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PubMed2006

Light-sensitive motile iridophores and visual pigments in the neon tetra, Paracheirodon innesi.

Although motile iridophores in the longitudinal stripes of neon tetra skin are under control of the sympathetic nervous system, they also respond to light directly and show circadian color changes. Using neon tetra skin, we found that the photoresponse of iridophores depends on light intensity, and that light near 500 nm is most effective. RT-PCR demonstrated the expression of mRNAs encoding rhodopsin and two kinds of cone opsins (Pi-green1 and Pi-green2) in neon tetra skin where the light-sensitive iridophores exist. These mRNAs are also expressed in the lateral eyes. The cone opsin genes, Pi-green1 and Pi-green2, show high similarity with the g101 and g103 genes of unique green cone opsins (belonging to the MWS/LWS group) of the blind Mexican cavefish. These results show that Pi-green1, Pi-green2, and/or rhodopsin may play important roles in the photoresponse of neon tetra iridophores, which are most sensitive to light near 500 nm.

Animals↗
PubMed2000

Retinoic acid produces rod photoreceptor selective apoptosis in developing mammalian retina.

PURPOSE: All-trans retinoic acid (ATRA) or 9-cis retinoic acid (9CRA), added to dissociated developing neural retinal cells, induces progenitor cells to adopt the rod cell's fate. Retinoic acid (RA) also produces apoptotic cell death in developing tissues. The effects of retinoids on mouse retinal development were examined. METHODS: Retinas were explanted on postnatal day (PN)1 and cultured with or without the retinal pigment epithelium (RPE) attached. Retinas were cultured for 3 weeks in the absence or presence of 100 or 500 nM ATRA or 9CRA. Morphologic development and apoptotic cell death were examined using cell-specific immunocytochemical markers, the TdT-dUTP terminal nick-end labeling (TUNEL) method, and a caspase assay. RESULTS: Retinal explants, with and without RPE, had similar age-dependent increases in opsin expression. In contrast, explants with RPE had less apoptosis during the first week than retinas without RPE. In explants with RPE, ATRA or 9CRA produced rod-selective apoptotic cell death in which 20% to 25% were lost by PN7 with no further loss by PN21. 9CRA-treated explants without RPE had a decreased number of apoptotic cells and a higher number of (rhod)opsin-positive cells at PN3. CONCLUSIONS: Factors in RPE appear to regulate rod apoptosis in developing retina. Retinoids produce rod-selective apoptotic cell death during normal rod differentiation. In contrast, retinoids accelerate the expression of opsin in retinas without RPE. These differential effects of RA on rod photoreceptors-apoptosis and differentiation-are similar to those observed in other developing tissues and play an important role in both normal and pathologic development.

Animals↗
PubMed2002

The retina of Spalax ehrenbergi: novel histologic features supportive of a modified photosensory role.

PURPOSE: The retina of the blind mole rat Spalax ehrenbergi was compared with other vertebrate photosensitive organs in an attempt to correlate its histologic organization with a presumptive nonvisual photoreceptor role. METHODS: The eyes of eight adult animals were analyzed by light and electron microscopy, using conventional staining and immunolabeling with antibodies against phototransduction proteins and calretinin. RESULTS: Rods accounted for most of the photoreceptor cells in the Spalax retina, although their morphology is dissimilar to that of sighted mammals, in that they contained only rudimentary outer segments. The latter showed strong rod-opsin and transducin immunoreactions. The phagosomes in the retinal pigmentary epithelium were also rod-opsin positive. Synapses were evident at the photoreceptor cells pedicles. Occasionally, several synaptic active sites were present, suggesting cone cell origin; however, cone-opsin was not immunodetected in the study samples. Synaptic ribbon fields, sometimes distant to the active sites, resembled those found in the vertebrate pineal. The other retinal layers were somewhat less organized than in sighted mammals. Some cells were displaced and the calretinin-positive inner plexiform layer had no sublayers. Calretinin immunolabeling was found in horizontal, amacrine, and ganglion cells. Folding of the retina produced rosette-like images similar to those reported before in the retina of nocturnal mammals and in the avian pineal gland. CONCLUSIONS: These data suggest that the retina of the mole rat has undergone evolutionary restructuring to a photoreceptive pineal-like organization. This supports the thesis that the photoreceptor cells of this unique organ have been reprogrammed during the subterranean adaptation of Spalax, from their original visual function to mediating photoperiodic regulation.

