Skeletal elements in the vertebrate eye and adnexa: Morphological and developmental perspectives

Abstract

Although poorly appreciated, the vertebrate eye and adnexa are relatively common sites for skeletogenesis. In many taxa, the skeleton contributes to internal reinforcement in addition to the external housing of the eye (e.g., the circumorbital bones and eyelids). Eyeball elements such as scleral cartilage and scleral ossicles are present within a broad diversity of vertebrates, albeit not therian mammals, and have been used as important models for the study of condensations and epithelial–mesenchymal interactions. In contrast, other elements invested within the eye or its close surroundings remain largely unexplored. The onset and mode of development of these skeletal elements are often variable (early versus late; involving chondrogenesis, osteogenesis, or both), and most (if not all) of these elements appear to share a common neural crest origin. This review discusses the development and distribution of the skeletal elements within and associated with the developing eye and comments on homology of the elements where these are questionable. Developmental Dynamics 235:1244–1255, 2006. © 2006 Wiley-Liss, Inc.

INTRODUCTION: AN OVERVIEW OF THE VERTEBRATE EYE

The vertebrate eye is a complex sensory organ. Whereas the eye can range in morphology from an oblate spheroid (in some teleosts) to bell-shaped and tubular (in deep-sea teleosts, owls, and nocturnal primates), in most taxa the eye is roughly spherical in shape. The relative simplicity of this superficial anatomy obscures the intricacy of the eye's development, physiology, and microanatomy. Furthermore, the eye is but one part of an integrated multicomponent visual system that includes mechanical support and protection, musculature, vasculature, and nerve supplies.

One of the first regions of the neuroectoderm to be defined is the eye field of the anterior neural plate (Lupo et al.,2002), beginning in mid-gastrulation. Morphologically, the earliest indication of the eye is a shallow sulcus that forms on the medial surface of the neural fold, before formation of the neural tube (around stage 10 in humans; Moore and Persaud,1993). After neural fold fusion and the formation of the forebrain, the optic sulcus continues to expand (evaginate) from the diencephalon leading to the formation of the optic vesicle, which is connected to the brain by means of the optic stalk (human, stage 12–13; chicken, Hamburger and Hamilton [HH] stage 9, 29–33 hr; zebrafish, 10 hr postfertilization; Hamburger and Hamilton,1951; Moore and Persaud,1993; Gilbert,2003; Glass and Dahm,2004). During subsequent development, the optic vesicle invaginates and becomes a two-layered structure with an inner neural retina and outer retinal pigment epithelium. As soon as the developing optic vesicle makes contact with the overlying ectoderm, it induces the ectoderm to thicken and form the lens placode (HH stage 14 chicken). The lens placode then also invaginates and pinches off from the ectoderm, to form the lens proper. The mesenchyme surrounding the developing optic cup differentiates into the sclera, the dense fibrous coat of the eye. A double-layered optic cup with pigmented retina and lens is present by approximately 35 days in humans (Robinson and Lovicu,2004), by 3–3.5 days (HH stage 20) in chicken embryos (Hamburger and Hamilton,1951), and by 72 hr postfertilization in zebrafish larvae (Glass and Dahm,2004).

Although their precise roles are undetermined, various transcription factors, e.g., Pax-6, Prox-1, Rx, Lhx-2, Otx2, Six3, are known to be involved in retina and/or lens development; others remain to be identified (Tomarev,1997; Jean et al.,1998; Bailey et al.,2004; Goudreau et al.,2004; and others). The formation of the eye is often presented as a classic example of a cascade of reciprocating and hierarchical inductive processes, with the inhibition of any one step arresting eye development and resulting in severe craniofacial abnormalities (see reviews by Coulombre,1965; Saha et al.,1992; Jean et al.,1998; Cvekl and Tamm,2004). Broadly stated, eye anatomy is roughly similar across vertebrate taxa, with variation largely restricted to shape and size of the components (e.g., tubular eyes of owls/falcons/eagles cf. spherical eyes of majority of other birds), internal cellular composition (e.g., numbers of cones and rods within the neural retina; size of ciliary muscle, etc.), and hence, functional capacity. Eye development in teleosts differs from that in tetrapods in several important aspects. For example, among zebrafish (Danio rerio), the rate of eye development is far more rapid than in model tetrapod taxa (Glass and Dahm,2004; Robinson and Lovicu,2004). Furthermore, the lens of zebrafish (and other teleosts) forms from delamination of cells from the lens placode; thus, there is no formation of a hollow lens vesicle, in contrast to the situation in mammals and birds (Glass and Dahm,2004).

Although less familiar, vertebrate eyes also differ with respect to the presence and distribution of investing and adnexal skeletal elements. Curiously, placental and marsupial mammals (unlike most lineages) do not develop intraocular elements (Table 1). Consequently, at least from the viewpoint of self-interest, skeletal elements of the eye are largely overlooked; thus, the mechanisms and process that govern them are virtually unknown. However, given their otherwise widespread distribution, they are important in the context of eye and craniofacial evolution and may play a significant role as accessible models for manipulation and experimentation.

