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concentration, hyperpolarisation of the photoreceptor plasma membrane and transmission to higher-order neurons in the retina. For photoreceptors to function, cGMP levels are tightly regulated at the levels of synthesis and degradation. In photoreceptor outer segments, cGMP is synthesised from GTP by membrane-bound retinal guanylate cyclases, (RetGC) (Fig. 7.1). Light stimulation of photoreceptor visual pigments activates a phosphodiesterase (PDE6) which hydrolyses cGMP to GTP (for reviews, see Arshavsky et al. 2002; Pugh et al. 1997). Inappropriate activity of either RetGC or PDE6 leads to an imbalance in cGMP levels, which is associated with multiple retinal disorders. Here, we outline human retinal diseases resulting from RetGC or PDE6 mutations, and describe the usefulness of the zebrafish to investigate these diseases.
Fig. 7.1 The phototransduction cascade; during dark phase, RetGC synthesises a basal level of cGMP, which keeps some CNG channels in an open configuration, and allows the influx of calcium, while ion pumps efflux calcium. An incident photon (hν) activates opsin, which (indirectly) derepresses phosphodiesterase 6, and breaks down cGMP. The CNG channels close as the cGMP level drops, and GCAP is derepressed, and stimulates RetGC-1. As the light impulse stops, the phosphodiesterase no longer breaks down cGMP, and RetGC-1 synthesises more cGMP, allowing some CNG channels to reopen, and the photoreceptor to depolarize
7.2Retinal Disorders Associated with Mutations in RetGCs and PDE6
The central role played by RetGC-1 in phototransduction implies that its absence or incorrect function will have severe consequences for visual function. A lack of cGMP is thought to lead to the inability of photoreceptors to depolarise, and an overabundance of cGMP may lead to constitutive opening of the CNG channels, keeping photoreceptors constantly hyperpolarised and unable to respond to light impulses (for review, see Duda and Koch 2002). The human gene encoding RetGC- 1 was the first gene implicated in Leber congenital amaurosis (LCA) (Perrault et al. 2000). LCA is an autosomal recessive, early-onset, retinal dystrophy and one of the
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most severe forms of inherited blindness in children. It is estimated that 20% of LCA cases are caused by RetGC-1 mutations, which predominantly result in inactive RetGC-1 (Perrault et al. 2000). Tucker and colleagues observed that mutations in RetGC-1 could lead to LCA, or to other, less severe retinal disorders, depending on the position of the mutation (Tucker et al. 2004). This strengthened the proposal that mutations in different regions of RetGC-1 may contribute to a spectrum of retinal disorders that were previously segregated due to differing severity of symptoms (Perrault et al. 2000).
Another severe retinal degeneration, linked in this case with dominant RetGC-1 mutations, is cone-rod dystrophy 6 (CORD6). Kelsell and colleagues show that CORD6 symptoms, including loss of central vision, photophobia, and “bull’s-eye” maculopathy are associated with mutations in the dimerisation domain of RetGC-1 (Kelsell et al. 1998). Despite the clinical analysis of the symptoms of CORD6, and a correlation between mutations in RetGC-1 and the severity of phenotype, there are few studies of the molecular effects associated with mutant RetGC-1 in the retina. It is not known whether dominant mutant RetGC-1 synthesises an excess of cGMP and is less easily inactivated in the retina as biochemical data suggest (Tucker et al. 1999; Ramamurthy et al. 2001), or if another cellular process leads to CORD6. There is a clear need to use in vivo models to better characterise the disease pathways induced by mutant RetGCs.
PDE6 is a holoenzyme composed of PDE6A, PDE6B and two PDE6G subunits in rods, and two PDE6C and two PDE6H subunits in cones (for review, see Ionita and Pittler 2007). Though the subunits work cooperatively, mutations in single subunits lead to distinct disease phenotypes. Rod-specific PDE6A mutations lead to autosomal recessive retinitis pigmentosa (arRP) (Huang et al. 1995), while mutations in PDE6B cause arRP or congenital stationary night blindness (Gao et al. 1996; Gal et al. 1994). Mutations in the cone-specific PDE6C are less well characterized, but are associated with cone photoreceptor loss and recessive achromatopsia (Chang et al. 2001; Chang et al. 2002). Mutations in the rod PDE6G subunit are associated with recessive retinitis pigmentosa (Tsang et al. 1996), while cone PDE6H subunit mutations lead to a recessive form of cone dystrophy (Piri et al. 2005). The heterogeneity of disease phenotypes reflects the spatial expression of different PDE6 subunits, and that disease-associated mutations are present in catalytic and inhibitory subunits of the PDE6 holoenzyme. As with retinal disorders associated with mutations in RetGC, the link between altered activity of PDE6 and photoreceptor degeneration is not well understood, and in vivo animal models will provide valuable data on these pathologies.
7.3Analysis of Teleost RetGC and PDEs in Retinal Function and Disorders
The infraclass Teleostei are contained in the class Actinopterygii, the ray-finned fishes, and contains a number of discrete species of fishes that are extremely useful in genetic analyses, including visual function, development and disease (Collery
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et al. 2006). In particular, teleost models have abundant cone photoreceptors mediating color vision and thus are advantageous for analyzing cone retinopathies (for review, see Goldsmith and Harris 2003). In medaka (Oryzias latipes), four discrete guanylate cyclase mRNAs are expressed in the retina: OlGC-R2 OlGC3, OlGC4 (OlGC-R1), and OlGC-C (OlGC5) (Hisatomi et al. 1999; Seimiya et al. 1997). In the retina, OlGC-C (OlGC5) is cone-exclusive whilst OlGC4 (OlGC-R1) and OlGC-R2, are expressed only in rods; and all are expressed in the pineal (Hisatomi et al. 1999).