Animals↗
PubMed2002

Ciliary neurotrophic factor as a transient negative regulator of rod development in rat retina.

PURPOSE: Ciliary neurotrophic factor (CNTF) has been shown to inhibit the developmental expression of rod differentiation markers in rat retinal cultures. The present study was undertaken to investigate whether CNTF transiently inhibits rod differentiation or induces irreversible changes in the developmental fate of rod precursors. METHODS: The effects of CNTF on rod differentiation were monitored in organotypic slice cultures from early postnatal rat retinas by quantification of opsin-immunoreactive cells. The in vitro formation of the photoreceptor layer was analyzed by light and electron microscopy. The developmental expression of the CNTF receptor in photoreceptors was determined by immunoblot analysis. RESULTS: CNTF did not interfere with the generation of rod precursors, their morphologic differentiation, or the formation of the outer nuclear layer. Inhibition of rod differentiation was reversible. In the continuous presence of CNTF the number of opsin-positive cells increased at a normal rate but with a delay of 3 to 4 days. Developing rods became resistant to CNTF, both in vivo and in vitro, and this correlated temporally with the downregulation of CNTF receptor expression. Receptor downregulation was inhibited by CNTF in a dose-dependent manner. At higher CNTF concentrations with sustained receptor expression, the CNTF-induced decrease in opsin expression was accompanied by an increase in the expression of a bipolar cell marker in rod precursors located in the photoreceptor layer. CONCLUSIONS: These data indicate that, although rod precursors exhibit some phenotypic plasticity in the presence of CNTF, the factor does not induce a switch in the developmental fate of rod precursors but plays a role as a transient and reversible negative regulator of rod differentiation.

Animals↗
PubMed2005

Detailed histopathologic characterization of the retinopathy, globe enlarged (rge) chick phenotype.

PURPOSE: The purpose of this study was to characterize the morphological abnormalities in the retinas of chicks (Gallus gallus) suffering from the autosomal recessive disease, retinopathy, globe enlarged (rge/rge). METHODS: rge/rge affected and age matched control retinas were examined from hatch up to 730 days of age. Thickness of retinal layers at six retinal regions was measured from plastic embedded sections. Morphological features were examined on semi-thin sections by light microscopy and on ultra-thin sections by transmission electron microscopy. Immunohistochemistry was performed using a panel of several different antibodies. Additionally, comparative counting of rod outer segments, rows of cells in the inner nuclear layer, and ganglion cells per unit length was performed. RESULTS: The earliest changes observed in rge/rge retinas were disorganization of the outer plexiform layer and abnormal location of the endoplasmic reticulum of the photoreceptors. In rge/rge retinas, cone pedicles were larger, irregular in shape, and usually contained multivesicular bodies. In addition, synaptic ribbons of the cone pedicles and rod spherules in rge/rge retinas were less numerous compared to controls. Large glycogen deposits progressively accumulated in the perinuclear cytoplasm associated with the abnormally located endoplasmic reticuli in accessory cones and rods. Total retinal thickness progressively decreased with age in rge/rge birds. This was accompanied by a decrease in the number of cells in the inner nuclear layer and a decrease in the number of rod outer segments (OSs). Several changes were detected in the rge/rge retinas using immunohistochemistry, including mislocalized opsin immunoreactivity of rod photoreceptors, a decrease in number and disorganization of opsin positive rod OSs (especially in the peripheral regions), a decrease in number of tyrosine hydroxylase positive neurites in the distal inner plexiform layer, and activation of macroglial and microglial cells. CONCLUSIONS: As we previously reported, the rge/rge chick has vision loss that is not the result of photoreceptor loss and is unusual in that electroretinographic responses, although abnormal, are maintained until well after vision loss has developed. The phenotype is associated with a developmental disruption of both rod and cone photoreceptor synaptic terminals that progresses with age. It is possible that these changes may be indicative of abnormal circuitry within the outer plexiform layer, and that they underlie the progressive loss of vision in rge/rge birds. Other early changes suggesting photoreceptor abnormality are dilation of photoreceptor cell bodies, abnormal positioning of endoplasmic reticulum in the perinuclear region that is associated with abnormal glycogen deposition, and mislocalization of opsin immunoreactivity in rods. The rge/rge birds develop globe enlargement after the morphological and electroretinographic abnormalities. Globe enlargement in chicks can be induced by a number of different environmental factors. It is possible that abnormal signaling of photoreceptors to inner retinal cells could induce excessive ocular growth in the rge/rge birds. Many of the morphological changes such as retinal thinning seen in older rge/rge birds may be partly the result of the considerable globe enlargement that occurs later in the disease process. Molecular genetic studies to identify the causal gene mutation should help explain the morphological features of the rge/rge phenotype and clarify their association with vision loss and electroretinographic abnormalities.

