Retinal Epithelium Research Articles

Zebrafish aussicht mutant embryos exhibit widespread overexpression of ace (fgf8) and coincident defects in CNS development.

During the development of the zebrafish nervous system both noi, a zebrafish pax2 homolog, and ace, a zebrafish fgf8 homolog, are required for development of the midbrain and cerebellum. Here we describe a dominant mutation, aussicht (aus), in which the expression of noi and ace is upregulated. In aus mutant embryos, ace is upregulated at many sites in the embryo, while noi expression is only upregulated in regions of the forebrain and midbrain which also express ace. Subsequent to the alterations in noi and ace expression, aus mutants exhibit defects in the differentiation of the forebrain, midbrain and eyes. Within the forebrain, the formation of the anterior and postoptic commissures is delayed and the expression of markers within the pretectal area is reduced. Within the midbrain, En and wnt1 expression is expanded. In heterozygous aus embryos, there is ectopic outgrowth of neural retina in the temporal half of the eyes, whereas in putative homozygous aus embryos, the ventral retina is reduced and the pigmented retinal epithelium is expanded towards the midline. The observation that aus mutant embryos exhibit widespread upregulation of ace raised the possibility that aus might represent an allele of the ace gene itself. However, by crossing carriers for both aus and ace, we were able to generate homozygous ace mutant embryos that also exhibited the aus phenotype. This indicated that aus is not tightly linked to ace and is unlikely to be a mutation directly affecting the ace locus. However, increased Ace activity may underly many aspects of the aus phenotype and we show that the upregulation of noi in the forebrain of aus mutants is partially dependent upon functional Ace activity. Conversely, increased ace expression in the forebrain of aus mutants is not dependent upon functional Noi activity. We conclude that aus represents a mutation involving a locus normally required for the regulation of ace expression during embryogenesis.

A new allelic series for the underwhite gene on mouse chromosome 15.

A new allelic series at the underwhite gene is described. Three of the alleles in the series--uw, uwd, and Uwdbr--arose as spontaneous mutations on different genetic backgrounds at The Jackson Laboratory. We report here the visible phenotypes and dominance hierarchy of these alleles, all of which are defined by a reduction of pigmentation in both eye and coat color. Electron microscopic analysis of retinal epithelium suggests that the primary defect is in the melanosome. The degree of severity of melanosome anomalies in the retina correlates with the degree of hypopigmentation in the coat. The perturbed gene and its gene product are unknown. We show that the uw locus is genetically distinct from Myo10, a suggested candidate gene for this mutation.

Independent regulation of anterior/posterior and equatorial/polar polarity in the Drosophila eye; evidence for the involvement of Wnt signaling in the equatorial/polar axis.

The Drosophila retina is made from hundreds of asymmetric subunit ommatidia arranged in a crystalline-like array with each unit shaped and oriented in a precise way. One explanation for the precise cellular arrangements and orientations of the ommatidia is that they respond to two axes of polarized information present in the plane of the retinal epithelium. Earlier work showed that one of these axes lies in the anterior/posterior(A/P) direction and that the polarizing influence is closely associated with the sweep of the Hedgehog-dependent morphogenetic wave. Here we present evidence for a second and orthogonal axis of polarity, and show that it can be functionally separated from the A/P axis. Further, we show that the polarizing information acting in this equatorial/polar axis (Eq/Pl) is established in at least two steps - the activity of one signaling molecule functions to establish the graded activity of a second signal.

Inflammation Measurement and Immunocharacterization of Cell Proliferation in an Experimental Model of Proliferative Vitreoretinopathy

