Pediatric Retina - StartseitePediatric retinal disorders include a wide range of highly diverse...

Pediatric Retina

James D. ReynoldsScott E. Olitsky (Editors)

Pediatric Retina

ISBN: 978-3-642-12040-4 e-ISBN: 978-3-642-12041-1

DOI: 10.1007/978-3-642-12041-1

Springer Heidelberg Dordrecht London New York

Library of Congress Control Number: 2010925788

© Springer-Verlag Berlin Heidelberg 2011

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James D. Reynolds, M.D.Professor and ChairmanDepartment of OphthalmologyUniversity at Buffalo Ross Eye Institute1176 Main StreetBuffalo, NY [email protected]

Scott E. Olitsky, M.D.Professor of OphthalmologySection of OphthalmologyChildren’s Mercy Hospitals and Clinics2401 Gillham RoadKansas City, MO [email protected]

v

Pediatric retinal disorders include a wide range of highly diverse disease processes. They range from the relatively straight forward, such as non-accidental trauma, to the very complex and confusing array of hereditary dystrophies. Few clinicians deal with all of these entities on a frequent basis, yet ophthalmologists who care for children will undoubtedly encounter nearly all of them occasionally. This fact makes a single comprehensive resource especially valuable. We believe this book represents that resource.

In developing a design for this text, we wished to address several points. The first issue was this need for a single comprehensive resource. But in dealing with this diverse pathology over the years, it was apparent that there often seemed to be a pedi-atric trained perspective distinct from the view of the retinal surgeon. Material written by authors from one group might be quite different from that written by members of a different group. We also noted that little published material attempted to synthesize basic science with clinical information. We wanted to create a text that would focus as much on pathogenesis as on natural history and that could merge laboratory and clinic. Thus the aim of this text is to provide a comprehensive single resource for all these diverse entities that would unite the different perspectives of pediatric and reti-nal surgeon, laboratory and clinic, and pathogenesis and clinical presentation.

We have attempted to accomplish this broad goal by careful author selection. Our contributors are a solid mix of pediatric and retina trained individuals. We also brought together many well known and well trusted clinicians, surgeons and clinician scientists with major laboratory research programs.

Each author or group of authors was given wide latitude in how they approached their assignment. Superficial consistency designed to make the chapters look alike was neither desired nor obtained. We did encourage comprehensiveness and real sci-ence. We appreciated uniqueness. This emphasis of substantive unity over the superfi-cial has produced a book with chapters of quite different looks. As an example, Dr. Gallie and company have produced a retinoblastoma chapter dramatically differ-ent from the norm. Each chapter will stand on its own. The book may be read cover to cover, but we expect few people will do this. We think most texts are used sporadically and in piecemeal fashion. Readers want to know about one disease at a time. And they would like to find it all in one place, including the wider primary references.

Even though our overriding concern was comprehensiveness, this was not always feasible. The critical reader will notice a difference in depth among the chapters. This is partly explained by the inclusion of material that focuses on a primarily adult dis-ease, such as diabetic retinopathy, in order to be comprehensive in breadth rather than depth. The breadth of the book is obviously also purposeful. We include conditions

Preface

ranging from the optic nerve to uveitis as well as extensive chapters on embryology, anatomy, physiology, and electrophysiologic testing.

We hope the reader will use this text as a frequent companion. If we have achieved our goals of a comprehensive text in both depth and breadth, of unifying the lab and the clinic, and in uniting the pediatric and retinal specialists’ perspective, the reader should come back again and again.

We would like to extend our thanks to all of our contributors. It has been a long, winding road and they have done a great job. We also appreciate the staff at Springer who have been extremely supportive. Our families deserve a thank you for support-ing, or at least tolerating, the additional time demands of an academic career. Finally, we would like to acknowledge the work of our ever present and never complaining assistant, Mrs. Elaine Taylor. Without her capable and dependable talent, this book could not have been done. She has always been our right hand. Thank you all.

Buffalo, NY, USA James D. Reynolds, MDKansas City, MO, USA Scott E. Olitsky, MD

vi Preface

vii

Contents

1 Development of the Retina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Federico Gonzalez-Fernandez

2 Anatomy and Physiology of the Retina . . . . . . . . . . . . . . . . . . . . . . . . . 39Göran Darius Hildebrand and Alistair R. Fielder

3 Electroretinographic Testing in Infants and Children . . . . . . . . . . . . . 67David G. Birch, Eileen E. Birch, and Rand Spencer

4 Retinopathy of Prematurity (ROP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85James D. Reynolds

5 Optic Nerve Malformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117Edward G. Buckley, Mathew Gearinger, Jin Jing, and Tamer Mahmoud

6 Inborn Errors of Metabolism Affecting the Retina . . . . . . . . . . . . . . . 147Scott E. Olitsky

7 Phacomatoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159Evelyn X. Fu and Arun D. Singh

8 Persistent Hyperplastic Primary Vitreous (PHPV) . . . . . . . . . . . . . . . 191Bruce M. Buerk, Mithlesh C. Sharma, and Michael J. Shapiro

9 A Language for Retinoblastoma: Guidelines and Standard Operating Procedures . . . . . . . . . . . . . . . . . 205Alejandra Valenzuela, Helen S.L. Chan, Elise Héon, and Brenda L. Gallie

10 Coats’ Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235Franco M. Recchia and Antonio Capone

11 Pediatric Hereditary Macular Degenerations . . . . . . . . . . . . . . . . . . . . 245Jack M. Sullivan, David G. Birch, and Rand Spencer

viii Contents

12 Generalized Inherited Retinal Dystrophies . . . . . . . . . . . . . . . . . . . . . . 295Shahrokh C. Khani and Airaj Fasiuddin

13 Vitreoretinal Dystrophies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315Magdalena F. Shuler, Jack M. Sullivan, Bernard R. Hurley, and J. Arch McNamara

14 Macular Choroidal Neovascularization and Defects in Bruch’s Membrane in Children . . . . . . . . . . . . . . . . . . 345Jonathan E. Sears

15 Proliferative Retinopathies in Children . . . . . . . . . . . . . . . . . . . . . . . . . 351Philip J. Ferrone and Steven Awner

16 Infectious Diseases of the Pediatric Retina . . . . . . . . . . . . . . . . . . . . . . 361Mohamed Hussein and David K. Coats

17 Abusive Head Trauma/Shaken Baby Syndrome . . . . . . . . . . . . . . . . . . 409Brian J. Forbes and Alex V. Levin

18 Pediatric Retinal Trauma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423Michael A. Samuel and Khaled A. Tawansy

19 Pediatric Uveitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433Christopher Hood and Careen Y. Lowder

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

1J. Reynolds and S. Olitsky (eds.), Pediatric Retina, DOI: 10.1007/978-3-642-12041-1_1, © Springer-Verlag Berlin Heidelberg 2011

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 con-cepts in developmental biology were first discovered, and continue to be uncovered by studies of retinal development in various systems ranging from droso-phila 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 syn-dromes. The emphasis taken, therefore, is to encourage the reader to understand the origin of the current con-cepts 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.

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 intro-duction of new techniques in systems such as zebrafish [2–5] and Xenopus [6–17] promises to further acceler-ate 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 sepa-rate times in the animal kingdom [18, 19], the remarkable functional conservation of master controller homeotic genes such as pax6 (see below) has challenged this posi-tion, arguing that all eyes derive from a common proto-type 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

Development of the Retina

Federico Gonzalez-Fernandez

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: [email protected]

1

1Charles Darwin (1809–1882)

2 F. Gonzalez-Fernandez

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 differ-ences 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 rho-dopsin forming a thermally stable metarhodopsin. The G-protein activation is simpler for invertebrates because unlike vertebrate rhodopsin, the active invertebrate pho-toproduct 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

Vitreousbody

Vitreousbody

Retina

Neural tissue

RetinaRetina

Retina

Lens

Optic nerve

Cuticularlens

abcde

fg

h

ij

k

l

e

h

ig

ck

dc

a

b

m

n

f

l

Lens

Lens-likebody

Microvillar photoreceptor cellCiliary photoreceptor cellEpidermal cells

Crystallinecone

a b

c d e f

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 posi-tioned 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 differen-tiate from the central nervous system, whereas that of cephalo-pod 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

31 Development of the Retina

second visual pigment. These marine invertebrates have a highly developed eye, which appears similar to verte-brate eyes in that it contains a single chamber with a single prominent lens (Fig. 1.1b). However, the cephalo-pod 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 trans-locates 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. How does this compare to the human retina? 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 neu-roectoderm 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 pig-ment 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 cili-ary 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 inter-photoreceptor matrix (IPM) and, therefore, are suscep-tible to detachment, a common clinical problem.

