eye Muscle Precursors Head paraxial mesoderm contains progenitors for many tissues in addition to skeletal muscle. These include cartilages and bones associated with the braincase, loose connective tissues such as meninges and adipocytes, and endothelial cells. In contrast to somites, wherein these progenitor populations are largely segregated, it appears based on mapping studies that these diverse precursors are either intermingled or contiguous in head mesoderm. The significance of this lies in the problem of generating diverse lineages. Somite cells are held in fixed positions relative to the dorsal and ventral parts of the adjacent neural tube (hindbrain and spinal cord) and overlying surface epithelium, all of which provide combinations of positive and negative regulators of early myogenesis and skeletogenesis.
A further complication – and one essential for the development of all craniofacial musculoskeletal systems – is the presence of a large, later-arising population of mesenchymal cells called the neural crest (Figure 2). Derived from neural folds either during (mammals) or shortly following (birds) closure of the cephalic neural tube (brain), these cells acquire a mesenchymal phenotype and quickly move peripherally, mostly atop underlying paraxial mesoderm. Neural crest cells from the rostral midbrain level move rostrally and caudally around the optic stalk and posterior part of the optic vesicle, then spread outwardly as the vesicle is transformed into the optic cup. After delineation of the lens from the lens placode, crest cells secondarily invade the space created anterior to the lens and establish the posterior epithelium (endothelium) and stromal populations of the cornea.
Differentiation and Morphogenesis of Extraocular Muscles
In the trunk, members of the wnt family of growth factors are secreted by surface ectoderm and provide essential positive stimuli for muscle differentiation. The same are released by head surface epithelium, but here their effects are to retard myogenesis of branchial muscles. Arriving neural crest cells separates branchial muscle progenitors from the source of these negative effects and, augmented by the release of additional myogenesispromoting factors, allows myogenesis to progress. The extent to which eye muscle progenitors follow a similar scenario is unclear. Some extraocular precursors, particularly the lateral rectus progenitors, are embedded deep within paraxial mesoderm and are therefore quite distant from both surface ectoderm and, at early stages, neural crest cells. This deep location places the lateral rectus precursors close to the neural epithelium at the level of the future metencephalon (pons). Several experiments have established that this location provides essential cues for lateral rectus formation. When newly formed trunk paraxial mesoderm cells were excised, before they had formed somites, then grafted into the head, in place of prospective lateral rectus mesoderm cells, the transplanted cells formed a muscle that expressed molecular markers unique to the lateral rectus and established proper anatomical connections with the braincase and sclera. Small changes in the location of the implants resulted in grafted cells contributing to the dorsal oblique or branchial arch musculature. Thus, the sites within head paraxial mesoderm at which each muscle primordium forms and is specified as to its identity are highly localized. Placing a barrier between the brain and paraxial mesoderm at this region does not prevent myogenesis, but the developing muscle cells lack molecular features that define their specific identity. Together, these experiments suggest that a rich tableau of local signals is necessary for early eye muscle differentiation, with both general myogenic and individual eye muscle-specific components.
Molecular Signatures and Muscle Differentiation All skeletal myoblasts use members of a closely related set of muscle-specific transcription factors to promote and coordinate their differentiation. Two of these, myf 5 and myoD, are cross-activating regulators that are among the earliest muscle-specific genes expressed. The upstream regulatory components of these genes are body regionand muscle group-specific, and serve to integrate the diverse micro-environments surrounding each muscle group with a highly shared set of outcomes, for example, activation of genes for desmin, myosins, muscle-specific actins, and junctional receptors.
37
Expression of myf 5 then myoD genes in eye muscle precursors generally is slightly later than expression in trunk axial muscles, but is simultaneous with that of branchial muscles (Figure 4). Expression of these regulatory genes coincides with the onset of aggregation of most muscle precursors (Figure 5), although it is not known whether these aggregates represent the totality of muscle precursors or only a subset of primary myoblasts. By these criteria, extraocular muscles appear similar to other head and also to trunk and appendicular muscles. However, as additional features of trunk and head myogenic regulatory networks have been identified, the number of differences has exceeded the similarities, and a heretofore underlying complexity has been revealed (Table 1). This area of investigation is rapidly expanding, and rather than detail each gene currently described, a few examples of categories of differences among muscle groups will be presented. Pax3 is a regulatory gene expressed in axial and appendicular muscle precursors, and null mutations of this transcription factor (e.g., Splotch mutation) result in severe depletion of trunk muscles. However, it is not expressed in head muscle precursors, and null mutations have no discernable effect on branchial or extraocular muscles. Another pronounced difference is in the hepatocyte growth factor (HGF) – cMet growth factor-receptor complex, which is functionally required for the correct dissemination and differentiation of appendicular and tongue muscle precursors. Again, this pathway has no known role in eye and branchial myogenesis, even though HGF is expressed in and around the precursors of branchial arch, lateral rectus, and both oblique muscles. The latter example further illustrates the considerable heterogeneity among extraocular muscles. The lateral rectus is particularly enigmatic, being the only head muscle that expresses the ubiquitous trunk paraxial mesoderm marker paraxis, and together with the dorsal oblique, the transcription factor lbx1, which is present in trunk hypaxial (thoracic and abdominal wall) muscle precursors. A further complexity arises from the often-changing patterns of gene expression during the early stages of head paraxial mesoderm development. The transcription factor pitx2, which is a key mid-level participant in the integrated formation of left-right asymmetry for the heart and mid-gut, is initially expressed symmetrically and ubiquitously throughout head paraxial mesoderm (Figure 6). However, a day later, during early myogenesis stages, its expression becomes more restricted but includes the first branchial arch, lateral rectus, and both oblique muscles in addition to periocular neural crest cells. Another regulatory gene, Tbx1, which is located in the region of chromosome 22 wherein deletions cause the DiGeorge syndrome, is similarly expressed over a wide domain of mesoderm (and pharyngeal endoderm) before becoming restricted to branchial arch and the lateral rectus muscles.
