Patterning of the R7 and R8 photoreceptor cells of Drosophila: evidence for induced and default cell-fate specification

Opsin gene expression in the R7 and R8 photoreceptor cells of the Drosophila compound eye is highly coordinated. We have found that the R8 cell specific Rh5 and Rh6 opsins are expressed in non-overlapping sets of R8 cells, in a precise pairwise fashion with Rh3 and Rh4 in the R7 cells of individual ommatidia. Removal of the R7 cells in sevenless, boss or sina mutants, disrupts Rh5 expression and dramatically increases the number of Rh6-expressing R8 cells. This suggests that the expression of Rh5 may be induced by an Rh3-expressing R7 cell, whereas Rh6 expression is most likely a default state of the R8 cell. We found that the paired expression of opsin genes in the R7 and R8 cells occurs in a sevenless and boss independent manner. Furthermore, we found that the generation of both Rh3- and Rh4-expressing R7 cells can occur in the absence of an R8 cell. These results suggest that the specification of opsin expression in the R7 cells may occur autonomously, whereas the R7 photoreceptor cell may be responsible for regulating a binary developmental switch between induced and default cell-fates in the R8 cell.     

Spatial and temporal expression of cone opsins during monkey retinal development

The primate retina requires a coordinated series of developmental events to form its specialized photoreceptor topography. In this study, the temporal expression of cone photoreceptor opsin was determined in Macaca monkey retina. Markers for mRNA and protein that recognize short wavelength (S) and long/medium wavelength (L/M) opsin were used to determine (1) the temporal and spatial patterns of opsin expression, (2) the spatial relationship between S and L/M cones at the time of initial opsin expression, and (3) the relative time of cone and rod opsin expression (Dorn et al. [1995] Invest. Ophthalmol. Vis. Sci. 36:2634-2651). Adult cone outer segments were recognized by either L/M or S opsin antiserum. Of all adult cone inner segments, 88-90% contained L/M opsin mRNA, whereas 10-12% contained S opsin mRNA. Fetal cones initially showed cell membrane as well as outer segment labeling for opsin protein, but cell membrane labeling disappeared by birth. No cones at any age contained markers for both S and L/M opsin mRNA or protein. S and L/M opsin protein appeared in the fovea at fetal day 75. Once opsin expression progressed beyond the fovea, both mRNA and protein for S opsin were consistently detected more peripherally than L/M opsin. Cones at the peripheral edge of S opsin expression had basal telodendria that appeared to reach toward neighboring cones. Because interactions between cone populations could organize the cone mosaic, the spatial relationship between S cones and the first cones to express L/M protein was analyzed quantitatively by using double-label immunocytochemistry. No consistent relationship was found between these two cone populations. Cones are generated at least 1 week before rods across monkey retina. However, rod opsin protein appears in and around the fovea at fetal day 66, 1 week before cone opsin protein. This suggests that independent local factors control differentiation in these two photoreceptor populations.     

A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin

Activation of the photoreceptor G protein transducin (Gt) by opsin, the ligand-free form of rhodopsin, was measured using rod outer segment membranes with densities of opsin and Gt similar to those found in rod cells. When GTPgammaS was used as the activating nucleotide, opsin catalyzed transducin activation with an exponential time course with a rate constant k(act) on the order of 2 x 10(-3)s(-1). Comparison under these conditions to activation by flash-generated metarhodopsin II (MII) revealed that opsin- and R*-catalyzed activation showed similar kinetics when MII was present at a surface density approximately 10(-6) lower than that of opsin. Thus, in contrast to some previous reports, we find that the catalytic potency of opsin is only approximately 10(-6) that of MII. In the presence of residual retinaldehyde-derived species present in membranes treated with hydroxylamine after bleaching, the apparent k(act) observed was much higher than that for opsin, suggesting a possible explanation for previous reports of more efficient activation by opsin. These results are important for considering the possible role of opsin in the diverse phenomena in which it has been suggested to play a key role, such as bleaching desensitization and retinal degeneration induced by continuous light or vitamin A deprivation.     

Chlamyrhodopsin represents a new type of sensory photoreceptor

In order to find optimal light conditions for photosynthetic growth, the green alga Chlamydomonas uses a visual system. An optical device, a rhodopsin photoreceptor and an electrical signal transduction chain that mediates between photoreceptor and flagella comprise this system. Here we present an improved strategy for the preparation of eyespot membranes. These membranes contain a retinal binding protein, which has been proposed to be the apoprotein of the phototaxis receptor. The retinal binding protein, which we named chlamyopsin, was purified and opsin-specific antibodies were raised. Using these antibodies, the opsin was localized in the eyespot region of whole cells during growth and cell division. The opsin cDNA was purified and sequenced. The sequence reveals that chlamyopsin is not a typical seven helix receptor. It shows some homology to invertebrate opsins but not to opsins from halobacteria. It contains many polar and charged residues and might function as a light-gated ion channel complex. It is likely that this lower plant rhodopsin diverged from animal opsins early in opsin evolution.     

