Progress in molecular biology and translational science, volume 134

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CONTRIBUTORS
Zsolt Ablonczy
Department of Ophthalmology, Albert Florens Storm Eye Institute, Medical University
of South Carolina, Charleston, South Carolina, USA
Leopold Adler IV
Department of Ophthalmology, Albert Florens Storm Eye Institute, Medical University
of South Carolina, Charleston, South Carolina, USA
S. Amer Riazuddin
Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland,
USA
Jeffrey H. Boatright
Department of Ophthalmology, Emory University School of Medicine, Atlanta, and Center
for Visual and Neurocognitive Rehabilitation, Atlanta VA Medical Center, Decatur,
Georgia, USA
Hannah E. Bowrey
Department of Ophthalmology, Albert Florens Storm Eye Institute, Medical University of
South Carolina, Charleston, South Carolina, USA
Nicholas P. Boyer
Department of Ophthalmology, Albert Florens Storm Eye Institute, Medical University
of South Carolina, Charleston, South Carolina, USA
Barbara M. Braunger
Institute of Human Anatomy and Embryology, University of Regensburg, Regensburg,
Germany
Ranjay Chakraborty
Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia,
USA
Chunhe Chen
Department of Ophthalmology, Albert Florens Storm Eye Institute, Medical University

of South Carolina, Charleston, South Carolina, USA
Seung-il Choi
Corneal Dystrophy Research Institute, Yonsei University College of Medicine, Seoul,
Republic of Korea
Micah A. Chrenek
Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia,
USA
Rosalie K. Crouch
Department of Ophthalmology, Albert Florens Storm Eye Institute, Medical University of
South Carolina, Charleston, South Carolina, USA

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xvi

Contributors

Ales Cvekl
Departments of Genetics and Ophthalmology and Visual Sciences, Albert Einstein College of
Medicine, Bronx, New York, USA
Lucian V. Del Priore
Department of Ophthalmology, Albert Florens Storm Eye Institute, Medical University of
South Carolina, Charleston, South Carolina, USA
Allen O. Eghrari
Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland,
USA
J. Fielding Hejtmancik
Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes
of Health, Bethesda, Maryland, USA

Mark A. Fields
Department of Ophthalmology, Albert Florens Storm Eye Institute, Medical University of
South Carolina, Charleston, South Carolina, USA
Rudolf Fuchshofer
Institute of Human Anatomy and Embryology, University of Regensburg, Regensburg,
Germany
James L. Funderburgh
Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
Eldon E. Geisert
Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia,
USA
Jie Gong
Department of Ophthalmology, Albert Florens Storm Eye Institute, Medical University of
South Carolina, Charleston, South Carolina, USA
John D. Gottsch
Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland,
USA
Hans E. Grossniklaus
Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia,
USA
Andrew J. Hertsenberg
Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
Shengping Hou
The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Lab of
Ophthalmology, Chongqing Eye Institute, Chongqing, PR China
Winston Whei-Yang Kao
Edith Crawley Ophthalmic Research Laboratory, Department of Ophthalmology, College
of Medicine, University of Cincinnati, Cincinnati, Ohio, USA
Aize Kijlstra
University Eye Clinic Maastricht, Maastricht, The Netherlands

Contributors

xvii

Eung Kweon Kim
Department of Ophthalmology, Vision Research Institute, Severance Hospital; Corneal
Dystrophy Research Institute, and BK21 Plus Project for Medical Science and Severance
Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Republic
of Korea
Masahiro Kono
Department of Ophthalmology, Albert Florens Storm Eye Institute, Medical University of
South Carolina, Charleston, South Carolina, USA
Yiannis Koutalos
Department of Ophthalmology, Albert Florens Storm Eye Institute, Medical University of
South Carolina, Charleston, South Carolina, USA
Hun Lee
Department of Ophthalmology, Vision Research Institute, Severance Hospital, and Corneal
Dystrophy Research Institute, Yonsei University College of Medicine, Seoul, Republic of
Korea
Chia-Yang Liu
Edith Crawley Ophthalmic Research Laboratory, Department of Ophthalmology, College
of Medicine, University of Cincinnati, Cincinnati, Ohio, USA
Wei Liu
Departments of Genetics and Ophthalmology and Visual Sciences, Albert Einstein College of
Medicine, Bronx, New York, USA
Peter Y. Lwigale
Department of Biosciences, Rice University, Houston, Texas, USA
Caitlin E. Mac Nair

Ophthalmology and Visual Sciences, and Cellular and Molecular Pathology Graduate
Program, University of Wisconsin—Madison, Madison, Wisconsin, USA
Rebecca McGreal
Departments of Genetics and Ophthalmology and Visual Sciences, Albert Einstein College of
Medicine, Bronx, New York, USA
Pia R. Mendoza
Department of Ophthalmology, Emory University School of Medicine, Atlanta,
Georgia, USA
Ravi Metlapally
UC Berkeley School of Optometry, Berkeley, California, USA
T. Michael Redmond
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, NIH, Bethesda,
Maryland, USA
Robert S. Molday
Department of Biochemistry and Molecular Biology, Centre for Macular Research,
University of British Columbia, Vancouver, British Columbia, Canada
Robert W. Nickells
Ophthalmology and Visual Sciences, University of Wisconsin—Madison, Madison,
Wisconsin, USA


xviii

Contributors

John M. Nickerson
Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia,
USA
Machelle T. Pardue
Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia,

