Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

Retinal Disease

Photoreceptors are the primary sensory cells within the visual system. There are two main types of photoreceptors within the vertebrate eye: rods, which are monochromatic and respond in low light levels, and cones, which respond to higher light levels and specific wavelengths within the visual spectrum. The system of light absorption, ion fluctuation, and neuronal transmission are processes that require significant energy and, thus, the retina is one of the highest-energy-consuming tissues in the body. The high level of oxygen consumption by photoreceptors makes them particularly susceptible to injury and perturbation often resulting in cell death. Retinal degeneration is a leading cause of blindness in the developed world. The most common form of degeneration is age-related macular degeneration, which first affects the cones within the central retina (macula) and then progresses to the periphery.

Retinal degeneration can also occur as a result of genetic mutations. Achromatopsia and Retinitis pigmentosa (RP) together define a large class of heritable diseases that affect vision in humans. Both of these diseases are caused by a wide variety of mutations that disrupt visual transduction and photoreceptor maintenance. Achromatopsia is characterized by defects in the cone photoreceptors while rods remain functional, resulting in extreme light sensitivity and color blindness or rod monochromacy. Achromatopsia symptoms are generally seen at birth and the vision loss is only rarely progressive. RP, in contrast, develops during childhood and in later stages of life starting with degeneration of the rod photoreceptors, and progressing in some cases to total blindness. RP is estimated to affect 1 in 10 000 people. Due to the diversity of genes associated with these disease states, most therapeutics have focused on the general prevention of cell death as a method for limiting progressive vision loss.

Animal Models of Vision

Many animal models are used to study the visual system, and each possesses various strengths and weaknesses. By using a variety of systems scientists are able to capitalize on the strengths of all of them. The two most common systems for retinal modeling have been the fruit fly, Drosophila melanogaster, and mice, with a variety of work occurring in related species such as rabbit or ferret.

Drosophilae have mainly been used to study ocular development and patterning. Surprisingly, despite the obvious structural differences between the insect compound eye and our own, many of the same signaling cascades are used to establish the structure and patterning of the Drosophila eye. Drosophilae have an extremely short

gestation period and well-established methods for rapid genetic manipulation, and these features have been used to understand the roles of multiple genes in eye development. However, there are still many limitations in the use of this organism, particularly in the modeling of retinal disease. Several differences exist between the insect and mammalian phototransduction cascade, and the structural differences of the compound eye also limit the applicability of this organism for disease studies.

The most commonly used mammalian animal model of the retina is the mouse. Mice have a number of advantages as a model system. There is an extensive literature on a variety of mutants that have been studied for many years. There are also multiple techniques for genetic manipulation, including the ability to modify genes and genomic loci and several techniques for retinal explantation for in vivo imaging. However, mice are nocturnal creatures and depend primarily on their olfactory system for foraging and predator identification. As a result, unlike the human retina, the mouse retina is dominated by rod photoreceptors and contains only 3% cone photoreceptors. While this makes mice ideal for studying rod photoreceptor disease and function, the study of cone physiology and function is less straightforward in this system. Additionally, mouse eye development occurs in utero, making it difficult to image and understand the early stages of eye development and retinal perturbation.

The Advantages and Techniques of the

Zebrafish Model System

The zebrafish system has a number of benefits for studying visual development and disease. First, zebrafish vision, similar to humans, is cone dominated. They rely upon their vision for food acquisition and have four different cone types: red, green, blue, and ultraviolet. In the zebrafish, ocular development occurs externally over the first 5 days postfertilization (dpf). During zebrafish development, the cone photoreceptors mature first between 3–5 dpf followed by the rods, which mature between 15–20 dpf. Thus, early zebrafish vision is dependent solely on cone-mediated vision. The rapid development of the visual system and early cone dominance have facilitated several genetic screens using young embryos to select specifically for cone related defects (see below).

Zebrafish were originally developed as a model system because they are inexpensive to keep, and take only 3 months to reach sexual maturity after which a mating pair will produce 200–300 eggs per week for at least a year. Further, development of many different genetic tools has added greatly to the versatility of this model for scientific study. In particular, mutagenesis protocols were optimized in the mid-1990s to introduce high frequencies of mutations. This made it possible to conduct

large-scale forward genetic screens. Hundreds of different mutants affecting many aspects of vertebrate development and function have been identified. The constantly improving annotation of the nearly completed genome makes identifying mutated genes straightforward, and an impressive collection of vision mutants is available.