Animals↗
PubMed2005

Knockdown of wild-type mouse rhodopsin using an AAV vectored ribozyme as part of an RNA replacement approach.

PURPOSE: To develop a hammerhead ribozymes (Rz) that might be exploited in a "digest and replace" gene therapy strategy for autosomal dominant retinitis pigmentosa (ADRP) caused by mutations in the gene for rhodopsin (RHO). METHODS: A ribozyme (Rz397) was designed to hybridize with an accessible region in rhodopsin mRNA. It was tested in vitro to determine the kinetics of cleavage of a target oligonucleotide. Following transfection of cultured cells, reduction of rhodopsin mRNA in response to Rz397 was measured RT-PCR. The gene for the ribozyme (Rz397) was inserted in an adeno-associated virus (AAV2) vector and packaged in AAV2 capsids. The virus was injected subretinally in the eyes of C57BL/6J (RHO+/+) and rhodopsin knockout hemizygous (RHO+/-) mice at postnatal days 6 (P6) and 30 (P30). Mice were analyzed by full-field electroretinography (ERG). The reduction of opsin protein was measured by western blot analysis and visualized by immunocytochemistry. Reduction of rhodopsin mRNA was assessed using in situ hybridization. Morphometric microscopy of fluorescent antibody-antigen complexes and autoradiography of retinas were used to quantify levels of rhodopsin protein and mRNA, respectively. RESULTS: Transient co-transfection of HEK 293 cells with a wild-type rhodopsin cDNA and Rz397 resulted in an approximately 60% reduction of RHO mRNA one day after transfection. RHO+/- -mice injected with AAV2-Rz397 at P6 showed a 50% reduction in b-wave amplitudes in injected eyes relative to saline injected contralateral eyes. However, injection of RHO+/- -animals at one month and of RHO+/+-animals at either age had no impact on ERG. Nevertheless, we detected an 80% reduction of opsin protein in ribozyme-injected eyes of hemizygous mice (by western blot) and a 50% reduction in opsin content in RHO+/+ mice (by morphometry). These reductions were confirmed by in situ hybridization. CONCLUSIONS: AAV2-Rz397 led to significant (greater than or equal to 50%) reduction of rhodopsin mRNA and protein in mice. It affected ERG amplitudes only when injected in hemizygous RHO knockout pups. This RNA inhibitor may prove useful in treating animal models of ADRP as part of an RNA replacement approach.

Animals↗
PubMed2006

Intraocular injection of kainic acid does not abolish the circadian rhythm of arylalkylamine N-acetyltransferase mRNA in rat photoreceptors.

PURPOSE: Melatonin synthesis in mammalian retinal photoreceptors is under photic and circadian control and regulated by changes in the activity of arylalkylamine N-acetyltransferase (AANAT). Recent studies have suggested that retinal dopaminergic neurons contain a circadian pacemaker, and dopamine is the neurotransmitter that drives circadian rhythmicity in the mammalian retina. METHODS: To investigate the role of inner retinal neurons, including dopamine neurons, in generating the rhythm of melatonin synthesis, rat retinas were lesioned with kainic acid (KA), which was shown previously to induce degeneration of neurons in the inner nuclear layer and to eliminate rhythmicity in the dopaminergic system. Aanat, rhodopsin, medium wavelength (mwl) opsin, short wavelength (swl) opsin, and period1 (Per1), and period2 (Per2) mRNA levels were measured using real-time quantitative RT-PCR in KA injected and control eyes. RESULTS: Our data show that intraocular injections of KA did not abolish the daily and circadian rhythms of Aanat mRNA in the photoreceptors, but it did shift the phase of the Aanat transcript rhythm in constant darkness. Surprisingly, KA injections reduced the levels and eliminated daily rhythms of mwl and swl opsin transcripts, but not of rhodopsin mRNA. Per1 and Per2 mRNA levels were rhythmic in saline injected and in KA-treated retinas, and Per2 mRNA levels were significantly reduced (20-50%) in KA-treated retinas. CONCLUSIONS: These findings demonstrate that the circadian clock generating melatonin rhythmicity is largely KA insensitive and likely to be located in the rod photoreceptors, although KA-sensitive neurons do influence its timing. More important, our data demonstrate that dopamine rhythmicity is not necessary for generating the circadian rhythm of Aanat mRNA in the photoreceptors. Our data also indicate that Per1 and Per2 are rhythmically transcribed in the rat retina and KA treatment has a dramatic effect on the overall levels of Per2 mRNA.