USA
Kevin Schey
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA
Robin H. Schmidt
Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia,
USA
Daniel Schorderet
IRO - Institute for Research in Ophthalmology, Sion; Faculty of Life Sciences, Swiss Federal
Institute of Technology, and Department of Ophthalmology, University of Lausanne,
Lausanne, Switzerland
Alan Shiels
Department of Ophthalmology and Visual Sciences, Washington University School of
Medicine, St. Louis, Missouri, USA
Deborah L. Stenkamp
Department of Biological Sciences, University of Idaho, Moscow, Idaho, USA
Felix L. Struebing
Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia,
USA
Ernst R. Tamm
Institute of Human Anatomy and Embryology, University of Regensburg, Regensburg,
Germany
Janey L. Wiggs
Harvard Medical School, and Massachusetts Eye and Ear Infirmary, Boston,
Massachusetts, USA
Christine F. Wildsoet
School of Optometry, University of California, Berkeley, California, USA
Charles B. Wright
Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington,
Kentucky, USA
Peizeng Yang

The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Lab of
Ophthalmology, Chongqing Eye Institute, Chongqing, PR China
Qingjiong Zhang
State Key Lab of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University,
Guangzhou, PR China
Yan Zhang
School of Optometry, University of California, Berkeley, California, USA


PREFACE
The only thing worse than being blind is having sight but no vision.
Helen Keller

The visual process is complex, depending on a combination of precise functions of the anterior segment, which serve to focus light precisely on the retina, and the posterior segment, which receives light signals, transforms them
into electrical signals, and performs preliminary processing before transmitting them through the optic nerve and pathways to the visual cortex. Each
component of this system must function precisely and dependably for correct vision. This requires that each part of the visual system undergoes appropriate developmental regulation, that each molecule in the various metabolic
and functional pathways functions correctly, and that they all interact seamlessly in carrying out visual perception. Finally, the biological systems that
support and maintain homeostasis of the cells making up the visual system
are required to protect and preserve vision over the lifetime of the individual
to prevent age-related causes of blindness such as age-related cataract and
macular degeneration.
One way in which to gain insight into the intricate processes supporting
vision is through the examination of inherited diseases affecting vision, and
from these the proteins and pathways which they affect. In addition to obvious candidates, such as rhodopsin for retinal degenerations and lens
crystallins for cataracts, the study of inherited visual diseases has identified
previously unsuspected pathways and processes critical for visual function,
including the role of complement and other immune regulators in the retina
and processes such as message sequestration and autophagy in the lens. However, the process works both ways: in order to study or even understand the
molecular genetics of vision, one must have a firm foundation in the basic
biochemistry, cell and developmental biology, and molecular biology of its

component parts. It is this interrelationship between the basic sciences and
the study of inherited diseases, and eventually the clinical application of
knowledge derived from both, that this book aspires to delineate.
In order to accomplish this intertwining of basic biology, genetics, and
clinical application, coverage of each component of the eye begins with its
developmental biology and progresses through its biochemistry and molecular biology before finishing with the molecular genetics of its inherited
diseases. The material aims at being approachable by a graduate student

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Preface

or even an advanced undergraduate, while at the same time being of sufficient depth to provide even an advanced researcher a concise overview of
each area. In that vein, while avoiding being cumbersome each chapter is
sufficiently referenced to provide access to the original literature in the area
which it covers. Finally, the chapters are written to maintain the interest of
the reader and hopefully will inspire young scientists to pursue a career in
vision research.
J. FIELDING HEJTMANCIK
JOHN M. NICKERSON


CHAPTER ONE

Overview of the Visual System
J. Fielding Hejtmancik*, John M. Nickerson†,1
*Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health,

Bethesda, Maryland, USA
†
Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia, USA
1
Corresponding author: e-mail address:

Abstract
This introduction provides an overview of the retina, in which we survey the fundus,
layers of the retina, retinal cell types, visual transduction cascade, vitamin A cycle, neuronal wiring of the retina, and blood supply of the retina.

The visual system includes the optical components of the anterior segment
of the eye: in order, the cornea, the aqueous humor, and the lens; and the
posterior segment, including the vitreous body, the retina, and the optic
nerve. Finally, the visual system includes the optic tracts and optic radiations,
transmitting neural signals to the visual cortex, and several additional nuclei
of the brain (Fig. 1). Each of these components is critical in receiving, transmitting, and interpreting visual information.
The optical components in the anterior segment of the eye focus light
onto the retina, which then transduces the light signal into neural signals.
In addition, the retina also carries out initial processing of the neural signals
before passing them through the optic nerves and tracts to central nervous
system components that carry out their elaborate processing and integration
with other senses. In addition, the oculomotor system, basically the efferent
arm of the visual system, controls stability of position of the eyes as well as
directing and coordinating movements of the eyes to objects of interest.
Light initially traverses the anterior chamber where it first passes through
the transparent cornea, aqueous humor, lens, and vitreous body (Fig. 1). The
speed with which light travels through each of these components is inversely
proportional to its density, with the ratio of the velocity of light in a vacuum
to the velocity in medium being the refractive index. Thus, light waves
striking the surface of the cornea at an angle are slowed differentially, so that

the light entering the cornea first is slowed more than that which travels longer through air. This bends the direction of the light called refraction. If the
components of the anterior segment, especially the cornea and lens, develop
Progress in Molecular Biology and Translational Science, Volume 134
ISSN 1877-1173
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2015 Elsevier Inc.
All rights reserved.

