Retinal Epithelium Research Articles

1057. Successful Gene Therapy for Retinal Degenerations by Lentiviral Gene Transfer

Our goal is to perform gene therapy by efficient integration into safe positions in the host genome. The integrase enzyme from the phage phiC31 directs site-specific integration into a small subset of native genomic sequences 1. Recent gene therapy studies using phiC31 integrase in mice have demonstrated site-specific integration of transgenes and long-term gene expression in liver, skin, and muscle (e.g., 2). Here, we demonstrate use of the phiC31 integrase as a simple and effective method for non-viral long-term gene transfer in the eye. We developed a method to achieve efficient non-viral delivery of plasmid DNA to rat retinal epithelium (RPE) in vivo. The method involves subretinal injection of plasmid DNA, followed by in situ electroporation (adapted from 3). Delivery of GFP by this method was evaluated in frozen sections using fluorescence microscopy. In subsequent experiments, a luciferase plasmid, with or without a plasmid encoding the phiC31 integrase, was delivered to rat RPE. Luciferase expression was followed over time by using in vivo luciferase imaging. Subretinal DNA injection followed by electroporation yielded abundant transgene delivery in rat RPE, as assayed by both GFP and luciferase expression. Expression was strongest 48 hours after delivery. In the absence of integrase, luciferase expression declined to near-background levels within 3-4 weeks after treatment. Co-injection of the integrase plasmid led to long-term stable transgene expression at all time points tested, up to three months post-procedure. Animals receiving integrase showed ~64-fold higher long-term expression than control animals. We conclude that subretinal injection followed by electroporation affords abundant transfer of plasmid DNA in mammalian RPE. Addition of phiC31 integrase confers robust long-term transgene expression, presumably by providing site-specific integration of the gene of interest into the genome of RPE cells. Having established non-viral permanent retinal gene transfer by site-specific genomic integration, we now plan to apply the technology to rodent models of retinal degeneration. Because site-specific integration provides strong, stable gene expression while reducing the probability of insertional mutagenesis, the phiC31 integrase system has great potential as a novel approach to non-viral gene therapy in the eye.

Neural stem cells in the mammalian eye: types and regulation.

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina. Recently, such cells have been reported to be present, in a mitotically quiescent state, in the ciliary epithelium of the adult mammalian eye. The retinal and ciliary epithelium stem cells/progenitors appear to share similar signaling pathways that are emerging as important regulators of stem cells in general. Yet, they are different in certain respects, such as in the potential to self-renew. These two neural stem cell/progenitor populations not only will serve as models for investigating stem cell biology but also will help explain the relationships between embryonic and adult neural stem cells/progenitors.

Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis.

Ezrin-Radixin-Moesin (ERM) family proteins organize heterogeneous sub-plasma membrane protein scaffolds that shape membranes and their physiology. In Drosophila oocytes and imaginal discs, epithelial organization, fundamental to development and physiology, is devastated by the loss of Moesin. Here, we show that Moesin is crucial for Drosophila photoreceptor morphogenesis. Beyond its requirement for retinal epithelium integrity, Moesin is essential for the proper assembly of the apical membrane skeleton that builds the photosensitive membrane, the rhabdomere. Moesin localizes to the rhabdomere base, a dynamic locus of cytoskeletal reorganization and membrane traffic. Downregulation of Moesin through RNAi or genetic loss of function profoundly disrupts the membrane cytoskeleton and apical membrane organization. We find normal levels and distribution of Moesin in photoreceptors of a Moesin mutant previously regarded as protein null, suggesting alternative interpretations for studies using this allele. Our results show an essential structural role for Moesin in photoreceptor morphology.

Familial adenomatous polyposis.