38
Differentiation and Morphogenesis of Extraocular Muscles
2 day
3 day
4 day
5 day
Move
Muscle group Dorsal (superior) rectus
Move
Inferior (ventral) oblique Inferior (ventral) rectus
Move
Medial rectus Move
Superior (dorsal) oblique
Move
Lateral rectus
Move
Branchial arch
Migr.
Tongue, laryngeal Epaxial (neck) Migr.
Wing Onset of myoD transcription
Aggregation of myoblasts move migr.
Key
Onset of myf5 transcription
Myoblast movements or migrations
Myosin proteins present
Figure 4 Timetable of gene activation in extraocular and other head and trunk muscles. The most consistent difference between trunk and head muscles is that the latter show a prolonged delay between the onset of myoD expression and the synthesis of muscle-specific proteins.
2.5 day
LR BA2 BA1 BA3
DO
VO LR DR
HGC
MyoD
6 day
4 day
BA2 BA3
LR DO
BA3 BA2
DO
EP
BA1 VO
VR MR
VR HYP
HGC
Myf5
DR
BA1
VO
Myosin
Figure 5 Early differentiation and morphogenesis of head muscles in chick embryos. BA1, 2, 3, branchial arches; DO, dorsal oblique; DR, dorsal rectus; Ep, epaxial muscle precursors; Hyp, hypaxial precursors, HGC, hypoglossal cord that forms tongue muscles; LR, MR, VR, lateral, medial and ventral rectus muscles; VO, ventral oblique.
At present the significance of these spatially and temporally dynamic expression patterns is unknown. It is possible that early expressions presage the later focal appearance of certain muscles, but it is equally plausible that each of these genes has multiple and distinct functions associated with each stage. As extraocular muscles mature, they exhibit a progression of fiber types, evidenced by changes in the myosin
isoforms and related contractile and energy metabolism proteins expressed. Emergence of these complex patterns requires a series of interactions among developing myofibers, surrounding connective tissues, and innervation. In the avian embryo, the primary myofibers of most extraocular muscles express embryonic slow myosin isoforms. However, one muscle, the quadratus nictitans, which is homologous to retractor bulbi muscles and is innervated
Differentiation and Morphogenesis of Extraocular Muscles Table 1
39
Summary of myogenic regulatory networks of head and trunk
Expression sites
Genes
Trunk
Limb and tongue
Branchial
EOMs
All muscle Trunk only
myf5, myoD noggin, ptc1 pax3, c-met barx2 lbx1 paraxis pitx2 tbx1 en2 pod1 hgf myoR
þ þ þ þ þ þ þ
þ þ þ þ þ þ (þ)
þ þ þ BA1 þ BA1 þ
þ þ LR, DO LR þ LR þ DO, VO DO, VO, L1
Trunk and Head
Head only
LR BA1 BA2
DO
Allantois Left (a)
Right (b)
Figure 6 At stage 8 ((a), dorsal view) Pitx2 is expressed symmetrically throughout head paraxial (and lateral) mesoderm populations, but only on the left within trunk mesoderm. By stage 21 ((b), 4 days) it is restricted to a subset of eye muscles, the first and second branchial arch muscle masses, and periocular mesenchyme.
by the accessory abducens nerve, the myofibers are either completely fast myosin expressing, as in the quail embryo, or mixed, as in the chick embryo (Figure 7). To explore the basis for these distinct, species-specific patterns, periocular neural crest cells of the chick were replaced with comparable populations from a quail embryo. The quadratus muscle in these chimeric embryos exhibits the quail donor phenotype (Figure 7), indicating that initial differentiation of fiber types requires interactions among myofibers and encompassing connective tissues.