Another advantage is that cellular behavior can be observed in the intact, living organism. During the first 2 weeks of development, zebrafish larvae can be maintained in a translucent state and visualized live using either exogenous or genetically encoded fluorescent markers (see examples in Figure 1). Zebrafish can be maintained alive and healthy in agar on the microscope stage for days. Thus, cells can be imaged over the course of development or degeneration without perturbing the extracellular environment. At this point, transgenic lines containing fluorescent markers in various cell types are simple to generate and maintain. Transient injection of plasmid DNA at the one-cell stage will also produce a mosaic expression of genes throughout the fish allowing for the study of isolated cells within the intact animal. Multiple promoters have been developed to drive expression in various types of cells within the eye.

While no procedures exist to manipulate specific genes within the genome, several methods have been developed which allow scientists to circumvent this limitation. Morpholinos are synthetic RNA-like oligonucleotides that can be injected at the one-cell stage or electroporated into the fish at later stages. These morpholinos bind to corresponding RNA sequences within the cell, resulting in the degradation of the RNA and loss of protein expression. The loss-of-protein function is not as complete as is seen in knockouts and is only transient, but it has allowed

researchers to study the effects of specific knockdowns during development. Additionally, several labs have developed a method known as targeting-induced local lesions in genomes (TILLING) in which high-throughput polymerase chain reaction (PCR) methods are used to identify specific gene mutations from libraries of randomly mutagenized fish.

Very recently, another method has been established in which nucleases are used to produce double-strand breaks at specific loci within the genome. These double-strand breaks are repaired by nonhomologous end joining, often resulting in deletions or insertions at the break site that can lead to frame-shift mutations in the target gene. The targeting specificity of these double-strand breaks is established by fusion to an array of zinc finger domains that bind to specific DNA sequences. Each zinc finger recognizes a 3-bp sequence and three zinc fingers are fused together to create a 9-bp recognition domain. Further, these zinc finger nuclease arrays must dimerize to activate the nuclease such that a total 18-bp recognition sequence is required. Currently, the technology for generating the zinc finger arrays is cumbersome, but soon this will be a rapid and convenient way to generate targeted zebrafish mutants.

Methods have also been developed to create chimeric fish allowing for the rapid evaluation of the cell autonomous nature of genetic phenotypes. To produce these fish, eggs are grown to the blastula stage and then cells from one egg are removed and inserted into another egg. This procedure does not affect fish development and wild-type chimeras grow normally. The production of chimeric fish can be used to determine the critical cell population for the phenotypic changes associated with a mutation. It can also be useful for the evaluation of neighboring and surrounding effects.

Zebrafish have one other major advantage for the study of disease, which is the aqueous environment in which they reside. Methods are being developed for rapid high-throughput drug screens using zebrafish by adding compounds to their water and evaluating the effects. Most of the current studies with this method use fluorescent cell markers to indicate the presence or absence of various cells. This technique has been used with fluorescent hair cells to identify compounds that prevent hair cell death in the presence of the ototoxic agent neomycin. As zebrafish are small and easy to maintain, they are the best vertebrate model for this type of shotgun approach to drug development.

Evaluating zebrafish vision

There are several ways of evaluating zebrafish vision. The simplest involves a manipulation of the fish instinct to maintain its position within a moving stream. In this test, groups of fish can be placed in a long dish with a series of moving bars along its side. As the bars move, the fish respond to the apparent current by swimming to maintain their position with the bars. Thus, shoals of fish can be tested simultaneously for their ability to see and respond to the moving lines. This is known as the optomotor response. A related test called the optokinetic response (OKR) is done with a single fish placed in a small dish in the center of a rotating drum decorated with vertical stripes. In this test, the fish’s eye will follow the stripes with periodic involuntary saccades. This is a very sensitive measure of an individual fish’s ability to perceive its visual environment. Several labs have identified blind fish in mutagenesis screens using the optomotor response and/or the OKR. These screens have yielded a variety of mutants that can be used as models for retinal disease.

Another method that has been used to measure fish vision is the electroretinogram (ERG), which is a stimulated measure of the electrical response of the eye to a flash of light. In these measurements, the fish are dark adapted and a small electrode is placed on the cornea. The eye is then stimulated with a light flash and the electrical response is recorded. This technique records both the primary photoreceptor response, which appears as a negative spike at the beginning of the recording known as the a-wave, and the response of the secondary neurons, which is a large secondary positive response following the a-wave, known as the b-wave.

