Ординатура / Офтальмология / Английские материалы / Development of the Ocular Lens_Lovicu, Lee Robinson_2004

14 Michael L. Robinson and Frank J. Lovicu

the lens, Remak recognised that the ectoderm (Hornblatt) thickened to form what we now refer to as the lens placode as it is contacted by the optic vesicle. Remak also described the fate of the lens vesicle in previously unparalleled detail in his 1855 book Untersuchungen uber¨ die Entwickelung der Wirbelthiere:

That the lens invaginates into the optic vesicle from the outside, Huschke, as we know, discovered. But what this investigator did not and in the state of the science at his time could not know, is the fact which I have discovered, namely that this invagination proceeds from the upper germ layer, which later furnishes the cellular (epidermal) coverings of the body. . . . The bulk of the wall of the lens, after being constricted off, consists of cylindrical, radially arranged cells, which also resemble strongly the cells of a columnar epithelium in that they appear very sharply delimited on their free surface facing the cavity. Each cell contains one, in rare cases even two, nuclei. These nuclei do not, however, lie at the same level. . . . This layer of cells is surrounded by a . . . very thin, apparently ‘structureless’ membrane. . . . This is the anlage of the lens capsule. . . . All fibers pass without visible interruption from the posterior wall of the lens capsule to the anterior almost parallel to the visual axis; the fibers are, consequently, shorter the farther away they lie from the visual axis. Their anterior and posterior ends are cut off sharply. At some distance from its anterior end each fiber contains a nucleus, but no trace of nuclei can be detected at the posterior end. . . . The posterior end of each fiber is directly in contact with the lens capsule; the anterior end, on the other hand, is separated from it by an epithelium consisting of nucleated cells which adheres to the capsule. Hence it follows that the cells of the posterior wall of the lens vesicle form the lens fibers, those of the anterior wall, on the contrary, form the epithelium. (Adelmann, 1966, pp. 1293–4)

With these observations by Remak, the basic descriptive embryology of lens formation was virtually complete, though many fine points, such as the origin of the lens capsule, would remain subject to debate and investigation for several more decades. Remak was a rather tragic figure in the history of nineteenth-century embryology. He received his medical degree in 1838 but was initially barred from teaching by Prussian law because of his Jewish faith. After graduation, he remained as an unpaid assistant in Johannes Muller¨ ’s laboratory, where he conducted basic research on the nervous system and supported himself with his medical practice. Remak was eventually granted a lectureship in 1847, becoming the first Jew to teach at the University of Berlin (Enersen, 2003). Remak’s descriptive work on the development of the eye was a very small part of his substantial body of research, but despite this he only succeeded in attaining the rank of assistant professor in 1859, six years before his death. Remark was the father of neurologist Ernst Julius Remak (1849–1911) and the grandfather of mathematician Robert Remak (1888–1942), who was killed in the Nazi concentration camp at Auschwitz (Enersen, 2003).

After Remak, who studied eye development in the chick, frog, and rabbit, others added details concerning lens formation in other species. For example, in 1877, Paul Leonhard Kessler described the development of the mouse lens in his work Zur Entwickelung des Auges der Wirbelthiere. Carl Rabl also published a marvelous book in 1900, Uber den Bau und die Entwicklung der Linse, which describes and illustrates lens development in fish, mammals, reptiles, amphibians, and birds. While these publications added details, they still built on the common theme elegantly described by Remak. Thus, at the close of the nineteenth century, the stage had been set for the descriptive embryology of the lens to give way to the experimental embryology of the lens that continues through the twentieth and into the twenty-first centuries. In particular, the groundwork had been laid for Hans Spemann (1869–1941) to perform his classic experiments revealing the induction of the

lens by the optic vesicle and establishing the theory of embryonic induction, which would become part of the foundation of developmental biology and lead to the discovery of the Spemann–Mangold organiser. Embryonic induction was an idea whose time had come.