Muscle Morphogenesis Except for muscles that remain closely associated with the vertebral column and skull, all muscle progenitors leave their sites of origin in paraxial mesoderm and disperse into peripheral tissues. Body wall muscles do so together with sclerotome-derived connective tissue precursors, and maintaining these close spatial relations is essential
for the morphogenesis of thoracic and abdominal muscles. For appendicular myogenesis, lateral myotome-derived cells move in sequential waves of primary and secondary myoblasts into nearby limb buds, where they form longitudinal bands of future dorsal and ventral groups before segregating into individual muscles. In the hindlimbs, some of these undergo secondary dispersal to form muscles of the perineal region. Branchial muscles are comparable to body wall muscles in that they initially exhibit a constant juxtaposition with the precursors of their connective tissues, which in the head are all derived from neural crest cells, and also with the motor nerves that innervate them. This maintained registration permits continuous exchange of signals among all three components during all stages of muscle differentiation and morphogenesis. This constant contiguity has most dramatically been demonstrated for the trapezius muscle, whose precursors arise among caudal branchial arch mesodermal populations and secondarily shift caudally to attach to the scapula. Mapping studies in both bird and mouse embryos revealed that the neural crest-derived connective tissues move with, and perhaps somewhat in advance of, these myoblasts and indeed contribute to the scapula. This recapitulates an ancestral condition in which the forelimb girdle articulated with the back of the skull, as is still present in fish. Again, however, the extraocular muscles exhibit a developmental scenario unlike any other muscles. As illustrated in Figures 5 and 8, these muscle precursors move towards, then around, the equatorial region of the developing eye to assume their definitive locations. During this process, each muscle leaves the company of surrounding mesoderm cells and becomes fully encompassed by neural crest cells, which secondarily penetrate the muscle mass and form internal (e.g., endomysium) as well as surrounding (perimysium, fascia, and tendon) connective tissues. These periocular crest cells need not have originated at the same axial level as the muscles. For example, the lateral rectus muscle, the neural crest cells that will form
40
Differentiation and Morphogenesis of Extraocular Muscles
D.R
D.R
Qd. N. Qd. N.
(a)
(c)
D.R
Qd. N. Qd. N.
(b)
(d)
Figure 7 Fiber-type determination in the quadratus nictitans (Qd. N.) muscle. (a, b) Sections through this muscle in chick and quail embryos processed with antibodies to slow myosin isoforms. The quail Qd. N. lacks slow fibers, whereas in the chick both fast and slow fibers are present. (c) A control embryo in which chick neural crest cells were transplanted into a chick host, and the Qd. N. developed normally. However, when quail crest cells were grafted into a chick host embryo (d), the exclusive fast donor phenotype resulted.
Mesencephalon
Isthmus Me cep tenhalo n LR n VR Myelencephalo MR
DR
DO
VO 1.0 mm
Telencephalon
Figure 8 The movements of the dorsal (yellow arrow) and ventral (green arrow) oblique muscles from their sites of origin to their terminal locations along the equatorial zone of the globe.
its connective tissues, and the abducens nerve that innervates it originate at three separate axial levels. Indeed, these primordia do not become intimately associated until each has independently approached the periocular environment. This negates the possibility of prolonged
interactions among contiguous progenitor populations, as occurs for branchial musculoskeletal systems. The mechanisms by which aggregates of extraocular muscle primordia change both absolute and relative positions remain enigmatic. There is no precedence elsewhere in the embryo for condensations of cells moving actively through surrounding tissues. However, several lines of evidence support a model based on passive displacement of eye muscle primordia. As was shown in Figure 2, the interface between neural crest and myogenic paraxial mesoderm is initially a flat plane. Changes in the relative positions of the eye due to flexures and differential growth of the brain and expansion of the optic cup introduce distortions in this plane, but the extent to which this might affect individual eye muscles has been difficult to define. In screening for a wide range of gene expression patterns, several were found that coincided with the patterns of movements taken by some extraocular muscles (Figure 9). These reveal a set of localized distortions of the neural crest-mesoderm interface. Finger-like projections of paraxial mesoderm expand dorsally and caudally around the optic cup, becoming interdigitated with periocular neural crest populations and passively bringing the dorsal and ventral oblique muscle primordia to their definitive
Differentiation and Morphogenesis of Extraocular Muscles
41
connective-tissue forming neural crest cells, which largely direct both the gross and microscopic differentiation of these muscles. BA1
See also: Congenital Cranial Dysinnervation Disorders; Extraocular Muscles: Extraocular Muscle Anatomy; Extraocular Muscles: Extraocular Muscle Involvement in Disease.
Further Reading Figure 9 Lateral view of a 3.5-day chick embryo showing the sites of expression of the MyoR gene. Note the finger-like projections (arrows) extending dorsal and caudal to the optic vesicle, along the sites at which the dorsal and ventral oblique are expanding.
locations. Subsequently, crest cells close behind each of these muscle primordia, creating the appearance of an island of myoblasts/myofibers amid a sea of crest cells. How these focal distortions are established is unknown. The cell surface adhesion molecule semaphorin 3A is expressed by mesodermal cells in these projections, but its role in not known.
