Epigenetic signals resulting from either cell-matrix or cell-cell interactions are critical to the regulation of cell differentiation and development. This is particularly true in the complex differentiation process that enables a lens epithelial cell to become a differentiated lens fiber cell and in the developmental events that direct a region of head ectoderm to invaginate, pinch off, and begin to form the lens. In this chapter we discuss the role of cell adhesion molecules in lens differentiation and development.

Extracellular Matrix

The molecular organization of the basement membrane can profoundly influence cellular behavior by providing information that can affect the genetic program of a cell. Its major components include proteins such as laminin, collagen type IV, fibronectin, and proteoglycans. These extracellular matrix (ECM) proteins direct differentiation-specific gene expression in most cell and tissue types (Bissell and Barcellos-Hoff, 1987; Bissell et al., 1982; Streuli et al., 1991). Their ability to orchestrate both cell differentiation and tissue development requires interaction with cell surface receptors (e.g., the integrins), by which they initiate specific intracellular signaling pathways (Giancotti and Ruoslahti, 1999). The expression and distribution of ECM proteins in the lens is well characterized, both throughout development and in the mature lens. The knowledge that has been gained, in combination with mutational and inhibitor studies, provides insight into the role of ECM molecules in the process of lens development.

Introduction

Regulation of the cell division cycle is an essential process by which the cell monitors its growth and differentiation. Maintaining the proper controls on these cellular processes is essential not only during embryonic development but also throughout the lifetime of an animal. During embryonic development, in a temporally and topographically distinct manner, a wide variety of cells exhibit the capacity to become quiescent, to proliferate, and to irreversibly withdraw from the cell cycle and undergo terminal differentiation. Thus, both the entry of a cell into the cell cycle from a state of quiescence and the exit of the cell from active cycling must be precisely regulated if normal cell growth and differentiation are to be maintained. Furthermore, these two distinct types of cell cycle regulation must be coordinated with the regulation of differentiation. Over the past decades, much has been learned about the mechanisms that control cell cycle progression in vitro, primarily as it relates to cancer. Only in recent years has an understanding of how the cell cycle is controlled in vivo in normal development begun to emerge.

The ocular lens has served as a model system for unraveling the roles of cell cycle regulatory genes in a developmental context. A relatively simple tissue with a well-described blueprint of cell division and morphogenesis, the lens has been ideal for studying the coordination of both cell growth and differentiation in vivo.

Introduction

The cornea and the lens are the principal refractive elements of the eye responsible for, respectively, stationary and variable refraction. However, while both the cornea and the lens must be transparent to function properly, the basis of their transparency is quite different. In general, the cornea relies on the continuous pumping of interstitial fluid across its semipermeable surface membranes and a supramolecular organization of collagen fibrils for clarity. In contrast, lens transparency is presumed to be the result of a highly ordered arrangement of its unique fiberlike cells, or fibers, and a gradient of refractive index produced by a variable crystallin protein concentration within the fibers. However, while lens gross anatomy has a major role in determining lens optical quality (variable focusing power), lens ultrastructure is the principal factor in determining lens transparency. Furthermore, while all vertebrate lenses have a similar form, or structure, their anatomy is not identical, and thus their optical quality varies from species to species and as a function of age. In fact, on the basis of structure, four types of lenses can be distinguished. Key differences in lens morphology, caused during specific periods of development and growth, result in quantifiable variations in optical quality. Furthermore, lens structural anomalies, caused during the same periods of development and growth, result in quantifiable degradation in optical quality. Thus, vertebrate lenses are a prime example of form following function and malformation leading to malfunction.

Introduction

The lens consists of two morphologically distinct cell types, an unremarkable cuboidal epithelium that covers the anterior surface and concentric layers of fiber cells that account for the remainder, and vast majority, of the tissue volume (Fig. 9.1). The fiber cells are unique in the body. They have an enormous aspect ratio, being no more than a few micrometers wide but often exceeding a thousand micrometers in length. In cross-sectional profile, they appear as flattened hexagons, and their sharply angled membranes enclose a transparent cytoplasm that lacks the organelles found in typical cells. It is striking that these cells of remarkable shape and composition are derived from the more typical cells of the overlying epithelium.