1.2. Comparative Ocular Anatomy

Over time, developmental biologists have focused much of their attention on understanding the mechanisms underlying cell and tissue differentiation and the orderly manner in which they grow, developing to the size and shape appropriate for the body’s requirements as well as forming in the correct anatomical relationship with each other. These events are thought to depend on inductive cell and tissue interactions. As will be appreciated in the following chapters, for over a century the lens of the vertebrate eye has provided numerous researchers with a means of examining inductive tissue interactions involved in tissue differentiation throughout development and growth. The lens has readily been adopted as a model for such studies because, as will be described later, it is a relatively simple tissue made up of cells from a single cell lineage. The lens retains all the cells that are produced throughout its life, and it is isolated from a nerve and blood supply. The positioning of the lens in the eye, on the surface of the body, has made it easily accessible for experimentation, but most importantly the lens has proved to be suitable for the study of cell differentiation in vitro and in vivo, as not only do differentiating lens cells synthesise uniquely defined proteins such as the crystallins, they also undergo very distinct morphological changes, including cell elongation, cell membrane specialisation, and the loss of cytoplasmic organelles and nuclei.

The remainder of this chapter will therefore be devoted to introducing the ocular lens by reviewing in brief its structural diversity in a range of organisms and highlighting its adoption as an ideal tissue model for cell and developmental biologists alike. Sections of this part of the chapter summarise the wealth of information documented by Sir Stewart Duke-Elder (1958) in “The Eye in Evolution” a book volume on the ontogeny and phylogeny of the invertebrate and vertebrate eye, which the reader is encouraged to read in its entirety.

For many animals, the sense of vision is the most important link to the environment. Animals have adapted for survival in a variety of climatic conditions and terrains and thus have evolved a diversity of eye designs. Despite this diversity, each of the visual organs has a common functional role, the perception of light. The sensation of light is the most fundamental of the visual senses. The acquisition and development of vision has stemmed from the dependence of living organisms on light, with light influencing many aspects of survival, such as general metabolism and the control of movement (characteristic of the most primitive of animals), as well as influencing the behaviour and consciousness (through the visual senses) of higher animals.

Unicellular organisms such as ciliate and flagellate protozoa (e.g., Euglena) provide us with examples of the earliest stage in the evolution of an eye. These organisms contain a small region of protoplasm that has differentiated into a photosensitive ‘eyespot’. This specialised light-sensitive area, partially covered by a pigmented shield close to the root of its motile flagella, not only can receive a visual stimulus but is also utilised to orient the organism and direct it to more favourable regions in its environment. With the evolution of multicellular organisms came the differentiation of light-sensitive cells that allowed these higher organisms to distinguish between light and dark and even determine the direction of light. From this, eyes went on to become more specialised, evolving further to detect motion, form, space, and color.

In more advanced organisms, these patches of photosensitive epithelium indent to form a depression, giving rise to the ‘cupulate eye’ or ‘cup eye’. This structural change had the functional advantage of allowing for the development of a primitive sense of direction. Cupulate eyes have several forms, depending on the degree of invagination of the lightsensitive epithelium. The most primitive form can be found in the larva of the common housefly (Musca), present as a shallow pit in the epithelium. A deeper invagination, together with an increase in the number of photosensitive cells, converts this depression into a cavity with a small opening. As the opening of the depression continues to narrow, a dark chamber with a pinhole opening is formed. Such an eye is found in the chambered nautilus (a primitive cephalopod). Although the photoreceptors of the nautilus are indented to form an optic cup, the cup does not contain a lens, and its operation is based on the same principles as a pinhole camera. The pinhole is used to focus images, and although this provides excellent depth of field, a lot of light is required to provide an image of any quality. The optic cup can be filled with sea water, as in the case of the nautilus, or with secretions, as found in the ear shell (Haliotis). The final form of the cupulate eye is characterised by the closure of the cavity by the growth of an overlying transparent acellular cuticle which will one day go on to form the lens. The enclosed secretory mass forms the vitreous body, as seen in Nereis, the marine polychaete worm. Improvements on this design are found in some insects. For example, hypodermal cells might form a thickened cuticular layer which acts as a refringent apparatus. The optical arrangements of such an eye may further be improved, as seen in Peripatus (a caterpillar-like arthropod), in which the hypodermal cells form a large lens in place of the vitreous. These hypodermal cells, usually continuous with the surface ectoderm or with the sensory cells of the cupula, may also edge themselves underneath the cuticle and go on to form a transparent refractile mass below the cuticular lens, thereby constituting a primitive lens or vitreous. Overall, the lens of a simple eye may be either acellular and cuticular or cellular.