Summary The early stages of extraocular muscle formation are well described but poorly understood mechanistically. They arise at discrete sites within unsegmented head paraxial mesoderm then launch into developmental programs that share some features with trunk and branchial muscles but are largely and surprisingly unique. Passive distortions of the mesoderm-neural crest interface bring these muscle primordia to their definitive locations around the ocular globe, where they become surrounded then infused by
Borue, X. and Noden, D. M. (2004). Normal and aberrant craniofacial myogenesis by grafted trunk somitic and segmental plate mesoderm. Development 131: 3967–3980. Bryson-Richardson, R. J. and Currie, P. D. (2008). The genetics of vertebrate myogenesis. Nature Reviews Genetics 9: 632–646. Evans, D. J. R. and Noden, D. M. (2006). Spatial relations between avian craniofacial neural crest and paraxial mesoderm cells. Developmental Dynamics 235: 1310–1325. Grenier, J., Teillet, M. A., Grifone, R., Kelly, R. G., and Duprez, D. (2009). Relationship between neural crest cells and cranial mesoderm during head muscle development. PLoS ONE 4: e4381. Marcucio, R. M. and Noden, D. M. (1999). Myotube heterogeneity in developing chick craniofacial skeletal muscles. Developmental Dynamics 214: 178–194. Noden, D. M. and Francis-West, P. (2006). The differentiation and morphogenesis of craniofacial muscles. Developmental Dynamics 235: 1194–1218. Noden, D. M., Marcucio, R. M., Borycki, A-G., and Emerson, C. P., Jr. (1999). Differentiation of avian craniofacial muscles. I. Patterns of early regulatory gene expression and myosin heavy chain synthesis. Developmental Dynamics 216: 96–112. Noden, D. M. and Schneider, R. A. (2006). Neural crest cells and the community of plan for craniofacial development: Historical debates and current perspectives. In: Saint-Jeannet, J. P. (ed.) Neural Crest Induction and Differentiation, pp. 1–23. Boston, MA: Landes Bioscience and Springer Science Media. Tzahor, E. (2009). Heart and craniofacial muscle development: A new developmental theme of distinct myogenic fields. Developmental Biology 327: 273–276. Yoshida, T., Vivatbutsiri, P., Morriss-Kay, G., Saga, Y., and Iseki, S. (2008). Cell lineage in mammalian craniofacial mesenchyme. Mechanisms of Development 125: 797–808.
Glossary Mesenchyme – Embryonic cells with a fibroblast-like appearance, surrounded by extracellular matrix, lacking tight junctions with their neighbors, and often capable of undergoing extensive migratory movements. These can be of several different embryonic origins, and include cells that will contribute to many lineages. Morphogenesis – It includes those processes that establish the correct locations and three-dimensional organization of tissues and organs. This includes the proper positioning of extraocular muscles around the globe and their attachments to the sclera and orbital skeleton. Myoblasts – Mitotically active cells committed to the skeletal muscle lineage but not yet expressing muscle-specific proteins such as desmin and myosins, which generally are not evident until after myoblasts fuse to form multinucleated myofibers. Myotome – The several regions of each somite that contain progenitors of skeletal muscle progenitors. Neural crest – Mesenchymal cells that are derived from neural fold tissues and that move peripherally along well-delineated pathways to form neurons and glia of the peripheral nervous system and, in the head region, the connective tissues of the midface and branchial regions. Paraxial mesoderm – Early embryonic cells that are located beside and beneath the developing brain and spinal cord, and include the precursors of most skeletal muscles.
Introduction Muscles that move and stabilize the eye have been highly conserved during vertebrate evolution. While a few remarkable adaptations have occurred, such as co-opting of the dorsal (superior) oblique muscle to generate protective heating for the brain in some fishes, these muscles have retained an anatomical organization linking axes of the body and the eye that arose hundreds of millions of years ago. Considering their ancient status, it is logical to assume that the early development of extraocular muscles would
34
similarly be well conserved among different species, and therefore amenable to comparative analyses that supplant the absence of direct examination in mammals, including humans. However, compared to trunk and limb muscles, our understanding of the origins of ocular muscles and the mechanisms that initiate then maintain their development is at best fragmentary. Myogenesis of skeletal muscles is a lengthy process, with several parameters continuing to be function-dependent throughout the life of an animal. Primary myogenesis spans the period during which populations of myoblasts – committed, mitotically active muscle progenitors – arise, emigrate to their sites of differentiation, fuse to form multinucleated innervated myofibers, and establish intimate connections with connective tissues. This population forms a scaffolding, including the delineation of global and orbital domains, within which secondary myogenesis occurs. During secondary myogenesis stages, previously sequestered latent myoblasts are activated to proliferate and differentiate, forming more than 90% of the myofibers present in mature muscles and generating region-specific specialized fiber types that in most species are present before or soon after birth.