The visual cycle in the vertebrate retina takes advan-tage of these anatomical arrangements. The photorecep-tors 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 excel-lent 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 contin-uum 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 biol-ogy 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 ret-ina, 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 mecha-nisms responsible for the creation of this specialized region? Finally, how can these concepts help us under-stand 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 per-formed by Hilde Mangold, a Ph.D. student in the labo-ratory 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 experi-ments that typically involved delicate and technically demanding micro-dissections. Delicate surgical scal-pels consisted of human hairs glued to glass rods as handles. One of the key experiments involved trans-planting 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 epi-dermal 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 pro-vided 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 develop-ment 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 under-stand organogenesis. The eye continues to provide one of the most powerful experimental systems to under-stand 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 begin-ning to understand the mechanisms responsible for specific disorders such as aniridia and other develop-mental 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 rela-tionships of the final product cannot be tampered with. How does the remarkable arrangement of these tis-sues 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 behav-ior 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

more tissues [52–54]. Key to understanding the inter-action 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 ecto-derm, 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 ecto-derm 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 actively-acquired condition dependent on the expression of specific homeotic genes such as pax6. Tissue interac-tions may also be inhibitory in nature. This is illus-trated 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 3Octavia E. Butler (1998)


are thought to trigger the expression of the transcrip-tion factor otx2 in the late gastrula ectoderm. Inducers from the neural folds from the anterior neural plate then induce pax6 expression in the anterior ectoderm. It is thought that the expression of these transcription factors is important to making the surface ectoderm competent to respond to the optic vesicle during the late neural stage. In turn, the optic vesicle secretes factors that induce the synthesis of sox transcription factors. These factors initiate the production of the lens. The inner layer of the optic cup becomes the neural retina and the outer layer the retinal pigmented epithelium (RPE). In Hans Spemann’s own words, “Though the results in detail may need to be retested and supplemented in many cases, this seems to be cer-tain: that in several if not all species of the Amphibia in the neural stage or shortly after the closing of the medullary fields, the rudiment of the lens is more or less firmly determined; that the epidermis possesses the potency for lens formation in different degrees; and, finally, that the optic cup possesses the ability to activate this potency for lens formation” [62].

1.4 Men are born with two eyes, but only one tongue, in order that they should see twice as much as they say4

The formation of two eyes has provided vertebrates with stereopsis as well as a broader view of the world. How does the developing embryo form two separate and identical eyes? The solution to this mystery is integral to the larger question of how the central nervous system accomplishes bilateral development of most of its struc-tures [63]. Our current understanding, which is emerg-ing through the combination of clinical observations, classical embryology, and molecular biology, is that a single eye field is bisected by signals from the underly-ing prechordal mesoderm. The early eye field therefore represents a larger area of neural ectoderm that is com-petent to respond to the inducer signal to form an eye. If the competence of the central eye field is not inhibited

Fig. 1.2 When Hans Spemann performed his elegant optic vesicle ablation studies over a century ago, it was known that the lens derives from the ectoderm (pink), while the optic vesicle gives rise to the retina. However, what triggers the lens to form at its correct location over the retina? (a) The micro-dissection studies were technically demanding especially given the small size of the amphibian embryos. In this photograph, the optic vesicle can be appreciated budging under the surface ectoderm (arrows). The broad area of head ectoderm that is competent to form a lens shaded pink. (b) Cross-sectional diagram in the plane represented

by the white frame in panel A. After pealing back a flap of ecto-derm, Hans Spemann used a hot needle to selectively destroy the optic vesicle on one side while preserving its overlying ectoderm. (c) The remarkable finding was that ablating the optic vesicle completely prevented formation of the lens (green) during subse-quent stages of development. These observations, which indicate that signals from the optic vesicle are able to trigger lens differen-tiation in the adjacent competent ectoderm, provided the first experimental evidence of tissue induction [62, 328]). This figure is an unpublished diagram from the author’s laboratory

4Charles Caleb Colton (1780–1832)


when induction begins, the entire field will respond with the formation of a single eye. Typically, the inhibitory signals to the central eye field are not entirely absent, and fused globes result. In fact, true cyclopia, i.e., a single eye, is vanishingly rare, with most cases showing two fused eyes (synophthalmia). A case of synophthal-mia in Patau’s syndrome is shown in Fig. 1.3 [64]. A histological study of the eyes of this case is shown in Fig. 1.4 including immunohistochemical characteriza-tion of the dysplastic rosettes of the retina [64].

The problem of cyclopia should be considered in the context of the larger problem of creating bilaterality in

the CNS and dorsal-ventral patterning [65, 66]. Indeed, cyclopia is part of the spectrum of the devel opmental abnormalities seen in holoprosencephaly (a signal cerebral hemisphere). The phenotype of holoprosen-cephaly is quite variable consisting of a spectrum from severe manifestations with major brain and face anom-alies to clinically normal individuals with only a single fused central incisor to clue in the observer (reviewed in [67–71]). Holoprosencephaly is the most common developmental defect of the forebrain in humans with an incidence as high as 1:250 during embryogenesis. However, due to intrauterine lethality, the liveborn

Fig. 1.3 Synophthalmia in Patau’s syndrome. (a) Thirty- two-week fetus showing partial midline fusion with single pro-boscis. Autopsy revealed holoproencephaly and multiple developmental abnormalities including transposition of the great vessels with pulmonary artery hypoplasia; interventricular and interatrial septal defects; fused cerebral hemispheres with a common ventricle; absence of the olfactory bulbs and tracts, and

basal ganglia. One thalamus was present with partial medullar and cerebellar fusion. (b) Closer view of the partially fused eye lids inferior to a single centrally placed proboscis. (c) Partial karyotype illustrating triploidy of chromosome 13. Compare with Fig. 1.4. Adapted from Chan et al. [64] with permission of BioMed Central


prevalence is 1:16:000. Holoprosencephaly is a mal-formation sequence in which impaired midline cleav-age of the embryonic forebrain is a fundamental feature [66, 72–76]. The prosencephalon fails to cleave sagitally into cerebral hemispheres, transversely into telencephalon and diencephalon, and horizontally

into olfactory and optic bulbs. Given the number and complexity of the cellular interactions that must occur in the developing forebrain, it is not surprising that a variety of genes (at least 12 different loci) and a variety of teratogens have been implicated in the pathogenesis of holoprosencephaly [66, 68, 69, 72, 76–87].