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J. Fielding Hejtmancik and John M. Nickerson

Figure 1 Overview of the eye including the anterior and posterior segments. The refraction of light rays from the fixation point at the tip of the arrow to the focal point on the
retina represents the summed effects of the anterior and posterior surfaces of the cornea and lens. The image of the arrow is projected in an inverted orientation on the
retina.

into the proper shape and optical density, and are appropriately transparent,
light rays originating from point source similarly are focused onto a single
point on the retina. This results in the image of an object being projected
in an inverted fashion onto the retina, so that the inferior visual field is projected onto the superior region of the retina, and the nasal visual fields are
projected onto the temporal retina. The entire biology of the anterior segment is oriented toward accomplishing the clear transmission and sharp
focusing of light onto the retina, and many of the genetic lesions of this part
of the eye interfere with this task.
In the retina, the light signals transmitted by the anterior segment are
converted to neural signals that undergo some initial processing before being

transmitted through the optic nerve and radiations to the brain. The retina
comprises two functional and structural parts: the retinal pigment epithelium
or RPE, which is the nonneural component, and the adjacent but distinct
neural or sensory retina. RPE cells contain melanin granules, which absorb
light passed through the retina, preventing its reflection by the sclera, which
would and degrade the quality of vision. In addition, the cells of the RPE aid
the photoreceptors by recycling visual pigments and phagocytizing shed
photoreceptor outer-segment tips. This requires that the outer segments
including the visual pigments are physically close to the RPE layer.


Overview of the Visual System

3

Conversely, the neural processing networks of the retina are the anteriormost structures, through which light passes before stimulating the photoreceptor cells in the posterior layer of the retina adjacent to the RPE (Fig. 2).
The neural retina is composed of six neuronal-cell types as well as nonneuronal glial Mu¨ller cells. These exist in three nuclear layers: from anterior
to posterior the ganglion cell, inner nuclear, and outer nuclear layers, separated by the inner plexiform and outer plexiform layers, in which synapses
occur. The photoreceptor cells, whose cell bodies lie in the outer nuclear
layer and whose outer segments lie adjacent to the RPE, carry out phototransduction, the biochemical process of transforming light to the electrical
energy of neural signals. There are two types of these highly specialized cells:
rod and cone cells. Rods contain rhodopsin and occur at greater density in
the peripheral retina. They mediate black and white vision and are able to
detect light under dim illumination, important for night vision. Cones are
densely packed in the central retina, especially the macula, and carry out precision and color vision under strong illumination, for example in daylight.
The photoreceptor cells synapse with horizontal and bipolar cells in the
outer plexiform layer. Bipolar cells correspondingly synapse with amacrine
and ganglion cells in the inner plexiform layer. The cell bodies of the
amacrine, bipolar, and horizontal, as well as interplexiform cells lie in the
inner nuclear layer, while the cell bodies of the ganglion cells lie in the ganglion cell layer. Finally, axons of the ganglion cells traverse the nerve fiber

Figure 2 The vertebrate retina. Schematic of the cells in the retina. ONL, outer nuclear
layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL,
ganglion cell layer; R, rod cell; C, cone cell; B, bipolar cell; H, horizontal cell; A, amacrine
cell; G, ganglion cell; RPE, retinal pigmented epithelium.


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J. Fielding Hejtmancik and John M. Nickerson

layer of the retina, collecting to form optic nerve, which leads through the
optic tracts to the optic radiations and visual cortex of the brain.
Each of these components including, its biology, function, and genetics,
is considered in detail in the following chapters. Because of the philosophical
approach of this volume as well as the limitations of space, general principles
are emphasized rather than the experiments and the results through which
our knowledge has been accrued. Students who wish additional information
regarding the experimental approach to visual science or a more detailed
description of the biology or genetics of the visual system should be able
to find it in the references, which are plentiful for each chapter.


CHAPTER TWO

Overview of the Cornea: Structure,
Function, and Development
Allen O. Eghrari, S. Amer Riazuddin, John D. Gottsch1
Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
1

Corresponding author: e-mail address:

Contents
1. Structure
1.1 Epithelium
1.2 Epithelial Basement Membrane
1.3 Bowman's Layer
1.4 Stroma
1.5 Descemet Membrane
1.6 Endothelium
2. Function
2.1 Epithelium
2.2 Epithelial Basement Membrane
2.3 Bowman's Layer
2.4 Stroma
2.5 Descemet Membrane
2.6 Endothelium
3. Development
3.1 Epithelium
3.2 Stroma
3.3 Descemet Membrane
3.4 Endothelium
Acknowledgments
References

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9
9

11
11
12
12
13
13
13
14
15
16
16
17
18
19
20
20

Abstract
The cornea is a transparent tissue with significant refractive and barrier functions. The
epithelium serves as the principal barrier to fluid and pathogens, a function performed
through production of tight junctions, and constant repopulation through differentiation and maturation of dividing cells in its basal cell layer. It is supported posteriorly by
basement membrane and Bowman's layer and assists in maintenance of stromal dehydration. The stroma composes the majority of corneal volume, provides support and
clarity, and assists in ocular immunity. The posterior cornea, composed of Descemet
membrane and endothelium, is essential for stromal dehydration, maintained through

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2015 Elsevier Inc.
All rights reserved.

