In development of the eye, as in other organs, the multiplication of cells as well as directional change in shape, structure, and function of the cells govern growth. Gene determination decides the direction in which a change can occur, whereas the reciprocal demands of the individual cells or parts determine how far that direction must be followed. Fundamentally, the process consists of these two activities: change in structure and shape due to relatively different rates of growth and also change in structure and function due to differentiation and functional specialization.

Induction of one ocular tissue by another and interrelations between these developing tissues have been extensively reinvestigated in many laboratories using various experimental techniques.^5–21 One example is the lens, which arises in direct response to induction by the optic vesicle. The developing lens, in turn, promotes normal morphogenesis of neural ectodermal and mesenchymal elements in the eye. It has an inducing influence on corneal differentiation and promotes vitreous growth. Moreover, a strong organogenetic connection exists between lens and iris. The reciprocal interactions between optic cup and lens bring about the functional adjustment of the ocular axes.

Although the neural retina grows and differentiates independently of the lens, the presence of the lens may influence the normal growth and change in shape of the pigment epithelium, choroid, and sclera. The pigment epithelium, however, directs the deposition of the mesenchyme around it; subsequently, all three layers grow in unison. The pigment epithelium also depends on the vitreous body for increase in its area.

Gastrulation (formation of the mesodermal germ layer) occurs early in gestation (day 7 in mice, day 20 in humans). The primitive streak forms as a longitudinal groove within the epiblast (future ectoderm) of the bilaminar embryonic disc. Epiblast cells migrate medially toward the primitive streak where they invaginate to form the mesodermal layer (Fig. 1). This forms the classic three germ layers: ectoderm, mesoderm, and endoderm. Gastrulation progresses in a cranial to caudal direction. Concurrently, cranial surface ectoderm proliferates forming bilateral elevations called neural folds (Fig. 2). Columnar surface ectoderm in this area now becomes neural ectoderm.

Experimental studies in mice using acute exposure to teratogens have demonstrated the significance of the period of gastrulation to later ocular development. Exposure to ethanol or retinoic acid during a short period equivalent to the third week of human gestation causes primary damage to the forebrain neural ectoderm.^22–24 This results in a spectrum of malformations including microphthalmia, anterior segment dysgenesis (Peters' anomaly), iris and optic nerve colobomas, and persistent hyperplastic primary vitreous.^25,26

As the neural folds elevate and approach each other (neurulation), a specialized population of mesenchymal cells, the neural crest, emigrates from the neural ectoderm at its junction with the surface ectoderm. In the development of the eye, the neural ectoderm (deriving from the neural plate and neural folds), the surface ectoderm, the neural crest, and, to a lesser extent, the mesoderm are of importance (Table 1).

TABLE 1. Embryonic Origins of Ocular Tissues

  Neural ectoderm (optic cup)

  Neural retina
  Retinal pigment epithelium
  Pupillary sphincter and dilator muscles
  Posterior iris epithelium
  Ciliary body epithelium
  Optic nerve

  Corneal endothelium
  Trabecular meshwork
  Stroma of cornea, iris, and ciliary body
  Ciliary muscle
  Choroid and sclera
  Perivascular connective tissue and smooth muscle cells
  Meninges of optic nerve
  Orbital cartilage and bone
  Connective tissue of the extrinsic ocular muscles
  Secondary vitreous
  Zonules

  Corneal and conjunctival epithelium
  Lens
  Lacrimal gland
  Eyelid epidermis
  Eyelid cilia
  Epithelium of adnexa glands
  Epithelium of nasolacrimal duct

  Extraocular muscle cells
  Vascular endothelia
  Schlemm's canal endothelium
  Blood

The cranial neural crest contributes most of the connective tissues of the eye and its adnexal structures.^{14,19,27–41} The hyaluronic acid-rich extracellular matrix influences migration and differentiation of the neural crest cells. This acellular matrix is secreted by the surface epithelium as well as the neural crest cells and forms a space through which crest cells migrate. Fibronectin secreted by the noncrest cells forms the limits of the mesenchymal migration. Interactions between the migrating neural crest and the associated mesoderm appear to be essential for normal crest differentiation. Many congenital malformations of the anterior segment and cornea probably arise from derangements in the axial migration of ocular neural crest.

Experimental embryologic studies have shown that the mesoderm actually contributes little to head and neck mesenchyme. The cranial correlates to the paired paraxial somites are called somitomeres. Seven pairs of cranial somitomeres have been identified in the mouse.^{33,40,42–51} In the eye, the mesoderm contributes only to the striated extraocular muscles and vascular endothelia. To these limited primary mesodermal elements come associated neural crest satellite cells (surrounding the striated muscles) and pericytes (surrounding the vascular endothelium). Circulating blood elements originate from mesoderm. The term mesenchyme broadly refers to any embryonic connective tissue and should not be confused with mesoderm. With respect to the head and neck, most of this connective tissue derives from the cranial neural crest, with the exceptions mentioned.

The optic primordium is a thickened zone in the differentiating central nervous system that forms the neural folds of the early embryo. Some of the neuroepithelium composing the optic primordium becomes the future optic cup and stalk; some cells may delaminate to contribute to the neural crest.²⁷ The optic sulcus or groove arises in the primordium at the time when the neural folds are still open in the forebrain (8 to 15 somite pairs, approximately 2 to 3.5 mm) (Figs. 3 and 4A). With enlargement of the sulcus, the optic evaginations and, later, the optic pits appear in the region of the future forebrain (see Fig. 4B). The portion of the evaginations adjacent to the midbrain contacts the mesencephalic neural crest cells, which will form the mesenchymal envelope isolating neural from surface ectoderm (see Fig. 4C).

At about the 24th day (2 to 4 mm) with the closure of the neural tube, the optic pits are pushed outward away from the central nervous system and toward the surface ectoderm. The two lateral bulges, caused by the outward extension of the growing optic pits, become pouch-shaped vesicles at about the 25th day of development (20 somite pairs) (Fig. 5; see Fig. 4D and E).

The optic vesicles become sheathed with cells of neural crest origin²⁷ that, except for a small region in the center of the bulge, separate them from the surface ectoderm (see Fig. 4E). The future primordium of the retina is present before closure of the neural tube, when the neural ectoderm is still open to the amniotic cavity. The optic stalk is formed by a constriction of the area between the vesicles and the future forebrain. At this time, all cells lining the inner surface of the vesicle's cavity are ciliated, and its outer surface, as well as the inner aspect of the surface ectoderm overlying it, is covered by a thin basal lamina.

The next event is invagination of the optic vesicles by differential growth and buckling to form the optic cup (Figs. 6 to 9). The temporal and lower walls move inward against the upper and posterior walls. This process also involves the optic stalk so that the optic (choroid/embryonic/retinal) fissure is formed where the two laterally growing edges of the cup and stalk meet. Mesenchyme (primarily neural crest) penetrates immediately into the cup by filling up the fissure.

The optic vesicle and optic stalk invaginate through differential growth and infolding. Local apical contraction⁵² and physiologic cell death⁵³ have been identified during invagination. This process progresses from inferior to superior so that the sides of the optic cup and stalk meet inferiorly in the optic fissure. The two lips of the optic fissure meet and initially fuse anterior to the optic stalk with fusion progressing anteriorly and posteriorly. Failure of normal closure of this fissure may result in inferiorly located defects (colobomas) in the iris, choroid, or optic nerve.