Table 1. Distribution of Skeletal Elements Associated with Vertebrate Eyesa

Taxon	Scleral cartilage	Scleral ossicles (bone)	Eyelid element (cartilage or bone)	Nictitating membrane lamina(e)	Other	Comments
a See text for details.
Teleost	Scleral ring	0 or 2	No	No	—	—
Chondrichthyan	Scleral cup	No	No	?	Optic pedicel	Scleral cartilage may calcify
Amphibian	Scleral cup (most)	No	No	?	—	No scleral cartilage in gymnophiones
Turtle	Scleral cup	Yes	No	Yes	—	—
Lizard	Scleral cup	Yes	Most with tarsal plate (cartilage) in lower eyelid, some with palpebral in upper eyelid	Yes	Osteoderms, parafrontal bones	—
Snake	No	No	No	No	—	—
Crocodylian	Scleral cup	No	Palpebral in upper eyelid	Yes	—	—
Bird	Scleral cup	Yes	Possible tarsal plate (cartilage and/or intermediate tissue)	Yes	Os opticus, scleral sesamoid, supraorbital(s)	—
Mammal	No (most)	No	Possible tarsal plate (cartilage and/or intermediate tissue)	Yes (hyaline and elastic cartilage)	—	Scleral cartilage present in monotremes

This review examines skeletal elements associated within and adjacent to the vertebrate eye. First, we discuss anatomical relationships between the various cartilaginous and osseous components, taxonomic distribution, modes of skeletogenesis, and homology-related issues. Second, we discuss details of the development of these skeletal elements (where these are available). As the majority of research on the development of the eye skeleton has until recently focused on the domestic chicken (Gallus gallus), this taxon forms a prominent part of our discussion. Investigations on eye development in mammals (e.g., mouse and rat) have been conducted (Fujiwara et al.,1994; Grindley et al.,1995; and others), but will not be discussed because they do not pertain specifically to the development of the skeletal system within and surrounding the eye. Similarly, studies on amphibians (e.g., Xenopus) appear to focus on either the signalling pathways and inductive mechanisms that occur during eye development or lens regeneration, with little attention given to the skeletal elements associated with the developing eye. For this reason, there is a bias toward literature on Gallus. Finally, we include some discussion of recent experimental manipulations in teleost larvae that suggest that the lens can influence growth and ossification of surrounding skeletal elements, as well as a study on cranial vault (calvaria) development in mammals, which has some parallels to scleral ossicle formation. We conclude that there is some evidence that mechanisms of both eye and bone development are different in teleosts and tetrapods and that further comparisons, especially those exploring developmental mechanisms and molecular signals, are needed.

THE EYE SKELETON

Among most vertebrates, the eye generally occupies a rostral or lateral position in the skull, although in at least one species of bird (American woodcock, Scolopax minor), the eyes are positioned caudolaterally such that it may be able to see behind its head. All vertebrate eyes are nested within a bony or cartilaginous cup-like depression, the orbital cavity. In most instances, the eye is capable of some degree of mobility within the orbit, buffered in part by adjacent tissue including orbital fat, fascia/ligaments, and musculature (extraocular and nonstriated). Additional mechanical protection and support for the eye can come from the surrounding circumorbital elements of the skull and the eyelids (discussed below). In taxa with a narrow cranial profile (e.g., birds), a skeletal interorbital septum separates the bilateral orbits.

The eyeball is composed externally of a fibrous connective tissue capsule. At the anterior pole of the eye, overlying the pupil and iris, this connective tissue tunic is known as the cornea, a smooth, transparent and distinctly convex covering (Fig. 1). Continuous with the cornea, the remainder of the eye is covered by a denser, more resilient fibrous coat, the sclera, often characterized by its distinct white coloration. Conjunctival epithelium overlies the scleral tissue. In addition to structural support, protection of the sensory apparatus (the retina and neurons) and maintaining intraocular pressure, the sclera also provides a site of insertion for the extraocular musculature. Recalling that the eye develops as an outgrowth of the forebrain (diencephalon) and that the optic nerve is not truly a nerve but rather a nerve tract, it is perhaps not surprising that the sclera of the eye is continuous with the dura mater (Tripathi and Tripathi,1984). The significance of this statement is discussed later in the context of skeletal elements within the sclera and cranial vault.

Figure 1. A diagrammatic section through a lizard eye, illustrating the position of all the major anatomical components discussed herein. Note the position of the scleral ossicles (black) above the equator of the eye at the corneal–scleral limbus, partially overlapping the anterior edge of the scleral cartilage cup (grey).

Among vertebrates, the skeleton is typically summarized as the sum total of elements composed of bone and cartilage, along with the minor contribution of various orally restricted dental tissues (viz., cementum, dentine, and enamel). As a tissue, however, cartilage has many discrete structural forms including hyaline (“glassy”), calcified, elastic, and fibrocartilage (Beresford,1993). Using routine light microscopy, Benjamin (1990) was able to identify nine different types of cartilage in the heads of various teleosts. For bone, diversity is typically expressed in terms of histological architecture: woven versus lamellar, compact versus cancellous. In addition, bone may be acellular (anosteocytic) or chondroid (a bony matrix invested with cartilage cells). Developmentally, bone is classified as either direct or indirect. Direct or intramembranous development occurs without a cartilage precursor and is characteristic of the dermal skeleton (e.g., the dermatocranium). Indirect development may occur from within (endochondral) or around (perichondral) a cartilage precursor, contributing to the aptly named replacement skeleton. Clearly, the idea of a cartilage–bone skeleton is an over simplification, and that a greater appreciation of skeletal development and biology relies on considering these tissues as points on a broader skeletal continuum.

Skeletal Elements Within the Sclera

As an optical system, the eye has a firm requirement for stable housing. Support begins with the sclera (Fig. 1). For many taxa, including humans and other placental and marsupial mammals, snakes, gymnophione amphibians (caecilians), lamprey, and hagfish, the dense fibrous connective tissue of the sclera lacks true skeletal tissues (Table 1) (Walls,1942; Caprette et al.,2004). For the rest, including most “fish” (chondrichthyans, teleosts, and non-tetrapodan sarcopterygians), reptiles (including birds, turtles, lizards, and crocodylians), and monotreme mammals, the connective tissue of the sclera is further reinforced by the development of cartilage and/or bone. Skeletal reinforcement of the eye is plesiomorphic for gnathostomes (Ritchie,1968; Donoghue et al.,2000; Burrow et al.,2005).