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Allen O. Eghrari et al.

tight junctions and endothelial pumps. Corneal development begins with primitive formation of epithelium and lens, followed by waves of migration from cells of neural crest
origin between these two structures to produce the stroma and endothelium.
Descemet membrane is secreted by the latter and gradually thickens.

1. STRUCTURE
The human cornea is an avascular tissue that measures approximately
11.5 mm horizontally and 10.5 mm vertically. Its relative transparency, with
average refractive index of 1.3375, and anterior radius of curvature centrally
of 7.8 mm makes this tissue responsible for three-fourths of the total refractive power of the human eye.

1.1 Epithelium
The corneal epithelium is composed of four to six layers of nonkeratinized,
stratified squamous epithelial cells, and in humans, it measures approximately 50 μm in thickness.
The most superficial two to three layers are flat and polygonal in shape1
with apical microvilli and microplicae, and covered by a charged
glycocalyx,2 which maximizes surface area with the mucinous layer of the
tear film. At the cell periphery, tight junctions provide a watertight seal
and assist in the prevention of pathogenic organisms from entering the
cornea.
Directly posterior, the wing or suprabasal cells contribute a two- to

three-cell thick layer and also demonstrate tight junction complexes
between cells.
Basal epithelial cells represent the posterior-most layer of the corneal epithelium. Perilimbal basal epithelial cells differentiate and migrate anteriorly
to repopulate the cornea; microvilli appear on the surface gradually during
this process of maturation. Basal epithelial cells utilize hemidesmosomes to
adhere to the underlying basement membrane and underlying stroma. The
hemidesmosome, anchoring fibril, and anchoring filament complex produce
an anchoring complex, which represents a common link between the intracellular cytoskeleton of the basal epithelial cell and the stroma posteriorly.3

1.2 Epithelial Basement Membrane
The epithelial basement membrane lies posterior to the epithelium and anterior to the corneal stroma and is laid down by basal epithelial cells.


Overview of the Cornea

9

Transmission electron microscopy reveals an anterior lamina lucida and posterior lamina densa, visible through transmission electron microscopy.4
While the lamina lucida is structured with laminins, the lamina densa is
largely composed of collagens, laminin, heparan sulfate proteoglycans, and
nidogens.5 Immunohistochemical studies reveal a heterogeneously distributed regional variation of collagen IV subtypes dependent on its location
in the central or peripheral cornea; these subtypes are composed of
heterotrimers from six alpha chains.5,6
Laminin is the most frequent protein besides collagen and is also composed of heterotrimers, with one alpha, one beta, and one gamma chain.
Structurally, it self-assembles into sheets and contributes to the embryological development of the epithelial basement membrane. Expression of laminin subunits varies over time during development,7 and knockouts
demonstrate severe dysfunction.8
The major heparan sulfate proteoglycan is perlacan, a protein distributed
in basement membranes throughout the body which mediates migration,
proliferation, and differentiation of other cells.4 Keratinocyte survival and
differentiation are regulated by perlacan, which has been shown to be critical

for the formation of epidermis,9 and upregulated after corneal stromal
injury.10
Nidogens are sulfated glycoproteins with three globular domains connected with rodlike or thin segments11 and are distributed throughout
the basement membrane with strong affinity to laminin and collagen IV.4
Nidogen-1 and Nidogen-2 each demonstrate distinct binding sites to collagen IV and laminin, respectively, reflected in inhibition assays and studies of
recombinant fragment binding.11

1.3 Bowman's Layer
Bowman’s layer is an acellular, nonregenerating layer posterior to the epithelial basement membrane, approximately 8–12 μm in depth and decreases
in thickness over time.
Its collagen fibrils are distributed such that their posterior surfaces merge
with the anterior stroma, leaving a smooth anterior surface. These fibers are
only half to two-thirds the thickness of collagen fibrils in the stroma.12

1.4 Stroma
The stroma contributes the majority of the cornea’s structural framework,
measuring approximately 500 μm in humans and representing approximately 90% of the corneal anterior–posterior axis, as seen in Fig. 1. This layer


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Allen O. Eghrari et al.

