The proliferative and apoptotic activities of E2F1 in the mouse retina.

The E2F1 transcription factor controls cell proliferation and apoptosis. E2F1 activity is negatively regulated by the retinoblastoma (RB) protein. To study how inactivation of Rb and dysregulated E2F1 affects the developing retina, we analysed wild-type and Rb(-/-) embryonic retinas and retinal transplants and we established transgenic mice expressing human E2F1 in retinal photoreceptor cells under the regulation of the IRBP promoter (TgIRBPE2F1). A marked increase in cell proliferation and apoptosis was observed in the retinas of Rb(-/-) mice and TgIRBPE2F1 transgenic mice. In the transgenic mice, photoreceptor cells formed rosette-like arrangements at postnatal days 9 through 28. Complete loss of photoreceptors followed in the TgIRBPE2F1 mice but not in the Rb(-/-) retinal transplants. Both RB-deficient and E2F1-overexpressing photoreceptor cells expressed rhodopsin, a marker of terminal differentiation. Loss of p53 partially reduced the apoptosis and resulted in transient hyperplasia of multiple cell types in the TgIRBPE2F1 retinas at postnatal day 6. Our findings support the concept that cross-talk occurs between different retinal cell types and that multiple genetic pathways must become dysregulated for the full oncogenic transformation of neuronal retinal cells.

Mutant rab8 Impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods.

Rab8 is a GTPase involved in membrane trafficking. In photoreceptor cells, rab8 is proposed to participate in the late stages of delivery of rhodopsin-containing post-Golgi membranes to the plasma membrane near the base of the connecting cilium. To test the function of rab8 in vivo, we generated transgenic Xenopus laevis expressing wild-type, constitutively active (Q67L), and dominant negative (T22N) forms of canine rab8 in their rod photoreceptors as green fluorescent protein (GFP) fusion proteins. Wild-type and constitutively active GFP-rab8 proteins were primarily associated with Golgi and post-Golgi membranes, whereas the dominant negative protein was primarily cytoplasmic. Expression of wild-type GFP-rab8 had minimal effects on cell survival and intracellular structures. In contrast, GFP-rab8T22N caused rapid retinal degeneration. In surviving peripheral rods, tubulo-vesicular structures accumulated at the base of the connecting cilium. Expression of GFP-rab8Q67L induced a slower retinal degeneration in some tadpoles. Transgene effects were transmitted to F1 offspring. Expression of the GFP-rab8 fusion proteins appears to decrease the levels of endogenous rab8 protein. Our results demonstrate a role for rab8 in docking of post-Golgi membranes in rods, and constitute the first report of a transgenic X. laevis model of retinal degenerative disease.

A functional rhodopsin-green fluorescent protein fusion protein localizes correctly in transgenic Xenopus laevis retinal rods and is expressed in a time-dependent pattern.

To study rhodopsin biosynthesis and transport in vivo, we engineered a fusion protein (rho-GFP) of bovine rhodopsin (rho) and green fluorescent protein (GFP). rho-GFP expressed in COS-1 cells bound 11-cis retinal, generating a pigment with spectral properties of rhodopsin (A(max) at 500 nm) and GFP (A(max) at 488 nm). rho-GFP activated transducin at 50% of the wild-type activity, whereas phosphorylation of rho-GFP by rhodopsin kinase was 10% of wild-type levels. We expressed rho-GFP in the rod photoreceptors of Xenopus laevis using the X. laevis principal opsin promoter. Like rhodopsin, rho-GFP localized to rod outer segments, indicating that rho-GFP was recognized by membrane transport mechanisms. In contrast, a rho-GFP variant lacking the C-terminal outer segment localization signal distributed to both outer and inner segment membranes. Confocal microscopy of transgenic retinas revealed that transgene expression levels varied between cells, an effect that is probably analogous to position-effect variegation. Furthermore, rho-GFP concentrations varied along the length of individual rods, indicating that expression levels varied within single cells on a daily or hourly basis. These results have implications for transgenic models of retinal degeneration and mechanisms of position-effect variegation and demonstrate the utility of rho-GFP as a probe for rhodopsin transport and temporal regulation of promoter function.

Identification of an outer segment targeting signal in the COOH terminus of rhodopsin using transgenic Xenopus laevis.

Mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. Here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ROS). Various green fluorescent protein (GFP)/rhodopsin COOH-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The fusion proteins were targeted to membranes via lipid modifications (palmitoylation and myristoylation) as opposed to membrane spanning domains. Membrane association was found to be necessary but not sufficient for efficient ROS localization. A GFP fusion protein containing only the cytoplasmic COOH-terminal 44 amino acids of Xenopus rhodopsin localized exclusively to ROS membranes. Chimeras between rhodopsin and alpha adrenergic receptor COOH-terminal sequences further refined rhodopsin's ROS localization signal to its distal eight amino acids. Mutations/deletions of this region resulted in partial delocalization of the fusion proteins to rod inner segment (RIS) membranes. The targeting and transport of endogenous wild-type rhodopsin was unaffected by the presence of mislocalized GFP fusion proteins.

Fluorescent photoreceptors of transgenic Xenopus laevis imaged in vivo by two microscopy techniques.

PURPOSE: To develop a method for imaging individual photoreceptors in an intact transgenic Xenopus eye, thus allowing in vivo observation of the effects of various transgenes on photoreceptor development, degeneration, or both. METHODS: Albino and pigmented transgenic Xenopus laevis that express enhanced green fluorescent protein (GFP) in the major ("red") rods were generated. The distribution of GFP throughout the retina and within the rods was evaluated by confocal microscopy of frozen sections and immunoelectron microscopy. In vivo images of photoreceptors were obtained using conventional fluorescence microscopes to image through the lens of the eye or a laser scanning confocal microscope to image through the hypopigmented iris of albino eyes. RESULTS: Confocal and immunoelectron microscopy of tissue sections showed that GFP was predominantly localized to the inner segments of the major rods; a smaller amount was in the outer segments. In a number of animals, not all the major rods expressed GFP. It was possible to identify these animals by obtaining fluorescence images of the retinas of intact, living tadpoles with conventional fluorescence microscopes, using the lens of the tadpole as part of the optical path. Confocal images of living animals could be used to visualize the distribution of GFP within the photoreceptors. CONCLUSIONS: The ability to observe individual photoreceptors noninvasively allows in vivo longitudinal microscopic analysis of photoreceptor development in transgenic Xenopus tadpoles.

A unique pattern of photoreceptor degeneration in cyclin D1 mutant mice.

Cyclin D1-deficient mice have small eyes with thin retinas. We observed that there was a lower level of retinal cell proliferation and a unique pattern of photoreceptor cell death. Death was first observed in scattered clusters of cells in the retina. It then appeared to spread from these few cells to nearby photoreceptors, eventually producing extensive holes in the photoreceptor layer. These holes appeared to be filled with interneurons from the inner nuclear layer. The death mainly occurred during the second to fourth postnatal weeks. Other models of photoreceptor degeneration in rodents differ in that they occur more uniformly across the retina, with death proceeding over a longer period of time until all, or nearly all, of the photoreceptors degenerate. We also tested whether expression of a bcl-2 transgene could prevent the death and found that it could not.

Apoptosis in the murine rd1 retinal degeneration is predominantly p53-independent.

PURPOSE: To determine if p53 mediates apoptosis in photoreceptors of retinal degeneration, rd1, mice. METHODS: The rd1/rd1 mice were interbred with p53 null mice to generate p53-/- rd1/rd1 and p53+/+ rd1/rd1 mice. Rates of loss and incidence of apoptosis in rod photoreceptors were analyzed at appropriate ages (postnatal days 12, 14 and 16). RESULTS: The extent and kinetics of photoreceptor cell loss in rd1 mice were nearly indistinguishable in the p53+/+ and p53 null mice. CONCLUSIONS: Photoreceptor cell apoptosis in the rd1 mouse model occurs by a predominantly p53-independent molecular pathway.

Post-Golgi vesicles cotransport docosahexaenoyl-phospholipids and rhodopsin during frog photoreceptor membrane biogenesis.