Fig. 1.4 Histological analysis of synophthalmic eyes from case presented in Fig. 1.3. (a) Horizontal section showing that the eyes consist of two partially fused globes. Although the lens is separate, the posterior chamber is common, and there is a single optic nerve. (b) The neural retina in many areas does not show the usual laminar histology but is composed of collections of neurons arranged in apparent cylinders often with a central lumen. The lumen of the cylinders is usually rimmed by a defi-nite line reminiscent of the external limiting membrane (ELM) of the normal retina. (c) Immunohistochemical localization of rod

opsin. (d) Immunohistochemical localization of interphotorecep-tor retinoid-binding protein (IRBP). (e) Immunohistochemical localization of cellular retinaldehyde binding protein (CRALBP) demonstrates Müller cell differentiation consisting of radial pro-cess extending between the rosette neuronal cells. The processes abruptly end at the ELM-like structure. Taken together, these findings indicate that the rosette structures seen in this case of trisomy 13 represent a dysplastic process rather than a differen-tiation of neoplastic cells as in retinoblastoma. Modified from Chan et al. [64]


1.5 Sonic Hedgehog and Revisiting Homer’s Odyssey

Significant advances have been made in recent years toward understanding the pathogenesis of cyclopia and holoprosencephaly. In particular, the “Sonic Hedgehog pathway” has emerged as having a central role in both of these pathological states [88–93]. “Sonic the Hedgehog” was a popular Sega Genesis video game character. The Sonic Hedgehog gene (Shh) was named after this hero when it was noted that mutations of this gene cause spiny backs in fruit flies (Mr. Sonic Hedgehog has blue spines down his back!). Sonic Hedgehog is a secreted protein that acts as a “cell fate switch.” Shh is the most extensively characterized ver-tebrate homolog and is involved in a wide variety of embryonic events. It can act as both a short-range, contact-dependent factor and a long-range, diffusible morphogen. Shh genes are highly conserved. In the human embryo, shh is expressed in the notochord, the floorplate of the neural tube, the gut, and the develop-ing limbs. Interestingly, Hedgehog proteins undergo autocatalytic processing and modification that are crit-ical for signaling activity [89, 94–97]. Autoprocessing of Hedgehog includes covalent attachment of choles-terol onto the carboxy terminus of its N-terminal domain. The N-terminal domain contains all known signaling capabilities, while the C-terminal domain is responsible for the intramolecular precursor process-ing, acting as a cholesterol transferase [98]. The cho-lesterol moiety is thought to direct Hedgehog protein traffic in the secretory cell [99]. Furthermore, binding of cholesterol to Sonic Hedgehog enhances its solubil-ity, allowing it to diffuse as a paracrine factor [100–105]. This ability to act at distance is critical to its function as a morphogen signal during embryogenesis (reviewed in [106]).

The importance of cholesterol to the Hedgehog sig-nal transduction pathway appears to explain the terato-genic effects of the steroidal-alkaloid compounds, jervine and cyclopamine, the later deriving its name from its tendency to induce cyclopia. These com-pounds are now known to cause cyclopia by inhibiting Hedgehog signaling [99, 107, 108]. The specific mech-anism of cyclopamine’s action is through binding the product of the Smoothen gene (see below) [99, 107, 108, 137]. Interestingly, both jervine and cyclopamine are found in the Veratum plant family. Pregnant cattle, goats, or sheep that graze on the corn lily plant Veratrum californicum early during pregnancy can

give birth to deformed offspring with cyclopia. Since Veratrum plants are found in the Mediterranean regions, it is plausible that the legendary Cyclops of Odysseus was not completely an invention of Homer’s imagination but may have been based on the occa-sional observation of cyclopic ewes in ancient times.

Sonic Hedgehog exerts its morphogenic effects by diffusing to cells that express the cell surface receptor Ptch, which is homologous to the Drosophila segment polarity gene Patched-1 (reviewed in [109]). Ptch con-trols Hedgehog-responsive genes through the tran-scription regulatory molecule Cubitus Interruptus (Ci) (reviewed in [110]). Key to this pathway is Smoothen, a membrane protein that binds to Ptch. In the absence of Hedgehog binding to Ptch, Smoothened is inactive, and Ci is tethered to cytoplasmic microtubules. While parked on the microtubles, Ci is cleaved, and a portion of Ci diffuses into the nucleus where it represses transcription. However, Hedgehog binding to ptch blocks the ability of Ptch to inhibit Smoothened. As a result, intact Ci can enter the nucleus to activate Hedgehog response genes.

In view of the complexity of the Hedgehog pathway, it is not surprising that blocking cholesterol modifica-tion of shh is not the only road to holoproscencephaly. In humans, mutational activation of Ptch and Shh can also result in holoproscencephaly with cyclopia [72, 74, 77, 78, 80, 83, 87, 91, 111–113]. How does disruption of the Hedgehog pathway lead to cyclopia? The answer to this question comes through a series of elegant clas-sical embryological and modern molecular studies.

The debate over how the cyclopic eye forms has been going on for more than a century. The arguments are closely connected with the mechanisms normally leading to the formation of the two separate retinal pri-mordia. The various models for formation of the reti-nal primordia are reviewed in [114]. In theory, cyclopia could result from the fusion of two originally separate eyes, or from the failure of a single primordium to sep-arate during development. Recent work has established that the latter possibility is correct. Microdissection studies have shown that removal of the prechordal mesoderm leads to the formation of a single retina in chick embryos and Xenopus explants [49, 114]. For example, Li et al. [114] noted that removal of the pre-chordal plate resulted in fusion of the forebrain as well as the retina. This result is illustrated in Fig. 1.5. The future retinas were identified by in situ hybridiza-tion with a chicken Pax-6 probe. In ~27% of embryos without the prechordal plate, a single retina, contin-uous from one side of the embryo to the other, was


formed. That only a fraction of embryos had cyclopia was probably due to incomplete removal of the pre-chordal plate. The same investigators went on to show that the prechordal plate expresses shh and was able to rescue the cyclopic phenotype in transplantation

studies. Such studies provide strong support for a role of the prechordal plate in the formation of two retinas. Interestingly, transplantation of the prechordal plate to the vicinity of the optic cup was able to suppress the expression of pax6 in the retina. The key role of pax in

HNHN

a b

c d

e f

Fig. 1.5 Elegant microdissec-tion studies such as this one performed by Li et al. [114] in the chick embryo have shown that removal of the prechordal mesoderm leads to the formation of a single retina. The major conclusion from these types of studies is that there is a single retina morphogenetic field that resolves into two retina primordial. This is accom-plished by suppression of retina formation in the median region of the field. The signal for this repression, which presumably is Sonic Hedgehog, comes from the prechordal plate. The data depicted here show the effects of prechordal plate removal on retina formation in chick embryos. (a–d) are ventral views. (a) A diagram of a stage 5 chick embryo showing the location of presumptive retina primordial (indicated by two gray circles) relative to the prechordal plate (marked as a blue line). The red dot symbolizes Hensen’s node. (b) A diagram of the region removed from the prechordal mesoderm (indicated by a box superimposed on the blue line). (c) A stage 13 chick embryo showing Pax-6 expression in the eyes. (d) Pax-6 expression in a stage 13 chick embryo from which the prechordal plate was removed at stage 5. (e) A transverse section of a stage 13 control chick embryo after in situ hybridization with the Pax-6 probe. Ventral is up. (f ) A transverse section of a stage 13 embryo which lacked the prechordal plate. The level of section is similar to that of the control embryo shown in (e). Ventral is up. Reproduced with permission from Development (Li et al, [114])


development is discussed further below. The important conclusion from studies such as these is that there is a single retinal morphogenetic field that resolves into two retina primordia. “This is accomplished by sup-pression of retina formation in the median region of the field. The signal for this repression comes from the prechordal plate.” As pointed out by Li et al. [114], this conclusion is consistent with the model suggested more than 75 years ago by Adelmann [115].

New concepts in development often go hand-in-hand with new insights into the mechanisms of evolu-tion. This is exemplified by intriguing studies of the Hedgehog pathway in blind cave fish by Yamamato et al. [116]. These investigators found that the embry-onic midline controls eye degeneration in blind cave-fish by overactivation of the Hedgehog pathway. Key to their experiments was comparing ocular develop-ment in a species of the teleost Astyanax mexicanus that has normal vision and lives at the water surface with that of the blind species that lives in caves. Eye primordia are formed during cavefish embryogenesis. However, these primordia arrest in development, degenerate, and sink into the orbits. Remarkably, trans-planting a surface fish embryonic lens into a cavefish optic cup can restore a complete eye. Compelling evi-dence is provided that in the cavefish, expression of shh and the related tiggy-winkle Hedgehog gene (twhh) is expressed in an expanded area along the anterior embryonic midline. This expanded Hedgehog signal-ing results in hyperactivation of downstream genes, lens apoptosis, and arrested eye growth and develop-ment. These features can be mimicked in surface fish by twhh and/or shh overexpression. The observations require that we modify our thinking regarding the evo-lution of eye regression. It had been generally assumed that the regression was caused by the accumulation of function mutations in eye genes, which accumulate without penalty due to conditions of relaxed selection for eyesight. The findings of Yamamato et al. [116] raise the alternative paradigm that control of eye regression is achieved by a gain of function in Hedgehog or related midline-signaling. Eye regression should therefore be viewed as being driven by natural selection for an adaptive trait [116]. The use of new experimental systems such as the zebrafish model promises to shed more light onto the mechanism of cyclopia [75, 76, 82, 83, 117–122].

