The Cornea and Lens
Together, the transparent cornea and lens make up the refractive unit of the eye responsible for focusing light onto the retina, which collectively have been termed the refracton (Piatigorsky,2001). In addition, the cornea serves as a protective barrier between the eye and the external environment. The relative refracting power of either the cornea or lens varies between species. For zebrafish and other aquatic animals, the lens provides relatively more focusing power than the lens of terrestrial animals. This is because, the refractive index of water, as compared to air, is better matched to that of the corneal cells and less refracting power is needed by the cornea (Greiling and Clark,2008). While the zebrafish lens provides the majority of light refraction, unlike some other teleosts, the zebrafish lens does not significantly change shape with focal point accommodation (Easter and Nicola,1996). In the following sections, we describe the anatomy and physiology of the zebrafish cornea and lens.
The vertebrate cornea is the transparent tissue covering the front of the eye. It is continuous with the opaque sclera that forms the outer shell of the eye globe. Five basic layers make up the cornea: the epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium (see Fig. 1). Like other vertebrates, the zebrafish cornea is avascular. The adult zebrafish cornea has been characterized histologically and ultrastructurally (Swamynathan et al.,2003; Soules and Link,2005; Zhao et al.,2006; Akhtar et al.,2008). When mature, it is ∼20 microns thick. The most superficial layer, the epithelium, is in direct contact with the aqueous environment. It is ∼3–6 cell bodies thick. These stratified squamous cells make up over half of the thickness of the zebrafish cornea–a larger proportion than that of the mammalian cornea. Zebrafish corneal epithelial cells show interdigitations and are connected with numerous intercellular junctions and the external layer of corneal epithelial cells show abundant microplicae and reticulations that form “microridges” (Collin and Collin,2000). However, unlike most mammals, birds and reptiles, microvilli are absent from bony fishes. En face, the microridges give the surface of these hexagonal cells a “fingerprint-like” appearance (Zhao et al.,2006). At the periphery of the cornea, mucus-secreting goblet cells are frequently interspersed within the epithelium (Soules and Link,2005). The microplicae of the superficial epithelial cells are thought to stabilize the glycocalyx produced by the goblet cells, as well as increase surface area to aid in the exchange of nutrients and metabolites. In other vertebrates studied, epithelial cells continually renew, being shed at the central region and generated in the peripheral limbal zone (Davies and Di Girolamo,2010). Corneal epithelial cell renewal and wound healing, however, has not been investigated in zebrafish. Bowman's layer, a thin extracellular deposition separates the epithelial cells from the stromal layer. The stromal layer is also primarily made up of extracellular matrix. The most prominent feature of the corneal stroma is the exquisite collagen organization (Fig. 1, right panel). Collagen fibrils are bundled into tightly packed units that run in parallel within a sublamina, but are orthogonal to those above and below. This organization facilitates transparency of the tissue and must be actively maintained (Benedek,1971). Spaces between the collagen bundles are enriched for proteoglycans and interspersed with flat keratocytes, which synthesize collagen (Akhtar et al.,2008). As a proportion of corneal thickness, the zebrafish stromal layer is relatively thin as compared with mammals. The stroma overlies a basal lamina termed Descemet's membrane. The innermost layer, the endothelium, is comprised of a single layer of junctionally connected, polygonal cells. The corneal endothelium is critical for metabolic homeostasis and maintenance of stromal collagen organization, and therefore transparency.
The adult zebrafish lens shows nearly all the morphological hallmarks that characterize lenses of other vertebrates (Soules and Link,2005; Dahm et al.,2007; Greiling and Clark,2008; Vihtelic,2008). The fish lens is more spherical than the typical vertebrate, but is comprised of an outer epithelial layer covering elongated fiber cells. Most lens epithelial cells are quiescent, except for a band encircling the marginal equator. These marginal zone lens epithelial cells proliferate and give rise to additional epithelial cells as well as differentiating fiber cells. The region of lens fiber cell differentiation is also called the Bow region or transition zone. Within this area, elongating fiber cells loose their internal organelles, which aids in transparency. The process of organelle disassembly seems to happen more abruptly in zebrafish than mammals (Dahm et al.,2007). The ongoing growth of the lens results in a concentric shelled organization, where the inner fiber cells are older and more differentiated than the outer fiber cells. Electron microscopy highlighted the onion-like nature of the lens and revealed interdigitating lateral protrusions, or ball-and-socket joints, between the fiber cells (Soules and Link,2005; Dahm et al.,2007; Vihtelic,2008).