Closure of the optic cup through fusion of the optic fissure allows establishment of intraocular pressure. Studies have demonstrated that, in the chick, the protein in the embryonic vitreous humor is derived from plasma proteins entering the eye by diffusion out of permeable vessels in the anterior segment.⁵⁴ After optic fissure closure, protein content in the vitreous decreases, possibly through dilution by aqueous humor produced by developing ciliary epithelium.

Table 2 lists the chronologic sequence of ocular development and comparative body-eye measurements in relationship to embryonic time intervals.

TABLE TWO. Revised Sequence of Human Ocular Development

As the lens placode invaginates, it forms a hollow vesicle (see Figs. 8 and 9D). The area of contact of the optic vesicle and the surface ectoderm determines the size of the lens vesicle, orbit, and palpebral fissure. The lens separates from the surface epithelium at about 33 days' gestation (7 to 9 mm; see Fig. 9D). The vesicle consists of a single layer of cells, covered by a basal lamina. Through appositional growth to its epithelial surface, the basal lamina acquires more layers that become the lens capsule. At first, the posterior capsule is more prominent than the anterior; the outer layers may have components from the mesodermal tissues forming the hyaloid vascular network.⁷² A zone of necrosis develops, displacing the lens placode from the surface ectoderm (see Fig. 9E and F). The process of lens vesicle detachment is accompanied by active migration of epithelial cells out of the keratolenticular stalk, cellular necrosis, and basement membrane breakdown.^73,74 Cup formation is achieved by contraction of the apical filaments. The process of induction is thus localized.

PRIMARY LENS FIBERS

The hollow lens vesicle consists of a single layer of epithelial cells with cell apices directed toward the center. Following detachment from the surface ectoderm, the lens vesicle is surrounded by a basal lamina, the future lens capsule. The cells lengthen (Figs. 10 and 11A) until the lumen of the vesicle is filled (45 days, 17 mm). These constitute the primary lens fibers. The apical ends of the newly formed fibers become firmly attached to the apical surface of the anterior lens epithelium.

The retinal anlage promotes primary lens fiber formation in the adjacent lens epithelial cells. Surgical rotation of the lens vesicle in the chick's eye by 180 degrees results in elongation of the lens epithelial cells nearest the presumptive retina, regardless of the orientation of the transplanted lens.⁵⁶ The retina thus develops independently from the lens, while the lens appears to rely on the retina for cytodifferentiation. This transformation of primary lens fibers is accompanied by ultrastructural changes in the nucleus and cytoplasm, decreased numbers of organelles, and increased numbers of fibrillar materials composed of the characteristic lens proteins.⁷¹ The primitive lens filled with primary lens fibers forms the embryonal nucleus, visible in the adult. This portion of the lens lacks sutures.

SECONDARY LENS FIBERS

The cells nearest the corneal primordium remain cuboidal and become the lens epithelium, which remains mitotic throughout life, giving rise to future lens fiber cells. Production of the secondary lens fibers is initiated by migration of the anterior epithelial cells toward the equator and their elongation at various degrees with a shift in their nuclear distribution, thus resulting in the lens bow (Fig. 12B, C, and F, and 13; see Figs. 11B and C). The basal ends of the fibers remain tightly attached to the basal lamina; their apical ends extend anteriorly to the center, thus forming the anterior suture. The tips of these secondary fibers are not yet tapered. A corresponding increase in cell volume and decrease in intercellular space within the lens accompany lens fiber elongation.⁶¹ The lens fibers exhibit surface interdigitations. They extend around the primary fibers beneath the capsule and meet in planes, the lens sutures, arranged essentially vertically to the surface. The basic anatomy of the lens is established after the first layer of secondary fibers has been placed (seventh week of gestation).⁷⁵

LENS SUTURES

Succeeding generations of cells extend anteriorly and posteriorly from the equator beneath the capsule. The anterior suture line is shaped like a Y that is inverted in the posterior aspect. The posterior suture is formed when the posterior central cells lose their nuclei, become separated from their basal lamina, and migrate inward.⁶⁶ Curved lens fibers result, with the superficial ones being the longest. Linear and triradiate sutures form, representing different stages in lens development.

MATURATION

The shape of the lens and its orientation with respect to the optic axis continually adjust to the developing eye. This is partly regulated by the neural retina and peripheral mesenchyme.¹⁰ Through the third month of gestation, the anteroposterior diameter is greater than the equatorial. Mainly because of the continued generation of secondary fibers, the equatorial diameter increases rapidly, thus making the lens more and more ellipsoid. The lens, still somewhat spherical at birth, grows throughout life.

A general structural densification occurs progressively during maturation. Fibrillar material is increased within the cytoplasm and cell organelles are decreased. The successive parallel layers of interdigitating, elongated lens fibers become tightly apposed (see Fig. 12D and E). Deeper nuclei become homogenous and dense. By the end of the third month, the innermost cells have lost their nuclei and simultaneously show disintegration of the chromatin and the ribosomes, leaving a finely filamentous cytoplasm.

CORNEA

Among the many publications on the morphogenesis of the cornea (Fig. 14) and the development of its constituents in various vertebrates, only a few can be cited in this general review.

Epithelium

When the lens cup separates from the surface ectoderm in embryos at about 33 days' postfertilization (7 to 9 mm in length), development of the cornea can be said to have begun. The surface ectoderm becomes continuous covering the optic cup and lens vesicle and later develops into the corneal epithelium.

Descemet's Membrane and Endothelium

During the next week, mesenchymal cells grow centrally between the basal laminae of the lens and corneal epithelium (Fig. 15; see 14A-C). Posterior to the basal lamina of the corneal epithelium, the mesenchyme has produced a double row of flattened cells, the future corneal endothelium (see Fig. 14A).

Descemet's membrane first appears at 8 weeks as a patchy accumulation resembling basement membrane material.^91,92 The patches become confluent and thickened owing to the synthetic activity of the endothelial cells. Evidence of organization is seen early during the fourth month, when four or five superimposed lamellae interspersed with collagen fibrils appear on the stromal side of the endothelial basal lamina. The endothelium has zonulae occludentes at the cell apices by the middle of the fourth month of development. Their appearance corresponds to the onset of aqueous humor formation.

Stroma

Following formation of the corneal endothelium, mesenchyme (neural crest) continues to migrate axially over the rim of the optic cup during the seventh week (17 to 18 mm) (Fig. 16). At 8 weeks (18 to 22 mm), migrating mesenchymal cells from the periphery invade the space between epithelium and endothelium. This mesenchyme, as well as that which will give rise to the sclera and iris stroma, is of neural crest origin.³⁰ The central portion of the future stroma is still acellular (see Fig. 14B). The endothelium merges with the stratified cells at the periphery over the lips of the optic cup. This mass of cells, in turn, is continuous with the cellular scleral condensation extending to the equator of the globe. The developing keratocytes begin to produce glycosaminoglycans.¹⁰⁴

In the early 8-week-old embryo, about 22 mm in length, the mesenchymal stroma consists centrally of five to eight rows of cells (Fig. 14C), within a fibrillar matrix containing collagen. Nerves have been identified within the corneal stroma and between epithelial cells at 3 months.^105–107

The most posterior layers of the corneal stroma are confluent peripherally with a condensed band of mesenchyme that is gradually spreading backward to enclose the eye. The mesenchyme destined to form the sclera is not distinct from that which will form the four oculomotor muscles.

The cornea at 2 months (about 20 mm) consists of an epithelium of outer squamous and basal columnar cells. The middle polygonal or wing cells of the adult do not appear until the fourth or fifth month. The stroma has about 15 layers of cells with rapidly developing collagen fibrils, most in the posterior portion. At 3 months, the endothelium of the central area consists of a single row of flattened cells that seem to rest on an interrupted basal lamina, the first indication of a thin Descemet's membrane. With the exception of Bowman's membrane, all corneal components are present (see Fig. 14D).