Figure 1 Normal human corneal structure as illustrated through anterior segment optical coherence tomography. The stroma composes over 90% of corneal volume. The epithelium, appreciated as a white band at the superior margin of the cornea, contributes
less than 10% of overall thickness. Descemet membrane is seen as an even thinner band
at the inferior edge.

is organized with a network of collagen fibers and ground substance, with an
extracellular matrix composed of water, inorganic salts, proteoglycans, and
glycoproteins.13

Stromal collagen is composed of a heterodimeric complex of Type I and
Type V collagen with a narrow diameter.13 Lumican and keratocan are the
major keratan sulfate proteoglycans in the stroma, which also include
mimecan. These demonstrate a bifunctional role to contribute structural
support; protein moieties bind to collagen fibrils to modulate their diameters, while glycosaminoglycan chains are highly charged and promote interfibrillar spacing.14 Loss of lumican in a mouse model results in corneal
opacity,15 and in addition to its structural role, lumican regulates neutrophil
migration during bacterial infections of the stroma.16 Decorin is a major
proteoglycan associated with dermatan sulfate and similarly surrounds
collagen fibrils. The concentration of proteoglycans varies with depth
in the anterior–posterior axis, such that there is greater hydration in the
posterior stroma.
Patterns of collagen lamellae also vary with depth. Anteriorly, the stroma
is marked by short, narrow sheets with extensive interweaving, while collagen posteriorly demonstrates long, wide, thick lamellae extending from
limbus to limbus, and without significant interlamellar connections.
Keratocytes, the major cells of the stroma, maintain the integrity
of this layer, producing collagen, glycosaminoglycans, and matrix
metalloproteinases.1
The corneal stroma represents one of the most highly innervated tissues
in the human body. Sensory nerves from the nasociliary branch of the ophthalmic division of the trigeminal nerve course radially toward the central
cornea through the anterior stroma. Between Bowman’s layer and the anterior stroma, these nerves form the subepithelial nerve plexus, and then perforate through Bowman’s layer to become the subbasal epithelial nerve
plexus, innervating the basal epithelial layer.17


Overview of the Cornea

11

1.5 Descemet Membrane
Descemet membrane is the basement membrane of the corneal endothelium
and measures approximately 3 μm in thickness in children, gradually thickening to 10 μm in adults. This membrane is composed of two layers: an anterior banded layer which is developed by collagen lamellae and proteoglycans

and detected in fetal corneas as early as 12 weeks of gestation,18 and a posterior nonbanded layer which is laid down by endothelial cells and thickens
over decades.
Structurally, Descemet membrane contains collagen type IV and VIII
fibrils. In contrast to other basement membranes throughout the body in
which the type IV subtype is common, collagen type VIII is relatively specific to Descemet membrane and forms ladder-like structures visible under
electron microscopy. In the anterior banded layer, collagen fibrils demonstrate a lattice-like configuration with periodic banding at 110 nm intervals;
an additional 0.3–0.4 μm extension of this layer borders the stroma and is
electron dense and homogenous.18,19
Posteriorly, the nonbanded layer is relatively homogenous with a fine
granular appearance and increases in thickness over time as it is laid down
by the corneal endothelium.
Similar to the stroma, laminin and fibronectin, as well as keratan sulfate,
heparan sulfate, and dermatan sulfate are also present, and appropriate hydration is required to maintain clarity.

1.6 Endothelium
The corneal endothelium is composed of a single layer of flat, polygonal cells
which line the posterior surface of the cornea, demonstrated in Fig. 2. These
cells, approximately 5 μm in depth and with diameter of 20 μm, maintain
the relatively dehydrated status of the stroma through ionic pumps in basolateral plasma membranes.
Actin filaments mediate cell migration and maintain cell shape and are
located as dense peripheral bands apically.20 Cadherin forms a thin pericellular band at apical cell junctions and extends along basolateral cell
borders; alpha-catenin, beta-catenin, and plakoglobulin follow a similar
pattern.21 Tight junctions are also located near the apical aspect of cell membranes, and corneal endothelial cells express the cytoplasmic complex
ZO-1,22 which is associated with tight junctions.
Anterior to tight junctions, gap junctions are also present in corneal
endothelium and contribute to the electrical coupling of endothelial


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Allen O. Eghrari et al.

Figure 2 Confocal microscopy of the human corneal endothelium reveals a monolayer
of flat cells distributed with a hexagonal pattern. Several larger cells contribute to slight
variability in size (polymegethism) and shape (polymorphism).

cells.23 The gap junction protein connexin-43 is expressed in the
corneal endothelium.24

2. FUNCTION
2.1 Epithelium
At the surface, the epithelium plays a central role in vision as the majority of
the refractive power of the eye occurs at the interface of air and the tear film.
Among the anterior layers of flat squamous cells, tight junctions provide a
surface barrier and assist to maintain the dehydrated state of the cornea, as
well as inhibiting entry of pathogens into the stroma.
Corneal epithelial cells regularly undergo apoptosis and desquamation
and are replenished by division from stem cells at the limbus, contributing
to a net movement of epithelial cells in an apical direction. On average, the
lifespan of epithelial cells is 7–10 days.25 Genetic expression varies in basal
epithelial cells as they shift from a perilimbal location to the central cornea,
with mouse studies demonstrating 100 differentially expressed genes
between basal epithelial cells in the two locations.26 Distinct patterns of
expression are evidenced by limbal-specific staining for ABCG2, K19,
vimentin, KGF-R, metallothionein, and integrin alpha9, whereas basal cells