Development of the zebrafish cornea and lens has been characterized by a variety of approaches including morphology, time-lapse microscopy, and by gene mutation analyses. Both structures develop from ectodermal cells of the lens placode. Thickening of the lens placode is visible by ∼16–18 hours post fertilization, hpf, (Schmitt and Dowling,1994; Li et al.,2000; Soules and Link,2005; Zhao et al.,2006; Dahm et al.,2007; Vihtelic,2008; Greiling et al.,2010). Shortly, after the ectodermal cells become more columnar, the lens anlage invaginates as a mass of cells. The zebrafish lens does not go through a hollowed vesicle stage. As the lens anlage completely delaminates from the epidermal surface, the remaining cells become the prospective corneal epithelium. Within the nascent lens, primary fiber cells begin to elongate and differentiate at the posterior of the cellular mass, while those in the anterior region differentiate into epithelial cells (Greiling et al.,2010). This entire process takes ∼10 hours and is coordinated with initiation of optic cup morphogenesis (Martinez-Morales and Wittbrodt,2009). Between 24 and 36 hpf, periocular mesenchymal cells of neural crest and head mesoderm origin migrate into the newly formed anterior chamber. Subsets of these cells contribute to the corneal endothelium, as has been shown for other vertebrates. However, it should be noted that definitive fate mapping studies have not been done for the differentiated zebrafish anterior segment structures. Following immigration of corneal endothelial cells, differentiation continues and a rudimentary stromal layer is apparent shortly after the endothelial cells form a monolayer at ∼48 hpf (Zhao et al.,2006). Ongoing differentiation continues until 1 month, when the cornea reaches the adult form (Soules and Link,2005; Akhtar et al.,2008). In contrast, adult lens morphology is reached at earlier stages, although fiber cell compaction continues until the 1-month stage. The simplicity of anatomy and compartmentalization of zebrafish lens development has facilitated its use for general insight into cell biological and gene regulation processes (Harding et al.,2008; Imai et al.,2010; Tittle et al.,2010).
The Iridocorneal Angle
The iridocorneal angle, the region of the ocular anterior chamber where the cornea meets the iris, hosts cells specialized in maintaining intraocular pressure. Intraocular pressure is balanced by aqueous humor production and drainage. In most mammals, tissues that mediate aqueous humor production and clearance run symmetrically around the iridocorneal angle (Gabelt and Kaufman,1997; Goel et al.,2010). For example, the ciliary epithelia cells that produce aqueous humor are organized circumferentially just posterior to the iris. Aqueous humor flows past the lens and through the pupil, where it filters through trabecular meshwork cells. Aqueous humor is ultimately collected through endothelial-lined collector channels of Schlemm's canal. Anatomical characterization of the anterior segment of zebrafish, however, suggests differences in tissue organization and a higher degree of dorso-vental specialization in fish (Soules and Link,2005). Aqueous humor tracer studies indicate that the ciliary epithelium of zebrafish, although lacking folds and processes, is the site of aqueous humor production (Gray et al.,2009). There appears to be enhanced production with the dorsal quadrant of the ciliary epithelium. In contrast, the outflow pathway is exquisitely localized to the ventral part of the angle (Soules and Link,2005; Gray et al.,2009; Fig. 2). At this region, an endothelial-lined canalicular network can be seen in histology or by electron microscopy. Interestingly, within this region aqueous humor can enter at both the iridocorneal angle and behind the iris through a break in the ventral ciliary epithelium. The canalicular network leads to a relatively large sinus cavity that is continuous with an episcleral venous plexus.
Another difference between the zebrafish iridocorneal angle and that of mammals is the presence of the annular ligament. This tissue has a hypertrophied appearance and seems to have a structural role in giving shape to the cornea and preventing collapse in the angle region. The annular ligament, while circumferential, does show differences in thickness in a nasal (shallow) to temporal (deep) manner (Yoshikawa et al.,2007). Although the overall tissue organization and presence of the annular ligament of the zebrafish iridocorneal angle is strikingly different from mammals, at the cellular and ultrastructural levels, the ciliary epithelium and cells of outflow pathway show a high-degree of conservation (Soules and Link,2005; Gray et al.,2009).
