Morphogenesis of the anterior segment in the zebrafish eye
© Soules and Link; licensee BioMed Central Ltd. 2005
Received: 27 April 2005
Accepted: 28 June 2005
Published: 28 June 2005
The ocular anterior segment is critical for focusing incoming light onto the neural retina and for regulating intraocular pressure. It is comprised of the cornea, lens, iris, ciliary body, and highly specialized tissue at the iridocorneal angle. During development, cells from diverse embryonic lineages interact to form the anterior segment. Abnormal migration, proliferation, differentiation, or survival of these cells contribute to diseases of the anterior segment such as corneal dystrophy, lens cataract, and glaucoma. Zebrafish represent a powerful model organism for investigating the genetics and cell biology of development and disease. To lay the foundation for genetic studies of anterior segment development, we have described the morphogenesis of this structure in zebrafish.
As in other vertebrates, the zebrafish anterior segment derives from diverse origins including surface ectoderm, periocular mesenchyme, and neuroepithelium. Similarly, the relative timing of tissue differentiation in the anterior segment is also conserved with other vertebrates. However, several morphogenic features of the zebrafish anterior segment differ with those of higher vertebrates. These include lens delamination as opposed to invagination, lack of iris muscles and ciliary folds, and altered organization in the iridocorneal angle. In addition, substantial dorsal-ventral differences exist within the zebrafish anterior segment.
Cumulatively, our anatomical findings provide a reference point to utilize zebrafish for genetic studies into the mechanisms of development and maintenance of the anterior segment.
The anterior segment of the vertebrate eye is comprised of the cornea, lens, iris, ciliary body, and highly specialized tissue at the iridocorneal angle. Two main functions are ascribed to the ocular anterior segment. The first is to focus incoming light onto the neural retina and the second is to regulate intraocular pressure. For mammals and other higher vertebrates, refraction of light entering the eye is accomplished by both the transparent cornea and lens. In many aquatic vertebrates, including fish, the lens is solely responsible for focusing incoming light [1, 2]. In all vertebrates, intraocular pressure is maintained by the balance between aqueous humor production and outflow . The dynamics of aqueous humor have been best characterized in mammals where ciliary epithelial cells produce the clear ocular fluid while the trabecular meshwork, which is situated at the iridocorneal angle overlying Schlemm's canal, regulates drainage.
The structures of the anterior segment arise from diverse embryonic lineages and there is exquisite coordination among the different compartments during development. Studies in avian and mammalian species have shown that tissues of the anterior segment derive from surface ectoderm, head mesoderm, neural crest and neuroectoderm [4–7]. Development of the anterior segment initiates with the invagination of the lens from surface ectoderm. With establishment of the lens vesicle, head mesoderm and neural crest cells migrate into a periocular location and eventually move into the anterior segment of the rudimentary eye between the surface ectoderm and the neural retina and lens. These mesenchymal cells differentiate into the corneal endoderm, structures at the iridocorneal angle, and iris and ciliary body stroma. The non-pigmented and pigmented epithelium of the iris and ciliary epithelium derive from the peripheral edge of the retinal neuroepithelium and retinal pigmented epithelium, respectively. Developmental anatomy of the anterior segment for many higher vertebrates has been well characterized and excellent reviews exist [8, 9]. However, the relevant cellular interactions between various structures of the anterior segment and the molecular basis of development is just beginning to be understood.
A detailed understanding of the mechanisms of development of the anterior segment can provide general insights into questions such as tissue induction, cell type fate determination, and the regulation of cellular morphogenesis. In addition, an understanding of ontogeny of the anterior segment has significance to several human diseases. Primarily, several forms of glaucoma are associated with anterior segment dysgenesis and genes which are essential for formation of this part of the eye can promote glaucoma [9, 10]. Corneal dystrophies and lens cataracts are additional examples of anterior segment disease. The zebrafish has many experimental advantages for studying both development and disease phenotypes, including those for glaucoma . These include the ability to conduct genetic screens for complex traits owing to high fecundity and genomic infrastructure. In addition, ease of transgenesis for target gene functional analysis, coupled with rapid and transparent initial development, facilitates cell behavior characterization via time-lapse microscopy. However, an overview of the development for the zebrafish anterior segment has not been described and the extrapolation of ocular anatomy from other teleost species is not favorable due the high morphological diversification of the bony fish . In this study we report the characterization by light and transmission electron microscopy (TEM) the morphogenesis of the zebrafish anterior segment. We find that while there is rapid initial establishment of the anterior segment, occurring within the first three days of embryogenesis, significant growth and morphogenesis continues until approximately 1 month when the mature morphology is attained. There are also significant differences in the elaboration of dorsal versus ventral regions within the zebrafish anterior segment. Importantly, both similarities and differences exist between the anatomy of the zebrafish ocular anterior segment and that of mammalian eyes.