The most common skeletal reinforcement of the eye is scleral cartilage, typically in the form of a cup-like element investing most of the scleral capsule (Figs. 1, 2A). Although usually described as hyaline, in chondrichthyans the scleral cartilage may become calcified (Walls,1942). Among amphibians, there are several different patterns of scleral cartilage distribution, including premetamorphic development and loss, premetamorphic development and persistence, or postmetamorphic development (Stadtmüller,1924; Walls,1942; Cloete,1961; Eyal-Giladi and Zinberg,1964). Within Actinopterygii, the distribution of scleral cartilage also differs; in teleosts (ray-finned fish), the scleral cartilage is restricted to a narrow ring encircling the equator of the eyeball; in Chondrostei (sturgeons, bichirs) and Amiiformes (bowfins), the sclera is composed of a cup of cartilage with a large posterior foramen (Walls,1942). Alternatively, some teleosts (gymnotid eels, pearlfish) reportedly lack scleral cartilage entirely (Walls,1942). Accordingly, it is believed that the scleral cartilage of all vertebrates is homologous and that in teleosts there was reduction of the cup to form a ring (Walls,1942).

Figure 2. A selection of skeletal elements found within or directly associated with the vertebrate eye. A: Dorsal view of a whole-mount stained eye of the turtle Chelydra serpentina, embryonic stage 26 (Yntema,1968), showing the scleral ossicles (arrow, Alizarin red), and the scleral cartilage (sc, Alcian blue). B: Lateral view of the tubular sclerotic ring of the owl Bubo bubo. Anterior (the cornea) is to the top. The arrowhead indicates the position of the scleral sesamoid bone, nested alongside the scleral ossicles. Used with permission from S. Karger AG, Basel, and Dr. G.A. Bohórquez Machecha. C: Skull of Gallus gallus in lateral view, demonstrating the position of the sclerotic ring (arrow) within the orbit. The scleral ring is composed of 13–14 scleral ossicles. D: Photomicrograph of Masson's trichrome-stained histological section through two adjacent embryonic Gallus (HH stage 46) scleral ossicles (sc) situated above the retina (r) and within the scleral mesenchyme (m). The left ossicle overlaps the right one in this figure. E: Radiograph of the teleost Diplectrum sciurus in the lateral view, demonstrating two large scleral ossicles anteriorly (arrow) and posteriorly in the eye. Produced with permission from the California Academy of Sciences, Ichthyology Department. F: Whole-mount Alizarin stained teleost Esox lucius in the lateral view, demonstrating small scleral ossicles anteriorly (arrow) and posteriorly in the eye. G: The os opticus from (left to right) the woodpecker Melanerpes erythrocephalus, hawk Buteo borealis calurus, and Gallus sp., each in the medial view (not to scale). Modified from Tiemeier (1939). H: Skull of a subadult Caiman crocodilus in dorsolateral view. Arrow indicates the palpebral, nested along the prefrontal bone of the circumorbital series. I: Photomicrograph of Mallory's trichrome-stained histological section through the integument above the orbit and frontal bone (f) of the lizard Tarentola mauritanica. Note the presence of osteoderms (o) within the dermis. J: Black and white photograph of a whole-mount Alizarin stained lizard Teratoscincus scinus (California Academy of Science 101437) in dorsal view, demonstrating parafrontal bones (op) dorsal to the orbit. K: Photomicrograph of hematoxylin and eosin–stained section through the dermis (d) above the orbit, the parafrontal bones (op), and prefrontal bone (pf) of the lizard Aristelliger praesignis. Scleral cartilage (sc) is also visible in this section adjacent to the retina (r). Scale bars = 0.9 mm in A, 0.5 cm in B, 100 μm in D,I,K, 5 mm in E, 0.68 mm in F, 2 mm in J.

Among many teleosts and reptiles (including birds, but excluding snakes and crocodylians), the sclera may also develop small osseous plates or scleral ossicles (ossicularis sclerae; Figs. 1, 2A–F). In reptiles, scleral cartilage underpins most of the globe, whereas scleral ossicles are limited to the anterior-most margin of the sclera and are thought to be involved in accommodation. In adults, each scleral ossicle either over- or under-laps adjacent ossicles to form an imbricated ring. This ring is situated at the corneal–scleral limbus, where the scleral and corneal tissues fuse. The absence of scleral ossicles in some taxa (e.g., mammals, amphibians, snakes, crocodylians) may represent secondary losses as basal Sarcopterygii had sclerotic rings composed of many (over 20) parts (Walls,1942). The number of scleral ossicles contributing to the sclerotic ring is variable and a commonly cited source of taxonomic information. In reptiles, the number of elements ranges from a minimum of 6 (in some turtles) to a maximum of 18 (in some birds).

In contrast, among modern teleosts, the number of scleral ossicles is highly constrained, with either two, situated anteriorly and posteriorly in the eye, or none (Nakamura and Yamaguchi,1991). Among those teleosts with scleral ossicles, these elements may range in size from small nubbins to large arcs, each forming half a complete ring around the eye (Fig. 2E,F). A preliminary analysis indicates that the scleral ossicles of teleosts and reptiles may not be homologous based on differences in morphology, positional relationships to the scleral cartilage elements, their development and modes of ossification (Franz-Odendaal, unpublished observations).

Scleral ossicles are particularly robust among members of the avian lineages Strigiformes (owls) and some Falconiformes (falcons and eagles; Bohórquez Mahecha and Aparecida de Oliveira,1998). In these taxa, the eyes are tubular (rather than spherical), with a mushroomed posterior pole (Bohórquez Mahecha and Aparecida de Oliveira,1998). To yield this unusual morphology, each ossicle is concavely deflected toward the posterior pole rather than remaining a flat plate (as in most other birds, e.g., Gallus; compare Fig. 2B with 2C). Diving birds typically have soft lenses to aid visual acuity under water and, hence, also have robust scleral ossicles to prevent eyeball distortion during accommodation (Walls,1942). A similar function has been proposed for these elements in teleosts (Walls,1942).