Animals↗
PubMed1984

Cycloheptatrienylidene analog of 11-cis retinal. Formation of pigment in photoreceptor membranes.

In biochemical and electrophysiologic studies employing the bullfrog (Rana catesbeiana) and the rat, the authors examined the interaction of opsin and an 11-cis-locked analog of retinal. In previously bleached preparations of bullfrog receptor outer segments (ROS) and isolated retinas, incubation with the aldehyde form (I) of the analog leads to the appearance of a pigment that is degraded slowly by hydroxylamine but is relatively resistant to photolysis. In the ROS preparation, the analog pigment (lambda max of difference spectrum congruent to 497 nm) also forms on incubation with NADP+ and the alcohol form (II) of the analog. In vitamin A deprived rats possessing only approximately 45% of the normal complement of rhodopsin, intraperitoneal injection of II leads within 1 day to the appearance of the analog pigment in the photoreceptors, at levels representing a major fraction of the opsin initially available for pigment formation. Formation of the analog pigment appears to have no significant effect on the sensitivity of electroretinographic b-wave responses recorded from the rat eye; furthermore, administration of II appears to suppress the sensitizing activity of all-trans retinol injected 1 day later. The data are discussed in relation to other studies examining chromophore-opsin interactions and electrophysiologic changes associated with the formation of rhodopsin in situ.

Animals↗
PubMed1994

Structure and function in rhodopsin. Requirements of a specific structure for the intradiscal domain.

We concluded previously from mutagenesis in the intradiscal domain of bovine rhodopsin that the formation of a tertiary structure comprising the N-terminal tail and the three polypeptide loops is essential to the in vivo assembly of the functional rhodopsin. We now report on more comprehensive mutagenic studies in the intradiscal domain to determine more precisely the requirement for the formation of the above-proposed tertiary structure. Three large deletions, two consisting of groups of 10 amino acids each, and the third of 34 amino acids, were carried out in the N-terminal loop. All the three mutant opsins only poorly formed the rhodopsin chromophore. In the BC loop, we carried out five 2 amino acid deletions, 2 single amino acid deletions, and three mutations in which short sequences in the loop were reversed. All the resulting mutant opsins had lost the ability to bind 11-cis-retinal. In the DE loop, where extensive mutagenesis had previously been carried out, we carried out 3 amino acid replacements (Asn, Thr, Tyr) at Cys187. None of these mutants bound 11-cis-retinal. In loop FG, we carried out four 2 amino acid deletions, 1 single amino acid deletion, 3 amino acid replacements, and one mutation in which the sequence of the 7 amino acids was reversed. All the mutants in FG loop partially formed the rhodopsin chromophore. All the mutants now described appeared to be retained in the endoplasmic reticulum: several that were examined in detail were complexed with non-opsin proteins, the chaperonins. Treatment with ATP-MgCl2 released the latter from the mutant rhodopsins. Our overall conclusion is that the formation of the specific structure in the intradiscal domain has highly stringent spatial requirements.

Amino Acid Sequence↗
PubMed1997

A new form of inherited red-blindness identified in zebrafish.

A red-blind zebrafish mutant, partial optokinetic response b (pob), has been isolated by measuring eye movements of larvae in a three-generation screen for recessive mutations affecting the visual system. pob larvae exhibit eye movements in response to rotating black and white stripes illuminated with white light, but they do not move their eyes when the stripes are illuminated with red light. Physiological, immunohistochemical, and in situ hybridization analyses of pob retinas showed a selective loss of red-sensitive cones at 5 days postfertilization (dpf). At 3 dpf, cells expressing red opsin are present, suggesting that red-sensitive cones form initially but then disappear rapidly, whereas other photoreceptors remain. Linkage analysis indicated that the mutation identified in the pob mutant is not at the red opsin locus. Because red opsin is the only known molecule unique to red cones, these data suggest that a novel gene is required for the maintenance or function of red cones.