The Visual System: Phosphodiesterase

and Phototransduction

Cyclic GMP (cGMP) phosphodiesterase (Pde) is an important enzyme in the process of phototransduction. During phototransduction the 11-cis retinal absorbs a single photon of light and alters the conformation of the opsin protein to activate the associated heterotrimeric guanosine triphosphate (GTP)-binding protein, transducin. The activated transducin removes the inhibitory gamma subunit from phosphodiesterase, which then degrades cGMP in the outer segments (for a schematic see Figure2). The lowering of cGMP levels stimulates the closure of cyclic-nucleotide-gated ion channels in the plasma membrane, causing hyperpolarization and a decrease in neurotransmitter release at the synapse, initiating light signaling to downstream neurons. Channel closure also interrupts Ca2+ influx leading to a decrease in intracellular [Ca2+]i. The drop in intracellular [Ca2+]i activates a Ca2+-sensitive guanylyl cyclase, restoring cGMP to presignaling levels. Both types of photoreceptors have a similar phototransduction cascade, but use different

genetically encoded enzymes to accomplish each task. Therefore, a mutation in the cone phosphodiesterase will not affect rod phototransduction.

The photoreceptor Pde (Pde6) is a multisubunit enzyme that differs slightly in rods and cones. In both cells the catalytic domain is a dimer that is bound and inhibited by the regulatory gamma subunit called Pg. During phototransduction, activated transducin binds to Pg and exposes the catalytic cGMP-binding site allowing the catalytic domain to cleave cGMP. In rod photoreceptors, two related but different proteins, Pa and Pb, form the Pde6 catalytic domain. In cones, the catalytic domain consists of a dimer of one protein Pa’, also known as Pde6c.

Retinal Degeneration in pde6 Mutants: Primary Degeneration

Mutations in the Pde gene (pde6) are found in several families with RP and similar mutations have been used for many years as a model for RP in mice. The oldest and most commonly used mouse model of RP is the retinal degeneration 1 (rd1) mouse that contains a mutation in the Pb rod-specific subunit of Pde6, the pde6b gene. In this model, rod degeneration is apparent by postnatal day 8 and nearly all of the rods are lost by 3 weeks of age. Despite many years of study, the process of primary photoreceptor degeneration in the rd1 mouse is not well understood. Initial work has implicated programmed cell death pathways. The death of rods is associated with the extreme DNA cleavage that accompanies apoptosis, and this DNA cleavage can be detected with the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay. However, several studies suggest that the standard apoptotic cascades, including a group of cysteine proteases known as the caspases, and other typical apoptotic effectors, are not involved. Instead, recent work implicates intracellular calcium concentration ([Ca2+]i) and the calpains, a calcium-activated set of proteases found in the mitochondria, as the relevant initiators of photoreceptor programmed cell death.

In rd1 mice, the absence of Pde6 results in elevated levels of cGMP even under dark conditions, and it has been hypothesized that this results in the greater open probability of the cyclic-nucleotide-gated ion channels leading to increased [Ca2+]i levels and apoptosis. However, little is known about the distribution of [Ca2+]i during the death of cones. An initial report using the Ca2+ channel blocker, D-cis-diltiazem, to inhibit [Ca2+]i accumulation showed a decrease in apoptosis. However, other labs using the same and other Ca2+ channel blockers have been unable to repeat these results. Due to the difficulty of imaging a mouse retina in vivo over long time periods, none of these studies has examined the levels of [Ca2+]i within the cells in vivo. The translucence

of the zebrafish retina enables in vivo imaging of both the morphological changes of degeneration as well as changes in levels of signaling molecules, such as Ca2+ or cGMP.

Retinal Degeneration in pde6 Mutants: Secondary Retinal Degeneration, the Bystander Effect – Models and Mechanisms

In humans, RP is characterized by a lack of night vision, but often progresses over time to tunnel vision and, in some cases, complete blindness. This progression of the disease is due a gradual death of cones by apoptosis. Cell death begins in the peripheral retina where the number of rods is highest and moves toward the central retina. The cone photoreceptors are fully functional and do not use the mutated genes for phototransduction. However, the death of the mutant rods causes healthy cones to die apoptotically. This transmission of death to healthy neighboring cells is a process known as the bystander effect. The death of these cones represents the most debilitating part of this disease, and has significant potential for therapeutic intervention.