Establishment of the anterior chamber
By one month, the cornea has further stratified (Figure 3G, H). The epithelium has lost its scalloped appearance and is no longer continuous with the outer epidermis. As compared to mammals, the corneal epithelium is relatively thick containing 3–4 layers of interdigitated cells (arrows, Figure 3H). An increase in the number of adherens juctions was noted between surface epithelial cells and subepithelial cells (Figure 3I, J). A thin extracellular deposition (Bowman's membrane) separates the subepithelial cells from the the corneal stroma layer. The stroma, although thin compared to mammals, has increased in thickness with development and maintains the orthogonal array of collagen fibrils (Figure 3K). Sparsely distributed, flattened cells can be seen upon electron microscopy within the stroma, particularly at the peripheral edges. The number of these keratocytes, as well as the thickness of the stroma, increases with age past 1 month. At the peripheral limbal region, the stroma splits into multiple layers. Just beneath the stroma is a single layer of cells overlying a second, thinner stromal layer with similar collagen organization (Figure 3K, ). Subjacent to these cells, an additional collagen-rich extracellular layer can be seen with electron microscopy (Descemet's membrane). The endothelium is also thin compared to mammals and is comprised of a single layer of flattened cells which extend over the iridocorneal angle covering the surface of the annular ligament. Mucus secreting goblet cells were frequently observed at the peripheral edges of the cornea (data not shown).
The iris and ciliary zone
Anterior chamber angle and annular ligament
A striking difference between the embryonic angle and that of the adult zebrafish, is the presence of the annular ligament. The annular ligament is a prominent, but highly variable feature between species of bony fish [1, 18]. The annular ligament is named for its ligament-like, fibrous meshwork appearance in aldehyde-fixed preparations. This meshwork fills much of the iridocorneal angle and runs circumferentially (thus annular) throughout the anterior segment. In zebrafish, this structure is visible by histology at approximately 17 dpf (Figure 7 and 8). It appears to differentiate from the mass of mesenchymal cells that are present during early developmental stages in the angle region. The shape and extent of the annular ligament varies greatly between dorsal and ventral regions of the eye. In the dorsal region, the inner surface of the annular ligament forms a deep "U" shape (Figure 7F). In the ventral region, the extent of the annular ligament is reduced and forms a "funnel" shape (Figure 8F). As part of this shape difference of the anterior segment angle, the iris projects slightly forward in the dorsal region, while in the ventral half, the iris bends posteriorly (Figure 9A). By histology, the annular ligament appears porous and devoid of nuclei. However, electron microscopy revealed that small, irregularly shaped nuclei are present in annular ligament cells (Figure 9C). Ultrastructurally these cells contain very large accumulations of non-membrane bound aggregates of what appears to be glycoprotein. In other teleosts, these cells have been described as glycogen aggregates , but in zebrafish, the fibrilar nature of the aggregated material appears to be glycoproteinatious in nature. Interspersed within the glycoprotein aggregates are islands of darker staining cytoplasm. The surface of the annular ligament facing the anterior chamber is lined by an endothelium. The endothelium, at the base of the annular ligament, becomes more villous with numerous cytoplasmic convolutions. The annular ligament itself thins at the edges and extends onto both the posterior surface of the cornea and the anterior surface of the iris at its base.
