Development of the Retina

Appreciating the mechanisms responsible for normal retinal development is critical to understanding disease processes affecting the pediatric retina. Remarkably, the retina develops over a relatively long period of time beginning from the early gestational period through the first years of life. Consider, for example, that the human macula, which although first evident at a gestational age of 11 weeks, is still very immature even at birth [1]. The relative long window of retinal development is a vulnerability leaving this intricate tissue susceptible to specific genetic and environmental insults over an extended period. Furthermore, many of the central concepts in developmental biology were first discovered, and continue to be uncovered by studies of retinal development in various systems ranging from drosophila to man. Thus, the study of retinal development continues to provide important insights not only into the pediatric retina, the main subject of this book, but also into the mysteries of developmental embryology, vertebrate evolution, and disease pathophysiology. The following pages are meant to provide a foundation to the subject rather than a compendium of genes and syndromes. The emphasis taken, therefore, is to encourage the reader to understand the origin of the current concepts that exist, most importantly to appreciate that we are only at the tip of the iceberg with most important discoveries and breakthroughs yet to be made.

F. Gonzalez-Fernandez

Departments of Ophthalmology, Pathology & Anatomical Sciences, and Neurosciences of the State University of

New York, and Ira G. Ross Vision Research Center, Ross Eye Institute; Medical Research Service, Veterans Affairs Medical Center, Buffalo, NY, USA

e-mail: fg23@buffalo.edu

1.1  To suppose that the eye . . . could have been formed by natural selection, seems, I freely confess, absurd . . .1

Before considering the development of the retina, it is useful to reflect on the fact that the vertebrate retina is fundamentally different from that of other members of the animal kingdom. Indeed, this difference is critical to appreciating the development of the human retina, its specialized physiology, and finally, its vulnerability to disease states. Despite the inherent differences in the eyes of various members of the animal kingdom, much of what we understand regarding the mechanisms responsible for the development of the human retina comes from a synergism of research studies utilizing organisms ranging from Drosophila to mice. The introduction of new techniques in systems such as zebrafish [2–5] and Xenopus [6–17] promises to further accelerate the pace toward understanding the development of the human retina and the pathogenesis of diseases that affect the retina in both children and adults.

Molecular embryology is also causing us to revisit fundamental questions related to the evolution of the eye. Although morphological observations have provided compelling evidence that eyes emerged at least 40 separate times in the animal kingdom [18, 19], the remarkable functional conservation of master controller homeotic genes such as pax6 (see below) has challenged this position, arguing that all eyes derive from a common prototype monophyletically [20–23]. Whatever the mechanism for origin and evolution of eyes, the diversity of structure between eyes of different creatures is tremendous. For example, the eyes of insects are typically arranged

1Charles Darwin (1809–1882)

Fig. 1.1  Comparative evolution and embryology of the retina. (a) Compound eye of an insect with a sector exposed. Each ommatidia contains a separate lens (b in drawing), and set of eight retinula photoreceptors (f in drawing). (b) The eye of a typical cephalopod. Although reminiscent of the vertebrate eye, the cephalopod retina is inverted with the photoreceptors positioned on the inner surface of the retina (compare with (f )). (c) The vertebrate innovation of the choroid/RPE-photoreceptor complex

is made possible by the invagination of the optic vesicle into a two-layered optic cup. (e) An arthropod compound eye. (f) A cephalopod lens-eye. In chordates, photoreceptor cells differentiate from the central nervous system, whereas that of cephalopod and arthropod eyes differentiate from the epidermis. Drawings (a, b) and (c, d) are modified from Duke-Elder [327], and Fernald [211] respectively with permission from Elsevier

in faceted partial spheres consisting of thousands of ommatidia, the basic unit of the compound eye (Fig. 1.1a) [24]. Each ommatidium contains eight retinula cells with visual pigment contained in microvilli that organized into rhabdomeres analogous to vertebrate outer segments.