The elegant complexity of the Hedgehog signal pathway reflects its central importance as a regulatory

system. This is underscored by the realization that mutations of ptch cause Gorlin syndrome or nevoid basal cell carcinoma syndrome [123–127]. This syn-drome, which is inherited in an autosomal dominant manner, is characterized by dental, skeletal, and radiographic abnormalities including falx calcifica-tion, bifid/fused ribs and altered vertebral segmenta-tion, and a predisposition to tumor development including early-onset basal cell carcinomas [128]. In fact, ptch mutations are common in sporadic basal carcinomas.

Ocular abnormalities are often present in Gorlin syn-drome, including the first patient with this syndrome examined by Dr Gorlin [123]. However, of the various developmental abnormalities seen in the syndrome, the ocular findings, which are present in 15–25% of patients, are less well characterized [129, 130]. The ocular findings that may be present in Gorlin syndrome include defects of organogenesis (microphthalmia, coloboma, ocular hypoplasia), cataract, and posterior segment abnormalities (inappropriate retinal myelina-tion, retinoschisis) [131–136]. It is therefore intriguing that the Hedgehog pathway appears to play an impor-tant role not only in establishing separate eyes, but also in the normal development the retinal structures themselves. Of particular importance is the observa-tion that shh is expressed in retinal-ganglion cells. This could set the stage for an influence of the gan-glion cells on normal organization of the remainder of the retina. Interestingly, ptch and gli are expressed in retinal neuroblasts, and astrocyte precursor cells in the optic nerve [137, 138]. These relationships are sum-marized diagrammatically in Fig. 1.6. In this model, retinal ganglion cell-derived shh expression is required for Hedgehog target gene induction in the retina and optic nerve. This induction plays a role in precursor cell proliferation, photoreceptor differentiation, and normal cellular organization [139–141]. To address the question of the role of the Hedgehog pathway and the ptch receptor in particular on the development of the mammalian retina, Black et al. [142] studied ocular development in mice heterozygotic for disruption of the ptch gene [143–146]. The retinas of PtchlacZ(+/-) mice exhibit abnormal cell cycle regulation, culminat-ing in photoreceptor dysplasia and Müller cell gliosis. Interestingly, the PtchlacZ(+/-) mice also show vitreo-retinal abnormalities resembling those found in patients with Gorlin syndrome. In these patients, an intrareti-nal glial response results in epiretinal membrane


formation. These membranes, due to their proliferative and contractile nature, cause significant visual loss, especially in older patients with the syndrome. The investigators hypothesize that alteration of Müller cell/ Hedgehog signaling may play a role in the pathogen-esis of the idiopathic epiretinal membranes and rosette formation in the retina of these patients. The role of the Hedgehog pathway in retina disease will be further uncovered by ongoing research in several laboratories aimed at clarifying its role in the normal patterning and differentiation of the retina [138–140, 147–152].

1.6 The Homeotic Genes: Master and Commander5

Orchestrating the formation of a complex structure such as the eye requires turning on some, and suppress-ing other defined sets of genes at specific times during development. Gene regulatory proteins that do just that by recognizing short DNA segments were first appreci-ated in the 1950s. One of the first regulatory proteins to be recognized was the lambda repressor. Encoded by the bacterial virus, bacteriophage lambda, this repres-sor shuts off the viral genes that encode for viral coat particles. Turning off the synthesis of the coat proteins allows the virus to multiply silently within the cell.

Understanding how gene regulatory proteins work would have to wait for X-ray crystal structures of higher resolution than that which lead to the original discovery of the DNA double helix structure. Indeed, for 20 years after its discovery, the helix was thought to have a monotonous structure of ten nucleotides spaced exactly at 36° helical twists completing each spiral turn. However, better X-ray structures and solution NMR replaced this idealized structure with a nonuniform spi-raling DNA helix consisting of “major” and “minor” groves. The groves can be appreciated in Figs. 1.7 and 1.8. The larger domain provided by the major grove allows a new set of proteins to interact with the DNA. These regulatory proteins were first identified in bacte-ria and were called helix-turn-helix DNA-binding pro-teins because they consist of two a helices bent at an angle. The C-terminal helix or recognition helix inter-acts with the DNA by fitting into the major groove. Remarkably, the recognition helix is able to specifically identify the different base pairs from their edges with-out the need to open the double helix! Variations in the amino acid residues that make up the recognition domain dictate the specificity of the homeodomain for the particular DNA sequence. Outside of the helix-turn-helix, the remainder of the transcription factor can vary greatly, allowing various helix-turn-helix motifs to present their DNA binding motifs in unique ways. Furthermore, the regions of the protein outside of the homeodomain may modify the DNA-homeodomain interaction by making their own contacts with the DNA, thus fine-tuning the interaction. Finally, it should be mentioned that each homeodomain transcription factor

shh

Ptch

axonslight ERM

NB

RGC

IPL

INL

OPL

ONL

RPE

mullercell

Embryonic Adult Adult(dysplasia)

Rosette

Fig. 1.6 Clues to the histogenesis of retinal dysplasia are com-ing from studies of the effect of disruption of the Hedgehog pathway on the lamination of the neural retina. Of particular rel-evance are studies of retinal dysplasia in Gorlin syndrome. This syndrome results from mutations of the PTCH gene on chromo-some 9q23.1, the human homologue of the Drosophila patched gene. PTCH is a transmembrane protein that functions as the receptor for members of the Hedgehog family of intercellular signaling molecules (see text). Black et al. propose that disrup-tion of the ability of ptch receptor to respond to Sonic Hedgehog elaborated by the ganglion cells prevents the normal lamination of the retina with the formation of retinal rosettes [142]. Their model is shown here in a set of diagrams of the developing and adult mouse retina. At late stages of embryogenesis, the retina consists of two layers, the retinal ganglion cell (RGC) layer and the neuroblast (NB) layer, which contains proliferating precur-sor cells. RGC axons are located on the surface of the retina and exit the eye at the optic disc to form the optic nerve. The adult retina is organized into: RGC, inner nuclear layer (INL), and the rod and cone-containing outer nuclear layer (ONL). The nuclear layers are separated by the inner and outer plexiform layers (IPL, OPL), which contain neuronal processes. Müller cells span the width of the retina. In the embryonic and adult retina, Shh is expressed in RGCs, and Ptch is expressed neuorblastic layer (embryonic) and Müller cells (adult). The dysplastic retina (far right) shows rosette formation in the ONL, and an epiretinal membrane (ERM) at the vitreoretinal interface. Here, Muller cell processes have recruited contractile cells leading to retinal trac-tion. Adapted from Black et al. [142] with permission of Oxford University Press

5Patrick O’Brian (1969)


binds as a dimer to the DNA recognition sequences [153]. These DNA sequences are therefore arranged as symmetrically half-sites. An important point is that this allows each protein monomer to make a nearly identi-cal set of contacts, increasing binding affinity and allow-ing cooperative interaction with other factors.

It was not long after these genes were recognized in bacteria that their counter parts in the fruit fly were found. This led to the discovery of a class of genes called homeotic selector genes. Homeotic genes were found to play an important role in orchestrating fly body-plan development. When the sequences of sev-eral homeotic genes were compared, a striking feature was noted: each contained a nearly identical stretch of 60 amino acid residues. The conserved region defines this class of proteins and is termed the homeodomain. Interestingly, when the three-dimensional structure of the homeodomain protein was determined, it was found to have a helix-turn-helix structure related to that already known in bacteria. This was the first indication that principles of gene regulation in bacteria may also apply to higher organisms. Indeed, homeodomain pro-teins are now well recognized from bacteria to man.