An experimental model of proliferative vitreoretinopathy was developed in the rabbit eye by injecting a solution of human platelet-rich plasma. In this model we evaluated the progression with time of intraocular inflammation and the rate and origin of cell proliferation. A sterile solution adjusted to 10⁷ platelets was injected into the right eye of a total of 46 pigmented and 14 albino rabbits. Animals were sequentially sacrificed at days 7, 14, 21 and 1 month after injection. Clinical evaluation of vitreoretinal proliferation, using a classification in six grades, and of anterior segment inflammation assessed by a Laser Flare Meter, were done for 1 month after injection, before histopathological analysis. Eighty percent of eyes developed tractional retinal detachment in 1 month. Histopathology showed intense cell migration and proliferation in the area of the ciliary body, as early as the seventh day, then further increasing rapidly. Infiltrates were composed of cytokeratin- and vimentin-expressing cells. Abnormal expression of vimentin was also found in ciliary and retinal epithelia and in Müller cells. Inflammation measured by the Laser Flare Meter was maximal at day 11 and then reached a plateau at significantly higher levels than controls. Albino rabbits showed significantly lower grades of proliferation, as compared to pigmented rabbits. This study thus clarified some characteristics of exper- imental vitreoretinal proliferations that proved similar to those in human diseases, such as the involvement of ciliary body and retinal pigment epithelium, the existence of inflammatory reactions preceding cell proliferation and strong changes in intermediate filaments. This may provide a simple and valuable model for antiproliferative assays and shed some light on the pathogenesis of intraocular proliferative disorders.

Linking Frizzled and Wnt signaling in Drosophila development.

Drosophila Frizzled-2 (Dfz2) has been identified as a putative fly Wingless (Wg) receptor. Although Dfz2 shows significant homology with Fz, a protein that operates in the mechanisms that establish planar polarity in Drosophila epithelia, any clear evidence for an involvement by Fz in a Wnt signaling pathway has hitherto been absent. Here we describe the planar polarity phenotypes of loss-of-function and overexpression of Fz in the developing Drosophila eye and find it almost identical to the loss-of-function or overexpression of Dishevelled (Dsh - a protein operating in Wnt second messenger systems). In addition, we show that overexpression of Shaggy (Sgg - another component of the Wnt pathway) in the eye also causes a phenotype similar to Fz and Dsh. To test further the link between planar polarity and Wnt signaling we misexpressed Wg in the developing eye and found it had a potent polarizing effect in the retinal epithelium. Since the overexpression of Fz in the developing eye gave a phenotype consistent with activating the Wnt pathway, we tested overexpression of Fz in the developing embryonic ectoderm and found that it phenocopied overexpression of Wg. To check that Fz was indeed able to activate a Wnt pathway we overexpressed it in Drosophila tissue culture cells and observed the characteristic phosphorylation of Dsh that occurs in response to Wnt signaling. Taken together our results significantly strengthen the case for Fz acting in a Wnt signaling pathway in Drosophila.

Genetic Testing in Hereditary Colorectal Cancer

<h3>CASE PRESENTATION</h3> A 29-year-old woman presented to her family physician with a 2-month history of 30 to 44 mL (2-3 tbsp) of bright red blood per rectum with every bowel movement. She was referred to a surgeon, who performed anoscopy and gave her a diagnosis of hemorrhoidal bleeding. She underwent hemorrhoidal surgery of unknown type. The patient again sought medical attention 3 months later, complaining of persistent and unchanged rectal bleeding. She had no change in bowel habits, weight loss, or family history of colorectal cancer or polyps. Examination of her skin did not show osteomas, lipomas, or epidermoid cysts. Direct ophthalmoscopic examination revealed no pigmentation of the retinal epithelium. Abdominal examination was normal. A stool sample taken during rectal examination tested guaiac positive. The results of an upper gastrointestinal tract barium series were normal, but air contrast barium enema showed multiple filling defects throughout the colorectum. Subsequent colonoscopy revealed

Pigmentary retinopathy associated with the mitochondrial DNA 3243 point mutation

Fourteen patients from four unrelated families were studied to determine the prevalence of retinal pigmentary abnormalities associated with the MELAS A to G 3243 point mutation. Neurologic and ophthalmic examinations, retinal photography, pattern shift visual evoked potentials, and electroretinography were performed in all patients. Eight of the 14 patients had retinal pigmentary abnormalities characterized by symmetric areas of depigmentation involving predominantly the posterior pole and midperipheral retina. None of the patients had optic atrophy and only one patient with pigmentary retinal abnormalities had impaired visual acuity. None of the diabetic subjects (n = 6) had signs of diabetic retinopathy. Fluorescein angiography demonstrated mottled hyper- and hypofluorescent areas indicating multiple window defects in the retinal pigmentary epithelium. Visual evoked potentials showed delayed P100 responses in four of the eight patients with retinal pigmentary abnormalities. We conclude that there is a high prevalence of retinal pigmentary abnormalities in patients with MELAS A to G 3243 point mutation. These abnormalities are usually asymptomatic and best detected by retinal photography.