AbstractFamilial adenomatous polyposis (FAP) is an autosomal, dominantly inherited disorder that predisposes to the development of hundreds to thousands of adenomatous polyps throughout the colon and rectum (MIM 175100). Left untreated, it is almost 100% certain that at least one of these polyps will become malignant, usually by the time the patient is 40 yr of age (1). Affected patients may also develop a range of extracolonic manifestations, including benign lesions such as congenital hypertrophy of the retinal epithelium (CHRPE), jaw cysts, and sebaceous cysts as well as desmoid tumors and other malignancies including periampullary tumors, thyroid cancers, adrenocortical tumors, brain tumors, and hepatoblastomas (1). The colorectal features, in association with extra colonic manifestations, are also known as Gardner’s syndrome. A small proportion of patients have an attenuated form of the disease characterized by less than 100 polyps and a later age of onset, known as attenuated adenomatous polyposis coli (AAPC) (2). Hereditary desmoid disease is a relatively rare condition characterized by autosomal dominant inheritance of multiple desmoid tumors but with no colorectal polyposis (3). In general, the clinical features of the disorder allow a diagnosis of FAP to be made relatively easily. However, the attenuated form of the disease can show similarities to hereditary non-polyposis colon cancer (HNPCC), and mutation analysis of the causative genes may be required to aid in the differential diagnosis.KeywordsFamilial Adenomatous PolyposisAdenomatous Polyposis ColiPolymerase Chain Reaction ConditionMagnesium ChlorideAdenomatous Polyposis Coli GeneThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Use of Gain-of-Function Study to Delineate the Roles of crumbs in Drosophila Eye Development

The cell polarity gene, crumbs(crb), has been shown to participate in the development and degeneration of the Drosophila retina. Mutations in CRB1, the human homologue of Drosophilacrb, also result in retinitis pigmentosa and Leber congential amaurosis. In this study, we used the gain-of-function approach to delineate the roles of crb in developing Drosophila eye. In the third-instar larval stage, eye development is initiated with photoreceptor differentiation and positioning of photoreceptor nuclei in the apical cellular compartment of retinal epithelium. In the pupal stage, differentiated photoreceptors begin to form the photosensitive structures, the rhabdomeres, at their apical surface. Using GMR-Gal4 to drive overexpression of the Crb protein at the third-instar eye disc, we found that differentiation of photoreceptors was disrupted and the nuclei of differentiated photoreceptors failed to occupy the apical compartment. Using hs-Gal4 to drive Crb overexpression in pupal eyes resulted in interference with extension of the adherens junctions and construction of the rhabdomeres, and these defects were stage-dependent. This gain-of-function study has enabled us to delineate the roles of Crb at selective stages of eye development in Drosophila.