Recent data shows zebrafish to express three membrane-bound retinal GCs: zGC1, zGC2 and zGC3 (Rätscho et al. 2009) (Fig. 7.2a). zGC1 and zGC2 are expressed in rods, while zGC3 is expressed exclusively in cones. zGC1 and zGC2 are equivalent to human RetGC-2 both by sequence homology and rod localization, while zGC3 is equivalent to human cone RetGC-1. The zebrafish zatoichi mutant has no cone-driven visual response owing to a null mutation in zGC3, and was identified through a lack of optokinetic or optomotor visual function (Muto et al. 2005). As zGC3 corresponds to human RetGC-1, the zatoichi mutant may be considered an animal model of LCA. Although zatoichi zebrafish have no visual function, there is no observed abnormal retinal histology at 5 days post fertilization (Muto et al. 2005). This at least initial preservation of photoreceptors in zatoichi zebrafish allows testing of in vivo therapies such as gene therapy, cell transplantation and pharmacological modifiers to recover functionality of extant photoreceptors. In addition, zatoichi photoreceptors can be utilized to examine the effects of the absence of zGC3/RetGC-1, and to understand better the molecular mechanisms behind the progression of LCA.
Orthologues of human PDE6C, -A, -G and -H have also been identified in zebrafish (Vihtelic et al. 2005), and PDE6B can be found through database homology searches (zPDE6B, XM_679910). Similarly, PDE6 subunit orthologues can be found in medaka libraries (OlPDE6B, ENSORLG00000009868; OlPDE6C, ENSORLG00000005135; no PDE6A orthologue is currently discoverable in medaka). Phylogenetic comparisons indicate that PDE6A orthologues are most closely related between species, followed by PDE6B orthologues (Fig. 7.2b). Cone-specific PDE6C orthologues differ more between species, which may reflect comparison of diurnal and nocturnal animals.
Functional work already done in zebrafish has examined the effects on photoreceptors of mutations in the PDE6C subunit. Stearns and colleagues examined lines with a pde6c frameshift mutation that likely leads to a truncated protein or an mRNA targeted for degradation, where insufficient breakdown of cGMP leads to cone death, followed by localized rod death (Stearns et al. 2007). Similarly, Nishiwaki and colleagues examined lines with a pde6 mutation that confers a single amino acid change within the cGMP-binding GAF domain, and found opsin mislocalization and cone death, though without accompanying rod death (Nishiwaki et al. 2008). It is currently unknown whether excess cGMP due to RetGC-1 mutations also leads to cone cell death. The death of cones associated with PDE6 components indicates the importance of the phototransduction cascade components in normal maintenance of photoreceptors, as well as visual signaling. The heterogeneity of disease phenotypes
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seen when comparing truncated and missense PDE6C zebrafish proteins illustrates how the zebrafish model system can be used to dissect how multiple mutations in a single gene can cause a range of symptoms. A loss of cones followed by rods when cone-specific PDE6C has a truncation mutation indicates that the rods die as a result of cone death; loss of cones without rod loss due to a single amino acid change in PDE6C indicates distinct mechanisms of degeneration. Work of this kind is useful to highlight potential routes for therapeutic intervention (e.g. cell death inhibitors vs cone cell transplantation).
In addition to using zebrafish to characterize altered visual function due to loss- of-function mutations in retGC or PDE6, transgenic zebrafish lines may be used to characterize gain-of-function disease phenotypes associated with dominant human visual diseases. We are currently assessing the pathology of expressing mutant human RetGC-1 in zebrafish cones to recapitulate CORD6, whose symptoms are well characterized (see Section 7.2), but where little is known about the in vivo effects of the mutant protein. Already, the misexpression of native GCs in the zebrafish has demonstrated the importance of correct levels and spatial expression of cGMP synthesis, as ectopic expression of zGC1 (gucy2f) leads to serious defects in the nervous system (Maddison et al. 2009).
Fig. 7.2 (a) Phylogenetic tree showing retinal guanylate cyclases from human (Homo sapiens), Rat (Rattus norvegicus), Mouse (Mus musculus), Medaka (Oryzias latipes) and Zebrafish (Danio rerio). (b) Phylogenetic Tree showing PDE6 Subunits from the above species. Functionally related proteins are demarcated by white and shaded ellipses. Sequences were aligned using ClustalW. Disease phenotypes associated with mutations in these genes are inset in rounded rectangles
In summary, current zebrafish methods allow researchers to make transgenic lines expressing mutant proteins to study the effects of dominant heritable diseases, and to reduce or eliminate native proteins to study recessive and null mutation diseases. Identification of zebrafish genes orthologous to RetGCs and PDE6 subunits means that zebrafish can be used to generate models of recessive retinitis pigmentosa, congenital stationary night blindness, dominant cone-rod dystrophy, and achromatopsia. In parallel with furthering knowledge about the onset and progression of these diseases, zebrafish can be easily used to assay molecular and