Exclusive to many birds, the eye may also develop an additional scleral element, the curious os opticus (os nervi optici, Gemminger's ossicle). Located at the posterior pole of the scleral cartilage, the os opticus surrounds the foramen through which the optic nerve fibers pass. At present, it has been documented in 219 species from 35 families, including perching birds, woodpeckers, hummingbirds, toucans, kingfishers, and falcons (Tiemeier,1950). Characteristically, the internal architecture of this bone reveals a cancellous marrow cavity with fatty tissue. Morphologically, this element varies in shape from a complete encircling element in some woodpeckers (e.g., Melanerpes erythrocephalus) to an abbreviated arc in Gallus (Tiemeier,1939,1950; Fig. 2G). Most commonly, however, the os opticus resembles an intermediate U-shape (e.g., Buteo borealis calurus; see Tiemeier,1950, for an exhaustive list of species that have this bone and their description). With few exceptions, the os opticus is a single element. A comparable bone has not been found in other recent classes of vertebrates. Although arguably contributing to rigidity at the entry point of the optic nerve, the function of this unusual element is unknown.

Skeletal Elements of the Ocular Adnexa

Numerous skeletal elements are positioned external to and in close proximity of the eye (Table 1). Although not part of the eyeball in a strict sense, these adnexal elements are included with our eye skeleton discussion as a matter of completion and functional interrelatedness.

Circumnavigating the orbit of teleosts (at least primitively) is a series of dermal bones known as the circumorbitals (ossa circumorbitalia). Among the majority of actinopterygians, the circumorbitals are usually characterized by the passage of the mechanosensory organs (lateral line system). Circumorbital bones are clustered into one of two major groupings: the suborbitals (infraorbitals), which lie below the eye, and transmit the infraorbital canal, and the supraorbitals situated above the eye and conveying the supraorbital canal (Schultze,1993). The supraorbital group of bones generally consists of one to three elements along the dorsal margin of the orbit, although deeply nested teleosts often have none and/or the supraorbital sensory canal may be absent (e.g., zebrafish; Cubbage and Mabee,1996). Although highly reduced among modern amphibians, homologues of the circumorbital bones, the lacrimal, prefrontal, postorbital, and jugal bones are commonly present in amniotes (Fig. 2H).

Peripheral to the housing of the orbit, the eye receives immediate protection and support from the eyelids (palpebrae). Eyelids are movable structures invested with specialized glands and a supportive connective tissue plate, the tarsal plate or tarsus (tarsus palpebralis). In most taxa (birds, mammals, lizards), the tarsal plate is described as a dense, fibrous connective tissue, possibly including cartilage, present within one or both of the upper and lower eyelids (Gauthier et al.,1988; Rieppel,2000). In humans, the tarsal plate of the upper eyelid is composed of collagens types I, III, and V, as well as glycosaminoglycans (chondroitin sulphate 4 and 6), aggrecan, and cartilage oligomeric matrix proteins but lacks collagen type II as well as chondrocytes (Milz et al.,2005). Thus, for humans, the upper tarsal plate represents neither a truly fibrous nor a truly cartilaginous element but instead one that is composed of a unique transitional tissue (Milz et al.,2005). In many birds, lizards, and Sphenodon (the tuatara), the upper eyelid has limited mobility and a putative tarsal plate is instead found within the lower eyelid (Underwood,1970; Gauthier et al.,1988). Among various reptiles, including modern crocodylians, one lineage of lizards (anguimorphans), and some herbivorous dinosaurs, the upper eyelid is invested with an ossified element, the palpebral (Fig. 2H). The most extreme development of the palpebral is found in the armored dinosaur Euoplocephalus tutus, where the bone forms a quarter-sphere and adducts to completely occlude the orbit (Coombs,1972; Vickaryous and Russell,2003). Notwithstanding the superficially similar position, palpebrals and tarsal plates differ both anatomically and developmentally and are not considered homologous (Gauthier et al.,1988).

Another unusual location for skeletal development is within the third eyelid or nictitating membrane (membrana nictitans). Although vestigial in humans (retained as the plica semilunaris; Tripathi and Tripathi,1984; Arends and Schramm,2004), the nictitating membrane is common throughout mammals (e.g., aardvark, platypus, many primates) as well as chondrichthyans, nonburrowing lizards, crocodilians, and birds (Walls,1942; Underwood,1970; Arao and Perkins,1968). Movement of the nictitating membrane across the cornea is under muscular control and acts to mechanically protect, cleanse, and moisten the surface. Interestingly, in many taxa, this eyelid is stiffened by one or more thin cartilaginous nictitating membrane laminae (cartilago intercipiens; Stibbe,1928). Research on domesticated mammals suggest that these laminae may consist of purely hyaline cartilage (cats and horses) or may be heavily invested with elastic fibers (dogs, pigs, and cows; Schlegel et al.,2001). Although not expected (Arends and Schramm,2004), cartilage development within the plica semilunaris of humans has been observed (Stibbe,1928).

More unusual still are the sesamoid-like elements found associated with the eyes of owls and at least one other nocturnal bird (the common potoo, Nictibius griseus; Bohórquez Mahecha and Aparecida de Oliveira,1998). Lying adjacent to the tendon of one of the muscles responsible for adducting the nictitating membrane (the pyramidal nictitating muscle) is the scleral sesamoid bone (os sesamoideum esclerae; Fig. 2B, arrowhead). During contraction, the scleral sesamoid bone acts to anchor the pyramidal nictitating muscle to the sclera and divert the tendon from projecting over the cornea. It remains to be determined whether this is indeed a true sesamoid (sensu; Vickaryous and Olson,2006), as the scleral sesamoid is actually invested within the synovial tendon sheath and not the tendon per se.