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.     

Several levels of EGF receptor signaling during photoreceptor specification in wild-type, Ellipse, and null mutant Drosophila

Dominant Ellipse mutant alleles of the Drosophila EGF receptor homologue (DER) dramatically suppress ommatidium development in the eye and induce ectopic vein development in the wing. Their phenotype suggests a possible role for DER in specifying the founder R8 photoreceptor cells for each ommatidium. Here we analyze the basis of Ellipse mutations and use them to probe the role of DER in eye development. We show that Elp mutations result from a single amino acid substitution in the kinase domain which activates tyrosine kinase activity and MAP kinase activation in tissue culture cells. Transformant studies confirmed that the mutation is hypermorphic in vivo, but the DER function was elevated less than by ectopic expression of the ligand spitz. Ectopic spi promoted photoreceptor differentiation, even in the absence of R8 cells. Pathways downstream of DER activation were assessed to explore the basis of these distinct outcomes. Elp mutations caused overexpression of the Notch target gene E(spl) mdelta and required function of Notch to suppress ommatidium formation. The Elp phenotype also depended on the secreted protein argos and was reverted in Elp aos double mutants. Complete loss of DER function in clones of null mutant cells led to delay in R8 specification and subsequently to loss of mutant cells. The DER null phenotype was distinct from that of either spitz or vein mutants, suggesting that a combination of these or other ligands was required for aspects of DER function. In normal development DER protein was expressed in most retinal cells, but at distinct levels. We used an antibody specific for diphospho-ERK as well as expression of the DER target gene argos to assess the pattern of DER activity, finding highest activity in the intermediate groups of cells in the morphogenetic furrow. However, studies of mutant genotypes suggested that this activity may not be required for normal ommatidium development. Since we saw distinct phenotypic effects of four different levels of DER activity associated with wild-type, null mutant, Elp mutant, or fully activated DER function, we propose that multiple thresholds separate several aspects of DER function. These include activation of N signaling to repress R8 specification, turning on argos expression, and recruiting photoreceptors R1-R7. It is possible that during normal eye development these thresholds are attained by different cells, contributing to the pattern of retinal differentiation.     

Overexpression of Bcl-2 does not rescue impaired B lymphopoiesis in IL-7 receptor-deficient mice but can enhance survival of mature B cells

IL-7 receptor-deficient (IL-7R(-/-)) mice are lymphopenic as a result of defective cell production at early steps in both B and T lymphopoiesis. In the bone marrow, there is an incomplete block in B cell development at the transition from the pro-B to the pre-B cell stage. As a consequence, peripheral lymphoid organs of IL-7R(-/-) mice contain abnormally low numbers of mature surface (s) Ig-expressing B cells and this is accompanied by a relative increase in immature sIg- B cells. Transgenic expression of the anti-apoptotic protein Bcl-2 in IL-7R(-/-) mice rescues the defect in T cell development and in mature T cell function. The present report shows that constitutive expression of Bcl-2 is incapable of rescuing B lymphopoiesis in IL-7R(-/-) mice but can enhance survival of those mature B cells which escape the developmental arrest. Thus the essential role of IL-7R signaling in B lymphoid cells cannot be replaced by Bcl-2, indicating that in B lymphopoiesis IL-7R signaling is necessary for promoting cell division and/or for inhibiting a Bcl-2-insensitive pathway to apoptosis.     

pH-dependent fluoride transport in intestinal brush border membrane vesicles

Ciliary neurotrophic factor (CNTF) exerts a multiplicity of effects on a broad spectrum of target cells, including retinal neurons. To investigate how this functional complexity relates to the regulation of CNTF receptor alpha (CNTFR alpha) expression, we have studied the developmental expression of the receptor protein in chick retina by using immunocytochemistry. During the course of development, the receptor is expressed in all retinal layers, but three levels of specificity can be observed. First, the expression is regulated temporally with immunoreactivity observed in ganglion cells (embryonic day 8 [E8] to adult), photoreceptor precursors (E8-E12), amacrine cells (E10 to adult), bipolar cells (E12-E18), differentiated rods (E18 to adult), and horizontal cells (adult). Second, expression is restricted to distinct subpopulations of principal retinal neurons: preferentially, large ganglion cells; subpopulations of amacrine cells, including a particular type of cholinergic neuron; a distinctly located type of bipolar cell; and rod photoreceptors. Third, expression exhibits subcellular restriction: it is confined largely to dendrites in mature amacrine cells and is restricted entirely to outer segments in mature rods. These data correlate with CNTF effects on the survival of ganglion cells and mature photoreceptors, the in vitro differentiation of photoreceptor precursors and cholinergic amacrine cells, and the number of bipolar cells in culture described here or in previous studies. Thus, our results demonstrate an exceptional degree of complexity with respect to the regulation of neuronal CNTFR alpha expression in a defined model system. This suggests that the same signaling pathway is used to mediate a variety of regulatory influences, depending on the developmental stage and cell type.     