The vesicular eye may be considered the final stage in the development of the simple eye. This type of eye is marked by the closure of the invaginated light-sensitive epithelium, which gives rise to an enclosed vesicle separated entirely from the surface ectoderm by mesenchyme. In its simplest form, the vesicular eye is spherical and lined with ectodermal cells, as found in the edible snail Helix pomatia. The vesicle has a specific polarity, with the more posterior cells being partly light sensitive and partly secretory while the more anterior cells remain relatively undifferentiated. The cavity of the vesicle is filled with a refractile mass of secreted material, homologous with the vitreous of higher organisms.

In a further stage of complexity, the vesicular eye takes the form of a camera-like eye through the addition of a lens and now resembles the eye of vertebrates. The best example of this can be found in cephalopods (e.g., octopus), which have the most elaborate eyes in the invertebrate kingdom. The eye vesicle of cephalopods is filled with a vitreous secretion. The posterior cells lining it form the retina while the anterior cells fuse with an invagination of the surface epithelium to form a composite spheroidal lens (see Fig. 1.8F). The posterior half of the lens is thus made up of vesicular epithelium while the anterior half is derived from the surface epithelium. Encircling the lens, the fusion of the vesicular and surface epithelium gives rise to a ‘ciliary body’, with an ‘iris’ derived from the surface epithelium. This type of cephalopod eye is highly complex, is capable of image formation, and has the ability to accommodate. In contrast to other invertebrates with fixed-focus lenses, cephalopods can focus for near and far vision by changing the position of the lens relative to the retina. Although at the morphological level these eyes rival those of vertebrates, they are simple

18 Michael L. Robinson and Frank J. Lovicu

Figure 1.9. (A) Scanning electron micrograph of an ant, showing its compound eye comprising relatively few ommatidia. (B) Scanning electron micrograph of a representative region of ommatidia from the compound eye of a moth. (C) Schematic diagram of a longitudinal section of a single ommatidium from a compound eye. Scale bar: (A), 100 µm; (B), 10 µm.

in type. They are derived solely from the epithelium and combine a vertebrate-like optical system with invertebrate photoreceptors.

1.2.2. The Compound Eye

The compound or faceted eye has evolved along a different path from that of the simple eye. Unlike the simple eye, characterised by functionally independent light-sensitive cells, the sensory elements of the compound eye are structurally and functionally associated in groups (Fig. 1.9). The sensory elements that make up the compound eye, each referred to as an ommatidium, are separated from their neighbours by a mantle of pigment cells. The number of ommatidia can vary greatly, from as few as 9, as found in some ants, to 30,000, as found in dragonflies. The visual field of the compound eye is composed of images from individual ommatidia, with each acting as a single retinal cell of the simple eye. Each image does not represent the same overall picture but a small portion of the visual field within the ommatidium’s angular range. These separate parts of the field are transmitted to the receptive cells of the ommatidium, where the overall image is fused; the large number of images increases the acuity of vision by a mosaic effect.

Each ommatidium has a relatively simple structure (Fig. 1.9C). Superficially, the cuticle is composed of corneal facets which fit into each other to form a mosaic (hence the name faceted eye). Underneath this lies a refractive device, the crystalline cone, which transmits light through the action of its two convex surfaces. The crystalline cone is made up of concentric lamellae, with its refractive index increasing from the periphery to its central axis.

This allows it to act as a lens cylinder in which incident light is progressively refracted until brought to its central axis. Unlike the lens of cephalopods and vertebrates, the crystalline cone has a fixed focus and is incapable of adjustment. The remainder of the ommatidium is made up of the sensory cells arranged in a tubular form, referred to as the retinule. These nerve cells (rhabdomeres) are supported by a fenestrated basement membrane and are radially arranged so that their differentiated inner borders join to form a centralised refractile rod, the rhabdome.