Origins of Extraocular Muscles Striated (skeletal) muscles throughout the body arise within paraxial mesoderm, which is located in close apposition to the embryonic brain and spinal cord. Exceptions to this are the avian striated ciliary muscle that is of neural crest origin, and possibly the striated muscles of the esophageal wall; however, both of these are involuntary. Among voluntary muscles, some of the more ventrally located branchial muscles arise from lateral mesoderm that is contiguous with paraxial mesoderm. Many early accounts of head myogenesis placed the embryonic origin of some eye muscles, especially the lateral rectus, in the same category as branchial (pharyngeal) arch muscles, and ascribed both to a lateral mesoderm origin. These claims were based on the sites at which muscle condensations are first grossly evident in the embryo. However, with the advent of mapping methods and assays for early muscle-specific gene expression patterns, separate and distinct sites of origin for all eye muscles within preotic (i.e., located rostral, in front of the developing inner ear) paraxial mesoderm was confirmed (Figure 1). The sites of origin of extraocular muscles parallel the sites of emergence of the three cranial motor nerves that
Differentiation and Morphogenesis of Extraocular Muscles
Prosencephalon
Extraocular Medial rectus Ventral rectus Dorsal rectus
1st arch Mandibular adductors Intermandibular
3rd arch Pharyngeals Stylopharyngeus Hypaxial Laryngeal Glossal Rectus capitis
Mesencephalon
Dorsal oblique
n. V
n. IV
Retractor bulbi Lateral rectus
Metencephalon
Pterygoideus 2nd arch Stapedial Digastricus, facials
n. III
Ventral oblique
Paraxial mesoderm Lateral mesoderm
35
n. VI
n. VII DO n. IX
Myelencephalon n. XII
1st som.
n. X
Epaxial Biventer, splenius complexus (a)
2nd som. 3rd som. (b)
Figure 1 Sites of origin of craniofacial striated muscles in avian embryos. Panel (a) shows, in dorsal view, the locations of each muscle primordia within paraxial and lateral mesoderm. Panel (b) illustrates one mapping method used in avian embryos, wherein a small bolus of replication-incompetent retrovirus injected into early mesoderm and the embryo harvested 2 weeks later and stained for the appropriate reporter gene, in this case a bacterial galactosidase. In this embryo, in which the eye has been removed to reveal underlying tissues, only the dorsal (superior) oblique muscle was labeled.
innervate them. However, in contrast to axial and branchial muscles, sites of myogenesis are not congruent with locations of motor nerve emergence. Indeed, each of these cranial nerves needs to elongate considerably through peripheral tissues before initial contacts with target muscles are established. Some axons, such as those of the abducens nerve, must extend longitudinally beneath the brain to reach their target lateral rectus muscles, while others, such as the oculomotor nerve fibers, project perpendicular to the floor of the brain before decussating to approach their several target muscles. In extant vertebrates, head paraxial mesoderm constitutes a sparse population of mesenchymal cells (Figure 2). This contrasts with the situation in the neck and trunk regions, where paraxial mesoderm first forms somites, which are segmentally arranged, cuboidal aggregates of epithelial cells. As each somite matures, it becomes delineated into distinct myogenic (myotome) and connective
tissue-forming (sclerotome) regions. The most cranial somite is located beside the hindbrain, immediately caudal to the otic vesicle, and paraxial mesoderm rostral to this site fails to form epithelial tissues and lacks overt evidence of segmentation. Head paraxial mesoderm is present adjacent to the prospective eye-forming regions of the rostral neural plate, but is largely displaced caudally as the optic vesicles emerge and expand lateral to the diencephalon. In the midline just in front of the notochord, this population of loose paraxial mesoderm cells is contiguous with a sparse and species-variable population of prechordal mesenchymal cells. Mapping experiments in avian embryos have shown that prechordal mesoderm contributes to the genesis of extraocular muscles innervated by the oculomotor nerve (Figure 3), but it is not known whether this contribution is exclusive of or supplementary to that of paraxial mesoderm. It is not known if the same occurs in mammalian embryos.
36
Differentiation and Morphogenesis of Extraocular Muscles
Neural crest
Optic vesicle
Mesencephalon
Pharynx Lateral mesoderm
Heart tube Neural crest
(a)
Paraxial mesoderm
(b)
Figure 2 Colorized scanning electron micrographs showing the early relations of neural crest (blue) to mesodermal (red) populations in dorsal (a) and transverse (b) views. Small arrows indicate the direction of movement of the neural crest cells.
(a)
(b)
(c)
Figure 3 Contributions of prechordal mesoderm to developing extraocular muscles is shown by labeling their precursors at stage 4–5 (early gastrulation) with DiI, a fluorescent membrane-binding tag (site ‘o’ in (a)), and fixing the embryos over a day later((b), stage 12, ventral view). Labeled cells in (c) are within the eye muscle-forming region of paraxial mesoderm.