Overview of the Cornea

13

of the corneal epithelium specifically stain for K3 and K12, Connexin 43,
involucrin, P-cadherin, nestin, and integrins alpha2, alpha6, and beta4.27
The expression of Keratin-12 is specific to the corneal epithelium.28
Dysfunction of this protein is appreciated with heterozygous mutations in
KRT12, which are causative for Meesman’s corneal dystrophy.
The barrier function of the epithelium is additionally important for
immune regulation and prevention of pathogen entry into the cornea. Epithelial cells maintain toll-like receptors and secrete proinflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α.29 Langerhans cells exist at the basal
layer of the corneal epithelium and subbasal nerve plexus30 and demonstrate
chemotaxis to the central cornea in response to epithelial-secreted IL-1.31

2.2 Epithelial Basement Membrane
The epithelium releases factors that modulate cell differentiation and apoptosis, including TGF-β1 and PDGF. As such, the epithelial basement membrane serves as a physical barrier to modulate the effect of these factors on
keratocyte function. Removal of the epithelial basement membrane has
been shown to accelerate processes of stromal wound healing32 and carries
implications for scar and haze formation in the stroma.

2.3 Bowman's Layer
Bowman’s layer may play a role in protection of the subepithelial nerve
plexus which courses through from the anterior stroma. Its absence, however, which occurs most commonly after phototherapeutic keratectomy in
humans, does not appear to result in loss of vision or significant structural
changes of the cornea overall. Moreover, among mammals, this layer is specific to primates.

2.4 Stroma
The stroma provides both structural support to the cornea and transparency
by facilitating the passage of light through the framework of collagen fibrils
in a manner that prevents scattering. The bifunctional role of proteoglycans
promotes a small distance between collagen fibrils, decreasing the opportunity for light to scatter. The refractive index of the cornea decreases as light
passes through the anterior–posterior axis due to a more hydrated posterior
cornea. Disruptions in this pattern of lamellae, whether through hydration

or abnormal deposits, disturb transparency and result in loss of vision.


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For proper stromal function in the passage of light, the cornea must overcome scattering from keratocytes, from which the scattering of light is
clearly appreciated during in vivo confocal microscopy. Similar to the lens,
in which water-soluble crystallins contribute to clarity, corneal crystallins are
water-soluble proteins which decrease light scattering. These include aldehyde dehydrogenase class 3,33 transketolase,34 and alpha-enolase.35 The former two comprise 50% and 10% of water-soluble proteins in the cornea,
respectively, and may play a role in reducing variability in the index of
refraction as light passes through the stroma.36
Avascularity of the corneal stroma is also needed to facilitate transparency, and the production of antiangiogenic factors assists to maintain a
balance with proangiogenic factors. These include the angiogenic properties
of the molecule vascular endothelial growth factor (VEGF), which are
effectively nullified by corneal secretion of soluble VEGF receptor-1
(sVEGFR-1 or sflt-1), trapping VEGF and blocking its activity.
Supportive evidence of this neutralizing effect is seen through suppression
of the soluble receptor by neutralizing antibodies or RNA interference
resulting in angiogenesis, and the fact that patients with aniridia are
deficient in this molecule.37 Additional angiogenic factors include
basic fibroblast growth factor (bFGF) and membrane type-1-MMP
(MT1-MMP).38 Thrombospondins may additionally play a role in regulating neovascularization.39
Stromal clarity is also dependent on appropriate keratocyte cell differentiation. Keratocyte-derived myofibroblast formation, which may occur with
exposure to TGF-β1 and PDGF, is associated with development of corneal
haze and disordered extracellular matrix production.40
In addition to surface immune regulation by the epithelium, the stroma
plays a pivotal role in corneal immunity. The central cornea contains immature and precursor-type dendritic cells, while the peripheral cornea demonstrates resident bone marrow-derived dendritic cells.41 Keratocytes may be
incited by TNF-α and epithelial release of IL-1α to produce IL-6 and

defensins.31 Macrophages are present in the posterior stroma.42

2.5 Descemet Membrane
As the basement membrane for the corneal endothelium, Descemet membrane assists in the maintenance of corneal dehydration. This is evident in
corneal hydrops, focal acute episodes of corneal edema experienced in
keratoconic corneas in which breaks occur in Descemet membrane.


Overview of the Cornea

15

Iatrogenic Descemet membrane tear or detachment after intraocular surgery
also results in corneal edema.
The posterior nonbanded layers continue to thicken throughout
life through endothelial secretion, in contrast to the anterior descemet
membrane (DM) which remains relatively constant.