Ventral angle specializations
Serial histological sectioning in both embryos and adults revealed specializations at the iridocorneal angle within a narrow region of the ventral anterior segment (Figure 10). These specializations include a network of canals and openings that lead to episcleral vasculature. Within this region, the iris and ciliary zone dramatically change in morphology. The tip of the iris forms a nodule and bends sharply towards the vitreous (Figure 10I–L). Another feature of this ventral specialization is the loss of the non-pigmented ciliary epithelium. At the ciliary zone, the iris thickens and forms a thumb-like structure that projects into the vitreous and contains melanophores and non-pigmented myoepithelial cells (Figure 10I, 11B). By electron microscopy, the non-pigmented myoepithelial cells show thickened, electron dense intracellular filaments and may have contractile function to regulate lens position via the zonules (Figure 11H). A ventral canal is present as early as 3 dpf and matures significantly into a canalicular network as the embryo grows. This complex structure appears to arise as a specialization of the embryonic fissure.
Within this defined ventral region at the ciliary zone, there is another major branch of the canal that penetrates through the base of the iris and also connects to the angular aqueous plexus (Figure 12). Similar to the canal at the iridocorneal angle, this passage is also endothelial lined. At 5 dpf, flattened endothelial cells line the narrow opening which occasionally contains undifferentiated and red blood cells (Figure 12C, D). In the mature fish, the endothelial cells that line this ciliary canal have further differentiated (Figure 12G). These cells line the opening to the canal and show large vacuoles and extensive cyctoplasmic extensions (Figure 12H). The dorsal angle completely lacks this canalicular network and does not have either the band of absorptive-looking tissue at the iridocorneal angle or the subjacent ciliary passage.
Zebrafish as a model for anterior segment development and disease
Zebrafish have several experimental advantages that have made this a valuable model organism for analysis of mechanisms of development and disease. Experimental advantages include the ability to efficiently conduct mutational analyses, including those designed to reveal complex genetic interactions. Ease of transgenesis facilitates gain-of-function and cell labeling studies. Cell labeling coupled with rapid, transparent, and external embryogenesis enable in vivo cell behavior studies during development. In our analysis, we have characterized the morphogenesis of the zebrafish anterior segment of the eye in order to establish an overview reference for future studies on the mechanisms of development and diseases of this structure. Ontogeny of the zebrafish anterior segment is protracted occurring over the first month of development. Some structures of the anterior segment such as the lens and cornea differentiate relatively early and show only peripheral growth. Other structures such as the iris, ciliary zone, and iridocorneal angle undergo dramatic morphological changes during this time. Our analysis of zebrafish anterior segment development has also identified differences between the dorsal and ventral regions. In particular, structures specialized for regulating aqueous humor dynamics are localized in a dorsoventral specific manner.
Comparing anterior segment development in zebrafish and mammals
Overall morphogenesis of the anterior segment in zebrafish is similar to mammals and other vertebrates but there are some differences. For example the lens, although derived from surface ectoderm, delaminates as opposed to invaginating. Periocular mesenchyme appears to contribute to the corneal endothelium, iris stroma and angle specializations like other vertebrates. However, there appears to be a proportionally smaller bolus of immigrating mesenchymal cells and migration does not occur in obvious waves as in the chick [4, 19]. Local proliferation appears to be a driving force for growth of the anterior segment in zebrafish, similar to that in the mouse [6, 20]. The relative timing of differentiation for the various components of the zebrafish anterior segment is like that for other vertebrates, suggesting conservation in tissue interaction and inductive events [19, 21].
While hallmarks of anterior segment development in zebrafish appear to be largely conserved with other vertebrates, anatomical specializations exist. For example, the iris stroma is non-contractile and devoid of muscle cells and the ciliary "body" does not contain processes or a circumferential band of muscle. For this reason, we refer to the region adjacent and posterior to the iris as the ciliary zone, and not the ciliary body. The ciliary zone, however, is expanded and has a morphology consistent with contraction in a small area of the ventral-most region. The ciliary epithelium also displays dorsoventral differences. The ciliary epithelium in the dorsal eye appears specialized for secretion of aqueous humor while the ciliary epithelium in the ventral most region does not show extensive ultrastructural specializations. Instead, the ventral ciliary zone appears specialized for aqueous humor drainage. Other anatomical differences with mammals can be found within the iris. Within the iris stroma, in the place of muscle cells are lamina of pigment cells of various types. These include melanophores, iridophores, and xanthophores. The exact function of these chromophores in the iris are unknown, but they likely serve to prevent light from entering the eye in regions outside of the pupil. Finally, similar to the mammalian iris, the zebrafish iris is highly vascularized (Figure 5 and 6).