Genetic dissection of signal transduction mediated by the sevenless receptor tyrosine kinase in Drosophila

The specification of the R7 photoreceptor cell fate in the developing eye of Drosophila depends on the local activation of the sevenless (sev) receptor tyrosine kinase by boss, a protein expressed on the membrane of the neighboring R8 cell. Constitutive activation of the sev receptor results in a dosage dependent increase in the number of R7 cells per ommatidium. Genetic screens have been used to identify mutations that alter the efficiency of signal transduction. Subsequent molecular characterization of the corresponding genes has led to the identification of a number of proteins involved in transducing the signal from the receptor to the nucleus. In contrast to the receptor and its ligand, these components are shared between different signal transduction pathways not only in Drosophila but are also homologous to components involved in signal transduction in other organisms.     

Improved adenovirus vector provides herpes simplex virus ribonucleotide reductase R1 and R2 subunits very efficiently

We have constructed a new adenovirus (Ad) expression vector, pAdBM5, that allows for the production of unprecedented levels of recombinant protein in the human 293 cell line using the Ad expression system. The main feature of this vector is a combination of enhancer sequences that increases the activity of the ectopic major late promoter (MLP) in recombinant Ad. In 293 cells infected with helper-free Ad recombinants generated with the pAdBM5 transfer vector, both herpes simplex virus (HSV) ribonucleotide reductase R1 and R2 subunits represent the most abundant polypeptides, accounting for as much as 15-20% of total cellular proteins. Our data suggest that this level of expression is probably very close to the upper limit of the system. Furthermore, when compared to the widely utilized baculovirus (Bac)/Sf9 expression system, the improved Ad vector showed a better performance for the production and purification of active HSV-2 ribonucleotide reductase R1 and R2 subunits. The R2 subunit was about 5-fold more abundant in recombinant Ad-infected 293 cells than in Bac-infected Sf9 cells while the R1 subunit was produced at roughly similar levels with either system. However, the amount of active soluble R1 obtained from recombinant Ad-infected 293 cells was at least 5 times higher because most of the R1 produced in Sf9 cells was insoluble.     

Lineage relationships of the fetal thymocyte subset that expresses the beta chain of the interleukin-2 receptor

The beta chain (p75) of the interleukin-2 (IL-2) receptor (IL-2R) is expressed on up to 5-7% of fetal thymocytes on day 16 of gestation, declining thereafter to a minute proportion of less than 1% around birth, and of 1-2% of adult thymocytes. A significant part of fetal IL-2R beta+ thymocytes are gamma delta cells. The precursor-progeny relationships of fetal IL-2R beta+ thymocytes to the alpha beta T cell lineage have not been previously studied, nor has their position within the developmental sequence been determined. Here we show that IL-2R beta is expressed on a subset of very immature cells, along with high amounts of Pgp1 and Fc gamma RII/III, partially preceding the expression of intracellular CD3 epsilon. IL-2-R beta disappears before expression of IL-2R alpha. IL-2R beta+ cells, purified by sorting on day 15 of gestation, efficiently reconstituted fetal thymic lobes depleted of lymphoid cells by treatment with desoxyguanosine. They developed into T cell receptor (TCR) alpha beta+, TCR gamma delta+, and CD4/CD8 double- and single-positive cells in similar proportions as did sorted IL-2R alpha+ day 15 fetal thymocytes. These data suggest that IL-2R beta expression marks a short period of very early thymocyte development, perhaps immediately after entry into the thymus.     