One of the earliest examples of the compound eye can be found in the long-extinct trilobite, an ancient marine creature. Trilobites had a faceted eye that was limited in its view: it could see sideways but not upward. Although extinct, the trilobite has many living arthropod relatives. While most arthropods have lenses made up of relatively soft, unmineralized cuticle (similar to the rest of their exoskeleton), the eye of the trilobite, which is continuous with its solid armour, is made up of the abundant mineral calcite. Trilobites used the transparency of clear calcite as a means of transmitting light. The trilobite eye varied from species to species, with the types differing in the number of lenses they contained (from one to over a thousand). One of the most common trilobites, Phacops, had eyes that contained lenses lined up in conspicuous rows, each lens distinguished as a tiny, perfectly formed sphere (or a slightly drop-shaped sphere). These lenses were often slightly sunken, with a wall between adjacent lenses, stopping light from overlapping onto neighbouring lenses. Other trilobite eyes contained larger biconvex lenses, designed to focus light.

To date, nature has provided animals with two very different types of image-forming eyes, which have developed independently: the compound eye and the camera-like eye of vertebrates. As described above, the compound eye is a fixed-focus eye composed of ommatidia, each responsible for different portions of the visual field. The camera-like eye of vertebrates contains a pigment-shielded cup, elaborated by the presence of a single lens as well as muscles for focusing. The lens (and the cornea, in land dwellers) has developed to focus an image on a continuous receptor surface, the retina. Common to these two types of image-forming eyes is the presence of a lens, whose role is to transmit and refract light. As the cornea is primarily responsible for this in land creatures, the lens also serves to adjust the focus for near and distant vision. There are exceptions to this. In the simple eye of invertebrates, for instance, the lens may be employed, not to form an image, but to concentrate light in order that the eye may function in low light. Furthermore, a lens is not essential for an image to be formed on the retina, for a pinhole can play this role, as it does in the chambered nautilus, described earlier.

1.2.3. The Vertebrate Lens

The vertebrate ocular lens is suspended in the eyeball, situated behind the cornea and iris, and is supported posteriorly by the vitreous body (Fig. 1.10). The slightly convex anterior surface of the lens is in contact with the pupillary margins of the iris, and its more convex posterior surface occupies the hyaloid fossa of the vitreous. In most vertebrates, the primary support of the lens is provided by numerous suspensory ligaments, commonly known as zonular fibers, that extend from the equatorial rim of the lens to the surrounding muscle of the ciliary body. With relaxation of the ciliary muscle, the lens flattens, losing some of its convexity. This is the basic principle underlying accommodation: focusing the lens to give a clear view of a specific object.

Although the structure of the lens at first glance appears relatively simple, it is remarkably complex (Fig. 1.11). The lens substance consists entirely of densely packed lens fibers and

20 Michael L. Robinson and Frank J. Lovicu

Figure 1.10. Schematic diagram showing the topographic anatomy of the eye.

epithelium, enclosed by a thick membranous lens capsule. The orderly aligned elongate fiber cells, which extend circumferentially from an anterior to a posterior point, make up the mass of the lens. These fiber cells are arranged in many layers, and fibers of any one layer, running in diametrically opposite meridians, meet end to end along radial planes known as lens sutures. The anterior half of the fiber cell mass is covered by a monolayer of cuboidal cells, the lens epithelium, which extends to the lens equator. The epithelial cells lie directly beneath the lens capsule. This basic structure is found in all vertebrates, and any variations are incidental in nature and have evolved essentially as adaptations to differences in function or habitat.