In this chapter, we examine what is known (and, more often, what is not) about the process of terminal differentiation in the lens. We propose a staging system that allows one to discriminate critical periods in the maturation of a lens fiber cell. Using this system, we follow a hypothetical fiber cell through the differentiation program, from the time when it is an unspecialized epithelial cell near the lens equator to the cessation of protein synthesis that occurs when it is a mature fiber cell buried in the lens core. We include speculations on how the differentiation program might act to influence the shape and thus the optical properties of the lens as a whole. Finally, it seems evident that in some cataracts at least, the differentiation program has been interrupted or corrupted.

Introduction

Just as the ocular lens gathers and focuses light, so too has it captured and focused the attention of developmental biologists. Since Spemann's first experiments introduced the concept a century ago, the vertebrate lens has served as a model for the phenomenon of embryonic induction. Figure 2.1 provides a diagrammatic representation of the major steps in vertebrate lens determination to illustrate the physical relationships among developing tissues during stages pertinent to this review. The figure is based on the chick embryo, as its relatively flat topology during the earliest stages of development is particularly convenient for illustrative purposes. The lens differentiates from a region of head ectoderm that early in development lies adjacent to the region of the neural plate from which the retina will form (Fig. A). As development proceeds, the region of presumptive lens ectoderm (PLE) is not in contact with the retinal rudiment, as the neural plate folds up into a closed tube (Fig. C), but it is brought into close proximity to the retinal anlage by virtue of the outgrowth of the optic vesicle (OV) from the forebrain (Fig. D). The first overt signs of lens formation appear only after the OV establishes close contact with the PLE. After contact is made, the PLE thickens to form a placode (Fig. E) that subsequently invaginates simultaneously as the inward collapse of the OV forms the double-layered optic cup (Fig. F).

Introduction

One of the most remarkable processes in nature is the process of replacing or regenerating damaged tissue. Some salamander species possess the capacity to regenerate a variety of tissues and organs as adult organisms. Other higher vertebrate species also possess regenerative abilities, but these are limited to early embryonic stages and⁄or tissues that can undergo renewal (Tsonis, 2000, 2001). Lens regeneration in the adult urodele amphibian represents one of these unique processes in which major cellular events such as dedifferentiation and transdifferentiation regulate tissue replacement. Dedifferentiation involves terminally differentiated cells reentering the cell cycle and losing the typical characteristics of their origin, whereas transdifferentiation allows a cell to change its identity and become a completely different cell type. During lens regeneration, the cells that undergo this transformation are the pigment epithelial cells (PECs) of the dorsal iris. This cell-type conversion is not usually observed in terminally differentiated cells that have followed a developmental path and had been determined in phenotype and function. Cancer cells share similarities with the PECs that undergo the regenerative process. In the former, during oncogenesis, the original phenotype is destabilized and the cells divide, resulting in uncontrolled growth, eventual invasion to other organs/tissues, and the production of tumors. During lens regeneration, there must be a mechanism or program that destabilizes the cell phenotype but at the same time carefully directs these cells to divide, reorganize, and redifferentiate to new cell types that will be responsible for replacing the lost parts.

Introduction

The study of lens development provides a useful experimental system for investigating fundamental processes in developmental biology. The vertebrate lens develops from a series of interactions between the surface ectoderm, the optic vesicle, and the surrounding mesoderm, and these interactions involve successive steps of bias, competence, specification, and differentiation (see chap. 2 of this volume; see also McAvoy et al., 1999; Hirsch and Grainger, 2000). In recent years, these cellular and morphogenetic processes have been subject to investigation focusing on the molecular events underlying them (Weaver and Hogan, 2001). In particular, important insights were gained through genetic studies performed on the development of the eye in Drosophila (Treisman, 1999) and by comparisons of gene expression and function between the eyes of invertebrate and vertebrate species (Hill et al., 1991; Quiring et al., 1994; reviewed in Wawersik and Maas, 2000; Wawersik et al., 2000). These studies have led to the identification of conserved regulatory pathways mediating eye formation in both the fly and vertebrates.