Bowman's Membrane

Arising relatively late in gestation (see Fig. 14E and F), Bowman's membrane is observed by light microscopy during the fifth month, but somewhat earlier by electron microscopy. It is always acellular, presumably formed by the most anterior fibroblasts of the stroma, which move posteriorly as Bowman's fibers and the ground substance are synthesize. The epithelium may play a partial role in the local polymerization of the collagen precursors presumably produced by the most anterior stromal fibroblasts.¹⁰⁸

Transparency

Perhaps the most important and unique corneal characteristic is its transparency, which also develops during fetal life. The early embryonic and fetal cornea is translucent rather than transparent and is more hydrated than in the adult.⁹⁴ Condensation begins in the posterior stroma during fetal maturation.⁹⁵ At about the time that the most anterior stromal lamellae are formed, corneal transparency reaches adult quality. During this development, the water content of the cornea is being reduced so that the adult level of corneal hydration is attained at the same time as transparency.

SCLERA

The sclera forms first anteriorly, by mesenchymal condensation at the limbus near the future insertion of the rectus muscles and grows gradually posteriorly. Fibrocytes are involved in the synthesis of the elastic foci in the sclera.¹⁰⁹ In contrast, the cornea lacks elastic components.

Inspection of the sclera at 60 to 65 mm or 12 weeks reveals it as a mesenchymal condensation that has reached the posterior pole of the eye and surrounds the optic nerve. Some cells have entered among the optic nerve fibers and are arranged transversely, forming the first stages of the connective tissue lamina cribrosa. The scleral spur appears at 4 months as circularly oriented fibers; at 5 months, it is visible behind the anterior chamber. At this time the sclera is well differentiated all around the eye.

Although the corneal and scleral cells are derived from the same mass of mesenchyme surrounding the anterior part of the optic cup, they behave differently when in their definitive position. Corneal fibroblasts form collagen faster than the scleral cells and differ in the rate and amount of noncollagenous protein that they synthesize.¹¹⁰

IRIDOCORNEAL ANGLE

Light and scanning electron-microscopic studies reveal the anterior chamber angle of the human eye to have a continuous endothelial lining during the third and fourth months (Figs. 17 and 18). The tissues in the angle later differentiate into a loose reticulum with large enclosed spaces near the iris and ciliary body; outside of this trabecular tissue, a tighter aggregation of cells is oriented toward the sclera.^111–115 With the growth of surrounding structures, Schlemm's canal comes to lie at the level of the apex of the angle. Descemet's membrane and the corneal endothelium still cover a portion of the trabecular meshwork, but the endothelial lining of the chamber has become discontinuous (Figs. 19 and 20). The loose reticular tissue of the earlier stages now occurs only in the deepest part of the angle, where it has large intercellular spaces (see Figs. 17C and 20).

Anterior chamber angle formation seems to occur through a combination of processes. Differential growth of the vascular tunic results in posterior movement of the iris and ciliary body relative to the trabecular meshwork and exposure of the outflow pathways.¹¹⁶ In addition, there is gradual cellular rearrangement and mesenchymal atrophy, as well as enlargement of numerous large spaces, until they become confluent with the anterior chamber.¹¹¹

Following initial separation into corneoscleral and iridociliary trabecular regions at 15 weeks' gestation, the corneal trabeculae enlarge and there is regression of the corneal endothelium covering the angle recess. The discontinuity of the cellular layer covering the angle and the many lacunae present in late gestation may be correlated with the normal development of an increase in the outflow facility of aqueous humor. Outflow facility of fetal eyes under constant pressure reveals progressive increase with the age of the fetus (0.09 μl/min/mmHg before 7 months to 0.3 μl/min/mmHg at 8 months).^117,118 It may be speculated that, if the splitting and rebuilding of the endothelial membrane lining of the early iridocorneal angle is arrested, a block to normal outflow may result. Persistence of the endothelial (Barkan's) membrane has been postulated to be of significance in the pathogenesis of congenital glaucoma.

TRABECULAR MESHWORK

Early during the fourth month, the primitive trabecular meshwork consists of a roughly triangular mass of undifferentiated mesenchymal cells with its apex between the corneal stroma and endothelium. The periphery of the corneal endothelium covers a portion of this primitive trabecular meshwork where it faces the anterior chamber (see Fig. 11D).

SCHLEMM'S CANAL

Studies using staining for neuron-specific enolase indicate that, although most structures of the iridocorneal angle are of neural crest origin, the endothelial lining of Schlemm's canal (like the vascular endothelia) is mesodermal.¹¹⁹

During the fourth month, a narrow Schlemm's canal is sometimes present (see Fig. 17A), possibly derived from extensions of a collector channel plexus, which will eventually become aqueous veins. Vacuolation of the endothelium around Schlemm's canal commences during the fourth month, and individual cells are connected by zonulae adherentes. During the following 3 months, the endothelium thins, with more vacuoles and tight junctions visible.¹²⁰

A thin basement membrane lines the inner (vitreous) aspect of the nonpigmented epithelium and retina. Apical cilia projecting into the intercellular space are seen at 4.5 months. There is also increased prominence of Golgi complexes and associated vesicles within the ciliary epithelial cells. These changes and the presence of “ciliary channels” between apical surfaces probably represent the first production of aqueous humor.¹²¹

IRIS

The iris develops by anterior growth of the optic cup. The iris stroma develops from the same population of mesenchyme (neural crest) that forms the corneal stroma, corneal endothelium, and pupillary membrane. The neuroectoderm of the optic cup differentiates into the pupillary sphincter and dilator muscles and posterior iris epithelium and induces differentiation of iris stroma. Closure of the optic fissure is normally completed by 33 to 35 days' gestation. Failure of fusion of the fissure may result in an inferior (typical) iris coloboma alone or with iris hypoplasia.

Tunica Vasculosa Lentis, Pupillary Membrane, and Iris Stroma

In the 17 to 18 mm embryo (7th week), vascular outgrowths are seen extending from the rim of the optic cup over the anterior lens surface (see Fig. 16). Mesenchyme migrating into the space between the lens epithelium and corneal endothelium becomes the pupillary membrane during the eighth week (21 to 26 mm; see Fig. 12A, G, and H, and Figs. 16 and 18).⁸¹ The anterior chamber is then bounded anteriorly by the avascular corneal endothelium and posteriorly toward the lens by a thin, vascularized mesenchyme, the anterior portion of the tunica vasculosa lentis. The anterior tunica vasculosa lentis is continuous with the pupillary membrane, which is supplied by means of branches of the long posterior ciliary arteries and the major arterial circle (54 to 75 mm; Figs. 21 to 23). By the end of the third month, there is a rapid forward growth of both walls of the optic cup between the folded region and its margin (see Fig. 17A and B and Fig. 22).

During the fifth month, a series of loops of vascular arcades reach centrally into the mesenchyme of the growing iris (see Fig. 23). These originate from branches of the long ciliary arteries. Immature tight junctions unite endothelial walls of the iris vasculature as soon as they are formed; there are no fenestrations.¹²²

The arteriovenous loops of the pupillary membrane come to be arranged over the sphincter region and are the basis for the formation of the collarette. During the sixth month there is resorption of the axial (pupillary) portion of the pupillary membrane with subsequent atrophy of the blood vessels. The rest of the pupillary membrane disappears during the seventh and eighth month, not so much by dissolution as by remodeling of its constituents. The mesenchymal frame of the pupillary membrane is incorporated into the prospective iris stroma. Programmed cell death and phagocytosis by macrophages are involved in regression of the pupillary membrane. Dysfunction of any of these processes may play a role in the persistence of the pupillary membrane.^123,124

Neuroectodermal Constituents of the Iris

The inner layer of anterior portion of the optic cup differentiates into the posterior iris epithelium (continuation of the nonpigmented ciliary epithelium; Fig. 24). Pigmentation proceeds gradually from the pupillary margin, beginning at midterm (Fig. 25), toward the ciliary region and is completed during the seventh month.