Post-Golgi vesicles budding from the trans-Golgi network (TGN) are involved in the vectorial transport and delivery of rhodopsin to photoreceptor rod outer segments (ROS). We report here that newly synthesized docosahexaenoyl (DHA) phospholipids are sequestered and cotransported by rhodopsin-bearing post-Golgi vesicles to ROS. Frog retinas were pulse-labeled with [35S]methionine/cysteine and [3H]DHA prior to ROS isolation and subcellular fractionation. After a 1-h pulse, relatively uniform [3H]DHA-lipid labeling (DPM/microg protein) was observed in all fractions enriched in post-Golgi vesicles, TGN, Golgi, and endoplasmic reticulum (ER) membranes. During the subsequent 2-h chase translocation of free [3H]DHA from ROS to the photoreceptor inner segment contributed to an additional overall increase in labeling of lipids. The specific activity (dpm/nmol DHA) in ER-enriched fraction was similar or higher than in other subcellular fractions after both the pulse and the chase, indicating that the bulk of [3H]DHA-lipids was synthesized in the ER. After the chase a 2-fold increase in labeling of lipids in the ER and Golgi and a 2.6-fold in lighter TGN-enriched fractions was observed. The highest labeling was in the post-Golgi vesicle fraction (4-fold increase), with [3H]DHA-phosphatidylcholine and [3H]DHA-phosphatidylethanolamine showing the greatest increase. At the same time, newly synthesized [35S]rhodopsin shifted from the ER and Golgi toward TGN and post-Golgi fractions. Therefore, sequestration and association of [35S]rhodopsin and [3H]DHA-lipids in a TGN membrane domain occurs prior to their exit and subsequent vectorial cotransport on post-Golgi vesicles to ROS. Labeling of ROS lipids was very low, with phosphatidylinositol and diacylglycerols displaying the highest labeling. This indicates that other mechanisms by-passing Golgi, i.e. facilitated by lipid carrier proteins, may also contribute to molecular replacement of disc membrane DHA-phospholipids, particularly phosphatidylinositol.

Apoptosis of the mammalian retina and lens.

Although retinal neurons usually last the entire lifetime of an individual, many innate genetic and developmental errors and external stimuli can reduce their longevity leading to loss of visual acuity or blindness. Similarly, the lens, largely composed of denucleated fiber cells must remain transparent for life if vision is to remain clear. Apoptosis of retinal neurons and newly generated lens fiber cells contributes to retinal degeneration and cataract formation, respectively, in both humans and experimental mammals. The apoptosis is triggered by many stimuli in addition to inherited mutations and may be amenable to pharmacologic amelioration. These studies not only provide new clinical insights but also the opportunity to investigate the molecular pathways leading to apoptosis in an organ that is not required for survival. The eye, becomes, therefore, an important organ for evaluation of theories of apoptosis in vivo.

Repression of hybrid dysgenesis in Drosophila melanogaster by heat-shock-inducible sense and antisense P-element constructs.

Sets of sense and antisense P-element constructs controlled by a heat-shock-inducible promoter were tested for their ability to repress manifestations of P-element activity in vivo. As a group, the antisense constructs repressed pupal lethality, a somatic manifestation of P activity, and this repression was significantly enhanced by heat shock. Three of the 11 antisense constructs also repressed gonadal dysgenesis, a manifestation of P activity in the female germ line; however, none had any effect on P-element-mediated mutability in the male germ line. Among the 13 different heat-shock-inducible sense constructs that were tested, those containing the KP and DP elements were strong repressors of pupal lethality, gonadal dysgenesis and P-element-mediated mutability; however, individual lines carrying these constructs varied in their ability to repress each of these traits, presumably because of genomic position effects. With the exception of the sense construct that contained a complete P element, none of the sense or antisense constructs repressed a lacZ reporter gene driven by the P-element promoter. Overall, the experimental results suggest that in nature, P-element activity could be regulated by P-encoded polypeptides and by antisense P RNAs.

rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis.