Analysis of anterior segment development in the zebrafish also suggests conservation with other vertebrates. Like other species studied, zebrafish periocular cells of neural crest and mesodermal origin migrate into the eye and contribute to the specialized structures of the anterior eye (Soules and Link,2005; Yoshikawa et al.,2007; Langenberg et al.,2008). Furthermore, when genes known to regulate anterior segment development in other vertebrates are disrupted in zebrafish, dysgenesis also occurs (Gestri et al.,2009; McMahon et al.,2009; Skarie and Link,2009; Verbruggen et al.,2010). Specific fate maps and lineage analyses of periocular cell contribution to each defined structure of the anterior segment has not been carried out. In principal, however, the zebrafish provides many experimental advantages for such studies.
The Neural Retina
Development and Anatomy.
The zebrafish possesses a canonical vertebrate retina composed of one glial and six neural cell types that are arranged in three nuclear layers, separated by two synaptic (plexiform) layers (see Fig. 3). This distinct layering is already apparent at 3 dpf.
The outer most cell layer contains the photoreceptors that ultimately convert the physical light stimulus into a biological signal. The outer zebrafish retina contains one-rod photoreceptor cell type and the full vertebrate complement of four cone photoreceptors cell types. These are classified by their absorption spectra and morphology. Red- and green-sensitive cones are fused at the level of the inner segment and form double cones, while the ultraviolet (UV) and blue sensitive cones are separate and function as short and long single cones, respectively. Therefore zebrafish are, in contrast to humans, tetrachromats that are sensitive to light in the UV range.
In the inner nuclear layer, the somata of bipolar, horizontal, and amacrine interneurons are located, as are the cell bodies of the Müller glia cells. Synaptic contacts between photoreceptors and the inner retina are formed in the outer plexiform layer. Closest to the lens is the ganglion cell layer, containing the cell bodies of displaced amacrine cells and ganglion cells. The long axons of the latter constitute the optic nerve and after crossing the midline form the optic tract. At larval stages most of these axons project to the optic tectum, a dorsal midbrain structure homologous to the superior colliculus of mammals, but also to nine additional arborization fields (Burrill and Easter,1994). The proportionally thick inner plexiform layer reflects the complexity of synaptic contacts between ganglion and amacrine cells and cells of the inner nuclear layer.
The development of the zebrafish retina is extraordinarily rapid facilitating numerous studies addressing retinal development and its genetic control (reviewed in Tsujikawa and Malicki,2004a; Fadool and Dowling,2008).
The first ganglion cells differentiate around 32 hpf, followed by cells of the inner nuclear layer (Schmitt and Dowling,1994; Hu and Easter,1999; Schmitt and Dowling,1999). Rod and cone photoreceptor outer segments become apparent around 55 dpf (Schmitt and Dowling,1999). Synaptic structures indicative of functional maturation (ribbon triads) arise within photoreceptor synaptic terminals at around 65 hpf, followed by bipolar cell ribbon synapses approximately at 70 hpf. Signal transmission from photoreceptors to second order neurons starts around 84 hpf and becomes fully functional at 5 dpf (Biehlmaier et al.,2003). This extraordinary fast maturation of the visual system is mirrored by electrophysiological properties and the wealth of visually guided behaviors that can be evoked at these early larval stages (Neuhauss,2010). In some respects, the zebrafish retina never stops developing, since the mature adult retina continues to proliferate. All cell types of the retina are constantly generated in the circumferential germinal zone at the ciliary margin (see Cerveny et al., 2012). Additionally, rod photoreceptors originating from rod precursor cells of the inner retina are added throughout life (Raymond et al.,2006).
The zebrafish retina has remarkable regenerative capacities. Müller glia cells of the inner nuclear layer are able to produce all retinal cell types in response to injury (Bernardos et al.,2007; Fimbel et al.,2007).
Physiology and Function.