Although anatomically very different from our eyes, insects use the same molecule, 11-cis retinaldehyde, to capture photons [25–27]. However, it is in the way the retina reisomerizes the chromophore that the differences become apparent (reviewed in [28, 29]). In the rhabdomere, as in the photoreceptors of all animals, vision begins with the photoisomerization of 11-cis to all-trans retinaldehyde. However, in insects, the all-trans

isomer remains bound through its Schiff base to rhodopsin forming a thermally stable metarhodopsin. The G-protein activation is simpler for invertebrates because unlike vertebrate rhodopsin, the active invertebrate photoproduct can be formed directly without Schiff base deprotonation. From this metarhodopsin, the original rhodopsin is regenerated through the absorption of a second photon [30, 31]. Thus, insects have an elegant version of the vitamin A cycle consisting of rhodopsin/ metarhodopsin photoequilibrium.

Like insects, the dibranchiate cephalopods, which include squid, cuttlefish, and octopus, reform their 11-cis chromophore photochemically, but do so using a

second visual pigment. These marine invertebrates have a highly developed eye, which appears similar to vertebrate eyes in that it contains a single chamber with a single prominent lens (Fig. 1.1b). However, the cephalopod retina, which is composed of visual and supporting cells, has an inside-out arrangement compared to that of vertebrates [32]. In fact, their long photoreceptor outer segments (rhabdoms) are located in the inner retina close to the lens (Fig. 1.1c–f). In the rhabdoms, 11-cis retinaldehyde bound to rhodopsin is photoisomerized to the all-trans isomer forming metarhodopsin. However, unlike the insect system, cephalopod metarhodopsin is unstable resulting in the hydrolysis of the Schiff base and release of all-trans retinaldehyde. The retinoid is picked up by retinaldehyde binding protein, which translocates it to the photoreceptor inner segment myeloid bodies to complex with retinochrome [33–35]. It is there that the photon capture returns the chromophore to the 11-cis configuration. The 11-cis retinaldehyde is returned to the rhabdoms regenerating rhodopsin.

Thus, the insect and cephalopod systems rely on photochemical mechanisms to reform the 11-cis isomer. Howdoesthiscomparetothehumanretina?Appreciating the difference requires understanding the embryology of the vertebrate eye. The development of the vertebrate eye is fundamentally different from that of invertebrates in that the eye arises from an out-pouching of the neuroectoderm called the optic vesicle. Involution of the vesicle results in a two-layered optic cup. The outer layer of the cup remains as a single layer, the retinal pigment epithelium (RPE). The RPE is continuous with the outer epithelial layer of the iris and ciliary body. The inner layer of the optic cup becomes the inner pigmented layer of the iris, the inner nonpigmented layer of the ciliary body, and the neural retina. At the level of the iris and ciliary body, the two epithelial layers are physically attached through junctional complexes. However, the neural retina and RPE are separated only by the interphotoreceptor matrix (IPM) and, therefore, are susceptible to detachment, a common clinical problem.

The visual cycle in the vertebrate retina takes advantage of these anatomical arrangements. The photoreceptors have access to both the RPE and the Müller cells through the IPM; the emerging picture is that the RPE is largely responsible for rod chromophore regeneration, while the Müller cells appear to have an important role in cone chromophore regeneration through a separate, but not independent vitamin A cycle [27, 36, 37].

The significance of these anatomical relationships and the genetic basis of the inductive interactions occurring

between them is now beginning to be appreciated and is discussed below. This review is not meant to provide an exhaustive description of the field, as references to excellent reviews are cited throughout. The goal is to illustrate some of the more global principles that have emerged over the years, with particular emphasis on the continuum of basic and clinical observations. It is instructive that much of what we know about the development of the human retina has originated from nonvertebrate systems including pioneering studies in the fruit flies [38–40].

1.2  Good order is the foundation of all things2

A central question in any area of developmental biology is: How is the diversity of cell types controlled during the genesis of a complex tissue or organ?

Indeed, in a highly organized structure such as the retina, how are the intended types of cells produced in the right number, at the right location, and wired to the right cell? Furthermore, there are regional differences in the retina such as the fovea. What are the mechanisms responsible for the creation of this specialized region? Finally, how can these concepts help us understand the various disorders of the pediatric retina described in the chapters of this book?

Our discussion of these questions can only begin with the groundbreaking experiment published in 1924 that uncovered embryonic induction as a fundamental principle of development [41]. The experiment was performed by Hilde Mangold, a Ph.D. student in the laboratory of Hans Spemann in Freiburg [42]. Spemann was eventually awarded the Nobel Prize in 1935 for the work in embryonic induction. Tragically, Hilde Mangold died in an accident in 1924, when her kitchen’s gasoline heater exploded. At that time she was 26 years old and her paper was just being published.