The smooth muscles of the pupillary sphincter and dilator muscles represent the only muscles in the body of neural ectodermal origin. In avian species, however, the pupillary muscles are striated and originate from stromal mesenchymal (neural crest) cells that migrate into the muscle bundles to become skeletal muscle cells.¹²⁵

The first sign of differentiation of the sphincter muscle is the appearance of basal infoldings in the anterior epithelial layer (continuation of pigment epithelium) near the rim of the optic cup (see Figs. 17B and 25). This change is followed by reduced melanogenesis. At 3 months' gestation, fine fibrillar material is present in the basal part of these cells. In the sixth month, connective tissue septa and capillaries invade the muscle bundles and separate them from their origin, except at the pupillary edge. The muscle comes to lie free in the posterior mesenchymal layer (see Fig. 17D).^126,127

The dilator muscle develops later than the sphincter with fibers identified in the sixth month. The first sign of their differentiation is the appearance of fine fibrils in the columnar cells of the anterior epithelium (see Fig. 17E). The myoepithelial cells have a spindle shape, are contractile, but remain attached to their anterior epithelial site of origin. A basal lamina covers cell surfaces facing the stroma. Capillaries or mesenchymal septa do not invade the sheet of the partially differentiated muscle, which continues to develop after birth.^127,128

Melanogenesis

Pigmentation varies according to individual or racial coloration. In the macaque, chromatophores are noted in the iris stroma until term, in contrast to the human in whom pigmentation occurs between 6 and 7 months' gestation.¹²⁹ Melanosomes in the human iris are mature at term.¹³⁰ Most chromatophores, as seen with the optic microscope, appear to develop postnatally. Pigmentation in the anterior border layer is insignificant. If the stroma has a scant collagen fibril content and is thin, it allows the pigment epithelium to peek through and a brownish color is noticeable. Blue irides have a transparent anterior border layer allowing interference or double refraction in the region of the stromal collagen.

In the newborn, the superficially flat iris is not fully developed. The stroma is very thin and delicate with poorly formed connective tissue sheaths around the vessels. The collarette is nearer the pupil, but the anterior leaf is more completely developed around the pupil and not so transparent. Collagen formation is enhanced during the ninth month of gestation; it occurs first in the anterior stromal layers near the sphincter and then proceeds peripherally. In the newborn, however, the anterior leaf remains narrow; it grows toward the periphery but does not reach the iris root, where most of the obliterated vessels end.

CILIARY BODY

The Ciliary Epithelium

The anterior margin of the two-layered neuroectodermal optic cup lags behind the retina in differentiation (Fig. 26). Some evidence suggests proximity to the lens is required for differentiation of iris and ciliary body. Late in the third month (at 50 to 54 mm), longitudinally oriented interdigitations commence in the outer, pigmented layer of the anterior portion of the forward-growing cup, behind the advancing margin (see Figs. 17A and 21). By 12 weeks (at 65 mm), the outer (pigmented) layer starts to form meridional ridges; to adhere to the inner nonpigmented layer and to fold with it (see Figs 17B and 22). These 70 to 75 radial folds and ridges are the precursors of the ciliary processes. The growing tip extends forward, carrying with it the folded portion, which increases in complexity. A smooth region (the future pars plana) involving both epithelial layers comes to lie equatorial to these primitive ciliary processes (Fig. 27).

Stromal (and Vascular) Components of Ciliary Body and Processes

With the accumulation of mesenchyme between the growing margins of the optic cup and surface ectoderm, differentiation of the stromal elements of the ciliary body begins (see Fig. 24). Primitive ciliary muscle fibers are visible in the mesenchyme between the infolding region and the scleral condensations late in the third month.¹³²

Parallel vessels surround the anterior part of the optic cup and give rise to an irregular capillary-venous network (Fig. 28). During the fourth month, branches penetrate the mesenchyme that forms the core of the growing ciliary processes. The invading buds consist of endothelial ridges that develop lumina arising from the confluence of their intracytoplasmic vesicles with intercellular spaces.¹³³ As soon as canalization is accomplished, pores appear in the endothelial wall, but the basal laminae are intermittent. Thus, each primitive ciliary process has a vascular branch connected to the capillary net in the associated mesenchyme. This predominantly venous network is formed from branches of the parallel vessels continuing forward from the anterior portion of the choroidal vascular investment. The small twigs within each process make an elaborate, mostly venous, tufted plexus.

During the fourth month, the long ciliary arteries have formed the major arterial circle (see Fig. 24), and by the end of the fifth month, recurrent branches from it are seen in the ciliary body region. Each of these processes, however, receives one arterial branch only during the eighth month. Anastomosis with vessels of the arterial layer of the choroid is then established.

In general, ultrastructural expression of physiologic barriers, such as the blood-aqueous and blood-retinal barriers, is established early in gestation, almost simultaneously with the recognizable differentiation of the cells with which this concept is associated (i.e., tight junctions in the retinal or iris capillary endothelia and the pigment epithelium). Fenestration of the choriocapillaris and capillary endothelium of the ciliary processes is observable soon after lumina occur in these channels, thus providing the basis for their permeability.

Fine Structure of Ciliary Epithelia

In the early fetus, the inner (vitreal) surface of the nonpigmented ciliary epithelium exhibits irregularities and conical filaments (see Fig. 24) covered by a basal lamina.

Ciliary channels have been observed in human fetuses between the fourth and sixth months.^131,133 They are enlargements of the intercellular spaces between the apposed apical surfaces of the pigmented and nonpigmented epithelial cells of the ciliary processes. These channels are presumed to correlate with the onset of aqueous secretion and to constitute a primary reservoir for the aqueous humor. Basal infoldings into the vitreal aspect of the nonpigmented epithelium facing the posterior chamber are noted prenatally in the nonhuman primates¹³⁴ and in the ninth month in humans.

Ciliary Muscle

The ciliary muscle (see Fig. 27) develops in situ and, during the fourth month, organizes into fibers and strands. The triangular meridional portions differentiate in the fifth month. The anterior ends of the fibers are continuous with the developing scleral spur (see Fig. 25), although the tendons are not formed until 7.5 months. Circular fibers appear on the inner anterior aspect of the meridional muscle. The bundles increase in size and organization during the seventh month but are still incompletely formed at birth. The muscle then consists of slender bundles no thicker than one or two cell layers, whereas the meridional part adjacent to the sclera is more fully developed. Muscle fibers increase during the first year of life, but the connective tissue between the bundles and the amount of stroma do not grow much. With growth of the eyeball, the pars plana region elongates (see Fig. 27) so that the ora serrata, which is even with the midpoint of the ciliary muscle at 7 months' gestation, comes to lie on a level with its posterior third during the ninth month. Muscle capillaries are lined by continuous endothelia interconnected by tight junctions from the time of their formation. Unlike those of the ciliary processes, they are not fenestrated.