The simplest vertebrate eyes belong to cyclostomes (e.g., lampreys); these contain a large primitive lens not attached to the walls of the eyeball but fixed in place by the cornea and the vitreous humour. As mentioned earlier, in terrestrial vertebrates, the cornea does most of the focusing, strongly refracting most of the incident light rays. In marine vertebrates, such as fish, this is not the case, and so all of the focusing is performed by the lens. As a result, the lenses of fish are very large and almost spherical and have a higher refractive index than that of any other vertebrate lens. In fish, lens sutures are present, although simplified as a single line. As the flattened cornea of fish is optically ineffective, the lens must serve to gather as well as focus light. Because of this, the lens needs to be situated far forward in the ocular globe, bulging through the pupil and residing close to the cornea. In contrast, in land vertebrates, the lens is less rounded (relatively flat in higher vertebrates) and is situated farther back in the eyeball. The spherical shape of the fish lens and its close association with the cornea persist in amphibians, but only in their larval stage (e.g., tadpole). With

Figure 1.11. Anatomy of the human lens. The lens capsule (C) or lens capsule with attached epithelium (Cep) has been peeled back to expose the anterior surface. The internal anatomy of the lens is evident following removal of a lens wedge. ep, lens epithelium; fib, lens fibers; En, embryonic nucleus; Fn, fetal nucleus; An, adult nucleus; Ac, adult cortex; gz, geminative zone; tz, transitional zone; As, anterior suture; Ps, posterior suture. (Adapted from Worgul, 1982.)

adaptation to land, the primary burden of focusing light fell to the cornea, and the lens became situated posteriorly, behind the iris, and also became somewhat flattened in an anteroposterior direction. Accommodation in amphibians is achieved by bringing the lens forward for close-up focus. This occurs through the action of a small ventral muscle which pulls the lens forward or backward, although anurans (frogs and toads) utilise a dorsal muscle as well. The lens of reptiles is also flattened anteroposteriorly and has a greater convexity posteriorly. The ability to change the shape of the lens, or accommodate, was first seen in reptiles. Reptiles accommodate for near and far vision by making the lens rounder (for near sight) or flatter (for far sight) through the utilisation of muscles that do not exist in amphibians or fish. It is this modification of the capacity to accommodate that is responsible for the incredible range of focus shared by birds and mammals.

Variation in the structure and positioning of the subcapsular lens epithelium occurs among vertebrates. In contrast to the lens epithelium of fish, which extends beyond the lens equator, the epithelium in amphibians conforms to the higher vertebrate plan, extending only as far as the equator. With reptiles, the lens epithelium is further modified: the equatorial epithelial cells lose their cuboidal morphology, elongating in a radial direction to form an annular pad (Fig. 1.12). As a result, the lens now abuts the ciliary body. Variations on this theme are observed in other reptiles, such as the ophidians, which include snakes. Instead of an equatorial annular pad, the lens of a snake contains an anterior pad, and the anterior epithelial cells are elongated instead of cuboidal (Fig. 1.12B). A further distinction between the lens of a snake and that of other reptiles is the presence of sutures. Reptiles (with the exception of

22 Michael L. Robinson and Frank J. Lovicu

Figure 1.12. Representative histological sections of eyes demonstrating the annular pad of a lizard lens (A, arrow), the anterior pad of a snake lens (B, arrow), and the annular pad of a bird lens (C, arrow). Scale bar: (A) and (B), 40 µm; (C), 160 µm.

most ophidians), like cyclostomes, do not usually have lens sutures; the fiber cells terminate in one circumscribed area anteriorly and posteriorly.

Birds are amongst the descendants of primitive reptiles and have evolved on diverging lines from mammals. Throughout their evolution, they have attained the highest degree of vertebrate eye specialisation, a distinction shared only with mammals. The eyes of birds are designed on the same general plan of reptiles. They contain a lens with a well-defined annular pad (Figure 1.12C), the size of the pad dependent on the species. The annular pad is retained, albeit in a much smaller version, in the lens of some mammals, including monotremes (e.g., platypus) and marsupials. In placentals, which make up the majority of mammals, no annular pad is found in the lens. Instead, the lens contains an anterior monolayer of cuboidal epithelial cells which end at the equator.