2.6 Endothelium
The corneal endothelium plays an essential role in preserving stromal dehydration, thereby maximizing the fidelity of light passing through the cornea.
Maintenance of this gradient of hydration depends on tight junctions among
endothelial cells and pump function associated with Na+/K+-ATPase and
bicarbonate-dependent Mg2+-ATPase. Endothelial failure is, in turn, marked by corneal edema and represents a net influx of aqueous fluid into the
cornea at a higher rate than the amount pumped out during a given period
of time.
Aquaporin-1 is expressed in corneal endothelial cells, and mice lacking
AQP1 demonstrate a reduced ability to counter corneal edema secondary to
hypotonic saline exposure.43
A minimum number of endothelial cells are required to provide adequate pump function, and endothelial cell density decreases from approximately 3000–4000 cells/mm2 at birth to 2500 cells/mm2 in late adulthood.
Confocal microscopy reveals endothelial cell loss over a lifetime at a rate of

10.92 cells/mm2 per year.44 A loss of corneal endothelial density to several
hundred cells per mm2, which may occur from intraocular surgery or other
trauma, generally results in corneal edema.
Unlike the epithelium, endothelial cells do not demonstrate mitosis
in vivo. Replacement of endothelial cell function from damage and cell death
occurs not through cell division but migration. A focal loss of cells centrally,
therefore, results in centripetal migration of adjacent endothelial cells, subsequent formation of tight junctions and restoration of pump function, and
then a graduation remodeling of endothelial structure from enlarged, irregularly shaped endothelial cells to a more hexagonal pattern.1
A study of central, peripheral, and paracentral endothelial cell density in
normal eyes and eye bank corneas demonstrated that endothelial cell density
in paracentral and peripheral zones was higher by 5% and 10%, respectively,
compared to the central cornea.45 The exact causes of this distinction are
unclear and may be due to patterns of migration or aqueous flow dynamics,
or a process of peripheral endothelial cell proliferation yet to be understood.


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3. DEVELOPMENT
The eye originates from the somatic ectoderm and neural tube and
induction by the chordamesoderm. Following development of the diencephalon from induction of the anterior neural tube by the chordamesoderm, the optic vesicle begins to protrude. As the optic vesicle extends
and meets the head ectoderm, it induces the thickening of surface ectoderm
and formation of the lens placode, which invaginates to develop the lens.
This process correlates with expression of the transcription factors Six3,
Pax6, and Rx1 in the anterior neural plate. Rx1 is essential for eye formation, and Rx1-null mice do not develop optic cups and therefore do not
produce eyes.46 Six3 activation precedes Pax6 in the mouse, and in early
inactivation of Six3, the surface ectoderm does not thicken to produce
the lens placode.66

Pax6 serves as a master regulator of eye development. In the mouse, Pax6
expression is detected in head surface ectoderm at day 8 of embryogenesis
(E8), and spatial differences in Pax6 expression are noted in surface ectoderm
by E9.547: a band of surface ectoderm in which Pax6 expression is switched
off gives rise to epidermis, and an adjacent narrow band in which Pax6 is
expressed commits to ocular development, subsequently resulting in formation of the lens and corneal epithelium.48

3.1 Epithelium
As the surface ectoderm above the neuronal optic cup invaginates, developing the crystalline lens, this primitive lens becomes round and induces formation of the overlying ectoderm into corneal epithelium. This process
occurs in humans at approximately day 33 of gestation, and a bilayered epithelium is present by the fifth week of gestation.
In the mouse, commitment of the ectoderm to corneal epithelium begins
at days E11–E12, when epithelial progenitor cytokeratins K5 and K14
replace ectodermal cytokeratins K8 and K18. At approximately days
E15.5–E17.5, cytokeratins K12 and K3 are detected and are specific to
the corneal epithelium.49
The mature corneal epithelium, which is composed of six to seven layers
of cells, develops upon opening of the eyelids, which in humans is at approximately 24 weeks of gestation. The posterior-most basal epithelial cells
morph from a flattened, ovoid shape to cuboidal and later a columnar


Overview of the Cornea

17

formation. These cells flatten as they migrate anteriorly into the suprabasal or
wing cells and then to the anterior corneal epithelium.
Corneal epithelial development is regulated at the molecular level by
dynamic changes in genetic expression throughout development. In mice,
microarray analysis of the expression of 8666 genes between postnatal day
10 and days 49–56 revealed 442 genes with distinct levels of expression

between the two time points.50
MicroRNA (miRNA) function is essential for epithelial development,
and targeted deletion of a ribonuclease necessary for miRNA function, Dicer,
results in poorly stratified corneal epithelium and microphthalmia.51 In
human-induced pluripotent stem cells, miR-450b-5p serves as a switch for
Pax6, inhibiting Pax6 expression and directing corneal epithelial fate; miR184 is expressed in the corneal epithelium, and knockdown results in
decreased Pax6 expression.52 Mutation of a single base pair in miR-184 is
responsible for EDICT syndrome in humans.53

3.2 Stroma
At approximately the seventh week of gestation in humans, a subpopulation
of neural crest cells, which have originated at the junction of the closing neural tube and ectoderm and populated the periocular space,54 develop mesenchymal cells and migrate into the space between lens and primitive
corneal epithelium and develop into the stroma, endothelium, iris, and trabecular meshwork.
During this seventh week, migration of mesenchymal cells occurs in
three waves. In humans, as well as reptiles, birds, and other primates, the first
wave contributes to development of corneal and trabecular meshwork
endothelium, and the second wave differentiates into keratocytes. In contrast, a single wave is present in rodents, cats, rabbits, and cattle.55 A third
wave contributes to iris development. Both TGF-β2 and FOXC contribute
to differentiation of neural crest cells into corneal stroma.
Forkhead box (FOX) proteins share homology with the Drosophila
forkhead transcription factor, which regulates expression of genes associated
with cell growth, proliferation, and differentiation. In the mouse, Foxc1null mice demonstrate disrupted stromal development.56 In humans, autosomal dominant mutations in FOXC1 are associated with anterior segment
dysgenesis resembling Axenfeld–Rieger syndrome and Peters anomaly.57
TGF-β2 modulates stromal development, and in knockout mice
targeting the three TGF-β isoforms, only mice lacking the TGF-β2 isoform