Multiple functions of the EGF receptor in Drosophila eye development

BACKGROUND: During animal development, cells need to make spatially and temporally regulated fate decisions. These decisions are largely controlled by intercellular signalling, often through receptor tyrosine kinases. One of these, the epidermal growth factor receptor (EGFR), regulates multiple cell fate decisions. Its importance in the recruitment of photoreceptors in the developing fly eye, a useful model for neural development, has already been reported. Other EGFR functions in the eye have not been characterised. RESULTS: We have examined the consequences of removing or activating the EGFR at different stages of eye development. The earliest stages of assembly occurred normally within EGFR- clones--the morphogenetic furrow was unimpeded and the R8 photoreceptor was specified. All subsequent photoreceptor recruitment was blocked. EGFR- clones had a characteristic shape indicating that they had undergone substantial cell death posterior to the furrow, where the differentiation program is normally activated; consistent with this, excess apoptosis was detected. We found that the receptor also regulates cell proliferation in the disc, has an early function at the disc margin (where the morphogenetic furrow initiates) and contributes to the regulation of spacing of the R8 precursors. Finally, we found that activation of the receptor is sufficient to trigger non-R8 photoreceptor development, even in cells in front of the furrow or in the absence of the proneural gene atonal. CONCLUSION: At least five distinct functions of EGFR signalling need to be integrated during fly eye development. These include roles in cell proliferation, survival and differentiation.     

A transient role for ciliary neurotrophic factor in chick photoreceptor development

Previous studies suggest that ciliary neurotrophic factor (CNTF) may represent one of the extrinsic signals controlling the development of vertebrate retinal photoreceptors. In dissociated cultures from embryonic chick retina, exogenously applied CNTF has been shown to act on postmitotic rod precursor cells, resulting in an two- to fourfold increase in the number of cells acquiring an opsin-positive phenotype. We now demonstrate that the responsiveness of photoreceptor precursors to CNTF is confined to a brief phase between their final mitosis and their terminal differentiation owing to the temporally restricted expression of the CNTF receptor (CNTFR alpha). As shown immunocytochemically, CNTFR alpha expression in the presumptive photoreceptor layer of the chick retina starts at embryonic day 8 (E8) and is rapidly down-regulated a few days later prior to the differentiation of opsin-positive photoreceptors, both in vivo and in dissociated cultures from E8. We further show that the CNTF-dependent in vitro differentiation of rods is followed by a phase of photoreceptor-specific apoptotic cell death. The loss of differentiated rods during this apoptotic phase can be prevented by micromolar concentrations of retinol. Our results provide evidence that photoreceptor development depends on the sequential action of different extrinsic signals. The time course of CNTFR alpha expression and the in vitro effects suggest that CNTF or a related molecule is required during early stages of rod differentiation, while differentiated rods depend on additional protective factors for survival.     

Photoreceptor recovery in retinoid-deprived rats after vitamin A replenishment

Dietary deficiency in the retinoid precursors of the visual pigment chromophore 11-cis retinal results in the synthesis of photoreceptor outer segments containing opsin in excess of the vitamin A available for rhodopsin regeneration. This suggests that vitamin A-free opsin may be incorporated into newly synthesized outer segment disc membranes. If this opsin is functionally intact, it should be possible convert it to rhodopsin in vivo by providing the appropriate retinoids, and the resulting rhodopsin should should be able to mediate visual transduction. Experiments were conducted to evaluate this possibility and to identify the rate-limiting steps in photoreceptor recovery from retinoid depletion. Rates were maintained on diets either containing or lacking retinoid precursors of 11-cis retinal for 23 weeks, at which time outer segment opsin content greatly exceeded the availability of visual cycle retinoids in the retina. The retinoid-deprived animals were then each given a single intramuscular injection of all-trans retinol. At various time intervals after retinol administration, electroretinograms (ERGs) were recorded on some rats, and retinal rhodopsin contents were determined in others. At similar time intervals, blood and retinal pigment epithelial (RPE) retinoid levels and photoreceptor outer segment size were also determined. No significant increase in retinal rhodopsin content was observed up to 8 hr after injection, despite the fact that by 3 hr, blood retinol levels had recovered to more than 30% of normal. By 1 day after injection, however, rhodopsin levels had recovered to 30% of normal and ERG responses showed increases in visual sensitivity commensurate with the recovery of rhodopsin. The lag in rhodopsin recovery was apparently due to delayed uptake of retinol from the blood by the RPE. Photoreceptor outer segment size was reduced by over 50% in the retinoid- deprived rats and did not begin to recover by 1 day. By 1 week, however, outer segment size had returned to an average of 65% of normal. Commensurate with this regrowth of the outer segments, both rhodopsin levels and visual sensitivity increased between 1 and 7 days after vitamin A administration. Because the rates of recovery in rhodopsin levels and visual sensitivity greatly exceeded the normal rate of new opsin synthesis at short time intervals after vitamin A repletion, it appears that the opsin incorporated into the disc membranes of retinoid-deprived rats is able to form functional rhodopsin in vivo when the chromophore is supplied. Regrowth of the outer segments back to their normal size is required for full recovery of visual sensitivity.     