The simple scheme described above does not do justice to the diversity and complexity of retinal cell types. Already the outer retina contains four-cone and one-rod photoreceptor cell type, distinguished by their morphology and absorption spectra. These two photoreceptor types are adapted to different illumination levels with the cones mediating vision at bright light levels, while rods being functional at low light conditions. These two systems develop asynchronously in the zebrafish. Larval vision is largely dominated by cones, while rod function starts to impact vision only at later stages starting at around 15 dpf (Bilotta et al.,2001). Intriguingly, zebrafish larvae turn off their visual system at night, as evidenced by loss of visual responsiveness, a near absence of electrical responses to light, and the disassembly of presynaptic structures used in neurotransmitter release during the subjective night (Emran and Dowling,2010; Emran et al.,2010).
The complexity of five different photoreceptor cell types is more than matched by the perplexing variety of inner retinal cell types. Multiple subtypes of horizontal, bipolar, and amacrine cells can be distinguished by morphology, neurochemistry, and physiology. The description of inner retinal cells of the zebrafish is certainly not complete and in particular, the functional characterization of the various subtypes is in its infancy.
Recent advances in transgenic technology hasallowed labeling of specific cell types, either by using cell types specific promoters or by fortuitous transgenic labeling. Subsequent morphological and electrophysiological analysis of labeled cells will help inthe characterization of unique subpopulations of retinal cells (e.g., Cederlund et al.,2011). Similar experiments will likely yield a much more complete functional wiring diagram in the near future.
Comparable to other teleosts, the zebrafish contains four distinguishable horizontal cell types, as identified by a number of labeling methods (Connaughton and Dowling,1998; Yazulla and Studholme,2001; Connaughton et al.,2004; Song et al.,2008; Li et al.,2009). One large field horizontal cell is rod-specific, while the two small field horizontal cells (H1 and H2) connect solely to cones, just as a large field horizontal cell (H3), which responds best to UV light (Li et al.,2009; Connaughton and Nelson,2010).
At least 17 bipolar cell types have been recognized by morphological criteria. These can be classified into three groups according to their dendritic termination pattern. These three groups containing cells of ON, OFF, and multistratified ON- and OFF-type reflect their functional properties as well (Connaughton and Nelson,2000; Connaughton et al.,2004).
ON bipolar cells hyperpolarize in response to glutamate released by photoreceptors. The mechanism of hyperpolarization by the archetypical excitatory neurotransmitter glutamate is largely undisputed in mammals. In mammalian species, bipolar cells hyperpolarization is thought to be mediated by the activation of a metabotropic glutamate receptor (mGluR6) that ultimately leads to the closure of a transient receptor potential like (TRP) channel (Masu et al.,1995; Shen et al.,2009).
At least in the teleost retina, there is growing evidence for an additional mechanism involved in the cone ON-response. Members of the excitatory amino acid transporter (EAAT) family mediate a chloride conductance activated by glutamate that likely underlies the cone ON response (Wong and Dowling,2005; Wong et al.,2005a, b). In teleosts and other vertebrates, there is now mounting evidence for the parallel existence of both mechanisms. However, firm evidence for an EAAT mediated hyperpolarization of mammalian ON bipolar cells is missing. Intriguingly, mammals have lost two of the full vertebrate complement of seven EAAT family members (Gesemann et al.,2010), which might explain the small or absent contribution of this transporter mediated mechanism in the mammalian retina.
As for most central nervous system synapses, the action of glutamate on depolarizing OFF-type bipolar cells is mediated by ionotropic glutamate receptors of the AMPA and kainate type (Dowling,1987).
We are only beginning to cope with the diversity of amacrine cell types in the teleost retina with up to 70 morphological and neurochemically distinct cell types (Wagner and Wagner,1988). In the larval zebrafish retina, 28 morphological subtypes have already been distinguished by virtue of an elegant transgenic labeling approach (Jusuf and Harris,2009). Similar experiments with different transgenic reporters, at more mature stages, will likely result in the identification of additional subtypes.
Little work has been done on the functional characterization of amacrine cells in the zebrafish retina, with the exception of interplexiform cells. These dopaminergic cells extend processes into both plexiform layers and are involved in feedback signaling from the inner to the outer retina. Due to the position of their cell bodies, they can arguably be referred to as amacrine cells. These cells receive input from the olfactory bulb (Zucker and Dowling,1987) and likely play a crucial role in network adaptation of the retina to light (Witkovsky,2004). Functional disruption of these cells or of their afferent olfactoretinal centrifugal pathway leads to defects in visual sensitivity (Li and Dowling,2000a, b).