Determination of Eye Muscle Precursors Head paraxial mesoderm contains progenitors for many tissues in addition to skeletal muscle. These include cartilages and bones associated with the braincase, loose connective tissues such as meninges and adipocytes, and endothelial cells. In contrast to somites, wherein these progenitor populations are largely segregated, it appears based on mapping studies that these diverse precursors are either intermingled or contiguous in head mesoderm. The significance of this lies in the problem of generating diverse lineages. Somite cells are held in fixed positions relative to the dorsal and ventral parts of the adjacent neural tube (hindbrain and spinal cord) and overlying surface epithelium, all of which provide combinations of positive and negative regulators of early myogenesis and skeletogenesis.
A further complication – and one essential for the development of all craniofacial musculoskeletal systems – is the presence of a large, later-arising population of mesenchymal cells called the neural crest (Figure 2). Derived from neural folds either during (mammals) or shortly following (birds) closure of the cephalic neural tube (brain), these cells acquire a mesenchymal phenotype and quickly move peripherally, mostly atop underlying paraxial mesoderm. Neural crest cells from the rostral midbrain level move rostrally and caudally around the optic stalk and posterior part of the optic vesicle, then spread outwardly as the vesicle is transformed into the optic cup. After delineation of the lens from the lens placode, crest cells secondarily invade the space created anterior to the lens and establish the posterior epithelium (endothelium) and stromal populations of the cornea.
Differentiation and Morphogenesis of Extraocular Muscles
In the trunk, members of the wnt family of growth factors are secreted by surface ectoderm and provide essential positive stimuli for muscle differentiation. The same are released by head surface epithelium, but here their effects are to retard myogenesis of branchial muscles. Arriving neural crest cells separates branchial muscle progenitors from the source of these negative effects and, augmented by the release of additional myogenesispromoting factors, allows myogenesis to progress. The extent to which eye muscle progenitors follow a similar scenario is unclear. Some extraocular precursors, particularly the lateral rectus progenitors, are embedded deep within paraxial mesoderm and are therefore quite distant from both surface ectoderm and, at early stages, neural crest cells. This deep location places the lateral rectus precursors close to the neural epithelium at the level of the future metencephalon (pons). Several experiments have established that this location provides essential cues for lateral rectus formation. When newly formed trunk paraxial mesoderm cells were excised, before they had formed somites, then grafted into the head, in place of prospective lateral rectus mesoderm cells, the transplanted cells formed a muscle that expressed molecular markers unique to the lateral rectus and established proper anatomical connections with the braincase and sclera. Small changes in the location of the implants resulted in grafted cells contributing to the dorsal oblique or branchial arch musculature. Thus, the sites within head paraxial mesoderm at which each muscle primordium forms and is specified as to its identity are highly localized. Placing a barrier between the brain and paraxial mesoderm at this region does not prevent myogenesis, but the developing muscle cells lack molecular features that define their specific identity. Together, these experiments suggest that a rich tableau of local signals is necessary for early eye muscle differentiation, with both general myogenic and individual eye muscle-specific components.
Molecular Signatures and Muscle Differentiation All skeletal myoblasts use members of a closely related set of muscle-specific transcription factors to promote and coordinate their differentiation. Two of these, myf 5 and myoD, are cross-activating regulators that are among the earliest muscle-specific genes expressed. The upstream regulatory components of these genes are body regionand muscle group-specific, and serve to integrate the diverse micro-environments surrounding each muscle group with a highly shared set of outcomes, for example, activation of genes for desmin, myosins, muscle-specific actins, and junctional receptors.
37
Expression of myf 5 then myoD genes in eye muscle precursors generally is slightly later than expression in trunk axial muscles, but is simultaneous with that of branchial muscles (Figure 4). Expression of these regulatory genes coincides with the onset of aggregation of most muscle precursors (Figure 5), although it is not known whether these aggregates represent the totality of muscle precursors or only a subset of primary myoblasts. By these criteria, extraocular muscles appear similar to other head and also to trunk and appendicular muscles. However, as additional features of trunk and head myogenic regulatory networks have been identified, the number of differences has exceeded the similarities, and a heretofore underlying complexity has been revealed (Table 1). This area of investigation is rapidly expanding, and rather than detail each gene currently described, a few examples of categories of differences among muscle groups will be presented. Pax3 is a regulatory gene expressed in axial and appendicular muscle precursors, and null mutations of this transcription factor (e.g., Splotch mutation) result in severe depletion of trunk muscles. However, it is not expressed in head muscle precursors, and null mutations have no discernable effect on branchial or extraocular muscles. Another pronounced difference is in the hepatocyte growth factor (HGF) – cMet growth factor-receptor complex, which is functionally required for the correct dissemination and differentiation of appendicular and tongue muscle precursors. Again, this pathway has no known role in eye and branchial myogenesis, even though HGF is expressed in and around the precursors of branchial arch, lateral rectus, and both oblique muscles. The latter example further illustrates the considerable heterogeneity among extraocular muscles. The lateral rectus is particularly enigmatic, being the only head muscle that expresses the ubiquitous trunk paraxial mesoderm marker paraxis, and together with the dorsal oblique, the transcription factor lbx1, which is present in trunk hypaxial (thoracic and abdominal wall) muscle precursors. A further complexity arises from the often-changing patterns of gene expression during the early stages of head paraxial mesoderm development. The transcription factor pitx2, which is a key mid-level participant in the integrated formation of left-right asymmetry for the heart and mid-gut, is initially expressed symmetrically and ubiquitously throughout head paraxial mesoderm (Figure 6). However, a day later, during early myogenesis stages, its expression becomes more restricted but includes the first branchial arch, lateral rectus, and both oblique muscles in addition to periocular neural crest cells. Another regulatory gene, Tbx1, which is located in the region of chromosome 22 wherein deletions cause the DiGeorge syndrome, is similarly expressed over a wide domain of mesoderm (and pharyngeal endoderm) before becoming restricted to branchial arch and the lateral rectus muscles.