In addition to retinal degeneration, the bystander effect has been seen in a variety of diseases, including cancerous tumors, and can be either beneficial or detrimental to therapeutic efforts. One of the first descriptions of the bystander effect occurred during studies of gene therapy for cancer in which malignant tumors were injected with viruses containing suicide genes, which convert a prodrug into a lethal compound inside cells. Researchers found that, although only a small population within the tumor expressed the detrimental genes, significant portions of the tumor mass still died, suggesting that virally transfected cells were able to induce death in untransfected neighboring cells. Recently, researchers have found a similar occurrence in cells exposed to radiation therapy. In this case, cells that have not been irradiated show the genetic instability associated with radiation exposure.

In general, apoptosis does not affect the health of neighboring cells and it is unclear why in some instances there is a spread of death across a population. There are currently several hypotheses for how healthy cells are induced to die. One possibility is that live cells release a trophic factor that stimulates the growth and differentiation of their neighbors and is required for their proper maintenance. For instance, it has been suggested that rods release a factor that stimulates the growth of cones. Another possibility is that dying cells release toxic factors that kill neighboring cells. A potential corollary to both of these hypotheses is that either toxic or trophic factors are released to neighboring cells through gap junctions. It is also possible that the immune response triggered by the

A

E

Figure 3 Schematic of some of the possible sources of the bystander effect. (a, b) Live cells release trophic factors (happy faces) that help neighboring cells and are lost when the mutant cells (in red) die. These factors could be released exogenously

(a) or through gap junctions between cells (b). (c, d) Dying cells release toxic factors (skulls) that kill neighboring cells, again either exogenously (c) or through gap junctions (d). (e) The immune response to the presence of dying cells could have a deleterious effect on the remaining healthy cells.

removal of apoptotic cells has a deleterious effect on the neighboring cells. See Figure 3 for a schematic of these possibilities. Understanding the source of the bystander effect will suggest other methods by which it might be prevented.

Levels of the Bystander Effect in Photoreceptor

Degeneration

The extent of bystander cell death is not the same for all cases of retinal degeneration. The death of cones in rod–cone dystrophies has been extensively studied in human patients and in a variety of animal models, including the rd1 mouse. Mutations in rod phototransduction, resulting in rod death, almost always lead to some cone degeneration. However, the number of cells that die within the central fovea varies significantly from patient to patient. Mutations in genes required for cone phototransduction and their effects on rods are less well studied. Cone–rod dystrophies are conditions in which degeneration of the cones causes a decrease in scotopic vision, low light vision that is produced exclusively by rod function. Little is known about the state of rods in these patients, but it is thought that there is some degeneration associated with the visual loss. In contrast, some patients with cone dystrophies have cone death without affecting the rods or scotopic vision.

Differences in the connections between cells in a population might account for the variable levels of bystander death. In particular, it is thought that gap junctions

between rods and cones in mammals may be predisposed to allow the flow of materials from the rods to the cones, but not from the cones to the rods. Thus, it may be possible that the flow of information would be asymmetrical between cell types and this could lead to differences in the effects on neighboring cells.

In order to better understand the bystander effect, an important first step is to determine how death progresses throughout the population and which populations of cells are capable of propagating apoptotic signals to healthy neighbors. For this type of analysis, zebrafish provide an ideal system. Not only are mosaic animals easy to generate, but the transparency of the embryo make it possible to analyze the transmission of death in vivo. This feature combined with the other tools described above provides a novel and powerful approach to examining the bystander effect within the photoreceptor population.

Zebrafish Models of Retinal Degeneration: Mutations in pde6c

Mutations in the zebrafish cone phosphodiesterase have been identified in two separate genetic screens for fish lacking OKR at 5 dpf. One of the mutations is a null mutant (w59), while the other is a missense mutation in a conserved amino acid (els). Recently, recessive mutations in pde6c were also identified in mice and in three human families with achromatopsia. Preliminary results indicate that in humans this form of achromatopsia may be progressive, suggesting the possibility of a bystander-associated death of rods.

pde6cw59

pde6cw59 is a mutation in the splice site between exons 11 and 12 in the pde6c gene. The abnormal splicing caused by this mutation introduces a premature stop codon generating a null mutant. Zebrafish, homozygous for the pde6cw59 mutation, are viable and form normal swim bladders, a marker of general fish health, but must be raised with higher-than-normal food concentrations, as their visual defects impair acquisition of food (Figure 4(a)). In these fish, cone photoreceptors initially develop normally. However, at 4 dpf, when fish first respond to light, the cone photoreceptors in the central retina begin to degenerate. The pde6cw59 mutants exhibit a flat ERG at 5 dpf, indicating a total lack of photoreceptor response to light and no signaling to downstream neurons. At this stage in development, zebrafish vision relies on cones, so a flat ERG is consistent with the rapid cone degeneration observed in these fish. As the cone photoreceptors die, they retract their synaptic connections and outer segments and become spherical. Time-lapse images of this process are shown in Figure 4(b). In the final stages of cell death, only the