Ergothioneine Distribution in Bovine and Porcine Ocular Tissues

Ergothioneine (ERT), is a low molecular weight, sulfur-containing antioxidant occurring in up to millimolar amounts in mammalian tissues. Using an improved HPLC assay, ERT levels have been measured and compared in bovine and porcine eyes and erythrocytes. The rank order of ERT levels in bovine ocular tissue was lens > retina = cornea > pigmented retinal epithelium (RPE) > aqueous humor (AQ) > vitreous humor (VIT) > sclera. In porcine ocular tissue, the rank order was retina > AQ > VIT > RPE > cornea > lens > sclera. ERT levels in bovine lens were about 250 × > that in porcine lens. Porcine erythrocyte levels were 5.5 × > bovine levels. Species differences were also observed in the retina, VIT and AQ where porcine levels were 2 to 10-fold greater than bovine levels. ERT in bovine lens and cornea was 35 and 14 times greater than the corresponding level of reduced glutathione (GSH). Porcine lens had 45 times more GSH than ERT. Values for ERT and GSH in other tissues from both species were of the same order of magnitude. These results are consistent with a role for ERT in prevention of oxidative damage to the eye.

Early embryonic birth of gemmal cells in the prehatching chick

This autoradiographic study in the chick embryo tested whether one possible source of cells comprising early-developing taste bud primordia arose from gemmal cell precursors, or their progeny, born during the first week of incubation. Following single in ovo injections of [ 3 H]thymidine on embryonic day (E) 2, E4 or E6, labeled gemmal cells were identified in developing, anterior lower beak taste buds on E19, which is 2 days after the first sign of bud primordium formation and 2 days before hatching. The greatest number of labeled cells/bud occurred after the E2 injection. Thus the embryonic gustatory peripheral receptor, in common with developing auditory, vestibular and retinal sensory receptor epithelia, contains at least postmitotic and/or quiescent cells whose precursors proliferated during the first week of gestation. These early pre-bud precursors could serve as tropic targets for local epithelial cells in the formation of bud primordia as well as guiding growing distal processes of the gustatory sensory ganglia whose neurons are already postmitotic by E5.

Genesis of the adult retina and outer optic lobes of the moth, Manduca sexta. I. patterns of proliferation and cell death.

We have examined the development of the adult retina and the outer optic lobes in the moth Manduca sexta. The adult retina is generated from a group of epithelial cells lying within the larval head capsule between the larval ocelli and antenna. Proliferation of these cells begins during the feeding larval stage but accelerates at the end of the final larval instar. Proliferation occurs in two zones of mitotic activity; these zones flank a furrow in the presumptive retinal epithelium. The furrow and flanking mitotic zones migrate from posterior to anterior across the presumptive retinal epithelium. Posterior to the furrow, presumptive retinal cells from clusters and extend axons into the larval optic nerve. We have also examined the temporal patterns of neuronal proliferation and cell death during genesis of the adult outer optic ganglia, the medulla and the lamina. The medulla and the lamina are generated by distinct populations of neuroblasts in the outer optic analage; the neuroblasts divide asymmetrically to generate ganglion mother cells. Ganglion mother cells later divide symmetrically to generate immature neurons. Generation of the medulla cortex starts with the onset of the final larval instar, and cell death within the medulla cortex begins after the end of the final larval instar. Generation of the lamina cortex is initiated with the arrival of retinal afferents at the optic lobes, and cell death in the lamina cortex begins 1 day later. Generation of the outer optic ganglia terminates with the abrupt cessation of mitotic activity followed by degeneration of the outer optic anlage.

Role of the morphogenetic furrow in establishing polarity in the Drosophila eye.