38
Differentiation and Morphogenesis of Extraocular Muscles
2 day
3 day
4 day
5 day
Move
Muscle group Dorsal (superior) rectus
Move
Inferior (ventral) oblique Inferior (ventral) rectus
Move
Medial rectus Move
Superior (dorsal) oblique
Move
Lateral rectus
Move
Branchial arch
Migr.
Tongue, laryngeal Epaxial (neck) Migr.
Wing Onset of myoD transcription
Aggregation of myoblasts move migr.
Key
Onset of myf5 transcription
Myoblast movements or migrations
Myosin proteins present
Figure 4 Timetable of gene activation in extraocular and other head and trunk muscles. The most consistent difference between trunk and head muscles is that the latter show a prolonged delay between the onset of myoD expression and the synthesis of muscle-specific proteins.
2.5 day
LR BA2 BA1 BA3
DO
VO LR DR
HGC
MyoD
6 day
4 day
BA2 BA3
LR DO
BA3 BA2
DO
EP
BA1 VO
VR MR
VR HYP
HGC
Myf5
DR
BA1
VO
Myosin
Figure 5 Early differentiation and morphogenesis of head muscles in chick embryos. BA1, 2, 3, branchial arches; DO, dorsal oblique; DR, dorsal rectus; Ep, epaxial muscle precursors; Hyp, hypaxial precursors, HGC, hypoglossal cord that forms tongue muscles; LR, MR, VR, lateral, medial and ventral rectus muscles; VO, ventral oblique.
At present the significance of these spatially and temporally dynamic expression patterns is unknown. It is possible that early expressions presage the later focal appearance of certain muscles, but it is equally plausible that each of these genes has multiple and distinct functions associated with each stage. As extraocular muscles mature, they exhibit a progression of fiber types, evidenced by changes in the myosin
isoforms and related contractile and energy metabolism proteins expressed. Emergence of these complex patterns requires a series of interactions among developing myofibers, surrounding connective tissues, and innervation. In the avian embryo, the primary myofibers of most extraocular muscles express embryonic slow myosin isoforms. However, one muscle, the quadratus nictitans, which is homologous to retractor bulbi muscles and is innervated
Differentiation and Morphogenesis of Extraocular Muscles Table 1
39
Summary of myogenic regulatory networks of head and trunk
Expression sites
Genes
Trunk
Limb and tongue
Branchial
EOMs
All muscle Trunk only
myf5, myoD noggin, ptc1 pax3, c-met barx2 lbx1 paraxis pitx2 tbx1 en2 pod1 hgf myoR
þ þ þ þ þ þ þ
þ þ þ þ þ þ (þ)
þ þ þ BA1 þ BA1 þ
þ þ LR, DO LR þ LR þ DO, VO DO, VO, L1
Trunk and Head
Head only
LR BA1 BA2
DO
Allantois Left (a)
Right (b)
Figure 6 At stage 8 ((a), dorsal view) Pitx2 is expressed symmetrically throughout head paraxial (and lateral) mesoderm populations, but only on the left within trunk mesoderm. By stage 21 ((b), 4 days) it is restricted to a subset of eye muscles, the first and second branchial arch muscle masses, and periocular mesenchyme.
by the accessory abducens nerve, the myofibers are either completely fast myosin expressing, as in the quail embryo, or mixed, as in the chick embryo (Figure 7). To explore the basis for these distinct, species-specific patterns, periocular neural crest cells of the chick were replaced with comparable populations from a quail embryo. The quadratus muscle in these chimeric embryos exhibits the quail donor phenotype (Figure 7), indicating that initial differentiation of fiber types requires interactions among myofibers and encompassing connective tissues.