(a)

WT

pde6cw59

Outer nuclear layer

Inear nuclear layer

Optic nerve

rounded apoptotic bodies remain (Figure 4(c), arrows). This cell debris travels out of the outer nuclear layer and is disposed of by the macrophages of the immune system.

Within the central retina of the pde6cw59 mutant, a majority of cones die by 5 dpf. Figures 4(d) and 4(e) show the central retina of a wild-type (d) and mutant

(e) eye, expressing membrane-tagged cyan fluorescent

protein (MmCFP) specifically in the cone photoreceptors. The MmCFP accumulates in the outer segments of the cones. In Figures 4(d) and 4(e), the eye is visualized by removing it from a day-6 zebrafish larva and inverting it onto a microscope slide. In the wild-type eye, the central retina is densely populated with cones that show a regular mosaic pattern (Figure 4(d)). In the pde6cw59 eye,

most of the cone photoreceptors have died and been removed from the retina, and many of the remaining outer segments appear dystrophic (Figure 4(e)).

Unlike humans or mice, fish continue to produce photoreceptor cells throughout their life. At the periphery of the eye is a region of cells known as the circumferential marginal zone, in which new photoreceptors are generated by multipotent stem cells. In this zone, young cones are constantly differentiating throughout the life of the animal. Even in adult pde6cw59 mutants, there are always cones in this region of the eye, indicating that there is no defect in cone morphogenesis or differentiation, but that cones die as they mature. The circumferential marginal zone is not visible in Figures 4(d) and 4(e) but can be seen in Figure 4(a) as a faint ring of fluorescence around the periphery of the pde6cw59 eye.

The rods in the central retina also deteriorate during early development in pde6cw59 mutants but later recover. At 7 dpf, the rods appear normal and are slightly more clustered than in wild type, but not reduced in number. At 8–9 dpf, the number of rods begins to decrease in the central retina. The outer segments of the remaining rods in the central retina appear dystrophic. The deterioration of the rods continues through at least 6 weeks postfertilization. These data were the first evidence that zebrafish can undergo a bystander effect in the retina (i.e., mutant cones kill neighboring healthy rods).

The multipotent stem cells in the circumferential marginal zones of the eye can differentiate into rods throughout the life of the animal. Additionally, in cases of damage or injury, the Mu¨ller glia can also enter a mitotic state and produce stem cells capable of forming all types of retinal neurons. In the pde6cw59 mutants, this continuous regeneration eventually replenishes the small number of rods that have died. Thus, by 3 months postfertilization, the retina is completely populated with rods. Interestingly the pde6cw59 mutants never develop a scotopic ERG or OKR, indicating that even the remaining rods are unable to form proper connections with the downstream neurons in the eye. Unlike human and mouse eyes, zebrafish do not have separate rod and cone bipolar cells. The bipolar cells that connect to rods also connect to cone photoreceptors. The lack of scotopic vision in the pde6cw59 mutant suggests that the cones are necessary for the proper connections of the rods with their bipolar targets. In support of this theory, the bipolar cells of the pde6cw59 embryos often show an altered morphology with axons that extend into the ganglion cell layer and dendritic branches that send filopodia into the photoreceptor layer.

The pde6cw59 mutant allows for exceptional visibility of photoreceptor degeneration. Due to the rapid degeneration of the cones and rods in the central retina, it is possible to monitor many aspects of cell death as they are occurring. However, in the human diseases of RP and Achromatopsia, retinal degeneration can be a slow

process occurring over several years. Thus, although it is easier to visualize cell death and develop drugs that will combat the rapid loss of photoreceptors in the pde6cw59 mutation, it would also be useful to have a mutant where degeneration occurred over a more protracted period of time. Recently the els mutant was identified as a mutation in pde6c that results in slow cone degeneration.