PDZ domain polarity complexes

What are they? Three protein complexes, each containing at least one PDZ protein–protein interaction domain, are critical regulators of cell polarity in animals. Each complex can be referred to by one of its primary components: the Par-3 or Baz complex, including Par-6 and aPKC; the Crb complex, including Sdt; and the Scrib complex, including Dlg. The Lgl protein, which has activities similar to the Scrib complex, seems to physically associate not with the Scrib but with the Baz complex. Are they conserved across species? Yes: orthologous complexes are found in worms, flies and vertebrates. Not only is the composition of the complexes conserved, but there is also remarkable conservation of their localization, particularly in epithelial cells. Nevertheless, the mutant phenotypes caused by defects in components of the complexes vary across species, suggesting that the story may be more complicated. How were they identified? Primarily through genetic screens in model organisms. Mutations of Crb, Sdt and Baz cause polarity defects in Drosophila embryonic epithelia, while those of Par-3 and Par-6 disrupt cytoplasmic partitioning in the early Caenorhabditis elegans zygote. A mammalian Par-3 homolog physically associates with aPKC, while Baz is the fly Par-3 homolog. Dlg and Lgl were first isolated as Drosophila tumor suppressors that cause overgrowth and disorganization of imaginal epithelia. Scrib and its worm ortholog Let-413 were identified in screens for epithelial shape defects. What cells are they active in? All three complexes are required for polarization of the Drosophila embryonic and ovarian epithelia, though some fly epithelia that require the Scrib complex, such as wing imaginal discs, develop well without either the Crb or Baz complex. Worm epithelia require the Scrib complex; requirements for the Baz and Crb complexes have not yet been reported. In zebrafish, mutations in aPKC and Sdt homologs cause disorganization of certain endodermal, mesodermal and retinal epithelia. Outside of epithelia, the Baz and Scrib complexes are both involved in polarization of Drosophila neuroblasts and, later, in organization of the neuromuscular junction. Finally, the Baz complex is required for polarization of both the one-cell worm zygote and the Drosophila oocyte. Where are they found in the cell? In epithelial tissues, the Crb complex is found along the apical-most region of the lateral plasma membrane, corresponding to the tight junction in vertebrates or the marginal zone in Drosophila. The Baz complex is found at either the tight junction or the zonula adherens, while the Scrib complex is found along the basolateral domain. In the fly neuroblast and worm zygote, the Baz complex is polarized along one side of the cell (apical or posterior, respectively). The Scrib complex is localized throughout the cortex of fly neuroblasts. In mature neurons, Scrib and Baz are restricted to synapses. Lgl is broadly associated throughout the cell membrane in most cell types. Do the complexes interact? The three complexes can coordinate polarizing activities through positive and negative cross-regulation, as shown in the fly embryo. This regulation may be mediated by physical interactions amongst members of different complexes, such as that between the mammalian Par-6 and Sdt orthologs. A particularly intriguing case is Lgl, which physically interacts with the Baz complex but whose subcellular localization is controlled by the Scrib complex. How do they function in polarity? The mechanism by which the complexes mediate polarity at the cell biological level is not understood. The PDZ proteins act as scaffolds, without clear effector activities. It is suspected that polarizing activity is mediated by effector partners, such as the kinase aPKC and its substrate Lgl, which may be involved in vesicle transport or cytoskeletal regulation. Other effector partners, particularly those that interface with basic cell biological machinery, remain to be identified. Might they be involved in human disease? Mutations in a human Crb gene, CRB1, cause retinal degeneration in one class of retinitis pigmentosa patients. Indirect evidence hints at a role of the complexes in oncogenesis: Par-6 can potentiate transformation, while Scrib complex proteins are targets of oncovirus-mediated protein degradation. A third possibility is raised by the circletail mutant mouse, which carries a mutation in Scrib and displays severe neural tube defects. Further implication of the PDZ polarity complexes in developmental or pathological defects awaits reverse genetic approaches in vertebrate organisms. Where can I find out more?

Ultrastructure of adult rd mouse retina.

To examine the ultrastructure of the adult rd mouse retina in order to determine what structures are altered or lost and thus to better interpret changes produced by photoreceptor and/or retinal transplantation in this model of retinal degeneration. rd mutant mice expressing a LacZ reporter gene in rod bipolars were used in order to identify these cells and their processes. Mice of age 6 weeks to 5 months were studied by electron microscopy, concentrating on the posterior pole where retinal transplants are usually placed. The adult rd mouse retina contains degenerating cones, cone outer segments, cone synaptic pedicles with synaptic vesicles and post-synaptic contacts. The major abnormalities occur in the subretinal space where all traces of rods are gone and the main structures are inner segments of cones. These inner segments are smaller than normal, contain fewer and smaller mitochondria, have organized arrays of microtubules, resembling those in cone axonal processes, and are completely engulfed by massive proliferation of apical processes of the retinal epithelium. The subretinal space is well defined by the external limiting membrane vitreally and the retinal epithelium choroidally. Muller cells extend globular rather than filamentous processes into the subretinal space which contact the apical processes of the epithelium. Rod bipolar cells survive and retain processes in the external plexiform layer. The adult rd mouse retains structural elements necessary for phototransduction and transmission of signals to the inner layers of the retina by the cone system. The major deficits are located in the subretinal space where all rods are lost and cone inner segments undergo a slow degeneration. Rod bipolar cells survive but appear to be de-afferented; there was no evidence that they contact residual cone processes in the external plexiform layer. The rd mouse is a logical model to study the effects of transplantation of photoreceptors because second- and third-order retinal neurons as well as degenerating cones survive in the adult retina.