Morphological specializations at the iridocorneal angle
Another difference with mammals is found at the iridocorneal angle. In mammals, the angle is lined circumferentially with a trabecular meshwork, a complex structure of extracellular "trabecular" beams and a "meshwork" of absorptive endothelial cells. The trabecular meshwork forms a resistant barrier for aqueous humor outflow and covers Schlemm's canal. Schlemm's canal is a network of small canalicular passages leading to episcleral vessels where aqueous humor is ultimately drained. In zebrafish, the iridocorneal angle is lined with the annular ligament. This structure does not appear to be functionally analogous to the trabecular meshwork of mammals. However, within the ventral region of the angle, a specialized canalicular network of endothelial-lined openings does appear to be functionally analogous to the aqueous humor outflow structures of mammals. The circumferential ring of villous endothelial cells overlying the annular ligament at the iridocorneal angle may also function in absorption of metabolic byproducts, ions, or proteins in the aqueous humor. The anatomy of the mature zebrafish anterior segment suggests that in this species aqueous humor is produced primarily by dorsal ciliary epithelial cells, flows around the iris and into the anterior chamber, and then exits via a canalicular network in the ventral-most region. Additional experiments will be required to investigate this possibility further.
Cumulatively, our anatomical findings provide a reference point for utilizing zebrafish in genetic studies of the mechanisms of development and maintenance of the anterior segment. We find that while the morphological details of anterior segment structures in the zebrafish differ from higher vertebrates in many aspects, these structures show an overall conservation in anatomy and develop from similar cellular lineages. As in other vertebrates, surface ectoderm, periocular mesenchyme, and the anterior margin of the retinal neuroectoderm contribute to structures of the anterior segment. The timing of morphogenesis is also conserved among vertebrates. For example the lens and cornea are first to mature, while the iridocorneal angle does not reach adult morphology until later stages. In addition to providing a baseline for genetic studies of anterior segment development, our observations coupled with previous physiological approaches , provide insights into how aqueous humor is regulated in zebrafish and support the use of this species for investigation of anterior segment development and disease.
Wild type zebrafish (Danio rerio) of the AB/AB and TL/TL backgrounds were reared under standard conditions with a light cycle of 14 h light/10 h dark . No differences in anterior segment anatomy were observed between these two stains. Specimens were collected at various times of development from 24 hours to 2 months post fertilization. All individuals were photographed to document the body length prior to histological analysis. Prior to fixation, fish were anesthetized in 0.2 mg per ml of ethyl 3-aminobenzoate methanesulfonate (tricane). All experiments were performed in compliance with the ARVO statement for use of animals in vision research.
Fish were fixed in primary fixative [2% paraformaldehyde, 2.5% glutaraldehyde, 3% sucrose, 0.06% phosphate buffer (pH 7.4)] at 4°C for at least 24 hours. Fish were washed in 0.1 M phosphate-buffered saline (PBS), dehydrated through an ethanol series and propylene oxide and then infiltrated with EMbed-812/Araldyte resin mixture. Traverse, semi-thin (1 μm), plastic sections were cut with a glass knife on a JB4 microtome. Serial sections were collected from the central retina, defined as maximum lens diameter at the optic nerve. Following heat fixation to glass slides, sections were stained with 1% Toluidine Blue in 1% Borax buffer. Images were captured using a Nikon coolpix 995 digital color digital camera mounted on a Nikon E800 compound microscope with a 60X oil-emersion objective.
Fish were fixed in primary fixative and washed as for LM. Specimens were then post-fixed with 1% Osmium Tetroxide on ice for 1 hour to preserve membranes. Fish were dehydrated through a methanol series and acetonitrile and infiltrated with EMbed-812/Araldyte resin mixture. Ultrathin sections (60–70 nm) were collected on coated grids and stained with uranyl acetate and lead citrate for contrast. Images were captured using Hitachi H600 TEM.
List of abreviations
TEM: transmission electron microscopy
hpf: hours post fertilization
dpf: days post fertilization
We greatly appreciate the technical expertise of Clive Wells for Electron Microscopy and Michael Cliff in maintaining the zebrafish stocks. We also thank Joseph Besharse, Simon John, Richard Smith, Elena Semina, and Sally Twinning for invaluable discussions. This work was supported by a Pilot Initiative grant from The Glaucoma Foundation (BL) and NIH grant R01EY16060 (BL).
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