Additional insight into these molecular events has been provided by the evaluation of mouse or human syndromes in which morphogenesis is defective (Freund et al., 1996; Graw, 2000). The eye is frequently affected by inherited eye disorders: roughly one—quarter of the phenotypes listed in Mendelian Inheritance in Man involve the eye (Boyadijiev and Jabs, 2000), and several candidate genes implicated in these phenotypes have so far been identified.

Introduction

The transparency of the lens is closely linked to the structure and function of its cell membranes. To minimize light scattering, lens fiber cells are packed together in a tightly ordered array so that the space between the cells is smaller than the wavelength of light. Achievement of this configuration requires cell membranes to have sets of proteins able to facilitate the formation of junctions between cells, while its maintenance requires the effective regulation of cell volume. For a long time, much of the work on lens membranes was primarily devoted to the identification and biochemical characterization of intrinsic and peripheral membrane proteins and of the membrane lipids in which they are embedded. The main emphasis of this early work was on how these molecules were modified during lens aging and cataractogenesis, often without knowledge of their functions. It is only in the last 10 years that significant progress has been made on the important contributions cell membranes and their embedded proteins make to the physiology of the lens. The greatest quantum step in this context has without doubt been the discovery that the lens generates a circulating current (Robinson and Patterson, 1983). This current is believed to generate a circulating flux of ions and water that percolates through the lens carrying nutrients deeper into the lens and returning waste products to the lens surface more efficiently than is conceivable by passive diffusion alone (Mathias et al., 1997).

Lens Anatomy and Development (Pre-1900)

The past decade has witnessed a tremendous increase in the basic understanding of the molecules and signal transduction pathways required to initiate embryonic lens development. Other advances in this time period have elucidated structural and physiological properties of lens cells, often in an evolutionary context, making it possible to frame many pathological conditions of the lens as errors of specific developmental events. All of these recent advances rest on the fundamental observations of talented investigators in previous decades and centuries. While several texts describe the history of ophthalmology as a clinical discipline, the conceptual history of basic eye research as a science, and in particular the history of lens development research, is a much less traversed subject. Though it is inevitable that we cannot include all of the many important experiments and personalities that have played fundamental roles in shaping the field of lens development, we hope to stimulate appreciation for those pioneers, both past and present, to whom we owe a debt of gratitude for their contributions to the field.

Throughout human history, the sense of sight has been both treasured and revered. Without doubt, visual loss resulting from lens dysfunction has always plagued the human family. In the early years of lens development research, investigations of the eye were intertwined with the genesis of the field of ophthalmology.

How the lens develops and acquires its distinctive morphology and growth patterns has been a major research focus for developmental biologists. Growth factors are known to play key roles in influencing cell behavior and cell fates during development. In recent years researchers have identified some of the growth factor families involved in regulating the processes of lens induction, morphogenesis, and growth. The aim of this chapter is to review the current state of knowledge in this key area of lens research.

The lens develops from head ectoderm that is associated with an evagination of the developing brain: the optic vesicle (Fig. 11.1). Soon after these two tissues become associated, the presumptive lens ectoderm grows and thickens to form the lens placode. Subsequent invagination of the placode forms the lens pit, which later closes to form the lens vesicle. Cells in the posterior segment of the lens vesicle, next to the optic cup, elongate to form the primary fibers, whereas cells in the anterior segment of the vesicle differentiate into epithelial cells. These divergent fates of embryonic lens cells give the lens its distinctive polarity. From this stage onwards, the lens grows by continued proliferation of epithelial cells and differentiation of fiber cells. Proliferation initially occurs throughout the lens epithelial compartment but during development becomes progressively restricted to a band of cells above the equator, known as the germinative zone. Progeny of divisions that shift below the equator enter the transitional zone and elongate to give rise to secondary fibers.