Animals↗
PubMed1998

Docosahexaenoic acid promotes differentiation of developing photoreceptors in culture.

PURPOSE: The purpose of this work was to study the effects of diverse fatty acids on the composition, metabolism, differentiation, and characteristics of opsin expression in retina photoreceptors. METHODS: Cultures of rat retinal neurons were incubated with or without 22:6 n-3, 22:5 n-3, 20:4 n-6, 18:1 n-9, and 16:0, labeled and unlabeled. RESULTS: In photoreceptor cells incubated with 22:6 n-3 and 22:5 n-3, the proportions of these fatty acids in phospholipids increased four- to sixfold. The remaining fatty acids did not change lipid acyl chain composition. The labeled fatty acids were all actively esterified in neuronal lipids, particularly in phosphatidylcholine. Addition of unlabeled 22:6 n-3 did not affect the distribution among lipids of the other fatty acids but displaced [3H]20:4 n-6 from phosphatidylcholine and phosphatidylethanolamine. These results suggest that retinal neurons have specific mechanisms for processing fatty acids of different lengths and degrees of unsaturation and that 22:6 n-3 incorporation takes priority. Of all fatty acids, 22:6 n-3 was the most effective in promoting photoreceptor differentiation. In 22:6-sufficient photoreceptors, new apical processes formed, the expression of opsin augmented, and its localization improved, concentrating in the apical processes of the cells. CONCLUSIONS: The advancement in differentiation selectively elicited by 22:6 correlates with the fact that 22:6 n-3, but none of the other fatty acids, delays significantly the onset of apoptosis in photoreceptors in culture. The synthesis of 22:6-containing phospholipid molecules could be required for the proper localization of opsin. This could contribute to furthering the differentiation of photoreceptors, preventing their apoptosis, and extending their survival.

Animals↗
PubMed1997

Evidence from normal and degenerating photoreceptors that two outer segment integral membrane proteins have separate transport pathways.

Detachment of the neural retina from the retinal pigment epithelium induces photoreceptor degeneration. We studied the effects of this degeneration on the localization of two photoreceptor outer segment-specific integral membrane proteins, opsin and peripherin/rds, in rod photoreceptors. Results from laser scanning confocal microscopic and electron microscopic immunolocalization demonstrate that these two proteins, normally targeted to the newly-forming discs of the outer segments, accumulate in different sub-cellular compartments during photoreceptor degeneration: opsin immunolabeling increases throughout the photoreceptor cell's plasma membrane, while peripherin/rds immunolabeling occurs within cytoplasmic vesicles. The simplest hypothesis to explain our results is that these proteins are transported in different post-Golgi transport vesicles and separately inserted into the plasma membrane. More complex mechanisms involve having the two co-transported and then opsin finds its way into the plasma membrane but peripherin/rds does not, remaining behind in vesicles. Alternatively, both insert into the plasma membrane but peripherin/rds is recycled into cytoplasmic vesicles. We believe the data most strongly supports the first possibility. Although the transport pathways for these proteins have not been fully characterized, the presence of peripherin/rds-positive vesicles adjacent to the striated rootlet suggests a transport role for this cytoskeletal element. The accumulation of these proteins in photoreceptors with degenerated outer segments may also indicate that their rate of synthesis has exceeded the combined rates of their incorporation into newly forming outer segment disc membranes and their degradation. The accumulation may also provide a mechanism for rapid recovery of the outer segment following retinal reattachment and return of the photoreceptor cell to an environment favorable to outer segment regeneration.

Animals↗
PubMed2005

Demonstration of a genotype-phenotype correlation in the polymorphic color vision of a non-callitrichine New World monkey, capuchin (Cebus apella).