Newt embryos were used for many of these experiments that typically involved delicate and technically demanding micro-dissections. Delicate surgical scalpels consisted of human hairs glued to glass rods as handles. One of the key experiments involved transplanting a structure on the dorsal side of the blastopore embryo (the dorsal lip) to the ventral side of another embryo. By grafting this tissue between differently pigmented Newt embryos, the fates of the grafted cells

2Edmund Burke (1729–1797)

could be followed. The grafted tissue is now known as the Spemann-Mangold organizer [43]. The organizer could induce the formation of neural tissues from ectoderm that would have otherwise assumed an epidermal fate (neuralization), and it caused dorsalization of the ventral mesoderm, leading to the formation of somites. This resulted in the formation of a second embryonic axis.

The important insight that these experiments provided is that cells can adopt their developmental fate according to their position when instructed by other cells [44–47]. As we shall see, this concept has as a central role in the normal and pathological development of the retina.

1.3  All that you touch you Change. All that Change Changes you3

The concepts put forward by Hans Spemann and Hilde Mangold establish a fundamental framework to understand organogenesis. The eye continues to provide one of the most powerful experimental systems to understand the complex processes involved in the three dimensional topographical changes required for organogenesis. Not only are the fundamental processes of ocular development emerging but we are also beginning to understand the mechanisms responsible for specific disorders such as aniridia and other developmental defects [48–51].

Organs including the eye are complex structures composed of different tissue types. The arrangement of these tissues requires a precise choreography to ensure the proper placement of specific tissues within the organ. Obviously, the precise morphological relationships of the final product cannot be tampered with. How does the remarkable arrangement of these tissues take place, particularly in an organ as complex as the eye?

The emerging picture is that the coordination is accomplished by one group of cells affecting the behavior of adjacent sets of cells. Such changes may include altering cell shape, mitotic rate, cell fate, and patterns of gene expression. These interactions, which are critical to organogenesis, occur at short range between two or

3Octavia E. Butler (1998)

more tissues [52–54]. Key to understanding­ the interaction is the history and properties of involved tissues. The ability of one tissue to influence another tissue in this way has become known as induction. Tissues that produce signals that change the behavior of a second tissue are referred to as “inducers.” The tissue being induced is the “responder” [45, 47, 55].

The importance of interactions between inducing and responding tissues in the development of the eye was first appreciated more than a century ago with the pioneering embryological manipulations performed by Hans Spemann and others. Using amphibian systems­ and microsurgical techniques, these early embryologists demonstrated a variety of tissue/tissue interactions critical to the formation of the eye. For example, when the optic vesicle of Xenopus is placed in an ectopic location underneath the cephalic ectoderm, it will induce that ectoderm to form lens tissue. Therefore, a broad area of anterior ectoderm is able to respond to the optic vesicle. Ectoderm that is able to respond by forming a lens is said to be “competent.” Although competence may extend to a broad region, it does not extend to the entire ectoderm. For example, in an experiment performed by Hans Spemann in 1912, ectoderm transplanted from the trunk of the embryo is not able to form a lens. That is, the lens forming bias or competence present in the head ectoderm does not extend to the trunk ectoderm (reviewed in [50, 56, 57]). As will be discussed further below, competence is not a passive state, but an activelyacquired condition dependent on the expression of specific homeotic genes such as pax6. Tissue interactions may also be inhibitory in nature. This is illustrated by the inductive interactions critical to bisecting the eye field into two distinct lateral zones. This induction, necessary for the formation of eyes as bilateral structures, was first studied by the classical embryologists. These pioneers noted that removal of the head mesoderm beneath the anterior part of the medullary plate often resulted in cyclopia. Reinvestigation of this phenomenon in recent years indicates that factors secreted from the prechordal plate suppress the competency of the central primitive eye field, effectively splitting it into two lateral fields (see below).

In summary, the vertebrate eye is formed through coordinated reciprocal inductions from three distinct embryonic tissues [56–61]. During the mid-gastrula stage, the endoderm and cardiac forming mesoderm