In addition to skeletal reinforcement of the eyelids, various lizards and birds have one or more bony elements dorsal to the orbital cavity that appear to be in addition to the circumorbitals proper. In many scleroglossan lizards, a mosaic of small, polygonal or irregular-shaped bones overlies the area dorsal to the orbit. Among the majority of taxa, these bones are osteoderms, elements that develop and remain entrenched within the integument, superficial to the skull (Fig. 2I). Alternatively, it has been demonstrated that for at least two genera of geckos, these bones over the eye develop deep to the dermis, within the same plane as the frontal bone (Bauer and Russell,1989). To distinguish them from the more common osteoderms, these latter elements have been termed parafrontal bones (ossa parafrontalia; Fig. 2J,K). In birds such as Falconiformes, there is a single additional element, the supraorbital (os supraorbitale, os supraciliare) that articulates with the prefrontal to project over the orbit. Reportedly, among tinamou (a South American partridge-like bird often characterized as the closest living relative to ratites), the area above the orbit is invested with numerous irregular bones (Jollie,1957).

One final element, nested deep within the orbit of most chondrichthyans, is the optic pedicel, or eye-stalk. Resembling a cartilaginous prop with a flat, expanded distal end, the optic pedicel is positioned caudally adjacent to the optic foramen and appears to help support the globe within the orbit. However, in various taxa it is either missing (chimaeroids, the ratfish and relatives) or does not directly contact the eye (Walls,1942; Maisey,2005). Reportedly, it may act as a passive-elastic mechanism, returning the eye to its original position within the orbit after retraction by the extraocular muscles (Walls,1942). Whereas among extant taxa it is restricted to chondrichthyans, there is evidence to suggest the presence of the optic pedicel among various other types of fossil “fish,” including early osteichthyans (bony fish) and the extinct group Placodermi (Maisey,2005).

DEVELOPMENT OF THE OCULAR SKELETON

Neural Crest Origins

In the domestic chicken, streams of neural crest (NC) cells from the posterior diencephalon, the mesencephalon, and the first two rhombomeres of the metencephalon converge on the optic vesicle where they overlap one another extensively (Creuzet et al.,2005). This territory of NC cells corresponds to the cephalic domain of the neural fold that gives rise to the entire facial skeleton (Couly et al.,2002) and, hence, also the skeletal elements of the ocular region. These streams proceed to invade different parts of the developing optic cup: those from the diencephalon invade the anterior and lateral aspects; those from the mesencephalon invade the dorsal and medial aspects; those from the metencephalon invade the lateroventral aspects. Removal of the mesencephalic NC of chicken embryos at the five- to six-somite stage (HH stage 8.5) results in embryos without a sclera or a scleral cartilage cup and, therefore, deformed eyes (Couly et al.,2002). When this experiment is performed earlier at the three- to four-somite stage (HH stage 7.5–8), the result is cyclopia (only one eye develops in the middle of the face) (Etchevers et al.,1999), indicating the existence of a transient crest-dependent molecular requirement (Creuzet et al.,2005). For normal scleral development, a minimum of a third of the cranial NC domain is required (Couly et al.,2002). Although it is not clear which stream(s) gives rise to the other ocular skeletal elements, most if not all appear to be NC derivatives. For example, among the adnexal elements, circumorbital bones are part of the NC derived dermatocranium, and eyelids and nictitating membranes develop from NC mesenchyme (ectomesenchyme; Creuzet et al.,2005). Thus, the NC provides the skeletal support for ocular structures. The ocular skeleton (at least in Gallus embryos) is also sensitive to ectopic Hox gene expression, with both Hoxa2 and Hoxb4 capable of preventing sclera formation, and Hoxa3 preventing NC cell differentiation into skeletogenic and pericytic phenotypes around the eye (Creuzet et al.,2002).

In zebrafish, migrating cranial NC are identifiable 12 hr postfertilization (hpf). The most anteriorly positioned NC cells migrate first, followed by more caudally located streams. The destination of these cells corresponds to their position before location (i.e., segmental origin aligns with hindbrain segments; Schilling and Kimmel,1994) and determination of their cell fate may be controlled by members of the Wnt family (Dorsky et al.,1998). Although a fate map of NC cell migration into the pharyngeal arch of zebrafish has been completed (Schilling and Kimmel,1994; Raible and Kruse,2000; Yelick and Schilling,2002; for review), to our knowledge, nothing is known about NC cell migration specifically into the eye region of teleosts.

Skeletogenesis

Among those skeletal elements located within the eye that undergo ossification, only the scleral ossicles of tetrapods develop intramembranously by means of an epithelial–mesenchymal interaction. The development of the scleral ossicles is independent from that of the scleral cartilage both in time and in the epithelium responsible for its induction (discussed below). Among other intra-ocular elements, including scleral ossicles of teleosts, and the os opticus and scleral sesamoid bone of birds, ossification occurs by means of cartilage replacement. Regrettably, in most instances specific details regarding the exact mode of ossification are lacking or remain contentious. Among ocular adnexa, elements such as circumorbitals, osteoderms, and parafrontals all form without a cartilage precursor. Development occurs intramembranously, although details of the epithelial–mesenchymal inductions are not known. However, osteoderms in lizards such as the gecko Tarentola, are reported to develop as a result of the direct transformation of the skin (specifically the dermis) into bone, an unusual mode of osteogenesis that involves metaplasia (Levrat-Calviac and Zylberberg,1986; Zylberberg et al.,1992).