The larval retina likely contains most of the functionally specialized amacrine cell types known from studies of the mammalian retina. For instance, evidence for the presence of motion sensitive amacrine cells of the Starburst type was gained by the analysis of a metabolic zebrafish mutant (Maurer et al.,2010).
The zebrafish ganglion cell layer contains at least 11 morphologically distinct ganglion cells types (Mangrum et al.,2002; Ott et al.,2007). This number likely underestimates the true diversity of ganglion cells, since the identified morphological types overlap not completely with ganglion cell types identified in similar studies of the rabbit retina (Rockhill et al.,2002; Ott et al.,2007). The physiological characterization of ganglion cell types is just beginning. Six classes can be distinguished by their response to full field stimulation, yielding similar ON-OFF, sustained and transient characteristics as has been reported for other vertebrates (Emran et al.,2007).
In contrast to the puzzling cellular diversity of the five neuronal cell types of the retina, there is only one major glial cell type intrinsic to the retina, the Müller glia cells. Their morphological uniformity is more than matched by the multiple diverse functions they play in retinal physiology. Among these, the essential functions are the maintenance of ionic homeostasis, metabolic support of neurons, uptake, and recycling of neurotransmitters from synapses (for a general reviewed see Bringmann et al.,2006).
Müller glia cells have been implicated in visual pigment regeneration of all-trans retinal back to 11-cis retinal following photoisomerization after photon capture. This recycling of visual pigment is crucial for continuous vision and takes place in a series of biochemical reactions variably called the visual or retinoid cycle (extensively reviewed in Lamb and Pugh,2004). Reactions of the canonical visual cycle take place in photoreceptors and the retinal pigment epithelium (RPE). Recently Müller glia cells have been implicated in an alternative visual cycle, likely exclusively serving cone photoreceptors (reviewed in Wang and Kefalov, in press). Studies in the zebrafish have shown a function for a Müller glia cell specific retinoid binding protein in recycling of cone visual pigments, proving a role for a Müller glia cell based recycling pathway involved in cone vision. Cone visual pigment is regenerated by both the canonical and the alternative pathway, while rod visual pigment only has access to the canonical pathway (Fleisch et al.,2008; Fleisch and Neuhauss,2010).
Müller glia cells in the zebrafish have become a focus of research due to their aforementioned ability to dedifferentiate to a pluripotent state and reconstitute all retinal cell types after injury (Bernardos et al.,2007; Fimbel et al.,2007). Understanding and adapting the cellular mechanism of regenerating injured retinal cells to the human retina has the potential to revolutionize our treatment options for degenerative retinal disorders.
The function of the neural retina hinges on the interaction with the RPE (reviewed in Strauss,2005). These cells form a single layer of cells directly abutting the outer retinal photoreceptors. Apart from their eponymous pigmentation, these cells are characterized by long cellular protrusions. These microvilli interdigitate the photoreceptor's outer segments. Such an intimate proximity to the photoreceptor's outer segments enables RPE cells to phagocytose and digest the (potentially damaged) tips of the outer segments, thereby maintaining photoreceptor integrity. RPE cells separate the retina from the blood vessels of the choriocapillaris and are thus ideally situated to regulate the transport of nutrients and oxygen from the blood to the retina and move metabolic waste products back to the blood circulation. Among other physiological functions, key steps of the canonical visual cycle take place in RPE cells.
The sclera is the outermost layer of the eye and is rich in extracellular matrix, particularly collagen, elastin, and proteoglycans. This tough, fibrous tissue gives the eye shape and serves a protective role. The sclera is also the site of attachment and insertion of the extraocular muscles. In most vertebrates, there are three principle layers of the sclera: the episclera, the stroma, and the lamina fusca (reviewed in Watson and Young,2004). The outermost episcleral layer is thin, but more cellularized than the other layers. The episcleral surface is decorated with microplicae, which helps form a glycocalyx that lubricates the eye. The stroma is made of interwoven groups of collagen bundles, interspersed with flattened scleral fibroblasts. Unlike the corneal stroma, which efficiently transmits light, the scleral stroma scatters light, giving it an opaque appearance. The innermost layer of the sclera is composed of various types of pigmented cells and is rich in elastin. In addition, many lower vertebrates, including zebrafish and some other teleosts, have a scleral ring or scleral ossicle (Walls,1942). This cartilaginous structure encircles the anterior part of the eye and is embedded within the sclera between the pigmented layer and the stroma, where it is thought to reinforce the sclera and provide further protection to the eye. Detailed anatomical characterization of the zebrafish sclera has not been carried out. However, light and electron microscopic analysis of peripheral nerve processes in the sclera and choroid has been done (Chapman et al.,2009). This study not only revealed that nerve bundles traverse both the choroid and sclera to innervate vascular smooth muscle of the choroid, but also showed general features of the peripheral sclera of zebrafish.