Regulated expression of gp130 and IL-6 receptor alpha chain in T cell maturation and activation

The functional receptor for the inflammatory cytokine IL-6 is composed of the ligand binding IL-6 receptor alpha chain (IL-6R alpha) and the signal transducing chain gp130, which is a shared component of multiple cytokine receptors. We analyzed the surface expression of gp130 and IL-6R alpha in thymocytes and peripheral T cells. While all thymocytes expressed gp130 throughout thymic maturation, they gained expression of IL-6R alpha at the CD4 or CD8 single-positive stage. Approximately 10-30% of the CD4-CD8+ and 40-50% of the CD4+CD8- thymocytes expressed IL-6R alpha. Within the CD4+CD8- population, the IL-6R alpha- subpopulation was cortisone sensitive, appeared immature according to the cell surface markers expressed and failed to proliferate after TCR cross-linking. Peripheral T cells were predominantly gp130+ and IL-6R alpha+, but down-regulated gp130 and IL-6R alpha expression upon TCR engagement in vitro and in vivo. Peripheral gp130low/-IL-6R alphalow/- T cells expressed surface markers characteristic of memory T cells. We show that gp130 and IL-6R alpha are expressed in a regulated manner in T cells, depending on the developmental and functional stage.     

Reduction of all-trans-retinal limits regeneration of visual pigment in mice

Absorption of photons by pigments in photoreceptor cells results in photoisomerization of the chromophore, 11-cis-retinal, to all-trans-retinal and activation of opsin. Photolysed chromophore is converted back to the 11-cis-configuration via several enzymatic steps in photoreceptor and retinal pigment epithelial cells. We investigated the levels of retinoids in mouse retina during constant illumination and regeneration in the dark as a means of obtaining more information about the rate-limiting step of the visual cycle and about cycle intermediates that could be responsible for desensitization of the visual system. All-trans-retinal accumulated in the retinas during constant illumination and following flash illumination. Decay of all-trans-retinal in the dark following constant illumination occurred without substantial accumulation of all-trans-retinal, generated by constant approximately equal to visual pigment regeneration (t1/2 approximately 5 and t1/2 approximately 7 min, respectively). All-trans-retinal, generated by constant illumination, decayed approximately 3 times more rapidly than that generated by a flash and, as shown previously, the rate of rhodopsin regeneration following a flash was approximately 4 times slower than after constant illumination. The retinyl ester pool (> 95% all-trans-retinyl ester) did not show a statistically significant change in size or composition during illumination. In addition, constant illumination increased the amount of photoreceptor membrane-associated arrestin. The results suggest that the rate-limiting step of the visual cycle is the reduction of all-trans-retinal to all-trans-retinol by all-trans-retinol dehydrogenase. The accumulation of all-trans-retinal during illumination may be responsible, in part, for the reduction in sensitivity of the visual system that accompanies photobleaching and may contribute to the development of retinal pathology associated with light damage and aging.     

Pathogenesis of neuroimmunologic diseases. Experimental models

Development of the retina, like that of other tissues, occurs via an orderly sequence of cell division and differentiation, producing the functional retina. In teleost fish, however, cell division and differentiation in the retina continue throughout the life of the animal in two distinct ways. Stem cells in a circumferential germinal zone at the periphery of the retina give rise to all retinal cell types and progenitor cells located throughout the retina in the outer nuclear layer (ONL) produce new rod photoreceptors. These processes in adult retina recapitulate in space the embryonic events responsible for forming the retina. Analysis of these events in an African cichlid fish, Haplochromis burtoni, confirmed that cone photoreceptors differentiate first, followed by rod photoreceptors. Correspondingly, at the margin of the eye, cone photoreceptors differentiate nearer to the margin than do rods. Control of photoreceptor production is not understood. Here we present the time of appearance and distribution pattern of GABA and vimentin which are candidates for the control of retinal cell division and differentiation. Antibody staining reveals that both GABA and vimentin exhibit unique patterns of expression during embryonic retinal development. Vimentin immunoreactivity is evident throughout the retina in a spoke-like pattern between developmental Days 4 and 7, as both cone and rod photoreceptors are being formed. GABA is expressed in horizontal cells between Days 5 and 7, corresponding to the onset of rod differentiation in time and in position within the retina. Moreover, the wave of GABAergic staining in the horizontal cells parallels the wave of rod differentiation across the embryonic retina of H. burtoni. Thus, GABA may play a role in the development of rod photoreceptors.