The onset of skeletogenesis in both ocular and adnexal elements is highly variable between and within taxa. As for the rest of the dermatocranium, circumorbital elements develop early during skeletogenesis and are some of the first bones to appear in the body. In all taxa developing both scleral cartilage and scleral ossicles, the cartilage element develops before the bones. The os opticus appears to develop relatively late during ontogeny, although exact timings for most avian taxa are lacking. In the English sparrow (Passer domesticus), it begins to develop on the 22nd day after hatching, and in the domestic chicken, it is only known from skeletally mature adults (Tiemeier,1950). At present, there is no information regarding the onset of development of elements such as tarsal plates, nictitating membrane laminae, palpebrals, or parafrontals. Similar to the os opticus, osteoderms develop late during ontogeny (posthatching).

The molecular regulation controlling chondrogenesis is not identical for all cartilages, although they all arise from undifferentiated mesenchyme (Eames et al.,2003). The transcription factor Sox9 switches on chondrogenesis from undifferentiated mesenchyme, although once switched on, the downstream transcription factors differ depending on whether permanent (e.g., Meckel's, nasal, and auricular cartilages) or replacement cartilages are being developed (Fig. 3). The former requires continued Sox9 as well as Sox5 and possibly Sox6 expression for differentiation, whereas the latter requires Runx2 and possibly Osx (Eames et al.,2003). Eames and Helms (2004) have also shown that Runx2 expression is present earlier, in the condensations of replacement cartilages, but is not expressed in the condensations of persistent cartilages until later in development. Within the ocular region, the only persistent cartilage is the scleral cartilage (and, in the case of chondrichthyans, the optic pedicel); the os opticus and scleral sesamoid bones are examples of replacement cartilages. The os opticus appears to develop from the edge of the scleral cartilage foramen, surrounding the optic nerve. Given this difference in the type of cartilage and the close proximity of the scleral cartilage and os opticus, their development merits further investigation and discussion.

Figure 3. A summary of the current knowledge of the development of the vertebrate ocular skeletal elements discussed herein. Factors involved in the development of permanent cartilage, replacement cartilage, and bone are from Eames et al. (2003). Osteogenesis of osteoderms does not involve any of these typical skeletogenic processes and osteoderms, therefore, are omitted. None of the factors involved in the induction of any of the other skeletal elements are known. Some of the tissues are known as indicated. See text for details.

It is unknown whether the initial development of the os opticus and scleral cartilage are similar on molecular and morphological levels, considering their close proximity. Given that the os opticus develops late in ontogeny compared with the early formation of the scleral cartilage (posthatching vs. embryonic HH stages 31–34, 7–8 days, for Gallus; Coulombre,1965), it would be interesting to investigate whether (1) the os opticus develops from its own chondrogenic condensation; and (2) when Runx2 is expressed in the presumptive os opticus region, that is, are there two distinct condensations with differing molecular markers developing within the sclera of some birds?

Scleral Cartilage Development in Gallus

In Gallus embryos, scleral cartilage is determined by day 4 of embryonic development (HH stage 23), although it does not differentiate until 3 to 4 days later (stage 31–34; Coulombre,1965). Tissue culture experiments demonstrate that explanting mesenchyme younger than 4 days never gives rise to cartilage (Weiss and Amprino,1940). Studies further indicate that the interaction of the retinal pigmented epithelium (RPE) with the periocular mesenchyme leads to its ability to chondrify (Reinbold,1968; Newsome,1972; Stewart and McCallion,1975); thus, the RPE may ultimately be responsible for its induction. After scleral cartilage is induced, morphogenesis of this tissue becomes independent of the RPE and instead results from interactions among scleral chondroblasts (Hall,2005). This finding was demonstrated by performing chorioallantoic mesenchyme grafts or organ cultures of prechondrogenic scleral mesenchyme; in both cases sheets (not nodules) of cartilage were formed (Newsome,1972; Smith and Thorogood,1983). Even dissociating chondrocytes within scleral cartilage, resuspending the cells, and culturing them in vitro, results in the formation of sheets of cartilage (Weiss and Amprino,1940). Thus, the RPE induces scleral cartilage, but once induction has occurred, continued development is independent of this epithelium. It is currently unclear whether the RPE of teleosts induces scleral cartilage development. However, given that the scleral cartilage element is restricted to the equatorial region, that the lens influences scleral cartilage ossification (discussed below; Yamamoto et al.,2003) and that some aspects of eye development (i.e., the lens) differs between tetrapods and teleosts (Glass and Dahm,2004; Cvekl and Tamm,2004), it is possible that a different inductive mechanism exists in teleosts. What is needed is to determine whether the onset of scleral cartilage formation coincides with RPE differentiation and when the RPE obtains its ability to induce cartilage. RPE fate is determined before melanin is actually visible (based on pigment gene expression in Gallus at around HH stage 19.5, 70–71 hr; Franz,1997) 1 day before scleral cartilage fate determination.

The morphology of the scleral cup is ultimately molded by the mechanisms of the growing eye and can be modified by tension applied in vitro (Coulombre,1965). The presence of the scleral cartilage restricts eye shape such that, on its appearance, the (developing Gallus) eye no longer expands as a sphere; the radius of the corneal curvature becomes less than the radius of the sclera (Coulombre and Coulombre,1958). The shape of the eye, thus, is directly influenced by the development and growth of the scleral cartilage. A more detailed analysis of teleost eye shape and development of scleral cartilage is required to understand if a similar process occurs in these vertebrates.

