In the retinopathy, globe enlarged (RGE) chicken, the mutation of GNB3 results in a profound phenotype featuring a significant loss of visual acuity beginning at the time of hatching, and photoreceptor degeneration and globe enlargement in adults (Montiani-Ferreira et al., 2003
, Montiani-Ferreira et al., 2005
, Montiani-Ferreira et al., 2007
). Given the significance of this presentation, we sought to characterize the expression pattern of GNB3 in the retinas of chicks, as well as in the retinas of different non-avian vertebrates. The expression of GNB3 in cone photoreceptors was consistent across species from teleost fish to primates. Furthermore, GNB3 expression was observed in a subset of bipolar cells, which express Islet1 and PKC in most species. Taken together, our findings suggest that patterns of GNB3 expression are highly conserved in cones and bipolar cells across vertebrate species.
GNB3 has been consistently observed in the outer segments of cone photoreceptors in different mammalian species, including mice, cows and monkeys (Lee et al., 1992
, Peng et al., 1992
, Huang et al., 2003
). However there have been no reports of GNB3 expression in rod photoreceptors. Similar to previous reports, we found GNB3 was exclusively expressed by cones among primate, murine and canine photoreceptors. However, in chicken, guinea pig, goldfish and frog retinas, GNB3 was detected in rod photoreceptors; albeit at levels lower than those observed in cone photoreceptors. The expression of GNB3 in rod photoreceptors was unexpected given previous literature indicating GNB3 expression is restricted to cone photoreceptors (Lee et al., 1992
, Peng et al., 1992
, Tummala et al., 2006
, Dutt and Cao, 2009
, Haider et al., 2009
). It is possible that GNB3 expression occurs in both rods and cones; however, the GNB3 antibody utilized in this study may lack specificity to cone β-transducin and could recognize rod β-transducin. Alternatively, GNB3 may be expressed by both types of photoreceptors. In the chick retina, GNB3 was detected in rod photoreceptors labeled with the 4D2 rhodospin monoclonal antibody. This antibody has been shown to label mid-wavelength green-sensitive cones where the photopigment shares the closest sequence homology with rhodopsin (Xie and Adler, 2000
). However, all GNB3-immunolabeling disappeared in RGE retinas, indicating that the antibody was not cross-reacting with rod β-transducin in the chick. In guinea pigs, cross-reactivity of the 4D2 antibody with green-opsin in cones may explain the observation of GNB3 in the outer segments of some rhodopsin-positive photoreceptors. Despite the presence of GNB3 labeling in the rod photoreceptors of some species, findings that GNB3 is expressed in cone photoreceptors in all species examined indicates the highly conserved nature of the protein and suggests the importance of GNB3 in cone-driven vision.
Examination of the photoreceptor axon terminals revealed the presence of GNB3 in cone pedicles in most species with the exception of rodents. In mice and guinea pigs, there was little or no labeling for GNB3 in the cone pedicles, compared to labeling observed in the outer segments. It is possible that the differential distribution of of GNB3 in the axon terminals of the photoreceptors may result from light-dependent trafficking of GNB3. Examples of light-dependent trafficking of transducin have been documented in rod photoreceptors, where rod α-transducin translocates from the outer segments under dark-adapted conditions to the axon terminal in the OPL following light exposure (Brann and Cohen, 1987
). There is also evidence of light-dependent translocation of rod β-transducin in the retinas of mice and rats (Sokolov et al., 2002
, Lobanova et al., 2007
). By comparison, there is little evidence of light-dependent translocation of phototransduction-related proteins in cone photoreceptors. Elias and colleagues reported that, unlike rod α-transducin, cone α-transducin does not translocate with varying light levels (Elias et al., 2004
). Rosenzweig and colleagues showed that the subcellular distribution of transducin was correlated to light-dependent changes in membrane affinity, with cone α-transducin remaining bound to the outer segment during light activation (Rosenzweig et al., 2007
). Furthermore, when cone α-transducin is ecotopically expressed in rod photoreceptors this protein is translocated to inner segments with changes in light levels, indicating differences in the cell-intrinsic trafficking mechanisms can (Rosenzweig et al., 2007
). Chen and colleagues reported that cone α-transducin partially translocates in rat retina after a brief exposure to high-intensity light (Chen et al., 2007
). Reports of cone β-transducin translocation are rare; however, McGinnis and colleagues reported migration of cone β-transducin with light exposure of dark-adapted retinas in mice (McGinnis et al., 2002
). Given this evidence, it is possible that we failed to detect GNB3 in the axon terminals of cone photoreceptors in mice and guinea pigs because of light-dependent trafficking and harvesting conditions.
In the inner retina, GNB3 expression was observed in presumptive cone ON-bipolar cells across species with one exception; goldfish retinas had low levels of GNB3 expression in bipolar cells. There is some prior evidence for GNB3 expression in bipolar cells. Peng and colleagues reported, in primate retina, that GNB3 is expressed in presumptive rod bipolar cells that were co-labeled for PKC. This study also noted that the synaptic terminals of the GNB3-positive bipolar cells terminated in sublamina b of the INL, suggesting that some these bipolar cells were cone ON-bipolar cells (Peng et al., 1992
). Furthermore, Huang and colleagues demonstrated GNB3 in the dendrites of cone bipolar cells and colocalization of GNB3 with PKC in rod bipolar cells in mouse retina (Huang et al., 2003
). In our study, cone ON-bipolar and rod bipolar cells were identified using antibodies to Islet1 in chicken, macaque, mouse, canine and guinea pig retinas. It has been previously reported that Islet1, a LIM-domain transcription factor, is required for the normal differentiation of ON- and OFF-bipolar cells in the rodent retina (Elshatory et al., 2007
). In chicken, macaque, mouse, canine and guinea pig, the GNB3-positive bipolar cells expressed Islet1. Supporting the notion that most of the GNB3-positive bipolar cells are cone ON-bipolars, the axonal processes of these cells terminate within the ON layer of the IPL, sub-lamina B. In Xenopus
retina, GNB3 and Islet1 did not overlap completely within bipolar cells, suggesting that GNB3 may be expressed by types of bipolar cells in addition to the cone ON-bipolar cells or that Islet1 is not exclusively expressed by cone ON-bipolar cells.