els

The els mutation produces a single amino acid change; methionine (M) 175 is mutated to an arginine (R) (M175R) in the first GAF domain of Pde6c. At 5 dpf, when zebrafish vision is cone dependent, fish that are homozygous for the els mutation have a flat ERG and no OKR, although initially the cones appear morphologically normal. This indicates that phototransduction is disrupted but surprisingly cell death has not been triggered. This finding suggests that the els allele is not null for the Pde6c protein, but that secondary defects within the els cones may be disrupting phototransduction, resulting in a flat ERG. One finding in support of this idea is that, although all four types of cones are initially present in the els mutant, the localization of various opsins within the photoreceptors is abnormal. Generally, opsins are found only in the outer segments, but in the els mutant, opsin proteins are found throughout the cell. At this stage, there is also a small but significant increase in the number of apoptotic cells in the els retinas. By 3 weeks postfertilization, the cones and rods in the central retina have begun to show an altered morphology, and the number of cone cells has decreased. However, despite their slightly altered morphology, rod maturation occurs in these fish and by 3 weeks postfertilization the fish respond to OKR under scotopic conditions.

As the els fish matures, the cones continue to deteriorate and by 6 months postfertilization the retina no longer contains cones, but consists entirely of rods. The rods are morphologically normal and, unlike those found in pde6cw59, functional. This suggests that the significantly slower death of cones in els compared to pde6cw59 fish allows rods to form proper synaptic connections with bipolar cells. Interestingly, the total number of cells in the inner retina is decreased in this mutant; however, the ratio of inner retinal cells to rods has increased over wild type, suggesting that the rods are forming more connections in the mutant than they do in the wild-type retina. The number of mitotically dividing cells in the retina is also increased in the mutant, suggesting that there is an increase in cellular proliferation probably resulting from the death of the cone photoreceptors.

Pde6 Structure

Recent work on the structure and function of Pde6c has shed some light on the potential effects of the els mutant

for the Pde6c holoenzyme. The catalytic subunit of the Pde6 protein consists of two domains: a regulatory domain with two GAF domains, one of which (GAF A) binds cGMP, and a catalytic domain that has phosphodiesterase activity. The inhibitory subunit Pg binds to the GAF A domain though its C terminus and the catalytic domain through interactions with its N terminus. Recent work with the rod Pde6 has suggested that the binding of cGMP to the GAF A domain may increase the binding affinity of the N terminus of Pg for the catalytic domain. Thus, the binding of cGMP to the GAF A domain may help to regulate the activity of Pde6c in vivo. Met175 is located within the allosteric cGMP-binding site of the GAF A domain of Pde6c (Figure 5(a)). A recently determined crystal structure of the GAF A domain from chicken Pde6c reveals that the side chain of Met175 (equivalent to Met179 in chicken Pde6c) is in close proximity to the cyclic phosphate group of cGMP, although it does not directly interact with the cyclic nucleotide. As in other cyclic-nucleotide-binding Pde GAF domains, the phosphate group of the ligand is stabilized through the positive dipole of helix a3 (Figure 5(b)). The mutation M175R (equivalent to M179R in chicken Pde6c) introduces a larger and positively charged side chain into the binding pocket and thereby changes the cGMP-binding environment and, potentially, the binding affinity for the allosteric regulator cGMP (Figure 5(c)). A model in which a methionine side chain is substituted for an

arginine side chain suggests three potential consequences of the M175R mutation. First, the R175 side chain clashes with helix a3, which may disrupt the dipole interaction between the helix and the phosphate group. Second, the R175 side chain clashes directly with the phosphate group of cGMP. Third, R175 might form a salt bridge with the phosphate group. In the former two scenarios, R175 would disrupt cGMP binding and lower the binding affinity of the GAF domain for cGMP, whereas in the latter scenario, R175 may cause a higher affinity for cGMP.

In either model, it is likely that the M175R mutation interferes significantly with cGMP binding and the sensitive regulatory mechanism of Pde6 through its GAF A domain. This, in turn, may lead to impaired Pde6 function and cause overor underexpression of Pde6 protein, thereby disrupting the cone photoreceptor function. No data are available on the stability or functionality of the els mutant protein. However, the phenotypic comparison with the w59 mutant suggest that the els mutant is not a null mutation, but creates a more subtle effect on the levels or activity of the Pde6c protein. The proximity of the mutation to the cGMP-binding site in the GAF A domain implicates a possible disruption of the intramolecular allosteric regulation of catalytic activity ascribed to this portion of Pde6c.