Scleral Ossicle Development in Gallus

As noted above, among reptiles, scleral ossicles are neural crest (ectomesenchymal) in origin and ossify intramembranously. They are induced by transient scleral papillae that arise as thickenings of the conjunctival epithelium starting at around 6.5–7 days in Gallus embryos (HH stage 30; Coulombre et al.,1962). Each thickening becomes a single papilla, with the number and position of papillae corresponding exactly to the number and future positions of the ossicles. Papillae do not arise simultaneously, although all are present by the eighth day of incubation in chicken (HH stage 34). Scleral papillae and scleral ossicles arise sequentially and in the same strict order. The first sign of ossification is present at day 11–12 (HH stage 37–38), preceded by a prominent preossicular condensation (at day 10, HH stage 36). By day 14 (HH stage 40), 6 days before hatching, the modal number of 13–14 (in Gallus) is present and overlapping.

Coulombre and his colleagues showed that papillae directly influence development of the ossicles between 7 and 9 days of incubation (Coulombre et al.,1962). The removal of a single papilla prevents the ossicle beneath it from developing. However, the sclerotic ring is not disrupted as neighboring ossicles enlarge to close the sclerotic ring. It is unknown how far one can strain this system, i.e., how many papillae can be removed to maintain a complete sclerotic ring? Pinto and Hall (1991; see below) demonstrated that an inductive signal, emanating from the papillae, is involved. The factors controlling ossicle (i.e., condensation) size are not known. Some studies have indicated cell proliferation as being primarily responsible for the size of the preossicular condensation (Hale,1956; Fyfe and Hall,1983; van de Kamp and Hilfer,1985), but cell migration into the condensation may also play a role (Franz-Odendaal, unpublished data). The size of the ossicles may also involve a growth factor gradient between (presumptive) ossicles similar to that seen in the development of cranial bones (discussed below).

Various details of this epithelial–mesenchymal induction are known. By recombining scleral mesenchyme with mandibular epithelium, Hall (1981) demonstrated that scleral mesenchyme can respond to mandibular epithelium (and vice versa) by initiating intramembranous osteogenesis in the form of bony ossicles typical of scleral ossicles. Transfilter tissue recombinations using Nucleopore filters of various porosities (0.03–0.8 μm) and thicknesses (5 and 10 μm) showed that direct cell–cell contact between scleral epithelium and mesenchyme is not required for this induction to occur (Pinto and Hall,1991). Furthermore, using dialysis membranes, which excluded molecules of three size ranges (2,000–3,500; 6,000–8,000; or 12,000–14,000 Da), Pinto and Hall (1991) demonstrated that a diffusible signal between 3,500 and 6,000 Da was involved. When combined with studies using Millipore filters of 150 or 300 μm thickness, these authors also showed that the epithelial component acted over distances of 150 to 300 μm. The identity of the molecular signal(s) involved is a topic of current investigation.

The basis for variation in ossicle number has also been investigated experimentally. Coulombre and Coulombre (1975) showed by intubating the eye of developing Gallus embryos and draining away intraocular fluid that the rate of eye growth was decreased. This work also demonstrated that the number of ossicles, their distribution within the sclera, and their pattern of overlap/underlap were all dependent on the growth rate of the eye—the slower the growth of the eye, the fewer ossicles form. It is unknown how far one can strain this system and at what point growth is too slow to allow ossicles to develop. Nothing is known currently about how papillae arise or are patterned.

The scaleless mutant chicken is so named because of a mutation that specifically affects epithelial derivatives such that scales, feathers, spurs, and footpads are altered or significantly reduced. The scaleless mutation also inhibits some but not all scleral papillae from developing—only 3 of the 13 (or 14) papillae that usually form develop. However, only one persists beyond the ninth day of incubation; consequently, only a single condensation and a single scleral ossicle develop. The ossicle that forms is always below the one surviving papilla, although curiously this is not always the same papilla in different individuals. It is known that the expression of the extracellular matrix molecule tenascin is abnormal in scutate scales of scaleless (Shames et al.,1991) and this molecule may therefore also be involved in scleral ossicles development (Franz-Odendaal, unpublished data). This mutation, therefore, warrants further investigation to elucidate the controlling mechanisms of papillae pattern formation.

Returning to the sclera briefly, it is worth recalling that the dura mater, the outermost meningeal layer enclosing the central nervous system (brain and spinal cord), is continuous with this fibrous tunic (Tripathi and Tripathi,1984). Thus, it is perhaps not surprising that, as the sclera is involved in skeletal induction, so too is the dura. In addition, both are capable of inducing chondrogenesis (dura beneath sutures; the majority of the sclera) and/or osteogenesis (dura beneath the calvarial elements; the scleral junction with the cornea; Hall,2005). For induction to occur, both the dura and sclera require contact with an epithelium (Hall,1981; Yu et al.,1997). The formation of dermal bones (e.g., calvarial elements and scleral ossicles) results from the condensation of mesenchymal cells, which expand radially from central foci (Alberius and Friede,1992, and own observations). At present, inducing factors that give the mesenchymal cells osteogenic potential have not been identified for either dura or sclera derivatives, although the list of likely candidates includes fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), sonic hedgehog, and transforming growth factor-beta. Opperman et al. (1996) have shown (in calvarial explants) that the dura mater secretes soluble, heparin binding factors that are required to maintain suture potency. Iseki and colleagues have shown that Fgfr1 and Fgfr2 are expressed at the sutures of these membrane bones, that beads soaked with these factors and implanted in suture area result in ectopic osteogenic cells, and suggest that an FGF ligand, which modulates Fgfr1 and Fgfr2 expression, is present in a gradient across sutural area (Iseki et al.,1997,1999). For scleral ossicles, we know that the factor responsible for giving the scleral mesenchyme osteogenic potential is a diffusible factor of unknown identity (Pinto and Hall,1991). It has been proposed that the approaching bone fronts during calvaria development set up a growth factor gradient between them, which initiates suture formation (Opperman et al.,1993; Roth et al.,1996). The dura is also required to stabilize these sutures (see Opperman,2000, for review of suture development). Whether this occurs between individual scleral ossicles during development is unknown at present. Comparing the sclera and dura in this way may help in our understanding of their developmental capabilities and processes.