The Drosophila retina is a crystalline array of 800 ommatidia whose organization and assembly suggest polarization of the retinal epithelium along anteroposterior and dorsoventral axes. The retina develops by a stepwise process following the posterior-to-anterior progression of the morphogenetic furrow across the eye disc. Ectopic expression of hedgehog or local removal of patched function generates ectopic furrows that can progress in any direction across the disc leaving in their wake differentiating fields of ectopic ommatidia. We have studied the effect of these ectopic furrows on the polarity of ommatidial assembly and rotation. We find that the anteroposterior asymmetry of ommatidial assembly parallels the progression of ectopic furrows, regardless of their direction. In addition, ommatidia developing behind ectopic furrows rotate coordinately, forming equators in various regions of the disc. Interestingly, the expression of a marker normally restricted to the equator is induced in ectopic ommatidial fields. Ectopic equators are stable as they persist to adulthood, where they can coexist with the normal equator. Our results suggest that ectopic furrows can impart polarity to the disc epithelium, regarding the direction of both assembly and rotation of ommatidia. We propose that these processes are polarized as a consequence of furrow propagation, while more global determinants of dorsoventral and anteroposterior polarity may act less directly by determining the site of furrow initiation.

The retinal pigmented epithelium is required for development and maintenance of the mouse neural retina.

During development of the vertebrate eye, there is a series of reciprocal cellular interactions that determine the fate of the eye components. Although evidence from organ culture suggests that the retinal pigmented epithelium (RPE) organizes the laminar structure of the differentiated neural retina, no role has been identified for the RPE in early eye development, nor has the later function of RPE been demonstrated in vivo. To investigate the role of RPE cells in eye development, we generated transgenic mice that carry the attenuated diphtheria toxin-A gene; this transgene was driven by the promoter of the gene encoding the tyrosinase-related protein-1, which is specifically expressed in pigment cells. Depending on the expression level of the transgene, the retinal epithelium was ablated before or after its differentiation into a pigmented cell layer. We show that an early ablation (embryonic day E10-11) resulted in disorganization of the retinal layer, immediate arrest of eye growth and subsequent eye resorption. A later ablation (E11.5-12.5) allowed the eye to be maintained during embryogenesis, but the laminar structure of the retina became disrupted by the end of gestation, the vitreous failed to accumulate the adults were anophthalmic or severely microphthalmic. In some microphthalmic eyes, a number of RPE cells escaped ablation and formed patches of pigmented cells; the laminar structure of the retina was maintained immediately adjacent to such pigmented areas but disrupted elsewhere. In both cases--early or late ablation of the RPE--the retina appears to be the primary affected tissue. We conclude that presence of the RPE is required for the normal development of the eye in vivo. Its presence early in development is necessary for the correct morphogenesis of the neural retina. After the neural retina has started to differentiate, the RPE is still necessary, either directly or indirectly, to maintain the organization of the retinal lamina.

Mutation of PAX2 in two siblings with renal-coloboma syndrome

PAX2 is a member of a multigene family containing a paired box domain that was initially identified in Drosophila and subsequently in vertebrates (1). PAX genes play a critical role in embryogenesis, as demonstrated by mouse mutants and fetal expression patterns (2). Mutations causing autosomal dominant syndromes in humans have been documented in three of the nine PAX genes. Mutations in PAX3 cause Waardenburg syndrome types I and III (3,4); mutations in PAX6 cause aniridia (5-7) and recently we have shown that PAX2 is mutated in a family with autosomal dominant optic nerve coloboma, renal anomalies and vesicoureteric reflux (8). The disease phenotype associated with PAX2 mutations in humans may show variability. Indeed, there have been no other reports of a syndrome in humans with exactly the same phenotype as the family in which the PAX2 mutation was found. The most closely related human syndrome is the renalcoloboma syndrome (MIM #120330) (also called the papillorenal syndrome), which involves optic nerve coloboma and renal anomalies, but not vesicoureteric reflux (9-13). Renal-coloboma syndrome is a newly recognized rare syndrome (9,13). This syndrome is characterized by autosomal dominant inheritance of optic nerve anomalies and thinning of the retinal epithelium, resulting in loss of visual acuity and defective visual fields. In some, or all, of the affected family members there may also be simultaneous, chronic renal failure, chronic glomerulonephritis and/or renal hypoplasia. To determine whether families with typical renal-coloboma syndrome have associated PAX2 mutations, we have analyzed the PAX2 gene in blood DNA from two brothers who have renal-coloboma syndrome (9), their unaffected mother and eight unaffected maternal cousins. The younger brother presented with severe progressive renal failure leading to renal transplantation and a bilateral visual field defect with optic nerve colobomas. The older brother presented with chronic mild renal failure, a visual field defect and optic nerve colobomas. The two brothers were the only affected family members. Both parents were previously found to be normal by ophthalmologic examination (9). PCR-SSCP analysis revealed a variant pattern for exon 2 in the two affected siblings, but not in the mother (Fig. 1A) or in the cousins (not shown). Segregation of the SSCP variant with the two brothers suggested that PAX2 exon 2 contained a mutation associated with this disease.