Color-vision polymorphism in New World monkeys occurs because of an allelic polymorphism of the single-copy red-green middle-to-long-wavelength-sensitive (M/LWS) opsin gene on the X chromosome. Because color-vision types can readily be estimated from allelic types of the M/LWS opsin gene, this polymorphic system offers researchers an excellent opportunity to study the association between vision and behavior. As a prerequisite for such studies, genetically determined color-vision types must be concordant with phenotypes determined directly by behavioral criteria (e.g., by a color discrimination test). However, such correlations between genotypes and phenotypes have been studied only for callitrichine species. Using genetic, electrophysiological, and behavioral approaches, we evaluated the color vision of brown capuchin monkeys (Cebus apella), a representative non-callitrichine model animal for physiology and behavior. Two allelic M/LWS opsins-P545 and P530-were identified in the studied captive population. Females had one or both of the alleles, and males had either one. The retinal sensitivity in P530 dichromats was short-wave shifted relative to that in P545 dichromats, whereas that in P530/P545 trichromats was between the two groups. In a discrimination task using Ishihara pseudo-isochromatic plates, P530/P545 trichromats were successful in discriminating stimuli that P530 and P545 dichromats were unable to discriminate. In a food-search task, P530/P545 trichromats were able to locate red targets among green distracters as quickly as among white distracters, whereas both types of dichromats took longer. These results demonstrate the mutual consistency between genotypes and phenotypes of color vision, and provide a solid genetic basis on which the ecology and evolution of color vision can be investigated.

Animals↗
PubMed2003

Not all butterfly eyes are created equal: rhodopsin absorption spectra, molecular identification, and localization of ultraviolet-, blue-, and green-sensitive rhodopsin-encoding mRNAs in the retina of Vanessa cardui.

Surveys of spectral sensitivities, visual pigment spectra, and opsin gene sequences have indicated that all butterfly eyes contain ultraviolet-, blue-, and green-sensitive rhodopsins. Some species also contain a fourth or fifth type, related in amino acid sequence to green-sensitive insect rhodopsins, but red shifted in absorbance. By combining electron microscopy, epi-microspectrophotometry, and polymerase chain reaction cloning, we found that the compound eye of Vanessa cardui has the typical ultrastructural features of the butterfly retina but contains only the three common insect rhodopsins. We estimated lambda-max values and relative densities of the rhodopsins in the Vanessa retina (0.72, P530; 0.12, P470; and 0.15, P360) from microspectrophotometric measurements and calculations based on a computational model of reflectance spectra. We isolated three opsin-encoding cDNA fragments that were identified with P530, P470, and P360 by homology to the well-characterized insect rhodopsin families. The retinal mosaic was mapped by opsin mRNA in situ hybridization and found to contain three kinds of ommatidia with respect to their patterns of short wavelength rhodopsin expression. In some ommatidia, P360 or P470 was expressed in R1 and R2 opposed receptor cells; in others, one cell expressed P360, whereas its complement expressed P470. P530 was expressed in the other seven cells of all ommatidia. P470-expressing cells were abundant in the ventral retina but nearly absent dorsally. Our results indicated that there are major differences between the color vision systems of nymphalid and papilionid butterflies: the nymphalid Vanessa has a simpler, trichromatic, system than do the tetrachromatic papilionids that have been studied.

Amino Acid Sequence↗
PubMed1989

Immunocytochemical reactivity of Xenopus laevis retinal rods and cones with several monoclonal antibodies to visual pigments.

Immunocytochemical reactions with several antibodies to visual pigments were used to study visual cells of the Xenopus laevis retina. Monoclonal antibodies to bovine opsin "E," 1D4, and 4B4 (reactive with the N- and C-terminus and with the loop connecting transmembrane segments 5-6, respectively) and to chicken visual pigments COS-1 and OS-2 (binding to mammalian red/green and blue cones, respectively), as well as a rabbit antifrog opsin serum 11-7, were applied to semithin and thin sections of the retina. The bound antibodies were detected with the peroxidase technique at the light microscopic level; a three-stage immunogold procedure was used for electron microscopic immunocytochemistry. The overwhelming majority of rods were labeled by monoclonal antibodies "E," 4B4, 1D4, OS-2, and serum 11-7. A small fraction (2-3%) of rods did not bind monoclonal antibodies "E" and 4B4, but this minor population of rods was strongly reactive with 1D4 and to a lesser extent with OS-2, indicating the presence of different visual pigment. These rods differ in shape from the major rod type; they are thinner, shorter, and may be comparable to the blue-sensitive ("green") rods of other amphibia. Cones were morphologically heterogeneous: double cones, large single cones, and small single cones were found, and the large single and the double cones were occasionally duplicated. Double cones and large single cones (as well as their duplicated varieties) strongly bound monoclonal antibodies COS-1 and were unlabeled by all other monoclonal antibodies, except OS-2. The small single cone was remarkably unreactive with COS-1 and "E," weakly labeled by 1D4 and 4B4, and most reactive with OS-2 and 11-7. This unique pattern of immunocytochemical reactions in the small cone type indicates the uniqueness of its visual pigment from other cone types in the Xenopus retina. The present study shows the existence of two different opsins in morphologically distinct (thick and thin) rod types and at least two cone pigments in the heterogeneous cone population.