Development of the Scleral Skeletal Elements in Teleosts

Development of the scleral ossicles is not clearly understood. Previous work has long held that teleost scleral ossicles undergo endochondral ossification (Patterson,1977; Hall and Miyake,1992). However, because perichondral ossification is more common in teleosts than endochondral ossification (Witten and Huysseune,2006), the latter would seem to be less likely. That they develop from the scleral cartilage is now confirmed and some understanding of their growth and mode of ossification has been obtained (Franz-Odendaal, unpublished data). What remains unknown is what is restricting ossification to only certain regions of the cartilage ring (viz. the anterior and posterior positions). Although the molecular regulation of bone formation is fairly well understood in mammals and other tetrapods, in teleosts little work has been conducted (Franz-Odendaal et al.,2006). Eames and Helms (2004) have demonstrated recently that, even within one organism, there are subtle differences in the molecular signaling pathways for making cranial versus appendicular skeletal elements.

Similar to reptiles, development of the skeletal elements of the sclera is dependent on the developing eye. Although teleosts are used currently as model organisms to study eye development (e.g., Yamamoto and Jeffery,2002; Vihtelic and Hyde,2002; Glass and Dahm,2004), very few have specifically investigated eye development together with the surrounding skeletal element by means of direct larval manipulation. The most recent work is by Jeffery, Yamamoto, and colleagues in Astyanax mexicanus, the Mexican tetra (Yamamoto et al.,2003). This species exists as two morphs—a sighted surface form and a blind cavefish form in which the lens undergoes apoptosis shortly after its development. The sighted surface morph has two large scleral ossicles with minimal scleral cartilage joining their ends, whereas the blind cavefish form has the ring of scleral cartilage but no scleral ossicles. By transplanting the embryonic lens from the surface form to the blind cavefish form (and vice versa) and by extirpating the lens, these researchers noted that ossification of the scleral cartilage was affected as well as the size and positioning of the circumorbital bones (Yamamoto et al.,2003). In cavefish that had received a surface fish lens, the cartilaginous sclera ossified, suggesting that, although the cavefish eye appears to have lost the ability to promote scleral ossification, the scleral cartilage is still capable of responding to signals from the restored eye. Thus, the developing eye (specifically the lens) appears to promote ossification of the scleral cartilage but inhibit ossification of the circumorbital bones. How the fully differentiated lens does this when it is not in direct contact with these elements is not understood at present. Evidence that factors (currently unidentified) can diffuse across the eye and through eye tissues is known from studies on lens regeneration. Lens regeneration can occur either from the iris or cornea and is signalled from the neural retina and/or vitreous humor (reviewed in Henry,2003). One of the BMPs responsible for the development and ossification of bones, BMP7, is expressed in the developing lens (lens placode and lens vesicle in mice; Jena et al.,1997) and may be expressed at later stages in the teleost lens.

The primary gene responsible for lens degeneration may be hedgehog (Hh) signalling at the midline, because overexpressing Hh in surface fish embryos results in reduced Pax-6 domains and fish with degenerating eyes (Yamamoto et al.,2004). In cavefish, natural selection may be acting at the gene level (i.e., positive selection for increased Hh expression; Yamamoto et al.,2004), on the eye as a unit or on the adult phenotype (Franz-Odendaal and Hall,2006). Concurrent with eye degeneration in blind cavefish, other sensory organs (taste buds and neuromasts) are expanded, and some of the genes involved in the development of all three of these sensory organs are shared (e.g., Prox-1 and Hh). The interrelationship between these sensory fields is discussed in detail in Franz-Odendaal and Hall (2006).

FUTURE DIRECTIONS

Despite their relatively commonplace development within virtually all vertebrate lineages, skeletal elements nested within/adjacent to the vertebrate eye have, with few exceptions (avian scleral cartilage and scleral ossicles), largely been overlooked. When recognized, they are often viewed as enigmatic curiosities, rather than taxonomically predictable, biologically evolvable entities. Remarkably, even for elements with well-documented distributions among hundreds of species (e.g., os opticus, eyelid elements), our understanding of development, histology, and evolutionary significance is, at best, limited. Elements of the eye skeleton offer an easily accessible system for longitudinal studies involving embryonic manipulation and/or tissue cultures that explore cartilage and bone development/induction, as well as a new model system for investigating the role of the neural crest in craniofacial ontogeny. Without question, what is most clearly lacking is an understanding of the molecular biology of this skeletal region. In striking contrast to (nonskeletal) eye development, virtually none of the genes, proteins, or transcription factors regulating the bones and cartilages of the vertebrate eye and adnexa are presently known. Whether these factors are the same across vertebrate groups needs to be determined.

Acknowledgements

We thank NSERC (Discovery Grant to Brian K. Hall) for funding this research, and Brian K. Hall (Dalhousie University), John Fallon (Developmental Dynamics, Reviews Editor), and two anonymous reviewers for valuable comments and discussions. We also thank A.P. Russell (University of Calgary) for providing us with access to specimens in his care; H. Jamniczky (University of Calgary) for assistance with the photomicrographs; S. Karger AG, Basel, and Dr. G.A. Bohórquez Machecha (Universidade Federal de Minas Gerais, Brazil) for permission to reproduce Figure 2B; the California Academy of Sciences, Ichthyology Department for permission to reproduce Figure 2E; and finally Developmental Dynamics for putting together this Special Issue on Craniofacial Development.