Expression of Tfap2d, the gene encoding the transcription factor Ap-2δ, during mouse embryogenesis

Tfap2d encodes a novel member of the AP-2 family of transcription factors, called Ap-2δ. Ap-2δ has a unique DNA sequence binding specificity and lacks a PY motif as well as several highly conserved residues in the transactivation domain that are essential for transcriptional activation for other AP-2 proteins. In this study, we characterized the expression pattern of Tfap2d in mouse embryos from E7.5 to E16.5 using in situ hybridization as a first step towards understanding the role of Ap-2δ during development. Overall, Tfap2d is expressed with a more restricted temporal and spatial pattern than the other Tfap2 genes, with signals observed in the central nervous system, retina, and, uniquely, in the developing heart. Signals were not detected in tissues such as the neural crest, facial mesenchyme, and limbs where the other Tfap2 genes are expressed. The expression of Tfap2d was first noted at E9.5, later than other Tfap2 genes, with signals from the heart and brain. The expression in the developing myocardium persisted through E10.5. Expression of Tfap2d was diffuse in the developing brain at E10.5 to E11.0, but became restricted to the roof of the third vesicle with two narrow columns extending forward along the wall of the third vesicle and the roof of the heopallial cortex in the forebrain from E12.5 onwards. Tfap2d expression was also observed in the spinal cord at E10.5. From E13.5 to E16.5, Tfap2d was expressed in the retinal epithelium. Based on Tfap2d's unique expression pattern and functional features, we believe that Ap-2δ plays a role during mammalian development that is non-overlapping with those of the other Ap-2 transcription factors.

Protein expression, genomic structure, and polymorphisms of oculomedin

Purpose: To elucidate protein expression, genomic structure, and genomic polymorphisms of a novel gene, ‘oculomedin’, that has been cloned as a mechanical stretch-response gene from human trabecular cells in culture. Methods: Polyclonal antibody was prepared by immunizing rabbits with a chemically synthesized 15-mer peptide of oculomedin. Protein expression was revealed by Western blot analysis after polyacrylamide gel electrophoresis of extracts of mechanically stretched trabecular cells and control trabecular cells in culture as well as retinal tissue. Protein localization was studied immunohistochemically in the human eye section. Genomic structure was determined by searching the GenBank database. Genomic polymorphisms of the coding region in 163 glaucoma patients and 50 normal subjects were detected by PCR amplification and direct sequencing. Results: Western blot analysis showed that oculomedin protein was expressed only in stretched trabecular cells, not in control trabecular cells in culture. Immunohistochemically, oculomedin protein was localized to the trabecular meshwork, Schlemm’s canal endothelium, retinal photoreceptor cells, and corneal and conjunctival epithelium. The oculomedin gene (OCLM) consists of two exons which are located inside an intron of a different gene of unknown function (C1orf27) on chromosome 1q25, near and telomeric to myocilin (MYOC). Two types of heterozygous nucleotide substitutions resulting in amino acid changes were found in two of 75 patients with primary open-angle glaucoma, but not at all in patients with other types of glaucoma or in normal subjects. Conclusions: Oculomedin may play a role in the function of the trabecular meshwork and also in the development of primary open-angle glaucoma.

Liquid chromatography/electrospray tandem mass spectrometric analysis of incurred ractopamine residues in livestock tissues.