CHOROID

The stroma of the future choroid is wide and of a loose texture, surrounded by denser scleral mesenchyme by the end of the third month of gestation. Collagen fibrils have developed, and the fibroblasts are abundant with distended endoplasmic reticulum indicative of active protein synthesis.¹³⁵ Experimental studies have demonstrated that the neural tube is essential for the appearance of choroidal melanoblasts. At a later age, after the elements of the neural crest have migrated and have reached the periocular tissue, this mesenchyme is capable of determining the choroidal pigmentation.¹³⁶ Uveal melanocytes have the same neural crest origin as dermal melanocytes, differing in this respect from the pigment epithelium, which is strictly neural ectodermal in origin. However, the method by which melanosomes develop is identical in both choroid melanocytes and pigment epithelium.¹³⁷

The structural foundation of the choroid is its vasculature (Fig. 28). Vessels originating from endothelial blood spaces appear early in the mesenchymal tissue in close proximity to the outer, pigmented layer of the newly formed optic cup.¹³⁸ Their channels coalesce to form the annular vessel at the rim of the optic cup. They drain into the two main blood spaces, the supraorbital and infraorbital venous plexuses. During the second month (10 mm), the embryonic choriocapillaris forms around the developing pigmented epithelium, continuous with a plexus around the rim of the neuroectodermal cup (see Fig. 28A). Near the end of the month, some larger channels of these sinusoids connect with small twigs from a few short precursors of the posterior ciliary arteries that reach the vascular choroid by 30 mm. Rudimentary vortex veins are formed by the confluence of collecting channels that drain the plexuses (see Fig. 28B). Arteries have narrow lumina and walls with two or more cell layers; veins are enclosed only by endothelium.

With growth during the third month, the capillary bed stretches, some components enlarge and form the outline of a second venous layer. The capillaries situated beneath them become closed and a definitive choriocapillaris emerges. Normal choriocapillaries develop only from mesoderm that has been in contact with pigment epithelium. Extensions from the short posterior ciliary arteries radiate into this vascular bed, branch repeatedly, and empty directly into the choriocapillaris, which thus contains both arterial and venous components and reaches from papilla to equator. More anteriorly, only the primitive venous choriocapillary system exists at this period of development (see Fig. 28C).

RETINA

Retinal morphogenesis in humans and other species has been the subject of many investigations.^139–158

Early Differentiation

The primordium of the retina is present at the optic pit stage early during the third week of gestation even before closure of the neural tube (see Fig. 4A and B). The anterior part of the optic vesicle, the retinal disc, is the future neural retina, and has a marginal nonnucleated layer in contact with the lens placode. The sides of the invaginating vesicle are destined to become the pigment epithelium (see Fig. 9C and D).

Following vesicle invagination to form the optic cup, the inner layer has an outer nuclear zone and an inner anuclear marginal zone. The outermost layer of cells of the nuclear zone (the germinating, or proliferative layer) projects cilia to the surface of the contacting outer layer, or future pigment epithelium. These cilia disappear during the seventh week. They are replaced by the precursors of the photoreceptor outer segments during the fourth month.

The outer layer of the cup has two to three layers of pseudostratified columnar cells that enclose pigment granules at 33 days' gestation (7 to 9 mm). This layer produces the earliest pigmentation in the body. Punctate tight junctions near their apical ends join the cells. The basal lamina that originally surrounded the optic vesicle remains continuous over the inner (vitreal) and outer surfaces of the optic cup.

The primitive retinal cells rest on a basement membrane that faces the inner future vitreal aspect and extend their apices toward the pigmented epithelial cells. In general, mitotic figures occur in the outer zone and prevail longest in the outer surface layer adjacent to the space representing the remnant of the primary optic cavity; and at the margin of the optic cup (future ciliary body-iris region). Mitosis first ceases in the central area; growth goes on longer in the periphery. Most cell division in the presumptive retina occurs before 120 mm (approximately 15 weeks). It is not established when mitosis ceases in the pigment epithelium. It is probably limited to the periphery in late fetal life.

Formation of Layers

Retinal differentiation commences when mitosis has practically stopped. It spreads from areas facing the future vitreous (marginal zone) toward the primary optic cavity, and from the center of the base of the optic cup (inner neuroblastic layer) toward its edge.¹⁵⁹ Retinal ganglion cells and Müller's cells generally develop almost simultaneously. Here also, however, a gradient exists, given that axons and dendrites of ganglion cells near the optic nervehead differentiate earlier than those situated at the periphery. By proliferation and migration of cells, the neural epithelium separates into inner and outer neuroblastic layers in the seventh week of gestation (13 to 17 mm; see Fig. 11E and F). A few days later, a definite narrow nerve fiber layer is established, occasionally traversed by the radial fibers of the Müller cells.

Immature ganglion cell bodies move into the inner neuroblastic layer along with other less mature cells, presumably future amacrines, creating in their wake a nuclei-free entanglement of processes, the transient fiber layer of Chievitz (Figs. 29A and 30A). With further realignment of cells, this layer is mostly obliterated by 8 to 10 weeks' gestation. At this period, the cells of the inner and outer neuroblastic layers intermingle by means of their cytoplasmic extensions. They fill up the previously acellular Chievitz layer; cell bodies shift positions, establishing a new, comparatively cell-free zone of intertwined processes, the inner plexiform layer (50 to 55 mm, approximately 10.5 weeks) (see Figs. 29B and 30B). With the emergence of the inner plexiform layer, an inner nucleated layer, consisting mostly of the cell bodies of ganglion cells, becomes separated from an outer neuroblastic zone. The cell bodies of the Müller's cells and the developing amacrines are located near the inner border of the outer neuroblastic zone. Bipolar cells differentiate mostly from the middle portion of this outer zone, whereas horizontal cells and photoreceptors arise from its outermost region (see Fig. 29B and C, and Fig. 30B and C). These developmental processes are well under way by 10 weeks to 12 weeks (approximately 60 to 80 mm), when an identifiable outer plexiform layer separates the immature horizontal and bipolar cell nuclei from those of the photoreceptors.

Synaptogenesis precedes development of photoreceptor inner and outer segments by almost 2 months. Lamellar synapses start to form early in the fourth month in cone axons and bipolar terminals, as well as conventional synaptic complexes associated with amacrine cells; when these phenomena are operable, the cells are still immature.

The newborn's retina has configuration and layers of the adult's. The photoreceptor outer segments are well developed and in contact with the pigment epithelium. Synapses of the outer plexiform layer are apparent (see Figs. 29D and 30D).

Early in retinal morphogenesis, limiting membranes are established. Junctions of the zonula adherens type, representing the external limiting membrane of the retina are present in the fifth week between the outer plasma membranes of adjacent neuroblasts. A thin basal lamina exists over the inner surface of the marginal layer even before the lens vesicle formation. Contribution of basal laminalike material from developing Müller's cell processes combines with it to form the primitive internal limiting membrane.

Photoreceptors

The first indication of inner segment differentiation is the appearance of cilia in the outer cells of the external nuclear layer of the 10-week-old fetus. Later, the cell membrane becomes involuted, envelops the centriole, and forms a cylindric cytoplasmic process facing the apical portion of the developing pigment epithelium. Outer segment formation commences at 5 months. Outer segments start to develop when the ciliary filaments provoke infolding of the plasma membrane. These folds multiply, entubate, and then separate from the plasma membrane to be randomly distributed within the cytoplasm. Finally, they flatten and rearrange themselves to assume a stepladder architecture as lamellar sacs.^142,160 Although the major retinal constituents are laid down by the beginning of the fourth month, horizontal cells are distinguishable only as an irregular row during the fifth month, paralleling the development of the incipient photoreceptor inner and outer segments. The amacrine and ganglion cells are in their definitive locations and are more differentiated at the same time.