Immigrant Glial Cells of the Eye
Multiple types of glia are found within the eye or in association with the optic nerve. In addition to the “intrinsic” Muller glial cells that develop from retinal neuroepithelia, several “non-native” glial cell types are generated outside of the eye, but establish an ocular residence. Retinal glia, like glia throughout the nervous system, functions in homeostasis processes and respond to injury. Retinal immigrant glia includes microglia/macrophages and reticular astrocytes that are found within the eye, as well as oligodendrocytes that myelinate and associate with the optic nerve. Unlike “neuroglia” such as astrocytes and oligodendrocytes, microglia is derived from mesodermal, hematopoietic stem cells that migrate into the developing nervous system during early embryonic stages (Streit and Xue,2009). At this point, they are broadly termed macrophages. Later, once the cells have taken up residence in the nervous system, they are typically called microglia. In zebrafish, microglia originate from anterior part of lateral mesoderm, differentiate in the yolk sac before the onset of blood circulation and invade the cephalic mesenchyme starting at 22 hpf (Herbomel et al.,1999). With regard to the eyes, the first microglia migrate through the ventral fissure and into the vitreous space by 25 hpf (Herbomel et al.,2001). As retinal lamination progresses, the embryonic microglia become enriched in the synaptic layers, although some can be found within the inner cell body layers. Direct observation of the microglia was facilitated by injecting Neutral Red, which accumulates in the phagocytic cells. Time-lapse observation showed that microglia continually patrol throughout the neural retina–most often migrating within the plexiform layers (Herbomel et al.,2001). More recently, transgenic zebrafish lines have been generated using either apoE or mpeg-1 regulatory sequence to drive GFP expression (Peri and Nusslein-Volhard,2008; Ellett et al.,2011). These lines should make detailed analysis of microglia/macrophage behaviors more convenient, particularly for adult studies or within diseased or mutant conditions and generally, microglia function in immune surveillance and neuronal homeostasis (Graeber and Streit,2010). They clear debris via phagocytosis and can coordinate reactivity and functions of other glia. Microglia also modulates synaptic connections by responding to cues of the complement pathway (Fourgeaud and Boulanger,2007).
Another class of immigrant glial cells are the reticular astrocytes that derive at least in part from Pax2-positive optic stalk neuroepithelial cells (Macdonald et al.,1997). In zebrafish, differentiated reticular astrocytes populate the optic nerve just posterior to the exit point from the eye (Maggs and Scholes,1990; Macdonald et al.,1997). These cells form a thin monolayer situated just beneath the basal lamina and thus ensheathe the optic nerve. Processes from the reticular astrocytes appear to extend into the nerve bundle and contact nodes of Ranvier–regions devoid of axon wrapping by the oligodendrocytes. In addition to the optic nerve, reticular astrocytes are found at the inner limiting membrane of the neural retina, with a particular high density at the optic nerve head. In contrast to mammals, zebrafish reticular astrocytes do not express Glial fibrillary acidic protein and instead are marked by cytokeratin-18 immunoreactivity. Like mammals, however, the zebrafish retinal reticular astocytes are often found in association with the nerve fiber layer and blood vessels (Koke et al.,2010). The function of reticular astrocytes in zebrafish has not been directly assessed. However, in mammals these cells are thought to mediate several diverse processes. During development, reticular astrocytes help pattern the retinal vasculature (Fruttiger,2007). Within the mature retina, reticular astrocytes can influence vascular tone through their interactions with endothelial cells, but also provide metabolic support to neurons (Attwell et al.,2010; Allaman et al.,2011).