Ractopamine HCl is a beta-adrenergic agonist (beta-agonist) recently approved by the U.S. Food and Drug Administration, but not other governmental agencies, for use in finishing swine. For these reasons, it was important to develop and validate mass spectrometric methods for the detection and confirmation of ractopamine residues in livestock marker tissues. Incurred tissues in cattle, sheep, turkeys, and ducks were generated during 7-day ractopamine feeding (20 ppm in diets) periods. Disposition of ractopamine residues in liver and pigmented retinal epithelium was determined in animals slaughtered with withdrawal periods of 0, 3, and 7 days. Ractopamine residues, purified using solid-phase extraction, were measured using liquid chromatography (LC) and electrospray with detection by tandem mass spectrometry (MS/MS) in the multiple reaction-monitoring (MRM) mode. Total ractopamine residues (parent ractopamine + hydrolyzed conjugates) in liver were detected in all species on withdrawal day 0 (2-97 ppb) and were greatly diminished in all species by withdrawal day 7 (<1 ppb). Bovine and ovine retina had lower levels of ractopamine (0.5-3 ppb) than liver, and occular residues increased with withdrawal time, suggesting redistribution into this tissue. Lower limits of quantification were found to be approximately 0.1 ppb in liver and retina. Incurred ractopamine residues were confirmed by the precise and accurate agreement of MRM intensity ratios of diagnostic fragment ions (m/z 284, 164, and 121) from the protonated molecule between ractopamine residues in incurred samples and an authentic ractopamine standard. The limits of confirmation in liver and retina using recognized acceptance criteria were below 1 ppb. The high sensitivity and specificity for measurement and confirmation of ractopamine residues suggests this method will be applicable for regulatory residue surveillance programs.

Control of photoreceptor cell morphology, planar polarity and epithelial integrity during Drosophila eye development.

We report that the hindsight (hnt) gene, which encodes a nuclear zinc-finger protein, regulates cell morphology, cell fate specification, planar cell polarity and epithelial integrity during Drosophila retinal development. In the third instar larval eye imaginal disc, HNT protein expression begins in the morphogenetic furrow and is refined to cells in the developing photoreceptor cell clusters just before their determination as neurons. In hnt mutant larval eye tissue, furrow markers persist abnormally posterior to the furrow, there is a delay in specification of preclusters as cells exit the furrow, there are morphological defects in the preclusters and recruitment of cells into specific R cell fates often does not occur. Additionally, genetically mosaic ommatidia with one or more hnt mutant outer photoreceptor cells, have planar polarity defects that include achirality, reversed chirality and misrotation. Mutants in the JNK pathway act as dominant suppressors of the hnt planar polarity phenotype, suggesting that HNT functions to downregulate JUN kinase (JNK) signaling during the establishment of ommatidial planar polarity. HNT expression continues in the photoreceptor cells of the pupal retina. When an ommatidium contains four or more hnt mutant photoreceptor cells, both genetically mutant and genetically wild-type photoreceptor cells fall out of the retinal epithelium, indicating a role for HNT in maintenance of epithelial integrity. In the late pupal stages, HNT regulates the morphogenesis of rhabdomeres within individual photoreceptor cells and the separation of the rhabdomeres of adjacent photoreceptor cells. Apical F-actin is depleted in hnt mutant photoreceptor cells before the observed defects in cellular morphogenesis and epithelial integrity. The analyses presented here, together with our previous studies in the embryonic amnioserosa and tracheal system, show that HNT has a general role in regulation of the F-actin-based cytoskeleton, JNK signaling, cell morphology and epithelial integrity during development.

Lentiviral transduction of green fluorescent protein in retinal epithelium: evidence of rejection