The els mutant represents a unique opportunity to understand more about the enzymatic functions of Pde6 and its role in retinal degeneration. The slower degeneration

of this mutation compared to the w59 allele more accurately reflects slower human forms of degeneration. These two zebrafish pde6c mutants will provide complementary tools for studying Achromatopsia and apoptosis due to phosphodiesterase deficiency.

Conclusion

Zebrafish have gained prominence as a model for retinal disease. Cone-based vision, visual translucence, inexpensive maintenance, and rapid external embryologic development help make this model particularly exciting for retinal studies. Several genetic screens for blind fish have identified a variety of mutants that mimic human retinal disease. Among these, the pde6c mutants are a particularly good example of a retinal model that has been studied for many years in mice, and will benefit from the types of study available in the zebrafish system. In particular, the potential for live imaging of cells in vivo in an intact animal presents a novel opportunity to visualize and understand the source of photoreceptor degeneration.

One of the largest differences between the eyes of zebrafish and humans is the continuous growth of the zebrafish eye, and its regenerative ability in response to damage. Although this regenerative potential can complicate the evaluation of zebrafish as a model organism, it also presents a novel possibility to understand and imitate a natural system of retinal stem cell regeneration. By studying the differences between the zebrafish and mammalian systems it may be possible to stimulate our own potential for retinal regeneration.

The zebrafish model also offers an unprecedented potential for high-throughput drug screening. Using fluorescent cell markers and fluorescent plate readers, it will soon be possible to do large-scale screening of drugs that affect the levels of retinal degeneration. This method can also be used to test permeability and the toxicity of drugs. This is the first vertebrate animal model that provides a method for this type of rapid drug development.

See also: Color Blindness: Inherited; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin; Primary Photoreceptor Degenerations: Terminology; Secondary Photoreceptor Degenerations; Zebra Fish– Retinal Development and Regeneration.

Further Reading

Brockerhoff, S. E., Hurley, J. B., Janssen-Bienhold, U., et al. (1995). A behavioral screen for isolating zebrafish mutants with visual system defects. Proceedings of the National Academy of Sciences of the United States of America 92(23): 10545–10549.

Cote, R. H. (2007). Photoreceptor phosphodiesterase (PDE6):

A G-protein-activated PDE regulating visual excitation in rod and cone photoreceptor cells. In: Beavo, J. A., Francis, S. H., and Houslay, M. D. (eds.) Cyclic Nucleotide Phosphodiesterases in Health and Disease, pp. 165–193. Boca Raton, FL: CRC Press/Taylor and Francis.

Doyon, Y., McCammon, J. M., Miller, J. C., et al. (2008). Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nature Biotechnology 26(6): 702–708.

Goldsmith, P. and Harris, W. A. (2003). The zebrafish as a tool for understanding the biology of visual disorders. Seminars in Cell and Developmental Biology 14(1): 11–18.

Hamada, N., Matsumoto, H., Hara, T., and Kobayashi, Y. (2007). Intercellular and intracellular signaling pathways mediating ionizing radiation-induced bystander effects. Journal of Radiation Research (Tokyo) 48(2): 87–95.

Martinez, S. E., Heikaus, C. C., Klevit, R. E., and Beavo, J. A. (2008). The structure of the GAF A domain from phosphodiesterase 6C reveals determinants of cGMP binding, a conserved binding surface, and a large cGMP-dependent conformational change. Journal of Biological Chemistry

283(38): 25913–25919.

Morris, A. C., Scholz, T. L., Brockerhoff, S. E., and Fadool, J. M. (2008). Genetic dissection reveals two separate pathways for rod and cone regeneration in the teleost retina. Developmental Neurobiology 68(5): 605–619.

Muto, A., Orger, M. B., Wehman, A. M., et al. (2005). Forward genetic analysis of visual behavior in zebrafish. PLoS Genetics 1(5): e66.

Nishiwaki, Y., Komori, A., Sagara, H., et al. (2008). Mutation of cGMP phosphodiesterase 6alpha’-subunit gene causes progressive degeneration of cone photoreceptors in zebrafish. Mechanisms of Development 125(11–12): 932–946.

Paquet-Durand, F., Johnson, L., and Ekstrom, P. (2007). Calpain activity in retinal degeneration. Journal of Neuroscience Research 85(4): 693–702.

Ripps, H. (2002). Cell death in retinitis pigmentosa: Gap junctions and the ‘bystander’ effect. Experimental Eye Research 74(3): 327–336.

Sancho-Pelluz, J., Arango-Gonzalez, B., Kustermann, S., et al. (2008). Photoreceptor cell death mechanisms in inherited retinal degeneration. Molecular Neurobiology 38(3): 253–269.

