Somes Sanyal

Absence of receptor outer segments in the retina of rds mutant mice

In mice homozygous for the newly reported rds (retinal degeneration slow) gene, a progressive loss of visual cells occurs throughout life. Histogenesis of the retina proceeds normally with the exception of the receptor layer which remains rudimentary. Electron microscopic observations of the retina at different stages of development have revealed that the outer segments of the visual cells in this mutant fail to develop and that the receptor layer consists of inner segments only. In the absorption spectra of the retinal extract from the mutant the characteristic rhodopsin peak is lacking.

Development and degeneration of retina in rds mutant mice: photoreceptor abnormalities in the heterozygotes.

Mice homozygous for the rds (retinal degeneration slow) gene fail to develop receptor outer segments and show a slow loss of visual cells that starts from 14-21 postnatal days and results in complete absence at 1 year. In the heterozygous rds/+ mice the development of receptor outer segments is initially retarded. Although a distinct layer of outer segments of moderate length is formed, the disc structures remain disarrayed and form irregular whorls. Autoradiograms of rds/+ retinas show reduced incorporation of [3H]-leucine. Scleral movement of label, resulting from the addition of newly formed discs, is also retarded and appears irregular in comparison with the normal. Phagosomes, containing newly shed disc structures, within the retinal pigment epithelium of rds/+ mice are much larger than normal. Counts taken at different times of the dark- and light periods have shown an abnormally high turnover of phagosomes in the pigment epithelium of the rds/+ mice, with higher than normal peak frequency near the end of the light period, in contrast with the peak frequency in the normal pigment epithelium recorded around the beginning of the light period. Starting at 2 months, a very slow loss of visual cells, much slower than in the homozygous mutants, progresses throughout life. As a result, the outer nuclear layer at the age of 18 months or more is reduced to less than half. Prior to the reduction of the outer nuclear layer, the relative frequencies of the rod and cone perikarya in the rds/+ retina are similar to the normal values. With loss of visual cells, a small increase in the relative frequency of the cone perikarya is recorded in older rds/+ mice. This increase is more noticeable in the central than in the peripheral retina. The significance of the partial expression of the rds gene in the retina of the heterozygous mice in comparison with the changes observed in the homozygous retina is discussed. It is concluded that dose-dependent variation in phenotypic expression is an essential feature in the working of the rds gene.

Comparative light and electron microscopic study of retinal histogenesis in normal and rd mutant mice

SummaryMice, homozygous for the mutant gene rd show selective degeneration of the photoreceptor cells after their initial differentiation. Phenotypic expression in the mutant and in normal mice was studied by light and electron microscopy. The sequential emergence of developmental deviations in the mutant retina falls into three categories. First, predegenerative differences are manifest within the photoreceptor cells during 4–8 days after birth in retarded growth of the inner segments, reduced outer segment production, delayed development of the outer plexiform layer and slower segregation of the perikarya. Next, degenerative changes are recognized from 6 day onwards with swelling and vacuolization of the Golgi cisternae in the inner segments followed by cytolytic alterations affecting the ultrastructure of the entire cell. Lastly, with increasing loss of photoreceptor cells post-degenerative effects are seen in deepening of the basal infoldings and microvilli of the pigment epithelium and increase of Müllers fibres. The progress of degeneration in the mutant retina corresponds to the phase of rapid growth of the Golgi apparatus and rod outer segments in the normal retina. The role of the Golgi apparatus in the differentiation of the photoreceptor cells and its relation to the expression of the rd gene are discussed.

Histotypic differentiation of neonatal mouse retina in organ culture

Retinae from neonatal mice were explanted in toto, with or without the retinal pigment epithelium (RPE) and adjoining mesenchymal cells, and maintained in organ culture for up to 3 weeks. The explants remained flat, rosette formation was minimal and histogenetic changes followed in the normal sequence. After 11, 14 and 21 days in vitro the three cellular layers--the outer nuclear layer including well differentiated rod and cone perikarya, the inner nuclear layer and the ganglion cell layer--with the intervening plexiform layers were comparable to those of the in vivo eyes. Electron microscopic analysis revealed that in the explants without RPE the nuclear layers developed as in vivo, but receptor outer segments (ROS) were not formed. When the RPE was present, receptor inner segments appeared normal and ROS including profuse disc structures were developed. Presence of synaptic elements was also recognized. Mesenchymal cells, when present differentiated into choroidal and scleral tissues and appeared to play a supportive role for the RPE cells. The system is described in detail and its suitability for the analysis of various cellular and metabolic factors in the development of the retina is discussed.