Animals↗
PubMed1995

Transducin-alpha C-terminal mutations prevent activation by rhodopsin: a new assay using recombinant proteins expressed in cultured cells.

We have measured the activation by recombinant rhodopsin of the alpha-subunit (alpha 1) of retinal transducin (Gt, also recombinant) using a new assay. Cultured cells are transiently transfected with DNAs encoding opsin and the three subunits of Gt (alpha t, beta 1 and gamma 1). In the microsomes of these cells, incubated with 11-cis-retinal, light causes the rapid activation of Gt, as measured by the ability of GTP gamma S to protect alpha t fragments from proteolytic degradation. The activation of Gt is also observed when all-trans-retinal is added to microsomes under constant illumination. Activation depends on both opsin and retinal. Opsin mutants with known defects in activating Gt show similar defects in this assay. alpha t mutations that mimic the corresponding mutations in the alpha-subunit of Gs also produce qualitatively similar effects in this assay. As a first step in a strategy aimed at exploring the relationships between structure and function in the interactions of receptors with G proteins, we tested mutant alpha t proteins with alanine substituted for each of the 10 amino acids at the C-terminus, a region known to be crucial for interactions with rhodopsin. Alanine substitution at four positions moderately (K341) or severely (L344, G348, L349) impairs the susceptibility of alpha 1 to activation by rhodopsin. All four mutants retain their ability to be activated by AIF-4. Two other substitutions (N343 and F350) resulted in very mild defects, while substitutions at the remaining four positions (E342, K345, D346 and C347) had no effect. In combination with previous observations, these results constrain models of the interaction of the C-terminus of alpha t with rhodopsin.

Amino Acid Sequence↗
PubMed1998

Involvement of Mash1 in EGF-mediated regulation of differentiation in the vertebrate retina.

It is believed that signaling through the epidermal growth factor (EGF) receptor plays a critical role in the development of Drosophila eyes. In the present study we have analyzed the role that EGF-mediated signaling plays in vertebrate retinal development. We have observed that during late retinal neurogenesis EGF delays rod photoreceptor differentiation and that this effect of EGF involves the modulation of expression of a homologue of Drosophila proneural genes, Mash1. EGF causes a significant decrease in Mash1 expression and an increase in the proportion of proliferating cells in the retina in vitro. The decrease in Mash1 expression is accompanied by a concomitant decrease in opsin expression, a marker for overt rod photoreceptor differentiation. Withdrawal of EGF leads to an increase in both Mash1 and opsin expression; however, the onset of expression of Mash1 precedes that of opsin. Our study identifies a proliferative intermediate precursor, characterized by Mash1 expression, that is the target of EGF-mediated suppression of rod photoreceptor differentiation. Based on the evolutionarily conserved roles of EGF- and Notch-mediated signaling in the delay of differentiation in proliferating precursors we propose that these distinct signaling mechanisms act in concert to ensure the fidelity of the strict temporal and spatial nature of cell fate determination in the retina.

Animals↗
PubMed1991

Circadian photoreception in the retinally degenerate mouse (rd/rd).

We have examined the effects of light on circadian locomotor rhythms in retinally degenerate mice (C57BL/6J mice homozygous for the rd allele: rd/rd). The sensitivity of circadian photoreception in these mice was determined by varying the irradiance of a 15 min light pulse (515 nm) given at circadian time 16 and measuring the magnitude of the phase shift of the locomotor rhythm. Experiments were performed on animals 80 days of age. Despite the loss of visual photoreceptors in the rd/rd retina, animals showed circadian responses to light that were indistinguishable from mice with normal retinas (rd/+ and +/+). While no photoreceptor outersegments were identified in the retina of rd/rd animals (80-100 days of age), we did identify a small number of perikarya that were immunoreactive for cone opsins, and even fewer cells that contained rod opsin. Using HPLC, we demonstrated the presence and photoisomerization of the rhodopsin chromophore 11-cis retinaldehyde. The rd/rd retinas contained about 2% of 11-cis retinaldehyde found in +/+ retinas. We have yet to determine whether the opsin immunoreactive perikarya or some other unidentified cell type mediate circadian light detection in the rd/rd retina.

Animals↗