One of the most significant improvements in the eyes of birds (and of mammals) is the positioning of the lens, which is brought forward toward the cornea, allowing for an increased image size on the retina. Furthermore, because of its flattened shape, the avian eye is capable of maintaining an entire visual field in focus at any one time – a major advantage over the rounder eye of mammals, where the point of focus is limited to a small area of the visual field. The mechanism of accommodation in reptiles and birds is essentially similar to that of mammals. Reptiles and birds have a well-developed ciliary body containing a ring of pad-like processes which make contact with the lens periphery. With contraction of the ciliary body musculature, these processes push on the lens, forcing it to acquire a more rounded shape, suited for near vision. In more advanced land mammals, the lens is suspended by zonular fibers that attach to the ciliary muscles, so the ciliary body is not in direct contact with the lens. Contraction and relaxation of the ciliary muscles modify the tension on the zonular fibers, altering the shape of the lens for near and far vision, respectively.

This brief and by far incomplete review of comparative lens structure and function indicates the extent of adaptive variation seen in vertebrate eyes. One thing that has remained constant amongst vertebrates is the manner in which the different parts of the eye – in particular, the lens – develop, differentiate, and continue to grow throughout life.

1.3. Development of the Vertebrate Lens

The establishment of the unique architecture of the lens in the embryo is thought to depend on a series of inductive interactions that initiate its differentiation from head ectoderm, as discussed in Chapter 2. These inductive influences are thought to emanate initially from the surrounding neural and non-neural tissues with which the ectoderm interacts throughout the course of development. Once established in the embryo, the lens is thought to maintain its distinct architecture and polarity throughout life by means of its interaction with the surrounding ocular media (Coulombre and Coulombre, 1963).

In this part of the chapter, we briefly describe the morphological changes that result in the transformation of a sheet of ectodermal cells into a highly specialised light-transmitting organ, the lens. This topic has been covered in great detail in many excellent books, including those by Mann (1928, 1950, 1964), Duke-Elder (1958), and Jakobiec (1982), which continue to serve as good resources. These texts primarily describe lens morphogenesis during human development. The goal of this text is to provide a more generalised and comprehensive view of lens development across a variety of the more commonly used animal models (see Fig. 1.13). The lens and the skin share a common embryonic origin; both are derived from the embryonic surface ectoderm. Consistent with this, the lens, like skin, continues to grow throughout life. Although skin is continually being renewed, with its superficial cells being replaced with the progeny of the more basally located stem cells, the cells of the lens are all retained, resulting in the lens becoming larger.

Unlike the eye in most invertebrates, which is solely derived from ectoderm, the vertebrate eye requires the contribution of three primordial tissues: the ectoderm of the neural tube, surface ectoderm, and mesoderm. The vertebrate lens is derived from the surface ectoderm, which also gives rise to many other tissues, including the corneal epithelium. In vertebrates, the eyes first appear as flattened areas (optic areas) at the anterior end of the embryonic plate on either side of the neural groove. At the stage when the neural groove deepens into the underlying mesoderm, the optic areas ‘dimple’ to form the optic pits. With the closure of the neural tube (presumptive central nervous system), the optic pits deepen, hollowing out to form the optic vesicles (presumptive neural retina and retinal pigmented epithelium). The optic vesicles are simply bilateral evaginations of the neural ectoderm of the forebrain (anterior end of the neural tube) embedded in mesoderm. This evagination process ultimately brings the outer surface of the optic vesicles into contact with the surface ectoderm (presumptive lens) at either side of the head. Thus the presumptive lens and retina originate from separate groups of cells situated within an ectodermal sheet. In the course of development, neural tube formation results in the folding of this ectodermal sheet so that the presumptive retina is internalised as part of the neural tube, with the basal aspects of the presumptive lens and neural cells now facing each other.

In most vertebrates, lens formation is initiated by the proliferation and palisading of the layer of ectodermal cells overlying the optic vesicle to form a thickened lens disc or plate (placode) at the area of contact. The lens placode remains as a single layer of columnar cells, although it appears stratified due to the staggered positioning of its cell nuclei. With further growth, the central part of the lens placode indents, invaginating from this small depression, together with the optic vesicle, to form the lens pit and double-layered optic cup, respectively. With increased cell crowding, the lens pit continues to deepen and remains open at the surface only by a small pore. This pore is closed off rapidly, creating a spherical lens vesicle of columnar epithelial cells attached to the overlying ectoderm via the lens stalk. The lens stalk gradually degenerates, completely separating the lens tissue from the