To appreciate the significance of homeotic genes to the development of complex structures such as the human eye, it may be helpful to digress momentarily to the early history of the field. In 1859, Charles Darwin, motivated by curiosity of the origin of diver-sity, noticed that repetition of elements along the length of the body was a common feature of many animals and that the variation of related structures contained in these elements contributes to diversity. It is now well recognized that serial homology is a feature of meta-meric (segmented animals), whose structures such as legs, nerve ganglia, appendages, blood vessels, and so on occur within each segment. Darwin considered that through natural selection, structures gradually change from one form to another. However, the zoologist William Bateson, who coined the term “genetics,” pointed out that evidence for intermediate forms was often lacking. Bateson was therefore particularly inter-ested in the phenomenon where the structures on a par-ticular segment were transformed to the structures normally present on another segment [154, 155]. In 1894, he defined the term homeosis as the process whereby one segment is transformed into the likeness of another. Examples of homeosis are present in both

Fig. 1.7 The significance of the X-ray crystal structure of the human Pax6 paired domain-DNA complex is that it provides a general model for Pax protein-DNA binding. This image, based on the structure reported by Xu et al (1999, Genes & Development. 13:1263-75) [153], is from the RCSB Protein Data Bank (www.rcsb.org/pdb; NDB ID: PD0050).


plants and animals. For example, Cuenot (1921) noted that amputation of the antenna of the stick insect caused regeneration of a leg instead of an antenna in its place [338]. This concept is particularly interesting in that it went against Darwin’s hypothesis that structures change gradually through evolution.

Almost 30 years would go by before Thomas Morgan in 1923 working on fruit flies in his crowded lab at Columbia University made the observation that homeo-sis is inherited and that the responsible genes appear to reside on the fly’s third chromosome [156]. Fifty more years would pass before the discovery of a complex of genes whose role in development is to define the appendages that characterize each of the three segments that make up the thorax [157, 158]. This insight into the

molecular basis of homeosis came from a set of muta-tions in Drosophila that cause strange changes in the body of the adult fly. For example, in the antennapedia mutant, legs replace antennae on the head. In the bitho-rax mutant, an extra pair of wings appears where nor-mally there should be small appendages known as halteres. These mutations fit Bateson’s definition of homeosis as the transformation of parts of the body into structures appropriate for another position.

The homeotic selector genes are all part of a multi-gene family and lie in two gene clusters, the bithorax and the antennapedia complexes. The bithorax complex controls differences in abdominal and thoracic segments, while those in the antennapedia complex control the dif-ference in the thoracic and head segments. Together,

Fig. 1.8 Stereo view of the X-ray crystal structure of the Pax6 paired domain-DNA complex. The N-terminal Pax6 ribbon is in blue. The image may be viewed in 3-D without specialized stereo glasses. Suggestions for viewing molecular stereo images are available at http://spdbv.vital-it.ch/TheMolecularLevel/0Help/StereoView.html. Structural coordinates of Xu et al (1999, Genes & Development. 13:1263-75) [153] (RCSB Protein Data Bank, NDB ID: PD0050), are visualized here using PyMOL (2007, DeLano Scientific)


both clusters are known as the HOX complex [159–167]. Hox complexes are now known to be present in all ani-mals from cnidarians (jellyfish and corals) to humans. As mentioned earlier, the products of all the homeotic selector genes are similar in their DNA binding region. This region is 60 amino acid residues in length and is termed the homeodomain. The corresponding segment in the DNA is referred to as the homeobox, from which the abbreviation HOX is derived [168–170].

As “master and commander,” the homeotic genes preside over sets of genes that must be activated or repressed in a coordinated, time-sensitive manner to generate complex structures at the right time and in the right place [169, 171–173]. This is wonderfully shown by the coordination of segmentation itself in the devel-opment of the fly embryo. These elegant studies, which led to the 1995 Nobel Prize in Medicine, vindicated the approaches of experimental embryology and estab-lished many of the principles that apply to the induc-tive interactions taking place in the development of the retina (for excellent reviews see [158, 174–177]).

One class of Drosophila segmentation genes, the paired-axial homeobox ( pax) genes, is particularly rel-evant to the normal and pathological development of the human retina. Of the nine pax genes characterized to date, only four have been shown to cause abnormal development of the ocular structures: Waardenburg’s syndrome ( pax3) [178–183], Aniridia ( pax6) [184, 185], Peter’s anomaly (pax6) [186–188], and renal coloboma syndrome ( pax2) [189–193]. The corre-sponding spontaneous mouse mutants are Undulated (pax1) [183, 194], Splotch (pax3) [181, 183, 195–197], Small eye ( pax6) [183, 185, 196, 198–202]. Recently, analysis of spontaneous and transgenic mouse mutants has revealed that vertebrate Pax genes are key regula-tors during organogenesis of the kidney, eye, ear, nose, limb muscles, vertebral column, and brain [183, 188, 194, 203–209]. Like their Drosophila counterparts, vertebrate pax genes are involved in pattern formation during embryogenesis, possibly by determining the time and place of organ initiation or morphogenesis.

The role of pax6 in development has stimulated new thinking as well as controversy regarding the evolution of eyes. In his Origin of the Species, Darwin admitted that a structure as complex as the eye being formed by natural selection was difficult to accept. To address this problem, he proposed that eyes evolved from an imper-fect eye prototype from which more advanced visual organs would have arisen gradually through natural

selection. Indeed, numerous intermediates between the most primitive eye and that of vertebrates have been described. Although these observations do at first appear to support Darwin’s model, the profound diversity of structure and function among different eyes in the ani-mal kingdom appears to go against a monophyletic ori-gin (here we define an eye as a light-sensitive organ capable of forming an image). To reconcile the remark-able diversity with a mechanism based on natural selec-tion, it has been proposed that eyes arose independently at least 40 separate times [18, 210–212]. This view has been challenged by the observation that expression of the mouse pax6 gene in drosophila can induce the for-mation of ectopic functional eyes at various locations, including the legs, wings, and antennae [213–215]. These observations suggest to some biologists that pax6 is the master control gene for eye morphogenesis [20, 21, 213, 216]. This has led to the proposal that the vari-ous types of eyes in the animal kingdom evolved from a single prototype [23, 213, 216–220], a notion supported by the high degree of conservation of pax6 between flat-worms to humans [197, 217, 221–224]. However, nei-ther of the above models for the evolution of the eye appears to adequately embrace the profound diversity of eyes in the animal kingdom and the central role of pax6 in eye determination. Possibly resolving the con-troversy is the concept of intercalary evolution. This idea, which has recently been applied to the problem of eye evolution, provides an interesting model that could account for the monophyletic origin of the eyes and explain their fantastic diversity (the reader is encour-aged to see [21, 22, 216]).

1.7 More than Meets the Optic Vesicle6

The molecular processes touched upon above provide some of the mechanisms for a set of reciprocal inductive interactions that culminate in the formation of the retina. In this choreography, whose participants are the surface ectoderm, neuroepithelium, and the neural crest-derived mesoderm, timing is everything. The performance has intrigued biologists for over a century [225].

The process begins with the formation of optic vesi-cles protruding from the neural tube bilaterally. As shown in the scanning electron photomicrographs of

6Margaret Saha et al. (1989) [56]


Fig. 1.10, the optic vesicle extends to eventually make contact with the surface ectoderm. The surface ecto-derm is induced to form the lens. As the lens expands, the optic vesicle involutes forming the optic cup. The lip of the cup forms the iris and ciliary body; remainder gives rise to the retina. This embryology explains the two-layer architecture of the adult eye. The lip of the cup, now the iris epithelium covering the posterior sur-face of the iris, consists of two pigmented epithelial lay-ers continuous as the pupil margin. Proximally, the layers become the two layers of the ciliary body. The inner (vitreal) layer loses its pigment and is continuous with the neural retina, while the outer layer remains pig-mented and is continuous with the retinal pigmented epi-thelium (RPE). Figure 1.11 shows the juncture between the developing ciliary body and retina.