Development and degeneration of retina inrds mutant mice: Effects of light on the rate of degeneration in albino and pigmented homozygous and heterozygous mutant and normal mice

The effect of light on the rate of visual cell loss in mice afflicted by the rds (retinal degeneration slow) gene was analyzed by comparing the changes in the thickness of the outer nuclear layer. Visual cell loss in the pigmented, homozygous mutant mice, maintained in cyclic light, is slightly slower than in the albino mutant mice. In the pigmented mutant mice, exposed to constant light, and in the albino mutant mice, reared in darkness, rate of cell loss is not significantly altered. In the albino animals exposed to constant light, the rate of cell loss is faster in the homozygous mutant than in the normal, and intermediate in the heterozygous mutant retina. The accelerated cell loss in the mutant retina progresses from the centre to the periphery, and affects the rods earlier than the cones. This resembles the photic lesion in the normal retina but is unlike the genetic lesion in the mutant retina which appears to progress from the periphery to the centre and affects both rods and cones. It is concluded that the visual cells in the retina of rds mutant mice are more vulnerable to photic damage than those in the retina of normal mice.

Development and degeneration of retina in rds mutant mice: ultraimmunohistochemical localization of opsin.

In normal retina the developing photoreceptor cells first show presence of opsin over the distal ends of the ciliary protrusions. In a fully differentiated cell intense activity is seen over the rod outer-segment discs; some activity is also seen over the Golgi zone and near the distal ends of the inner segments but the other parts of the receptor cell appear negative. In the pigment epithelium opsin is seen only over phagosomes containing rod outer segment debris. In the homozygous rds mutant retina, developing receptor cells show opsin activity over the ciliary protrusions as in the normal. These ciliary protrusions grow in size and show increased opsin activity and presumably constitute the site of phototransduction in the mutant retina. Although typical disc structures remain lacking, variable amounts of immunopositive, irregular, membranous structures are occasionally observed. The inner segments in the mutant cells show very little immunoreactivity but the perikarya and the spherule terminals show increased immunoreactivity in comparison with the normal. At the onset of degeneration, some of the receptor cells in the mutant retina show extrusion of small, membrane-bound vesicles which are immunopositive for opsin. Some receptor cells undergoing lysis disintegrate and also add to the opsin-positive vesicular structures in the interphotoreceptor space. The vesicles are phagocytized by pigment epithelial cells. In older mutant mice at an advanced stage of degeneration, the receptor cells show reduced opsin activity. In heterozygous mutant mice the outer segments are reduced in length and the discs are abnormal in form. However, the intensity and the pattern of opsin localization in the outer segments and at other sites are similar to normal.

Development and degeneration of retina in rds mutant mice: The electroretinogram

In mice, homozygous for the retinal degeneration slow (rds) gene, the photoreceptor cells lack outer segment disc structures, contain low amounts of rhodopsin, and degenerate slowly, while the inner retinal layers remain intact. The electroretinogram (ERG) from 1-month-old mutant mice shows a lower than normal amplitude but the time-to-peak is normal. The ERGs from mutant mice of 2-3 months and 6-7 months of age show a further decline in response amplitude as the receptor cell population is depleted with progress of degeneration while the time-to-peak increases. The ERG is absent in 1-year-old mutant mice which have lost their receptor cells completely. The possible morphological correlations of the ERG and its components in the rds mutant mice are discussed.