This paper demonstrates lentiviral transduction of the humanized form of the Aequoria victoria gene for green fluorescent protein (GFP) into human fetal retinal pigment epithelium (RPE) in vitro and rabbit RPE in vivo. In vitro GFP expression of cultured human fetal RPE begins within two to three days after 12–16 h of maintained exposure to the virus at titers of 10 8–10 9 infectious units (IU)/ml. Both stationary and dividing cells are transduced using a lenti viral vector with a cytomegalovirus (CMV) promoter. Expression remains stable for at least three to four months without evidence of toxicity and continues through cell division. In vivo expression is followed non-invasively in rabbit eye using a scanning laser ophthalmoscope (SLO), which can detect single fluorescing retinal cells. In vivo expression begins within a few days after a viral solution is introduced into the subretinal space. A solution of 10 9 IU/ml produces fluorescence within three to four days. Less concentrated solutions lead to slower and less expression. No expression is detectable at concentrations of 10 6 IU/ml. Within one to two weeks after introduction of the viral solution, there is evidence of rejection seen by SLO as a loss of GFP fluorescence and disruption of the RPE. Histology shows damage to the RPE layer and monocytic cell infiltrates in the choroid and subretinal space within the area receiving the viral solution. Strong GFP expression leads to rejection within two weeks. With less expression, rejection is delayed and in some cases undetectable for at least six months. If the GFP gene is not included in the viral vector or if the viral concentration is insufficient to produce detectable GFP expression, rejection is not seen. Using a rhodopsin promoter or injecting the virus intra rather than subretinally produces weak expression and no rejection. Lentivirus can induce expression of a foreign gene in the RPE. Viral induced transduction and GFP expression have no effect on the viability of the RPE in vitro. Continued expression of GFP after cell division implies chromosomal integration of the gene. In vivo expression of GFP in RPE encounters rejection. Rejection may not occur with low GFP expression. The latter occurs with low viral titers, a rhodopsin promoter or intra-retinal injection of viral solution. The results are relevant to gene therapy in retina when gene transduction leads to the expression of foreign proteins.

Depletion of Mab21l1 and Mab21l2 messages in mouse embryo arrests axial turning, and impairs notochord and neural tube differentiation.

The nematode mab-21 gene specifies sensory ray cell identity and was first isolated because of its mutant sensory ray defects. Vertebrate Mab21 orthologs have since been identified in mammals and amphibians. In this report, we characterized in detail two Mab21 orthologs in mouse, Mab21l1 and Mab21l2. We examined the genomic organizations of Mab21 genes and used northern blot and in situ hybridizations to assay their temporal-spatial expression pattern. Their embryonic functions were revealed by specific attenuation of Mab21 messages with antisense oligos in cultured embryos. Mab21l1 and Mab21l2 have very similar protein make-up and gene structures. Both genes were expressed in overlapping domains of actively differentiating embryonic tissues. In addition, Mab21l1 had unique expression in the lens vesicles and genital tubercle whereas Mab21l2 was expressed in the retinal epithelium and umbilical cord. Mab21l1 and Mab21l2 depleted embryos had severe defects in notochord, neural tube, organogenesis, vasculogenesis, and axial turning. The findings demonstrate that both Mab21 genes are required in developing embryos for embryonic turning, formation of the notochord, neural tube, and other organ tissues.

Cell proliferation, survival, and death in the Drosophila eye

The Drosophila retina has a precise repeating structure based on the unit eye, or ommatidium. This review summarizes studies of the cell proliferation and survival episodes that affect the number of cells available to make each ommatidium. Late in larval development, as differentiation and patterning begin, the retinal epithelium exhibits striking regulation of the cell cycle including a transient G1 arrest of all cells, followed by a ‘Second Mitotic Wave’ cell cycle that is regulated at the G2/M transition by local intercellular signals. Reiterated episodes of cell death also contribute to precise regulation of retinal cell number. The EGF receptor homolog has multiple roles in retinal proliferation and survival.

Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development.

In zebrafish (Danio rerio) pigmentation is initiated during embryogenesis and begins in the retinal epithelium and in the melanophores. The pigment cells develop rapidly, and within hours they constitute a prominent feature of the embryo. In order to improve signal detection by whole mount in situ hybridization, confocal microscopy, or expression of GFP, embryos may be treated with 1-phenyl 2-thiourea (PTU) during embryogenesis. PTU inhibits melanogenesis by blocking all tyrosinase-dependent steps in the melanin pathway but can be toxic at high concentrations. The embryos remain transparent as long as the PTU treatment is continued. However, PTU treatment must be initiated before the initial pigmentation because it does not remove already formed pigment. Here we provide a protocol for generating transparent zebrafish while avoiding the toxic and teratogenic effects of PTU treatment.