Consistent with the hypothesis that GNB3 is expressed by cone ON-bipolar cells, we observed significant overlap of GNB3 and PKC. PKC has been shown to be expressed by both rod bipolar cells and cone ON-bipolar cells in multiple species (Negishi et al., 1988
, Greferath et al., 1990
, McCord et al., 1996
, Vaquero et al., 1996
, Caminos et al., 1999
). In the chicken retina, labeling for PKC and GNB3 revealed 3 different sets of bipolar cells; (1) PKC-negative/GNB3-positive cells, (2) intense PKC-positive/GNB3-positive cells, and (3) weak PKC-positive/GNB3-positive bipolar cells. These findings suggest that these bipolar cells were presumptive cone ON-bipolar (weak PKC-positive/GNB3-positive cells) and rod ON-bipolar cells (intense PKC-positive/GNB3-positive), whereas the identity of the PKC-negative/GNB3-positive bipolar cells remains uncertain. In the mammalian retinas, GNB3 was observed in PKC-immunoreactive bipolar cells. The overlap of expression of PKC and GNB3 in bipolar cells suggests that cone ON-bipolar and rod bipolar cells utilize GNB3 for signal transduction. In the rodent retina, some of the axon terminals of the GNB3-positive bipolar cells were clearly segregated from some of PKC-positive terminals in the IPL, supporting the notion that GNB3 is expressed by more than one type of bipolar cell. Similar results were observed in guinea pig and dog retinas, where GNB3-positive/PKC-negative and GNB3/PKC-positive bipolar cells were observed. Our observations indicate that the GNB3-positive bipolar cells in the chick retina express Islet1 and many of these cells express low levels of Lim3. The GNB3-negative bipolar cells that express high levels of Lim3 are likely to be OFF-cone bipolar cells. Taken together, these findings suggest that GNB3 is expressed by cone ON-bipolar cells and in some rod ON-bipolar cells, indicating the importance of GNB3 signal transduction in the ON pathway.
In the current study, immunofluoresence combined with Western blot analysis, clearly indicate that GNB3 protein is entirely lost from the retinas of RGE mutant chicks. The presence of mRNA in both WT and RGE retinas indicates that the transcriptional mechanisms for the GNB3 expression are intact in the RGE chicken. A previous report has suggested that the deletion of a single aspartic acid (D153del) from GNB3 in the RGE chicken reduces levels of expression by about 70%, as determined by a immuno-slot-blot preparation (Tummala et al., 2006
). The discrepancy between these studies likely resulted from the use of different GNB3 antibodies and the application of different immunological techniques. Our findings indicate that the loss of GNB3 in the RGE chicken does not overtly affect the formation and maturation of the retina during embryonic and early postnatal development. This is consistent with a previous report demonstrating the normal immunohistological profile of retinas in young (<P30) RGE chicks (Montiani-Ferreira et al., 2005
Clearly, the loss of GNB3 underlies the phenotype of the RGE-/- chicken which involves the loss of visual acuity at the time of hatching, and eventually (after P45) globe enlargement and retinal degeneration (Montiani-Ferreira et al., 2003
, Montiani-Ferreira et al., 2005
, Tummala et al., 2006
). The mechanisms underlying the progressive retinopathy and subsequent excessive ocular growth in this animal model remains unknown. We failed to find GNB3 expression in ocular tissues, with the exception of the retina. Thus, the ocular phenotypes of the RGE chicken, including uncontrolled eye growth, flattening of the anterior chamber and progressive retinopathy, result from a complete loss of GNB3 from retinal bipolar cells and photoreceptors.
The onset of expression of GNB3 during embryonic development coincides with the late-onset of genes in both photoreceptors and bipolar cells. The maturation of photoreceptors is delayed for at least 1 week after terminal mitosis and fate specification in the chick retina (Fischer et al., 2008a
, Ghai et al., 2008
). The onset of GNB3 expression in bipolar cells occurs before the onset of PKC expression (Caminos et al., 1999
), and GNB3 expression in photoreceptors appears before the onset of cone opsin expression, at about E15 (Bruhn and Cepko, 1996
). Despite an absence of GNB3 expression, the retinas of RGE -/- chicks show no gross retinal abnormalities, with the exception of subtle synaptic changes in the OPL (Montiani-Ferreira et al., 2005
). Moreover, the late onset of GNB3 in the embryonic chick retina may explain why no severe ocular abnormalities are observed at hatching in the RGE phenotype; an early onset of expression might be expected to have more severe developmental impact. However, the survival of photoreceptors is compromised in RGE retinas beginning at about P45, indicating that proper GNB3 function is required to maintain the photoreceptors in adult animals (Montiani-Ferreira et al., 2005