Stearns, G., Evangelista, M., Fadool, J., and Brockerhoff, S. E. (2007). A mutation in the cone specific pde6 gene causes rapid cone photoreceptor degeneration in zebrafish. Journal of Neuroscience 27(50): 13866–13874.

Wissinger, B., Chang, B., Dangel, S., et al. (2007). Cone phosphodiesterase defects in the murine cpfl1 mutant and human achromatopsia patients. Investigative Ophthalmology and Visual Science 48(5): 4521.

Zhang, X. J., Cahill, K. B., Elfenbein, A., Arshavsky, V. Y., and Cote, R. H. (2008). Direct allosteric regulation between the GAF domain and catalytic domain of photoreceptor phosphodiesterase PDE6. Journal of Biological Chemistry

283(44): 29699–29705.

Glossary

BrdU (bromodeoxyuridine) labeling – The synthetic nucleoside which is incorporated into nascent DNA during replication and is detectable by antibodies to indicate what cells have divided since exposure to BrdU.

CMZ (circumferential marginal zone) – The region of the retina distal to the optic stalk and proximal to the ciliary margin and lens. Here, retinal cells are continually born throughout the life of the zebrafish. CMZ cells express genes found in the neuroretina of the developing zebrafish embryo.

Homeobox transcription factors – The proteins that pattern tissue in that they regulate gene transcription by binding to a specific DNA sequence, the homeobox, in the target gene promoter. Morpholino – Similar to RNA, this polymerized oligomer with a morpholino (rather than ribose) backbone, can base pair with ribonucleic acid (RNA) molecules, and persists in the cell as it is not easily degraded by RNases. Morpholinos interfere either with ribosomal processing of the messenger RNA into protein or spliceosome processing of premessenger RNA into mRNA, thus depleting the amount of protein produced.

Notch signaling – The plasma membrane-bound receptor that regulates the cell fate choice of individual neurons. Intracellular cleavage product can act as a transcription factor and regulate the expression of pro-neural genes such as basic helix–loop–helix (bHLH) transcription factors.

Pcna (proliferating cell nuclear antigen) –

A marker for DNA replication in that it is a protein that functions as a trimer to promote DNA polymerase d processivity.

Shh (sonic hedgehog) signaling – The Shh family members act as morphogens to pattern tissue. Signaling pathway proteins and pathways are reutilized in more specific cell fate specification as tissues are patterned.

TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) – A hallmark of apoptotic cells is genomic DNA fragmentation by specific nucleases that cleave DNA between histones. This process results in semi-uniform lengths of DNA with overhanging hydroxyl groups. Terminal transferase efficiently polymerizes labeled nucleotides to DNA

hydroxyl groups without the need of a template. TUNEL is used to detect the genomic fragmentation of an apoptotic cell.

Introduction

Zebrafish has rapidly become a leading model system to study a variety of developmental processes, due to its large clutch size, external development of transparent embryos, and the rapid development of the embryo. Large and small genetic screens identified hundreds of mutants that affect the development of various tissues, including the retina. The ease in generating transgenic zebrafish lines has permitted a detailed cellular analysis of organ development without harm to the embryo. For example, transgenes that label specific cells are used to follow cell fates in the transparent embryo during tissue development. Alternatively, transgenes that express molecules that lead to cell death have been used to ablate cells to study either the disruption of development or organization of a tissue. Furthermore, the nearly complete sequence of the zebrafish genome has allowed comparative analyses with other vertebrate genomes to predict the presence of orthologous genes and developmental processes. Combined with the ability to direct the transient reduction in expression of desired proteins, it is possible to functionally analyze the potential role of different signaling pathways in the development of various tissues. Our understanding of zebrafish retinal development, from a sheet of neuroepithelial cells to a laminated and functional neural tissue, has benefited significantly from all of these approaches.

In addition to being an excellent model system to study early development, zebrafish has quickly become the premier model system to study tissue regeneration. In addition to exhibiting rapid and functional regeneration of the fin, liver, and heart, zebrafish also regenerate neuronal tissues, including the spinal cord, brain, and retina. Using mutants and transgenic lines that are readily available in zebrafish, a detailed comparison of the genes, molecules, and process that are required for retinal development and retinal regeneration is starting to be generated. While it may initially seem reasonable that regeneration would recapitulate the mechanisms that are involved in retinal development, recent studies revealed that regeneration may utilize the same genes and proteins as