A molecular phenotype atlas of the zebrafish retina.

The rasborine cyprinid Danio rerio (the zebrafish) has become a popular model of retinal function and development. Its value depends, in part, on validation of homologies with retinal cell populations of cyprinine cyprinids. This atlas provides raw and interpreted molecular phenotype data derived from computationally classified sets of small molecule signals from different cell types in the zebrafish retina: L-alanine, L-aspartate, L-glutamine, L-glutamate, glutathione, glycine, taurine and gamma-aminobutyrate. This basis set yields an 8-dimensional signature for every retinal cell and formally establishes molecular signature homologies with retinal neurons, glia, epithelia and endothelia of other cyprinids. Zebrafish photoreceptor classes have been characterized previously: we now show their metabolic profiles to be identical to those of the corresponding photoreceptors in goldfish. The inner nuclear layer is partitioned into precise horizontal, bipolar and amacrine cell layers. The horizontal cell layer contains at least three and perhaps all four known classes of cyprinine horizontal cells. Homologues of cyprinid glutamatergic ON-center and OFF-center mixed rod-cone bipolar cells are present and it appears likely that all five classes are present in zebrafish. The cone bipolar cells defy simple analysis but comprise the largest fraction of bipolar cells, as in all cyprinids. Signature analysis reveals six molecular phenotypes in the bipolar cell cohort: most are superclasses. The amacrine cell layer is composed of approximately equal 64% GABA+ and 35% glycine+ amacrine cells, with the remainder being sparse dopaminergic interplexiform cells and other rare unidentified neurons. These different amacrine cell types are completely distinct in the dark adapted retina, but light adapted retinas display weak leakage of GABA signals into many glycinergic amacrine cells, suggesting widespread heterocellular coupling. The composition of the zebrafish ganglion cell layer is metabolically indistinguishable from that in other cyprinids, and the signatures of glial and non-neuronal cells display strong homologies with those in mammals. As in most vertebrates, zebrafish Müller cells possess a high glutamine, low glutamate signature and contain the dominant pool of glutathione in the neural retina. The retinal pigmented epithelium shows a general mammalian signature but also has exceptional glutathione content (5-10 mM), perhaps required by the unusually high oxygen tensions of teleost retinas. The optic nerve and the marginal zone of the retina reveal characteristic metabolic specializations. The marginal zone is strongly laminated and its nascent neurons display their characteristic signatures before taking their place in the retina proper.

Ocular pANCA antigens are expressed in nonpigmented ciliary body epithelium and are conserved in multiple mammalian species

PURPOSE: pANCA marker autoantibody is expressed by a subset of patients with anterior uveitis. A recombinantly isolated pANCA monoclonal antibody, Fab 5–3, identifies ocular expression of corresponding pANCA antigens in human ciliary body and retina. In this study, Fab 5–3 was used to explore pANCA antigen expression in ocular tissues of multiple mammalian species and identify the ciliary body cell type expressing the pANCA antigen. METHODS: Ocular tissues were obtained from several mammalian species and evaluated for expression of the pANCA (Fab 5–3) antigen(s) using immunohistochemistry and Western analysis of tissue extracts. Additionally, primary cultures of nonpigmented and pigmented rabbit ciliary body epithelium were analyzed for pANCA expression using immunofluorescence and Western analysis. RESULTS: Ocular pANCA (Fab 5–3) antigen expression was observed by immunohistochemistry only in the cytoplasm of retinal ganglion cells and ciliary body epithelium. Retinal antigen expression was conserved in all species examined. Ciliary body expression was observed in human, rabbit, rat, and mouse, but not in pig or cow. Antigen expression in the rabbit ciliary body was restricted to the nonpigmented layer as defined in primary cultures of nonpigmented and pigmented ciliary body epithelium. Immunoreactive proteins in both the human and rabbit included a 32–33 kDa doublet (histone H1), and novel 80 and 100 kDa proteins. CONCLUSIONS: This study identifies ocular pANCA antigen expression in multiple mammalian species localized to the retinal ganglion cell layer and the non-pigmented ciliary body epithelium. The present study also establishes novel 80 and 100 kDa proteins which may correspond to the cytoplasmic antigens detected in situ and can be further characterized biochemically and immunologically using small animal model systems.