A developmental study of interphotoreceptor retinoid-binding protein (IRBP) in single and double homozygous rd and rds mutant mouse retinae

Interphotoreceptor retinoid-binding protein (IRBP) was studied using immunochemical and immunocytochemical techniques in retinae of mice with allelic combinations at the rd and rds loci at different stages of development and degeneration. Until postnatal day 7 (P7), IRBP is located intracellularly in developing retinae of the different genotypes. Thereafter, IRBP is present mainly in the interphotoreceptor matrix. As previously noted, cell death is slowest in the heterozygous +/+,rds/+ mutant with loss increasing in order in +/+,rds/rds, rd/rd, rds/rds and rd/rd,+/+ animals. The IRBP content of the total retina also approximates this pattern, with lowest amounts by far in rd/rd, rds/rds and rd/rd,+/+ mutants (after P14). Interestingly though, IRBP loss significantly precedes visual cell loss in the rd/rd,rds/rds retina. In all the mutants, the remaining rod cells in the outer nuclear layer exhibit synthesis of intracellularly located IRBP at late stages of degeneration. In the single homozygous rd/rd,+/+ and the double homozygous rd/rd,rds/rds mutants, IRBP is present intracellularly during the entire degenerative process with somewhat less intracellular IRBP in the rd/rd,rds/rds mutant. Retinae of homozygous +/+,rds/rds and heterozygous +/+,rds/+ animals exhibit a normal distribution pattern of IRBP immunoreactivity until loss of photoreceptor cells becomes pronounced at later stages of the disease. Many of the remaining cells at this time are probably cone elements although they are structurally changed. Double labeling with IRBP and S-antigen demonstrates, in many but not all, the presence of both proteins in the same cell body. Immunocytochemistry clearly demonstrated the presence of IRBP in remaining photoreceptor cells at late stages of the disease. Thus, the biochemically measured loss of IRBP appears to be a complex process neither directly dependent on the loss of photoreceptor outer segments and reduced interphotoreceptor matrix space (e.g. there is a sustained IRBP level in rodless rds mutants) nor simply due to cell death (e.g. in the rd/rd,rds/rds mutant, IRBP loss significantly precedes cell loss). That this IRBP is mainly intracellular, however, may indicate an abnormality in secretion which, combined with other factors, induces a degenerated and less differentiated phenotype.

Development and degeneration of retina in rds mutant mice: Light and electron microscopic observations in experimental chimaeras

Mice, homozygous for the rds gene, fail to develop the receptor outer segments and show a slow reduction of the outer nuclear layer. A series of 13 chimaeric mice was produced by combining morulae from albino rds/rds and pigmented normal (+/+) mice. At 3-4 weeks, variable stretches of visual cells without outer segments were observed together with stretches of visual cells with normal outer segments. The location of these areas was unrelated to the genotype of the overlying pigment epithelium. Phagosomes containing outer segment debris were present in albino pigment epithelial cells, located over normal outer segments, indicating normal functional properties of rds/rds pigment epithelial cells. At 9 months, regions with visual cell loss were observed underlying both types of pigment epithelial cells. Regions showing normal and intermediate thicknesses of the outer nuclear layer were seen more often than regions showing rds/rds type distribution. In another series of eight chimaeras, consisting of albino rds/rds and pigmented rd/rd genotypes, the eyes examined at 22 days showed more pronounced visual cell loss than in the rds----normal retinas at 9 months. Regions of the outer nuclear layer, containing a single row of cone perikarya, were similar to the rd/rd phenotype and differed from the phenotype of the double homozygous rd/rd rds/rds retina, which has a slower rate of degeneration than in rd/rd mice. Visual cell loss in these chimaeras at 9 months was similar to that in the rds/rds retina of the same age. The findings show that the expression of the rds gene, resulting in failure of outer segment development and eventual death of visual cells is unrelated to the genotype of the overlying pigment epithelial cells and suggest that the gene acts within the neural retina and possibly intracellularly in the visual cells.

Immunoassay of rod visual pigment (opsin) in the eyes of rds mutant mice lacking receptor outer segments

In 020/A mice, homozygous for the retinal degeneration slow (rds) gene, the photoreceptor cells fail to develop outer segments, and in the absorption spectra of retinal extracts the rhodopsin peak is lacking. Application of an enzyme-linked immunoassay using antisera against bovine opsin shows, however, that opsin is present in the homozygous mutant retina (0.010 nmol/eye) at 3% of the level of the normal retina (0.38 nmol/eye) of Balb/c mice. In the retina of heterozygous mice the opsin level (0.19 nmol/eye) is about half of the normal. Detection of opsin in the rds mutant retina demonstrates the functional basis for the reported electroretinographic response and light-mediated reduction in cyclic nucleotide levels in this mutant.