How the different regions of the optic vesicle become specified to form the above structures is largely unknown although inductive interactions appear to be central to the process. We now appreciate that the sur-face ectoderm of the head region becomes biased to form the lens through a series of early inductive inter-actions. For this reason, this surface ectoderm is referred to as the “prelens ectoderm” [50, 56, 57, 226]. At the point of contact between the two tissues, the cells of the prelens ectoderm thicken and palisade to form the “lens placode” (see Fig. 1.10d). At the reciprocal point of

contact in the optic vesicle, the cells at the tip of the vesicle also begin to palisade to form the retinal disc. Interestingly, this contact is essential to the specifica-tion of the neural retina [227]. The prelens ectoderm is a source of fibroblastic growth factor (FGF) [228–230]. Furthermore, FGF-mediated signaling can substitute for the prelens ectoderm in specifying the neural retina [231, 232]. The tightly apposed prelens ectoderm and optic vesicle then invaginate to eventually form the early lens and optic cup, respectively (Fig 1.10).

A fundamentally important question is what are the mechanisms that bring about the involution of the optic vesicle to form the optic cup? Although reciprocal interactions are key to specification of the lens and neu-ral retina, recent studies suggest that formation of the optic cup from the optic vesicle does not require assis-tance from the lens, as was previously thought [233]. This is not to say that the initiation of optic vesicle invagination does not require association of optic vesi-cle tissue with prelens ectoderm during a discrete tem-poral period. In an elegant set of experiments utilizing the developing chick embryo, Hyer et al. (2004) surgi-cally removed lens tissue at various stages of develop-ment, from the prelens ectoderm stages to the invagination of the lens placode and optic cup [233]. Their findings are summarized in Fig. 1.12. Removal of the prelens ectoderm resulted in persistent optic

Fig. 1.9 Immerging model of the molecular basis of embryonic lens induction. Ogino et al. (2008) provide evidence that inte-gration of signals from homeodomain protein Otx2 (pink) and SuH, a nuclear signal transducer of Notch signaling lead to lens expression in the presumptive lens ectoderm. These inputs converge to activate the major enhancer of Lens1, a gene essen-tial for lens formation (green). (a, b) The head region, by expressing Otx2 is competent to respond to the Notch signaling. (c) According to the model, Otx2 and SuH although binding to the Lens1 enhancer, remain in a quiescent state until receiving

input through Notch signaling. Delta2 a Notch ligand, which is expressed in the adjacent optic vesicle, activates Notch signaling by releasing the intracellular domain of Notch (blue rectangle). This domain separates from Notch and moves to the nucleus. The Notch intracellular domain complexes with SuH promoting the activation of Lens1 transcription. The localized Delta / Notch signaling therefore defines lens formation at focal regions of the competent (Otx2 expressing) ectoderm. These unpublished dia-grams of the author are based on the work of Ogino et al. Development 135, 249–58 (2008) [338]


vesicles. That is, the lens did not form and the vesicles failed to invaginate into cups. Interestingly, these vesi-cles did show neural retinal differentiation, despite the failure to invaginate. If the surgery is conducted at a later stage, that is after the formation of the lens pla-code, again the lens failed to, but the vesicle did go on to invaginate normally. These results indicate two impor-tant points. First, the optic vesicle neuroepithelium requires a temporally specific association with prelens

ectoderm to undergo neurogenesis. Second, the optic cup can form in the absence of lens. In summary, the prelens ectoderm induces the optic vesicle to form an optic cup (Fig. 1.12). Although the molecular signaling pathways involved are not known, the role of retinoic acid signaling in this process may be a fruitful avenue of future research [235, 236].

As the optic vesicle invaginates, the outer layer dif-ferentiates into the RPE, while the inner layer becomes

Fig. 1.10 Scanning electron photomicrographs of the develop-ing mouse retina. (a) Fronto-lateral surface view of the head region at gestational age 8.5 days (corresponds to ~25 days in humans). Note that the left optic vesicle has created a lateral budge on the embryo. (b) Cut away of through the center of the bulging optic vesicle shown in (a). Note that the optic vesicle impinges upon the surface ectoderm (far left surface). (c) Cut away at gestational age day 9 (~28 days in humans). Note the formation of the optic stalk/optic vesicle. (d) Cut through thick-

ening lens placode and the adjacent portion of the optic vesicle, which is beginning to invaginate. Thickening of the ectoderm and beginning of the invagination of the optic vesicle (GA day 10; ~29 days in humans). (e) The invaginating lens placode forms the lens vesicle, which here is about to pinch off from the surface ectoderm. The invagination of the optic vesicle has formed the bilayered optic cup. Note that at this stage, the optic cup remains connected to the forebrain via the optic stalk. Images contributed by Dr. Kathleen K. Sulik


the neural retina. Species differences exist in the overall structure of the optic vesicle. For example in teleosts, instead of a hollow vesicle, the retina arises from flat wing-like protrusions [237]. The RPE is a highly specialized epithelium that is a multifunctional and indispensable component of the vertebrate eye. Although a great deal of attention has been paid to its transdiffer-entiation capabilities and its functions in neural retina development, little is known about the molecular mech-anisms that specify the RPE itself. Recently, advances in our understanding of the genetic network that controls the progressive specification of the eye anlagen in

vertebrates have provided some of the initial cues to the mechanisms responsible for RPE patterning. The emerging picture is that there are specific transcription factors, including otx2, mitf, and pax6, and a few signal-ing cascades that accomplish the onset of RPE specifica-tion in vertebrates (reviewed in [238]).

The cells that compose the early optic vesicle are indistinguishable from each other as they all express transcription factors otx2, pax6, rx1, and six3 [239]. As a result, any region of the optic vesicle is at first competent to give rise to neural retina, optic stalk, or RPE. As development proceeds, this capability is regionally restricted by signaling molecules that coor-dinate expression of a limited number of specific tran-scriptional regulatory pathways. In particular, fibroblast growth factor (FGF) suppresses RPE specification. The FGF signal may arise from the lens placode [230, 240, 241]. FGF signaling is transduced through tyrosine-kinase type FGF receptors, which can activate a wide variety of signal transduction cascades. In contrast, other signals promote RPE differentiation. These sig-nals, such as the tumor-derived growth factor (TGFb) family member activin A, appear to come from the sur-rounding extraocular mesenchyme [242–244]. Of cen-tral importance is the microphthalmia-associated transcription factor (mitf), which encodes a transcrip-tion factor of the basic helix-loop-helix and leucine zipper family. Mitf has a conserved and fundamental function in the development of melanin-producing cells and is activated through the receptor tyrosine kinase (RTK) pathway (reviewed in [245, 246]). Interestingly, the microphthalmia mouse was found to be deaf and had a white patch of fur, features resem-bling the human syndrome Waardenburg syndrome, type II. Using a candidate gene approach, the mouse mitf gene was instrumental in isolating its human homolog, which led to the identification of Waardenburg mutations in a DNA binding protein encoded by the human MITF locus [247–249]. Cells of the optic vesi-cle destined to become neural retina express the tran-scription factor chx10. One of the functions of chx10 is to inhibit mitf expression [250]. Interestingly, human microphthalmia is associated with mutations in CHX10 [251]. Finally, the orthodenticle-related transcription factors (otx), which are homeodomain-containing tran-scription factors with an important role in anterior head formation, are also initially expressed throughout the entire optic vesicle. However, their expression becomes restricted to the presumptive RPE during optic cup for-mation. Finally, the paired-box transcription factors

Fig. 1.11 Photomicrograph of the rat retina at postnatal day 2. In rodents, differentiation of the retina occurs primarily postna-tally. The lower panel is a higher magnification of the region designated by the white asterisk showing details of the RPE/neural retinal interface. Arrowheads, retinal pigment epithelium (RPE); arrow in top panel, inner layer of the cilliary body; arrow in bottom panel, mitotic figure in neural retina. Note that gan-glion cell layer has already differentiated (black asterisk, upper panel). Unpublished images from the author’s laboratory


(pax) appear to make an important contribution. Pax2 is a negative regulator, while pax6 is a positive regula-tor of RPE specification. In summary, the optic vesicle is a dynamic structure. As it invaginates to form the optic cup, it segregates itself regionally into domains that will form the inner layer of the cup including the neural retina and domains that will form the RPE. This specification of different regions of the vesicle is accomplished through the interplay of a limited num-ber of regulatory pathways. Disruption of these path-ways is responsible for Waardenburg syndrome and some forms of microphthalmia.