The mouse fidgetin gene defines a new role for AAA family proteins in mammalian development.

The mouse mutation fidget arose spontaneously in a heterogeneous albino stock. This mutant mouse is characterized by a side-to-side head-shaking and circling behaviour, due to reduced or absent semicircular canals. Fidget mice also have small eyes, associated with cell-cycle delay and insufficient growth of the retinal neural epithelium, and lower penetrance skeletal abnormalities, including pelvic girdle dysgenesis, skull bone fusions and polydactyly. By positional cloning, we found the gene mutated in fidget mice, fidgetin (Fign), which encodes a new member of the 'meiotic' or subfamily-7 (SF7; ref. 7) group of ATPases associated with diverse cellular activities (AAA proteins). We also discovered two closely related mammalian genes. AAA proteins are molecular chaperones that facilitate a variety of functions, including membrane fusion, proteolysis, peroxisome biogenesis, endosome sorting and meiotic spindle formation, but functions for the SF7 AAA proteins are largely unknown. Fidgetin is the first mutant AAA protein found in a mammalian developmental mutant, thus defining a new role for these proteins in embryonic development.

Paradoxical Effects of Mineralocorticoids on the Ion Gated Sodium Channel in Embryologically Diverse Cells

PCR analysis and Western blotting revealed the expression of the mineralocorticoid receptor (MCR) and the epithelial sodium channel (ENaC) genes at the level of RNA, DNA, and protein in several leukemic cell lines, fibroblasts from human cornea, and epithelial cells from ocular tissues. Following immunofluorescence, the MCR appeared to be primarily nuclear whereas the ENaC was almost exclusively membrane-bound. Paradoxically, the MCR-specific antagonist ZK 91587 actually stimulated the multiplication of human erythroblastic leukemia cells, contrary to the inhibitory effect of the antagonist RU 26752 on the multiplication of corneal fibroblasts; both effects were opposed by aldosterone. In quantitative PCR, both basal and aldosterone-induced levels of ENaC were diminished by ZK 91587 in the corneal fibroblast, in contrast to the stimulation observed in the retinal pigmentary epithelium. Thus, contrary to the existing notions, (a) antimineralocorticoids can act both as agonists and antagonists, and (b) the receptor-mediated action of mineralocorticoids on the sodium channel is not restricted to the epithelial cell.

Vimentin intermediate filament protein as differentiation marker of optic vesicle epithelium in the chick embryo

For the study of the differentiation process of optic vesicle epithelium into neural retina, pigment epithelium and pars caeca retinae, vimentin intermediate filament protein in retinal epithelial cells was detected immunohistochemically in chick embryo at stages 11-21. In the late stage of optic vesicle development (stage 14), optic vesicle epithelium was classified into the following 3 different portions on the basis of vimentin staining intensity: latero-central epithelium under the lens placode, medio-central epithelium facing the latero-central epithelium, and peripheral epithelium connecting the latero-central and medio-central epithelia. Latero-central epithelium, the future neural retina, exhibited strongest staining of vimentin of the 3 portions. In contrast, medio-central epithelium, the future pigment epithelium, showed weakest staining. Moderate staining was observed in peripheral epithelium, the future pars caeca retinae. These differences in levels of vimentin expression were observed during optic cup formation. The present results clearly demonstrate that differentiation of retinal epithelium into neural retina, pigment epithelium and pars caeca retinae occurs in the late stage of the optic vesicle, and that retinal differentiation is reflected by the amount of vimentin in epithelial cells.