Small GTP-binding protein rab8 regulates transport from the TGN to the basolateral plasma membrane in epithelial cells and to the dendritic plasma membrane in cultured hippocampal neurons. In our approach to identify proteins involved in rhodopsin transport and sorting in retinal photoreceptors, we have found, using [32P]GTP overlays of 2D gel blots, that six small GTP-binding proteins are tightly bound to the post-Golgi membranes immunoisolated with a mAb to the cytoplasmic domain of frog rhodopsin. We report here that one of these proteins is rab8. About 50% of photoreceptor rab8 is membrane associated and approximately 13% is tightly bound to the post-Golgi vesicles. By confocal microscopy, antibody to rab8 specifically labels calycal processes and the actin bundles of the photoreceptor inner segment that extend inward to the junctional complexes that comprise the outer limiting membrane. Anti-rab8 shows a striking periodicity of high density labeling at 1 +/- 0.12 microns intervals along the actin bundles. Rhodopsin-bearing post-Golgi membranes cluster around the base of the cilium where rab8 and actin are also co-localized, as revealed by confocal microscopy of retinal sections double labeled with anti-rab8 and phalloidin. Microfilaments have been implicated in rod outer segment (ROS) disk morphogenesis. Our data suggest that rab6, which we have previously localized to the post-Golgi compartment, and rab8 associate with the post-Golgi membranes sequentially at different stages of transport. rab8 may mediate later steps that involve interaction of transport membranes with actin filaments and may participate in microfilament-dependent ROS disk morphogenesis.

Apoptosis or retinoblastoma: alternative fates of photoreceptors expressing the HPV-16 E7 gene in the presence or absence of p53.

A transgenic mouse model for retinoblastoma was produced previously by directing SV40 T antigen expression to retinal photoreceptor cells using the promoter of the interstitial retinol-binding protein (IRBP) gene. This gene becomes active prior to the terminal differentiation of photoreceptors. Because T antigen-transforming activity is attributable, at least in part, to the inactivation of the retinoblastoma (pRb) and p53 tumor suppressor proteins, we addressed the role of p53 in the development of retinoblastoma in mice. Transgenic mice expressing HPV-16 E7 under the control of the IRBP promoter were generated to inactivate pRb in photoreceptors while leaving p53 intact. Rather than developing retinoblastomas, the retinas of these mice degenerate due to photoreceptor cell death at a time in development when photoreceptors are normally undergoing terminal differentiation. The dying cells exhibit the histological and ultrastructural features of apoptosis and contain fragmented DNA. p53 is required for the induction of apoptosis in this model, because mice expressing E7 in a p53 nullizygous background develop retinal tumors instead of undergoing retinal degeneration.

Alpha A- and alpha B-crystallin in the retina. Association with the post-Golgi compartment of frog retinal photoreceptors.

alpha A- and alpha B-Crystallins are significant contributors to maintaining the transparency of the vertebrate lens. We have found that both alpha A- and alpha B-crystallins are also present, at approximately equimolar concentrations, in frog retinal cells. They were identified by sequencing portions of each polypeptide, by immunochemical cross-reactivity with antibodies to bovine alpha-crystallins, and by their relative mobility in two-dimensional gel electrophoresis. Retinal alpha-crystallins form macromolecular multimeric complexes similar to those found in the lens, and they are abundant both in soluble and membrane-associated forms. A surprising finding is that alpha-crystallins bind specifically to the photoreceptor post-Golgi membranes that mediate transport of newly synthesized rhodopsin. Upon treatment of post-Golgi membranes with urea or Triton X-114, a portion of the bound alpha B-crystallin remains tightly associated, indicating that the alpha B-form may mediate membrane binding of an alpha-crystallin multimeric complex. Both subunits are synthesized in vitro by isolated frog retinas, but alpha B-crystallin appears to have a higher renewal rate. Newly synthesized alpha-crystallins become associated with the post-Golgi membranes concurrently with newly synthesized rhodopsin. Association of alpha-crystallins with newly synthesized rhodopsin suggests that they may participate in photoreceptor outer segment membrane renewal. Our findings implicate an important function for both alpha A- and alpha B-crystallins in the same, extralenticular, tissue.

Loss of diurnal arrestin gene expression in rds mutant mouse retinas.

Despite the important advances in the molecular analysis of retinal degenerations, the causes of photoreceptor cell death in these conditions are unclear. To explore the metabolic impact of the rds mutation, we have continued our investigations on the expression of arrestin. Normal BALB/c mouse retinas have a diurnal variation in arrestin gene expression and protein biosynthesis, with low levels in dark-adapted and high levels in the light-adapted retinas. In contrast, arrestin is expressed in rds retinas at high levels throughout the diurnal cycle. A lack of a distinct diurnal cycle and continuously high expression of arrestin in rds retinas might be a reflection of metabolic derangements in these cells which are not an obvious consequence of the underlying molecular defect in the rds/peripherin gene.