Integrins and the Development of Three-Dimensional Structure in the Drosophila Compound Eye

A stereotyped, three-dimensional network of cell-cell contacts mediated by adherens junctions and cell-extracellular matrix contacts mediated by focal adhesions defines the architecture of the Drosophila ommatidium. Developmental reconstruction shows that this network is built in an incremental and generally conservative sequence; contacts established early in eye development typically persist into adulthood. Reconstructions show that photoreceptor apical surfaces are involuted into the retinal epithelium and are subsequently elaborated to form the photosensitive rhabdomeres. Rhabdomeres become aligned to the ommatidial optical axis via their anchorage to the retinal floor at the cone cell plate, a specialized nexus of cell-cell and cell-extracellular matrix contacts. Parallel reconstructions of retinal development in integrin mutants show that several eye phenotypes trace their origin to the structural failure of the cone cell plate.

Epithelial planar polarity in the developing Drosophila eye

Experiments with the insect ectoderm have suggested that planar polarity in epithelia results from the local orientation of cells to the slope of a gradient of positional information. Here we show that planar polarity in the Drosophila eye is inverted when the morphogenetic wave that sweeps through the presumptive retinal epithelium is induced to move in the reverse direction. We suggest that the movement of the morphogenetic wave may be causal in establishing the planar polarity of this epithelium.

Exclusion of the cone-specific ?-subunit of the transducin gene in Stargardt's disease

Stargardt's disease is an autosomal recessive infantile macular degeneration of unknown origin whose gene has been recently mapped to chromosome 1p21-p13 by linkage analysis in eight multiplex families. Since the cone-specific alpha-subunit of the transducin gene (GNAT2) has been mapped to chromosome 1p13, we tested GNAT2 as the disease-causing gene in our series. Using a novel intragenic polymorphism, we show here that GNAT2 is most probably located centromeric to the genetic interval encompassing the disease gene (D1S424-D1S236, location score = 3.54). In addition, single-strand conformation polymorphism and sequence analyses of the eight exons of the GNAT2 gene was performed in our probands. No evidence of a deleterious base substitution was observed in any affected individual. Taken together, these results support the exclusion of GNAT2 as the causal disease gene of Stargardt's disease. come. The pathogenesis is unknown and the treatment is limited to ultraviolet ray protection with dark glasses. Stargardt's disease has been recently mapped to chromosome 1p21-p13 in the genetic interval defined by loci D1S424 and D1S236. In addition, our previous study has suggested that Stargardt's disease is genetically homogeneous (Kaplan et al. 1993). With respect to the physical mapping of the proteins specific to the retinal pigmentary epithelium or to cones we noted that the cone-specific alpha-subunit of the transducin gene (GNAT2) has been mapped to chromosome 1p13 (Wilkie et al. 1992). The human GNAT2 is 9967 bp in length and consists of eight exons with seven introns (Morris and Fong 1993). In the present study, we examined this gene as the candidate gene in eighteen unrelated patients affected with Stargardt's disease.