The formation of the optic cup brings the inner and outer layers in close apposition. In fact, at the level of the iris and ciliary body, the two epithelial layers are physically attached by junctional complexes. At the level of the retina, the neural retina and RPE remain separated by a unique extracellular matrix that fills the subretinal compartment or space. Thus, the double lay-ered retina is an innovation bringing into physical proximity the photoreceptors, RPE, and Müller cells [252–254]. Each of these cells borders the subretinal

compartment, which is filled with an interesting extra-cellular material termed the IPM [255, 256]. The IPM is a complex structure consisting of interphotorecep-tor retinoid-binding protein (IRBP), growth factors [257, 258], metalloproteases [259], hyaluronan and hyaluronan binding proteoglycans [260], and sulfated glycosaminoglycans [261]. The IPM appears to medi-ate many of the critical interactions among the photo-receptors, RPE, and Müller cells, including retina/RPE adhesion, outer segment phagocytosis, outer segment structural stability, and nutrient exchange.

The possibility that the IPM has a significant role in development deserves further attention. Indeed, the development of the vertebrate retina depends on retina-RPE interactions [262, 263]. The presence, as men-tioned above, of growth-promoting substances in the IPM is consistent with this notion. Furthermore, the expression pattern of some IPM components suggests a role in the development. Interestingly, IRBP, which is thought to function in the adult retina to transport retinoids in the vitamin A cycle, accumulates in the subretinal space before the retinoid cycle is operational

11 12 13 13+ 14

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Fig. 1.12 How does the optic vesicle form? The emerging picture is that the prelens ectoderm, and not the lens, provides a critical signal to induce involution of the optic vesicle to form the optic cup. This diagram summarizes recent elegant microdissection studies of Hyer et al. [233]. The normal sequence of steps during the normal formation of the optic cup from the optic vesicle are shown: contact between the optic vesicle tissue and prelens ecto-derm occurs from stage 11 to 13, after which the corresponding retinal placode and lens placode form at stage 13, and finally, the

lens vesicle begins to invaginate concomitantly with the distal tip of the stage 14 optic vesicle. Amazingly, if the prelens ectoderm is removed at stage 12 or earlier, the optic vesicle will not invagi-nate. However, if the prelens ectoderm is removed slightly later, at stage 13, the optic vesicle is capable if invaginating, forming a cup without a lens, leading to the conclusion that early communication between ectoderm and optic neuroepithelium provides the funda-mental information for early optic cup formation. Reproduced from Hyer et al. [233] with permission of Academic Press


[264–268]. The gene for IRBP is expressed early during rodent retinal development and is up-regulated before that of opsin [266, 269–273] (Fig. 1.13). Perhaps IRBP participates in retinal development by facilitating the transport of retinoids or nutrients between the RPE and developing retina. Targeted disruption of IRBP results in early photoreceptor degeneration in transgenic mice [274]. In the Xenopus embryo, IRBP is first expressed

by photoreceptors in the central retina, and a central-to-peripheral gradient of IRBP appears to be established by diffusion of IRBP through the subretinal space [275]. Such a gradient could allow IRBP to transport retinoids and fatty acids from the RPE to the developing periph-eral retina. The potential role of the IPM and its compo-nents in modulating interactions between the RPE and neural retina is a promising area for future research.

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Fig. 1.13 Comparison of the emergence of the interphotorecep-tor matrix (IPM) with photorecep-tor differentiation. The graph summarizes quantitative densito-metric analysis of autoradiograms at multiple exposure times. The integrated density of opsin and IRBP mRNA bands relative to actin mRNA is plotted against postnatal age. Densitometric measurements were taken from films exposed from three different lengths of time as detailed in [271]. The corresponding drawings depicts the postnatal development. Shortly before birth, junctional complexes between the RPE and neuroblastic epithelium are released. At P0 (day of birth), the neural retina is separated from the RPE by a thin extracellular matrix (stippled, area not drawn to scale). By P5, the matrix has greatly enlarged as inner segments protrude into the subretinal space. At P10, primitive outer segments are present over most of the inner segments. By P20, the matrix has accumulated to accommodate the expanded volume of the subretinal space. Arrowhead, external limiting membrane. Reproduced from Gonzalez-Fernandez et al (1993) with permission of Academic Press [271]


1.8 Retinal Histogenesis: A Controlled Explosion

In a short period of time, the retina generates in an almost explosive fashion more than the sufficient num-ber of cells to comprise the mass of the retina. Simultaneously, in a highly coordinated manner, the retina achieves a wide array of different cell types located at the right position, in the right ratio, and con-nected to the right neurons [276, 277]. Accomplishing all of these goals is truly an amazing feat! Although we are far from having a complete picture of how this is orchestrated, some of the underlying mechanisms are beginning to emerge. The following paragraphs are meant to provide only an overview of what is one of the most exciting arenas of developmental neurobiology.

Once again, the retina shines as a model system providing one of the most elegant models not only into the development of its own complexity, but also into that of the central nervous system in general. A fundamental question in developmental neurobiol-ogy is how are cell fates in the CNS determined? A significant achievement of recent years has been a

better understanding of the relative contribution of nature vs. nurture to the process of cell fate determina-tion. The emerging picture is that the different cell types are not produced from predefined lineages [278, 279]. Postmitotic retinal neurons and Müller glia are produced from a pool of cycling progenitors. As development proceeds, specific cell types leave the cell cycle in an orderly fashion. However, various cel-lular approaches using different species are converg-ing on a model for cell-fate determination. This model combines the role of extrinsic as well as intrinsic regu-lators in controlling the cell-fate choices. Central to the model is the concept that the progenitor cell passes through intrinsically determined competence states [52, 280]. While passing through these states, the pro-genitors are capable of giving rise to a limited subset of cell types under the influence of specific extrinsic signals (reviewed in [281]) (Fig. 1.14).

The first component of this model is the temporal pat-tern of the appearance of the various retinal cell types. Cell birth dating performed over two decades ago pro-vided important key early observations into the process [282, 283]. These impressive studies showed that the

Progenitor cells

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Ganglion Cone Horizontal Amacrine Rod Müllerglia

Bipolar

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Mouse E11.5 E13.5 E15.5 E17.5 P0 P2 P4 P6

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Fig. 1.14 Time dependent changes in the competence of retinal progenitor cells plays an important role in determining the birth order of the different types of retinal cells. Reprinted by permis-

sion from Macmillan Publishers: Dyer and Bremner. Nat. Rev. Cancer (2005) [329]


various retinal cells are generated in a defined sequence. The order in which cells are born can be defined as the day on which they undergo their last S phase. This can be monitored by using [3H]thymidine labeling and auto-radiography. In humans, as well as many other species, ganglion cells are the first to be born. This is illustrated in Fig 1.14, which shows a photomicrograph of the developing rat retina at postnatal day 2. At this stage, the only retinal neurons that have clearly differentiated are the ganglion cells, which have formed a clearly defined layer within the inner layer of the primitive retina. It is also important to point out that at these early stages of retinal development, the RPE is clearly present (Fig. 1.11). Figure 1.15, 16 show immunohistochemical staining for cellular retinaldehyde-binding protein (CRALBP). In the developing retina, CRALBP is pres-ent within the RPE (Fig. 1.14b). In the adult rat retina, CRALBP is also expressed by the Müller cell glia within the retina (Fig. 1.15). However, at postnatal day 2, the Müller cells have not differentiated, and CRALBP is noted only in the RPE (Fig. 1.15). Thus the primitive neural retina, which is sandwiched between formed RPE and ganglion cell layers, is positioned to be under the inductive influence of soluble morphogens elaborated by the RPE on one side and the ganglion cells on the other.