Macular Development

Differentiation of this specialized area of the retina, the macula, commences relatively late and involves accumulation and redistribution of the neuronal elements. This subject has been reinvestigated in research in a series of monkey fetuses and neonates.¹⁶¹ The earliest evidence of maculogenesis is the localized increase of superimposed nuclei in the ganglion cell layer at the posterior pole, temporal to the disc, during the fifth month. At 6 months' gestation, the center of the macula has eight to nine rows of nuclei and bulges slightly above the inner surface of the retina surrounding it. Deep to the thickened ganglion cell layer, the layer of Chievitz is present, which persists until after birth, in the macular region. The wider outer nuclear layer consists mainly of immature cones.

During the seventh month, peripheral displacement occurs in ganglion cells. The thin layer within the incipient foveal depression combines with elements of the inner nuclear layer. The synaptic contact established among photoreceptors, bipolar cells, and ganglion cells in the human central retina between the 10th to 15th weeks is maintained despite this shifting of nuclei. The cones develop long basal axons to accommodate these displacements.¹⁶¹ Changes in the shape of the foveal cones progress until after birth, involving a decrease in the width of the inner segments and lengthening of the outer segments, thus allowing an increase in foveal cone density. In the 8-month fetus, there are two layers of ganglion cells over the slightly depressed central area; these are reduced to a single layer in the newborn (Fig. 31). A thin inner nuclear layer is still present. By 4 months' postpartum, both layers have retreated to the fovea slopes, leaving the cone nuclei practically uncovered in the center of the depression.

Retinal Periphery

Between 4 and 6 months, the ciliary body and retinal regions become distinct with a well-delineated ora serrata nasally (see Figs. 24, 25, and 27). At this time, a thin nerve fiber layer is present in the peripheral retina. The formation of the ora serrata goes on concurrently with that of ciliary process development. At 8 to 9 months, the temporal ora is complete (see Fig. 27D). Scalloping at the ora is increased after birth, presumably because of disproportionate postnatal growths of the pars plana and ora serrata zones compared with parallel growths during fetal life. The region from the ora to the equator of the retina continues to enlarge until 2 years of age. The surface area of the newborn retina is approximately 589 mm2.¹⁶²

Retinal Vascularization

Retinal angiogenesis has been extensively studied in laboratory animals^163–177 and reexamined in humans.^178,179 In humans, at approximately the 5 mm stage, the terminal portion of the primitive ophthalmic artery, a branch of the internal carotid artery, invades the optic fissure from below. After closure of the fissure between the fourth and fifth weeks, the vessel remains within the cavity of the optic cup (see Fig. 9F). It is now termed the hyaloid artery and its intraocular branches soon spread between the marginal zone of the primitive retina and the lens vesicle. The hyaloid artery supplies the nutritive requirements of both the lens and the growing retina before the latter acquires its own vasculature. At 65 to 70 mm (approximately 12 weeks), vessels derived from the ophthalmic artery accompany the hyaloid artery for some distance.

As the hyaloid artery regresses during the fourth and fifth months, retinal vessels develop. Primitive retinal vessels emerge near the hyaloid artery as it enters the optic disc.¹⁸⁰ Spindle-shaped mesenchymal cells, apparently derived from the wall of the two venous channels at the disc, form aggregations around the hyaloid vessels.^179,181 Buds or strands of cells thereafter push into the nerve fiber layer (Fig. 32). The proximal intraneural portions of the hyaloid vessels subsequently become the central retinal artery and vein. During the fifth and sixth months, lumina with occasional red blood cells appear within the solid cords. These may anastomose with adjoining cords, or primitive vessels, thus forming a polygonal network. Branches spread in depth to the outer border of the inner nuclear layer by the ninth month (see Fig. 29D).

As the primitive capillaries push toward the retinal periphery, they reach the ora serrata by the eighth month of gestation.¹⁸⁰ From the fourth to the seventh months, the growth rate of these new vessels is about 0.1 mm/day. Because the mature pattern is attained at approximately 3 months after birth, the retinal vasculature is sensitive to postnatal developmental disturbances.

In tissue culture experiments, it has been demonstrated that the retinal capillary endothelial cells retain their embryonic potential and can revert to more primitive cell types that can then redifferentiate into endothelial, fibroblastic, or muscle cells. Therefore, it is possible that endothelial cells, pericytes, and muscle cells may have a common origin. Late in gestation, the vascular endothelium of the retina is continuous, with single or multiple points of fusion between the cells.

The effect of oxygen on developing retinal vessels is contingent on the developmental sequence in angiogenesis. A relatively large capillary-free zone lies immediately adjacent to the retinal arteries and a similar zone, although much less evident, surrounds the veins. This capillary-free zone results from retraction and atrophy of the channels adjacent to the growing vessels, which is a more active process near the arteries. Such zones are found in fetuses at 6 to 9 months. This periarterial capillary-free space can be caused to widen by increased oxygen concentration in kittens,¹⁷⁰ thus accelerating capillary retraction and atrophy. Anoxia has the reverse effect.

PIGMENT EPITHELIUM

The outer wall of the optic cup is composed of a mitotically active pseudostratified columnar epithelium. The inner surface bears cilia that disappear with the advance of melanogenesis at 33 days (7 to 9 mm). From between 6 to 6.5 weeks, the prospective pigment epithelium is a monolayer of cuboidal cells (see Fig. 30A) the apical surfaces of which are reflected into short projections against the future photoreceptor outer segments. Its total surface area is 240 mm2 at the fourth month, which increases to 800 mm2 by 2 years of age. On surface view, the cells are hexagonal. This is the first tissue in the body to exhibit melanogenesis. Pigment granule formation is similar to that occurring within the epidermal melanocytes. The melanin is deposited on the folded inner membranes of vesicles that are probably of Golgi body or outer nuclear membrane origin.¹⁴⁷

The common origin of the inner and outer layers of the optic cup is demonstrated in mouse mutants exhibiting dysplasia of the retinal pigment epithelium resulting in formation of a second layer of neurosensory retina.¹⁸²

The sequence of pigment epithelial differentiation is inferred from the cytoplasmic organelles seen at various stages. These indicate involvement initially with protein synthesis (ribosomes), then membrane and polysaccharide synthesis.^183–185 The deeper lateral and basal infoldings, the latter associated with transport, also become prominent during gestation.¹⁸⁶ There is a continuous, although at a slower rate, addition of new pigment epithelial cells during fetal life. No mitotic figures are observed in the postnatal retinal pigment epithelium. The individual cells simply enlarge (hypertrophy rather than hyperplasia) to cover the large area created by further growth of the eyeball.¹⁸⁷

OPTIC STALK

The optic stalk forms a connecting channel between the vesicular cavities and that of the forebrain. (2 to 4 mm at 24 days; see Fig. 4E).^27,189 Its involution commences simultaneously with the collapse of the vesicles into the optic cup stage at about day 29 (5 to 7 mm). A shallow groove is formed in the stalk (see Fig. 9F), extending from the optic (choroid) fissure almost to the forebrain. In the 5 mm embryo, the hyaloid artery is within this depression. Some drainage from the sinusoids surrounding the optic cup occurs through a tributary of the primitive maxillary vein. This channel, also located within the optic stalk groove, is probably the precursor of the future central retinal vein. Direct continuity exists between the inner layers of the cup and stalk (Fig. 33A); the region of the disc is outlined by the neuroepithelial tissue of the primitive papilla.