Muscle Morphogenesis Except for muscles that remain closely associated with the vertebral column and skull, all muscle progenitors leave their sites of origin in paraxial mesoderm and disperse into peripheral tissues. Body wall muscles do so together with sclerotome-derived connective tissue precursors, and maintaining these close spatial relations is essential
for the morphogenesis of thoracic and abdominal muscles. For appendicular myogenesis, lateral myotome-derived cells move in sequential waves of primary and secondary myoblasts into nearby limb buds, where they form longitudinal bands of future dorsal and ventral groups before segregating into individual muscles. In the hindlimbs, some of these undergo secondary dispersal to form muscles of the perineal region. Branchial muscles are comparable to body wall muscles in that they initially exhibit a constant juxtaposition with the precursors of their connective tissues, which in the head are all derived from neural crest cells, and also with the motor nerves that innervate them. This maintained registration permits continuous exchange of signals among all three components during all stages of muscle differentiation and morphogenesis. This constant contiguity has most dramatically been demonstrated for the trapezius muscle, whose precursors arise among caudal branchial arch mesodermal populations and secondarily shift caudally to attach to the scapula. Mapping studies in both bird and mouse embryos revealed that the neural crest-derived connective tissues move with, and perhaps somewhat in advance of, these myoblasts and indeed contribute to the scapula. This recapitulates an ancestral condition in which the forelimb girdle articulated with the back of the skull, as is still present in fish. Again, however, the extraocular muscles exhibit a developmental scenario unlike any other muscles. As illustrated in Figures 5 and 8, these muscle precursors move towards, then around, the equatorial region of the developing eye to assume their definitive locations. During this process, each muscle leaves the company of surrounding mesoderm cells and becomes fully encompassed by neural crest cells, which secondarily penetrate the muscle mass and form internal (e.g., endomysium) as well as surrounding (perimysium, fascia, and tendon) connective tissues. These periocular crest cells need not have originated at the same axial level as the muscles. For example, the lateral rectus muscle, the neural crest cells that will form
40
Differentiation and Morphogenesis of Extraocular Muscles
D.R
D.R
Qd. N. Qd. N.
(a)
(c)
D.R
Qd. N. Qd. N.
(b)
(d)
Figure 7 Fiber-type determination in the quadratus nictitans (Qd. N.) muscle. (a, b) Sections through this muscle in chick and quail embryos processed with antibodies to slow myosin isoforms. The quail Qd. N. lacks slow fibers, whereas in the chick both fast and slow fibers are present. (c) A control embryo in which chick neural crest cells were transplanted into a chick host, and the Qd. N. developed normally. However, when quail crest cells were grafted into a chick host embryo (d), the exclusive fast donor phenotype resulted.
Mesencephalon
Isthmus Me cep tenhalo n LR n VR Myelencephalo MR
DR
DO
VO 1.0 mm
Telencephalon
Figure 8 The movements of the dorsal (yellow arrow) and ventral (green arrow) oblique muscles from their sites of origin to their terminal locations along the equatorial zone of the globe.
its connective tissues, and the abducens nerve that innervates it originate at three separate axial levels. Indeed, these primordia do not become intimately associated until each has independently approached the periocular environment. This negates the possibility of prolonged
interactions among contiguous progenitor populations, as occurs for branchial musculoskeletal systems. The mechanisms by which aggregates of extraocular muscle primordia change both absolute and relative positions remain enigmatic. There is no precedence elsewhere in the embryo for condensations of cells moving actively through surrounding tissues. However, several lines of evidence support a model based on passive displacement of eye muscle primordia. As was shown in Figure 2, the interface between neural crest and myogenic paraxial mesoderm is initially a flat plane. Changes in the relative positions of the eye due to flexures and differential growth of the brain and expansion of the optic cup introduce distortions in this plane, but the extent to which this might affect individual eye muscles has been difficult to define. In screening for a wide range of gene expression patterns, several were found that coincided with the patterns of movements taken by some extraocular muscles (Figure 9). These reveal a set of localized distortions of the neural crest-mesoderm interface. Finger-like projections of paraxial mesoderm expand dorsally and caudally around the optic cup, becoming interdigitated with periocular neural crest populations and passively bringing the dorsal and ventral oblique muscle primordia to their definitive
Differentiation and Morphogenesis of Extraocular Muscles
41
connective-tissue forming neural crest cells, which largely direct both the gross and microscopic differentiation of these muscles. BA1
See also: Congenital Cranial Dysinnervation Disorders; Extraocular Muscles: Extraocular Muscle Anatomy; Extraocular Muscles: Extraocular Muscle Involvement in Disease.
Further Reading Figure 9 Lateral view of a 3.5-day chick embryo showing the sites of expression of the MyoR gene. Note the finger-like projections (arrows) extending dorsal and caudal to the optic vesicle, along the sites at which the dorsal and ventral oblique are expanding.
locations. Subsequently, crest cells close behind each of these muscle primordia, creating the appearance of an island of myoblasts/myofibers amid a sea of crest cells. How these focal distortions are established is unknown. The cell surface adhesion molecule semaphorin 3A is expressed by mesodermal cells in these projections, but its role in not known.
Summary The early stages of extraocular muscle formation are well described but poorly understood mechanistically. They arise at discrete sites within unsegmented head paraxial mesoderm then launch into developmental programs that share some features with trunk and branchial muscles but are largely and surprisingly unique. Passive distortions of the mesoderm-neural crest interface bring these muscle primordia to their definitive locations around the ocular globe, where they become surrounded then infused by
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