Following the retinal ganglion cells, the next cells to differentiate in the developing vertebrate retina are the cone photoreceptors, horizontal cells, and amacrine cells. Following these cells is a second wave consisting of the rod photoreceptors, bipolar cells, and Müller cells. The relative appearance of these cells during the histogenesis of the retina is summarized in Fig. 1.14. The final result is the highly organized vertebrate ret-ina. A photomicrograph of the normal human retina shown in Fig. 1.17.

The second component of the current model for the histogenesis of the neural retina is the linear relation-ships among the retinal cells. Elegant lineage analysis studies in various species using either intracellular injec-tion of tracers or retroviruses appear to converge on a similar mechanism. The retinal progenitor cells them-selves appear to be multipotent. In fact, even in the last mitotic division of the progenitor cell, diverse cell types can still be produced [278, 279, 284–287]. Thus, the specific cell types that comprise the neural retina can be born at the same time. These observations suggest that specific extrinsic cues are important to direct the final cell fate. In vitro culture, systems have proved valuable information to identify the factors and the competence of the progenitors [288–290].

Fig. 1.15 Continuity of the developing peripheral retina with the ciliary body and iris epithelia. The rodent retina is a particularly useful experiment system as much of the development of the retina occurs during the 2 weeks following birth. Albino strains such as the one used here circumvent the problem melanin mask-ing immunoperoxidase or immunofluorescence staining. The photomicrographs shown in this figure correspond to the periph-eral rat retina at the second postnatal day of life. (a) H&E stained section showing that the neural retina is still largely undifferenti-ated at this age (white asterisk) except for the ganglion cells, which are clearly present (black asterisk). The ganglion cells have a larger nucleus compared to the undifferentiated retino-blasts and are restricted to their own layer in the inner retina. The RPE is continuous with the outer layer of the ciliary body (white arrows). At this location, the optic cup consists of the inner (black arrows) and outer epithelial layers of the ciliary body and iris. Both epithelial layers consist of a single row of cells. The two rows extend to the iris to line its posterior surface (far left pair of white and black arrows). The photomicrograph also shows the primitive angle and corneal endothelial layer (arrow heads). (b) Section similar to that in (a) except that due to processing artifact, there is compression of the angle with displacement of the lens (L) against the ciliary body. The section was probed with an antibody against cellular retinaldehyde-binding protein (CRALBP) and counterstained with toluidine blue, which has little affinity for the cytoplasm. The immunospecific staining for CRALBP labels the cytoplasm of the RPE and the outer ciliary body epithelial layer (arrows; brown reaction product). Sections treated with nonimmune serum showed no staining (data not shown). This staining for CRALBP in the ciliary body, although useful to show the continuity of the epithelial layers, is transient in the ciliary body disappearing in the adult retina (see also refer-ences [26, 330–333]). The antibody used for this immunohis-tochemical study was provided by Dr. Jack Saari. The images represent unpublished data from the author’s laboratory


There appears to be a state of competence allowing cells to respond to environmental cues [291]. This competence is controlled by intrinsic cues and cannot be modified [292]. In contrast, how competent cells will respond depends on a combination of positive and negative cues (reviewed in [287, 293–295]). Insight

into the connection between birth order and compe-tence comes from studies where the cell cycle is exper-imentally altered in the developing peripheral Xenopus retina. As shown in Fig. 1.18, the peripheral retina of the Xenopus is less differentiated than that of the cen-tral retina. In most vertebrates, retinal differentiation proceeds from a central-to-peripheral direction. Furthermore, in many animals, such as fish, frogs, and birds where the eye continues to enlarge significantly beyond the postnatal period, continued retinoblast pro-liferation allows the retina to expand together with the enlarging eye. The addition of mature retina is largely due to the proliferation of stem cells located in the peripheral retina. This proliferation with continual cell cycle exiting of some of the progenitor daughters pro-vides a wonderful system to study the relationship between external cues and cellular competence in the control of cell fate [296]. Ohnuma et al (2002). took advantage of this feature in the developing Xenopus retina to explore the coordination between birth order and the relative timing of cell cycle exit [54]. To con-trol cell cycle exit, these investigators missexpressed specific cell kinase inhibitors. Using this strategy, they found that early cell cycle exit enhances the retinal ganglion cell promoting activity of Math5 [54, 297]. Furthermore, inhibiting cell cycle exit biases the cell toward later retinal neuron cell fates. Taken together, these data suggest a model of the histogenesis in which early cell cycle withdrawal enhances the activity of factors that promote early cellular fates. Conversely, late cell cycle withdrawal inhibits proneural function and pushes cells toward later fates. In summary, an interplay between cell cycle control and cellular deter-mination helps to coordinate retinal histogenesis (see also [298–305]).

1.9 Focusing on the Fovea: A Marvel of Development

In view of the importance of the fovea to vision, it is remarkable that so little is known regarding the mechanism of its formation. The fact that the struc-ture is virtually restricted to primates may be part of the problem. As a result, the techniques and the experimental approaches proven so powerful in sys-tems such as Drosophila and mice may not be appli-cable to understanding histogenesis of a structure not

Fig. 1.16 Comparison of the expression of CRALBP in the adult and developing retina. The photomicrograph is from a 2.5-month-old albino rat retina probed with antibody directed against CRALBP. At this age, immunospecific reactivity for CRALBP is present not only in the RPE but also in the Müller cell glia (com-pare with Fig. 1.15). The Müller cells span the entire thickness of the neural retina; sclerad, their villous processes protrude into the subretinal space; vitread, the Müller foot processes form the inner limiting membrane, a true basement membrane lining the inner retinal surface. The inner limiting membrane does not take up the tolulidine counter stain well. Its location is designated by the slanted arrowhead. Insert shows the pattern of CRALBP expression in the developing retina at postnatal day 2. Mitotic divisions are present at the outer edge of the neuroblastic epithe-lium (white arrows). Note that although the RPE expresses CRALBP, at this age, no expression is present in developing neu-ral retina. This differential expression of CRALBP in the embry-onic retina compared to that in the adult retina is due to the fact that the RPE differentiates early during retinal development, while the Müller cells emerge relatively late (compare with the histogenesis birth order summary in Fig. 1.14). Black arrowhead, separation artifact of the neruoblastic epithelium from the apical RPE surface; White arrowheads, RPE nuclei; Black arrow, gan-glion cell nucleus. The antibody used for this immunohistochem-ical study was provided by Dr. Jack Saari. The images represent unpublished data from the author’s laboratory


present in those animals. Nevertheless, many of the principles of retina development discussed above will certainly have central roles. For example, pax has a key function as mutations in this gene disrupt the nor-mal development of the fovea [306–308]. A wealth of background information on its structure, function, and

development may be found in a number of thoughtful reviews [309–314].

The fovea is characterized by a high density of cone photoreceptors centered within its depression, a unique neuronal circuitry, and absence of a local retinal-blood supply. Remarkably, the location of the future fovea is

Fig. 1.17 Structure of the normal adult human retina (periph-eral macula region). The retina is a highly ordered structure with its various cell types arranged in discrete lamina. The layers are typically named according to their relative position with respect to the vitreous. Layers closer to the center of the eye are termed “inner,” and those closer to the sclera as “outer.” Within each layer, various cell types can be identified. For example, cone cell nuclei line up on the outer edge of the ONL. The remainder of the nuclei in the ONL belongs to the rods (compare with higher magnification photomicrographs in Fig. 1.18). Note that the

domain from RPE through ONL is avascular. For detailed description of the anatomy of the human retina with wonderful photomicrographs and drawings, the reader is referred to Hogan’s atlas of the human eye [334]. Labels: Scl sclera; C chor-oid; RPE retinal pigment epithelium; BM Bruch’s membrane; OS outer segments; IS inner segment; ONL outer nuclear layer; INL inner nuclear layer; GCL ganglion cell layer. The section was stained with hematoxylin and eosin. Image from the author’s laboratory and Ophthalmic Pathology Service of the Ross Eye Institute

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