Closure of the optic fissure commences during the fourth week, between 7 and 9 mm, with fusion of the central part of the optic cup. Its inner and outer margins fuse subsequently; closure of the cup is complete at 5 weeks (Fig. 34; see Fig. 33A). The lips of the optic stalk begin to close over the hyaloid artery at 12 to 17 mm, starting from the region near the forebrain and gradually extending distally (see Fig. 33A and B). Thus, fusion of the margins of the stalk lags behind that of the optic cup. The opening within the stalk through which the hyaloid artery enters is closed by 19 to 20 mm.

The margins of the optic fissure are covered by basal lamina. Breakdown of the basal lamina, inversion of the outer layers of the cup and stalk, degeneration of the superfluous cells, and eventual reconstitution of the basal lamina are essential events in normal closure.¹⁹⁰ It is suggested that cell death helps to control the growth rate of optic cup and fissure.¹⁹¹ Given that cell degeneration precedes invagination, it may serve to retard or inhibit it locally, or to integrate the series of infoldings in the dorsal optic cup and optic fissure.^192,193

MIGRATION OF NERVE FIBERS INTO THE INNER STALK LAYER

Some cells in the inner wall of the optic stalk vacuolate and receive axons from the ganglion cells of the retina. The fibers force their way through these spaces; by 19 mm, the fused optic stalk is almost completely filled by nerve fibers that surround the hyaloid artery. The primitive epithelial papilla with the hyaloid artery in its center is isolated by the confluent axons that course toward the brain. There is a potential space between the basal lamina around the hyaloid, which is covered by glia, and the basal lamina of the retinal surface, where the hyaloid artery enters from the papilla. This space between the artery and the glial sheath becomes accentuated with the atrophy of the hyaloid system. The segregated cells are converted to glia, which become the constituents of the primitive optic disc. Some of these glia are destined to form a conical formation around the hyaloid artery, called Bergmeister's papilla (Fig. 35). The cells of this last structure proliferate; by 4.5 months, there is a mantle around the regressing artery. The extent of the degeneration of these cells late in gestation defines the limit of the excavation at the disc.^194–196

LAMINA CRIBROSA

The collagenous fibers of the sclera progress from the perilimbal region posteriorly, where they encircle the developing optic nerve, thus forming the scleral foramen. In the fourth month, connective tissue fibers penetrate the optic nerve, running between groups of glia-covered axons to reach the hyaloid vessel. Thereafter, a network of collagenous and elastic fibers forming a sievelike scaffolding, the lamina cribrosa, bridges the scleral foramen. The latter attains its mature structure during the seventh month. It should be emphasized that the openings filled with axons are present first, and the “sieve” subsequently invades around them. A faint suggestion of the optic nerve sheath commences in the seventh week (before 20 mm), but it is precisely defined only in the fifth month (see Fig. 35). The sheaths are derived from the cranial neural crest mesenchyme. By the end of the third month, the optic nerve is 1.2 mm in diameter and 7 to 8 mm long (see Fig. 33C).¹⁹⁷

MYELINATION

Myelination starts in the fetus near the chiasm about the seventh month, progresses distally and stops at the lamina cribrosa about 1 month postpartum.^198–202 In the newborn, the myelin is extremely thin and seems to contain more cholesterol in the portion near the brain. During childhood, the number of myelin layers around the axons increases.

PRIMARY VITREOUS

Hyaloid Vasculature

The primary vitreous mostly consists of the hyaloid vasculature with some minimal associated matrix and cellular (neural crest) components. Vascular endothelia are of mesodermal origin. The matrix may be a combined secretion of the vascular and neural crest tissue.

The primitive dorsal ophthalmic artery gives off the hyaloid artery, which passes through the optic fissure during the fifth week of gestation (see Fig. 33) and branches within the cavity of the primary optic vesicle (Fig. 36A; see Fig. 9A). Arborization of the hyaloid artery produces terminal branches around the posterior lens capsule (the tunica vasculosa lentis). Other branches surround the lens equator and anastomose with the annular vessel around the outer edge of the optic cup (see Fig. 36B). The annular vessel sends loops forward and centrally, which compose the anterior tunica vasculosa lentis. Near the margins of the cup, anastomoses occur between the annular vessel and terminal branches of the hyaloid artery.

The hyaloid vasculature reaches its greatest development at about 9 weeks (33 to 40 mm; see Fig. 36B). Venous drainage from the vessels of the tunica vasculosa lentis and the pupillary membrane is accomplished through vessels that assemble into a net in the region where the ciliary body will subsequently arise. This plexus eventually communicates with venules of the choroid. No hyaloid vein is present.

SECONDARY (DEFINITIVE) VITREOUS

Development of secondary vitreous occurs during the seventh to eighth weeks, after closure of the optic fissure. A finer, more compact fibrillar network of monocytes and a small amount of hyaluronic acid characterize it.^{203–208,213,215,216} This newer vitreous also contains cells, the primitive hyalocytes, which most likely originate from the vascular primary vitreous (mesoderm) and the neural crest mesenchyme, rather than the retina. Collagen fibrils are produced by the hyalocytes and result in expansion of the secondary vitreous volume. Vessel walls of the hyaloid system consist of endothelial cells with a discontinuous mural cell covering.^210,211 The nonfenestrated endothelium is underlined by a continuous basal lamina. Expansion of the vitreous is associated with an overall increase in the volume of the globe through active production of aqueous humor.⁵⁴ Failure of closure of the optic fissure prevents this normal establishment of intraocular pressure necessary for globe expansion. This is one mechanism of (colobomatous) microphthalmia.

REGRESSION OF HYALOID VASCULATURE

Beginning first with the atrophy of the distal branches of the hyaloid, followed by that of the capillaries of the tunica vasculosa lentis, and finally by that of the hyaloid artery itself (at end of the fourth month), the primary vitreous retracts with the atrophying vessels (see Fig. 36C). By 160 mm (in the fifth month), atrophy of the vasculature posterior to the lens creates the funnel-like expansion of Cloquet's canal. Before the capillaries disappear, they are occluded by macrophages.²¹⁷ Persistence of the primary vitreous and failure of the posterior tunica vasculosa lentis to regress results in persistent hyperplastic primary vitreous (PHPV).^214,218,219

TERTIARY VITREOUS AND ZONULAR FIBERS

The secondary vitreous in the anterior peripheral region at the rim of the cup contains thicker, presumably aggregated collagen fibers. Some of these fibers abut the proliferating mesenchyme near and between the lens and the rim of the cup by the end of the third month (65 mm). These fibers form the marginal bundle of Drualt (Fig. 37; see Figs. 21, 22, 24, and 36C). Here, the vitreous is firmly attached to the internal limiting membrane of the retina. This constitutes the embryonic aspect of the vitreous base.

Regression of the peripheral branches of the hyaloid vasculature precedes zonular fiber appearance. During the sixth and seventh months of development (200 to 300 mm), the stainable vitreous regresses to its base on the pars plana and to its attachment to the lens (capsulohyaloidal ligament). Late in gestation, the fibers of the suspensory ligament (zonules) appear to originate from precursors in the valleys between the ciliary processes just anterior to the ora serrata. Early zonular fibers seem to be a continuation of the internal limiting membrane, which thickens over the nonpigmented epithelial layer covering the ciliary muscle.^220,221

EYELIDS

The eyelid territory is determined by the optic vesicle during the fourth week of gestation. Thus, microphthalmia that originates at the optic vesicle stage is associated with a small palpebral fissure whereas colobomatous microphthalmia may be associated with a more normally sized palpebral fissure. The upper and lower eyelids develop from mesenchymal condensations referred to as the frontonasal (paranasal) and maxillary (visceral) processes. The ectoderm of the skin proliferates in the region of the future upper lid at the outer canthus at 11 to 14 mm (6 to 7 weeks). In the second month (approximately 20 mm), both eyelid prominences are visible (Fig. 38A and B; see Fig. 26). Mesenchyme beneath the epithelium proliferates and is provided with blood vessels and accompanying macrophages. During the next few days, the basal lamina beneath the epithelium thickens. Invading nerve fibers and newly deposited collagenous fibrils are seen.²²⁴

Lid Adhesion

The lid folds not only move together by differential growth above and below the developing eye but also elongate laterally. The lid margins contact each other during the third month of gestation (approximately 35 to 40 mm; see Fig. 38C and D). Muscle cells originating from the mesoderm of the second visceral arch migrate to the face and eventually reach the area around the eye. After lid fusion at 10 weeks (45 mm; see Fig. 38E and F), rudiments of the orbicularis palpebrae muscle are present in the mesenchyme between the dermal and palpebral surfaces.