Distribution and elimination of clenbuterol in tissues and fluids of calves following prolonged oral administration at a growth‐promoting dose

A pharmacokinetic study is described in which Friesian calves (n = 30) were treated orally with clenbuterol at 10 times the therapeutic dose. The study was designed to establish the distribution and elimination of clenbuterol from edible tissues, the major compartments of the eye and body fluids. Animals (n = 24) were dosed (10 micrograms/kg body weight) twice daily with clenbuterol for 21 days and slaughtered in groups of five (one untreated control animal per group) at 6 h and 1, 2, 4, 8 and 16 days after cessation of treatment. At slaughter, samples of diaphragm muscle, liver, kidney, bile, urine and both eyes were obtained. One of the eyes was separated into constituent tissues: aqueous humour, vitreous humour, cornea, lens, retina (without pigmented epithelium), choroid (with pigmented retinal epithelium; choroid/PRE) and sclera. All samples were stored at -20 degrees C. Clenbuterol concentrations were higher in liver than kidney, bile and urine from day 2 of withdrawal onwards. Concentrations in choroid/PRE were at least 10 times higher than in liver at all periods following cessation of treatment and 52 times higher 16 days after treatment. The concentrations of clenbuterol in the constituent tissues of the eye were in the order choroid/PRE > cornea > > retina > aqueous humour/vitreous humour > or = lens. Concentrations of clenbuterol in choroid/PRE taken from eyes frozen whole were generally lower than those in choroid/PRE separated before storage. Choroid/PRE stored by either method contained clenbuterol at more than 100 ng/g 16 days following cessation of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinal pigment epithelial fine structure in the American crow (Corvus brachyrhynchos).

The fine structure of the retinal epithelial (RPE) region has been investigated by light and electron microscopy in the American crow (Corvus brachyrhynchos). In this species the RPE consists of a single layer of cuboidal cells which display numerous deep basal (scleral) infoldings and plentiful apical (vitreal) microvillar processes which surround photoreceptor inner and outer segments. The RPE cells are joined laterally by a series of tight junctions (Verhoeff's membrane) located in the mid to basal region. Within the epithelial cells, smooth endoplasmic reticulum is very abundant while rough endoplasmic reticulum (RER) is scarce. Mitochondria of various shapes are abundant basally while polysomes are plentiful and widespread. In the light-adapted state RPE nuclei are large and vesicular and basally located while the melanosomes of these cells are predominantly located with the apical processes indicating photomechanical movements. Myeloid bodies are large and numerous and often have ribosomes on their outer surface. Bruch's membrane (complexus basalis) is typical of avian species in that it is pentalaminate and the lamina densa is displaced near the choriocapillaris. The endothelium of the choriocapillaris is thin facing Bruch's membrane but is only moderately fenestrated. Some of these fenestrations display a double-layered diaphragm while the majority show the more typical single-layered diaphragm noted in most species.

Fine structure of the retinal pigment epithelium in the Port Jackson shark (Heterodontus phillipi)

The structure of the retinal epithelium (RPE), choriocapillaris and Bruch's membrane (complexus basalis) has been studied by light and electron microscopy in the Port Jackson shark (Heterodontus phillipi). In this elasmobranch the RPE consists of a single layer of low cuboidal cells which show basal (scleral) infolding and apical (vitreal) processes that enclose photoreceptor outer segments. Laterally these epithelial cells are joined by a series of apically located tight junctions. The RPE cells display a large vesicular nucleus, abundant smooth endoplasmic reticulum as well as numerous polysomes and mitochondria. Phagosomes are present, rough endoplasmic reticulum is scarce and myeloid bodies were not observed. Melanosomes are absent over the choroidally located tapetum lucidum, but are not abundant even in extratapetal areas. This paucity of melanosomes probably makes retinomotor movements unimportant. Bruch's membrane or complexus basalis is a pentalaminate structure. The endothelium of the choriocapillaris is thin but minimally fenestrated.

Patch culturing and transfer of human fetal retinal epithelium

Human retinal pigmented epithelium (RPE) can be cultured by removing small patches of this layer from the choroid of the fetal eye. Such RPE patches give rise to healthy, epithelioid monolayers in vitro within 1-2 weeks without contamination from retinal or choroidal cells. The viability and proliferative capacity of these cultures is independent of the initial polarity of the patch. These RPE monolayers develop apical/basal polarity and a basal lamina and rest on a field of collagen fibers; they are capable of phagocytizing outer segments. A patch can be lifted off a confluent monolayer and transferred to another culture dish without risking the viability of either the old or the new culture. This provides a means of transplanting an organized, polarized patch of human RPE from one place to another.