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Allen O. Eghrari et al.

demonstrated corneal abnormalities; these included thin corneal stroma,
absent corneal endothelium, corneolenticular fusion, and excessive hyaline
cells in the vitreous.58 Although this did not appear to involve apoptosis,
stromal thinning was associated with fewer keratocytes and lumican,
keratocan, and collagen I were significantly decreased.
During stromal development, glycosaminoglycans bind to proteins and
fill spaces between collagen fibers, and keratan sulfate proteoglycans accumulate in this space. Lumican is essential for corneal clarity, and mice homozygous for a mutation in lumican develop bilateral corneal opacification and
thick collagen fibrils visualized with transmission electron microscopy.15
Keratocan expression during development correlates directly with lumican
expression,59 and mutations in KERA are associated with cornea plana in
humans.
Migration of neural crest cells appears to be modulated by a combination
of factors expressed by the lens and neural crest-derived cells that have initiated migration. Removal of the lens results in premature invasion and
abnormal corneal differentiation. Semaphorin3A (Sema3A) is expressed in
the lens placode and epithelium throughout eye development and inhibits
periocular neural crest migration in vitro. Periocular neural crest expresses
neuropilin-1 (Npn-1), and levels of expression decrease in cells that migrate
to become corneal endothelium and stroma. A peptide which inhibits
Sema3A/Npn-1 signaling results in early entry of neural crest cells over
the lens.54

3.3 Descemet Membrane
The collagen of Descemet membrane is secreted by corneal endothelial cells
and detected in utero as early 12 weeks of gestation, at which point an
electron-lucent zone bordering the corneal endothelium lies adjacent to
an electron-dense zone bordering the stroma.18 These measure less than
40 nm in thickness, which is comparable at that age of gestation to the epithelial basement membrane. Descemet membrane becomes thicker with the
addition of multiple layers, resulting in a multilayered structure. This process
continues throughout gestation, from 1 layer at 12 weeks to 10 layers at

approximately 27 weeks, and 30–40 layers by birth.18
By 16 weeks, early signs of differentiation are present, with the
development of linear filaments measuring 170 nm in length and 40 nm
in width, aligned perpendicularly between the electron-dense layers.
These become uniformly distributed in a 110 nm banding pattern


Overview of the Cornea

19

characteristic to the anterior banded layer.18 At the time of birth, the membrane is largely composed of the anterior banded layer, measuring 3 μm in
thickness.
In contrast, the posterior nonbanded layer continues to thicken over
one’s lifetime, at a slower rate and lacking the striated appearance of the
anterior layer.
Mutations in COL8A2 are associated with a markedly thickened anterior
banded layer of Descemet membrane, more than three times thicker than
normal.60

3.4 Endothelium
Formation of the corneal endothelium, derived from the neural crest, occurs
alongside development of the stroma, and factors which disturb stromal
development frequently affect both layers. Foxc1-null mice demonstrate a
lack of corneal endothelium in addition to disruption of stromal
development,56 and TGF-β2-null mice demonstrate an absent corneal
endothelium associated with stromal thinning. In humans, miR-184associated EDICT syndrome results in both stromal thinning and a
beaten-metal appearance to the posterior cornea.53
The corneal endothelium develops as a monolayer and maintains this status through development and adult life, arrested in the G1 phase of development. Prevention of progression through the cell cycle is modulated by
p27kip1, a G1-phase inhibitor, and cell–cell contact inhibits cell proliferation.23 Contact inhibition is likely signaled from adherens junctions to the

nucleus,61 and disruption of adherens junctions may affect proliferation
through β-catenin or p120 catenin.
Reversal or modulation of processes of corneal endothelial development
may carry implications for wound healing and potentially for future therapies in repopulating corneal endothelium. In response to wound formation,
fibroblast growth factor-2 plays a key role in an endothelial-to-mesenchymal
transition, degrading p27, facilitating synthesis of type I collagen, inducing a
change in endothelial cell shape into a fibroblastic morphology, and resulting
in loss of the endothelial monolayer,62 modulating this process through
IL-1β and NF-κB.63 In cultured human endothelial cells, exposure to
EDTA alone disrupts cell junctions, and addition of bFGF results in
both proliferation and endothelial-to-mesenchymal transition, with a
fibroblastic-type appearance, and relocation of N-cadherin to the cytoplasm
from its original location in the cell junction; addition of TGF-β1 induces


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Allen O. Eghrari et al.

a fibroblastic state but without proliferation.64 Use of a selective inhibitor
of TGF-β, SB431542, counteracts the fibroblastic phenotype in cultured
human corneal endothelial cells.65

ACKNOWLEDGMENTS
This work was supported by NIH Grants K12 EY015025 (A.O.E.) and R01 EY016835
(J.D.G.).

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