Rab6 is associated with a compartment that transports rhodopsin from the trans-Golgi to the site of rod outer segment disk formation in frog retinal photoreceptors.

The biogenesis of light sensitive membranes in retinal rod photoreceptors involves polarized sorting and targeting of newly synthesized rhodopsin to a specialized domain, the rod outer segment (ROS). We have isolated and characterized the population of post-Golgi membranes that mediate intracellular transport of rhodopsin. In the present study we have examined the association of small (20-25 kDa) GTP-binding (G) proteins with these membranes. We found that one of the small G proteins, rab6, behaves like an integral membrane protein of the post-Golgi vesicles, although approximately 30% of rab6 is soluble. The distribution of the membrane-associated and the soluble forms is highly polarized. By confocal and EM immunocytochemistry it can be seen that most of rab6 is associated with the photoreceptor trans-Golgi cisternae, trans-Golgi network (TGN) and post-Golgi vesicles. The photoreceptor axon and synaptic terminal are unlabeled, but dendrites of deeper retinal layers are labeled. The distribution of rab6 across sucrose density gradient fractions parallels the distribution of sialyltransferase (a TGN marker) activity. About 9% of membrane-bound rab6 is associated, however, with the rhodopsin-bearing sialyltransferase-free post-Golgi vesicles, which represent a very small fraction (< 1%) of the total retinal membranes. Rab6 is absent from the mature ROS disk membranes but it is present at the sites of new ROS disk formation and in the ROS cytoplasm. This suggests that rab6 becomes soluble upon disk membrane formation. Therefore, rab6 may function not only as a component of the sorting machinery of photoreceptors that delivers rhodopsin to its appropriate subcellular domain but may also participate in some aspects of ROS disk morphogenesis.

Expression of opsin and IRBP genes in mutant RCS rats.

The retinal pigment epithelium of RCS rats bearing the autosomal recessive rdy mutation fails to ingest shed rod outer segment tips. Accumulation of disk debris in the subretinal space of the maturing mutant retina causes a secondary degeneration of photoreceptor cells. Two hypotheses have been offered as possible explanations of the death of photoreceptor cells in this disorder: (1) photoreceptors are starved for amino acids, retinal, oxygen, etc; and (2) that IRBP levels and synthesis may be decreased and interfere with retinal transport and this deficiency is lethal to these cells. To test these hypotheses, we have studied the effect of this mutation on the levels of expression of opsin and IRBP genes, and gene products and on rates of synthesis at various ages in dystrophic RCS p+ rats and compared the results to those obtained with normal Long Evans rats. The mutant rats and normal controls had comparable amounts of opsin and IRBP mRNA transcripts and rates of synthesis up to post-natal day 45 (P45) but opsin transcripts were barely detectable at P60 and thereafter. IRBP mRNA levels were also very low after P62 although somewhat higher than opsin mRNA. Opsin could be detected immunochemically, albeit at lower levels, at all the ages studied up to P310, but IRBP levels fell below detection after P45. We localized opsin and IRBP in the retina by post-embedding EM immunocytochemical procedures and found that opsin is present in the remnants of rod outer segment debris, even at P390, long after detectable opsin synthesis had ceased. These data suggest that expression of opsin and IRBP genes is not influenced by the shape and state of the outer segments, and that the rdy mutation does not influence the expression of the opsin and IRBP in these retinas until the photoreceptor cells are profoundly damaged. Thus, neither hypothesis about the causes of cell death in this disorder is supported.

Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa.

Thirteen mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa (ADRP) have been produced by transfection of cloned cDNA into tissue culture cells. Three mutants [class I: Phe-45----Leu, Gln-344----termination (deletion of C-terminal positions 344-348), and Pro-347----Leu] resemble wild-type rhodopsin in yield, regenerability with 11-cis-retinal, and plasma membrane localization. Ten mutants [class II: Thr-17----Met, Pro-23----His, Thr-58----Arg, Val-87----Asp, Gly-89----Asp, Gly-106----Trp, Arg-135----Leu, Arg-135----Trp, Tyr-178----Cys, and Asp-190----Gly] accumulate to significantly lower levels, regenerate with 11-cis-retinal variably or not at all, and are transported inefficiently to the plasma membrane, remaining primarily in the endoplasmic reticulum. These data suggest that there are at least two distinct biochemical defects associated with different rhodopsin mutants in ADRP.