Oligodendrocytes represent a third class of glial cells generated outside of the eye. Oligodendrocytes wrap the axons of the optic nerve and provide the components that make the myelin sheath. Oligodendrocytes of the CNS in general derive from ventricular zone neuroepithelial cells (Rowitch and Kriegstein,2010). These cells first give rise to neurons and later to oligodendrocyte precursor cells (OPCs). In zebrafish, like other vertebrates studied, these OPCs are Olig2- and Sox10-positive (Takada et al.,2010). OPCs migrate away from the ventricular zone to differentiate in association with the axons they will wrap. The exact ventricular zone location, which gives rise to optic nerve oligodendrocytes, has not been described in zebrafish or other vertebrates. However, as development progresses, mammalian optic nerve OPCs reside centrally within the fascicles of the optic nerve (Jennings et al.,2002). Currently, the only established function of oligodendrocyctes is to provide insulation to retinal ganglion cell axons and facilitate saltatory propagation of action potentials (Baumann and Pham-Dinh,2001).
Development of the Vasculature of the Eye
The process of intraocular vascularization is initiated by a population of mesodermal and neural crest cells that have migrated around the optic cup to enter the eye through the choroid fissure to form the periocular mesenchyme (POM). This mesenchyme gives rise to the first hyaloid vessels. Rapidly, the vessels organize in a hemispherical basket at the back of the lens forming the hyaloid vasculature, which is composed entirely of arterial vessels (Gariano,2003; Kitambi et al.,2009).
This embryonic hyaloid vasculature is eventually replaced by the mature retinal vasculature that is tightly associated with the retina ganglion cell (RGC) layer. In humans, the hyaloid vasculature is a transient structure that regresses and is replaced by the mature retinal vasculature. In zebrafish, this transition is less dramatic and occurs without replacement of the hyaloid vasculature (Alvarez et al.,2007). Failure of regression of the hyaloid vasculature in humans is associated with a developmental disorder known as retinopathy of prematurity (ROP). The vasculature of the mature eye consists of independent retinal and choroidal vessels. The retinal vasculature is less extensive in zebrafish than humans; the vessels associate with the RGC layer, but do not branch to form subretinal plexi within the inner and outer plexiform layer as in the human retina. Despite the difference in the distribution of this vascular network, ultrastructural analysis shows that the zebrafish vessels share similar features to mammalian retinal vasculature including a muscular coat containing vascular smooth muscle cells (Alvarez et al.,2007). In mammals, retinal vascular development is controlled by interactions between sensors of hypoxic stress including retinal ganglion cells, glial cells (retinal astrocytes and Muller glia), and endothelial cells (Dorrell and Friedlander,2006; Fruttiger,2007; Sapieha et al.,2008). The cell populations involved in stimulating retinal vasculogenesis in fish are still to be determined (Maggs and Scholes,1990; Alvarez et al.,2007; Koke et al.,2010).
The choroidal vasculature is a second circulatory system that surrounds the retina, is a dense capillaries network closely associated with the RPE and supplies oxygen and nutrients to the photoreceptor cell layer. As for the hyaloid vessels, it is believed that endothelial cells originate from the mesodermal component of the POM while stromal cells, melanocytes, and pericytes are derived from neural crest cells. Although the molecular mechanisms are largely unstudied, the development of the choroidal vasculature appears to depend on the presence of differentiated RPE (Saint-Geniez and D'Amore,2004). Defects in this set of vessels are associated with age-related-macular degeneration (AMD).
In addition to these two canonical vasculature systems, most fish have an extra vasculature system located behind the retina, the choroid rete mirabile. This is a counter current capillary exchange system responsible at least in part for the maintenance of a high partial pressure of oxygen in the retina (Barnett,1951; Wittenberg and Wittenberg,1974).
The final step in the development of a functional vasculature is the formation of the blood retinal barrier (BRB), which has an endothelial component formed by vascular endothelial cells lining the retinal vessels and an epithelial component formed by the RPE. Increased vascular permeability and breakdown of BRB is associated with loss of vision and can be induced by hypoxic conditions. The recent development of a transgenic fish line that visualizes the BRB (Xie et al.,2010) will facilitate studies aimed at understanding the formation and maintenance of this important barrier in both health and disease.