Lid Disjunction

According to observations made by electron microscopy, the two lids are temporarily joined by desmosomes during their adhesion, thus isolating the eye from the amniotic fluid.²²³ The period of lid disjunction varies but usually occurs through breaking of the connecting epithelial bridges (desmosomes) during the fifth month (150 to 170 mm) anteriorly, and at about 180 to 200 mm near the posterior surface (in the sixth month). The main causes of this process are attributed to keratinization and the appearance of keratohyalin granules, which, in turn, are preceded by lipid manifestation in the Meibomian anlage. The adherence of the lids probably prevents the corneal and conjunctival epithelium from keratinizing.

LACRIMAL GLAND

About 25 mm, the developing lacrimal gland is seen in the form of epithelial buds arising from the basal cells of the conjunctiva covering the temporal portion of the upper fornix. The resultant solid cords are the core around which the surrounding mesenchyme condenses and proliferates. At around 3 months (approximately 60 mm), the central cells of the cords start to vacuolate and lumina appear. The growing tendon of the levator palpebrae divides the gland during the fifth month. Full development is reached by 3 to 4 years postnatally.

EYELASHES

The first cilia appear at the lid junction at about 40 mm. As the surface epithelial cells proliferate, they protrude, together with their basal laminae, into the underlying mesenchyme. Hair follicles of the cilia arise on both lid margins in an anteroposterior direction. The first row on the lower lid is completed before the second row on the upper lid.²²² The glycogen and acid phosphatase content of most epithelial cells is high at this time.

CONJUNCTIVAL AND LID GLANDS

Mucus-secreting goblet cells in the conjunctival sac are visible at 10 weeks (52 mm). The Meibomian (sebaceous, holocrine) gland precursors are seen at 80 mm as epithelial buds and down-growths from the basal cells of the inner edge of the adhering lid margins (Fig. 39). The apocrine glands of Moll have their onset early during the fourth month (approximately 80 mm); their ducts arise from the walls of the ciliary hair sacs (see Fig. 39). The sebaceous glands of Zeiss appear at about 90 to 100 mm as lateral outgrowths on the epithelial invaginations constituting the first row of cilia. Soon after lipid production begins in these cells, a lipid-filled canal empties through the prospective hair shaft to the lid junction surface at 4 months (100 mm). The canals of the cilia keratinize at 110 to 120 mm. Hairs (cilia) are formed from the overlying epidermis and penetrate downward through the lipid-filled spaces. Keratinization of the walls of the Meibomian glands takes place at 5.5 months (170 mm). During the development of the cilia and Meibomian glands, their thickened basal lamina and the collagen fibers related to the orbicularis muscle fibers represent the future tarsus.

BONY ORBIT

The lesser wing of the sphenoid is initially cartilaginous, derived from the base of the skull, while the greater wing of the sphenoid and the rest of the bones are membranous processes that ossify between the sixth and seventh months. Osseous structures of the orbit are mostly derived from the cranial neural crest cells, which migrate to surround the developing eye and additionally form the frontonasal and maxillary processes. The maxillary process contributes to the floor and lateral wall of the orbit, and the nasal process provides the lacrimal and ethmoid bones. The air sinuses develop mainly postnasally. The ethmoid is the first to take shape, between the sixth and eighth weeks. A fibrous trochlea is seen at 37 to 40 mm. During the third month, orbital walls are differentiated and later become incompletely ossified. The angle between the orbital axis starts at nearly 180 degrees, diminishes to about 105 degrees at 3 months, and reaches 71 degrees at birth. The adult condition is 68 degrees. This decrease results from growth of the tissue behind and lateral to the eyes.

At term, the morphology of the orbital structures approaches that of the adult. The orbit fits closely to the eye at first and is nearly hemispherical. It grows as the orbital contents increase. If the eye does not grow, the orbit remains small, about 90% of normal size. The eye reaches its adult size by the age of 3, but the adult dimensions of the orbit are attained subsequently, sometimes as late as 16 years.

EXTRAOCULAR MUSCLES

The orbital contents consist of fat, connective tissue septa, and eye muscles. The former two are of neural crest origin. The human skeletal muscles of the trunk and limbs arise segmentally from each somite, as do the nerves.^{33,40,42–50,225,226} The mesoderm of the head region is in the form of somitomeres. In avian models, somitomeres 1 and 2 give rise to the dorsal, medial, and ventral recti, and to the ventral oblique muscles. Somitomere 3 forms the dorsal oblique and the lateral rectus arises from somitomere 5.⁵¹ There is a prechordal mass (first head somite) that gives rise to the premandibular condensation, from which the four oculomotor muscles develop (third cranial nerve).²²⁵ The other two muscle primordia arise in the maxillomandibular mesoderm and give origin to the lateral rectus (from the third head somite) (sixth cranial nerve) and the superior oblique (from the second head somite, fourth cranial nerve). Mesenchyme within the orbit is the source of in situ differentiation.^226,227 The cranial nerves grow from the brain into their respective mesodermal condensations in the following sequence: oculomotor, abducens, trochlear. In addition to these primordia, there are four other condensations around the outer rim of the optic vesicle. These are the future insertion sites of the rectus muscles, and they also participate in scleral morphogenesis.

Condensation of the future fascia bulbi (Tenon's) is present at 45 mm (10 weeks, approximately). Near the end of the third month (60 mm), the tendons of the rectus muscles fuse with the sclera in the vicinity of the equator of the bulb. The levator muscle forms from the dorso medial aspect of the superior rectus muscle at about 22 to 30 mm and grows laterally and over the superior rectus toward the upper eyelid. It is complete and in its permanent position during the fourth month.

NASOLACRIMAL DRAINAGE APPARATUS

At about 32 days (8 to 9 mm), the maxillary processes, which contact the paraxial mesoderm surrounding the eye, extend forward to the nasal pit and processes. Their surface ectoderm covering is thicker over the grooved interface separating these processes. As the maxillary mesenchyme grows forward and upward over the thickened ectodermal strip and fuses with the lateral nasal processes and the paraxial mesoderm, it buries the ectodermal cells that line this rudimentary fissure. The origin of the lacrimal tract is attributed to this irregular ectodermal cord of cells, which separates from the surface ectoderm and extends caudal and cephalic branches into the mesenchyme beneath it.²²⁸ These cells come to lie between the future medial canthus and nasal cavity during the sixth week.

The lacrimal sac anlage is derived from the adjacent upper portion of this nasolacrimal cord when it begins to thicken and bulge. Partial canalization is observed during the fourth month. The lacrimal puncta open after separation of the eyelids. The inferior extremity of the nasolacrimal canal fuses with a superiorly directed outgrowth of cells that originate from the nasal fossa during the sixth month or later.

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