Retinal Phenotype Research Articles

Infantile and childhood retinal blindness: A molecular perspective (The Franceschetti Lecture)

Much progress has been made in the past five years in the understanding of Leber congenital amaurosis (LCA) and allied early-onset retinal dystrophies, various forms of stationary sensory retinal blindness, and genes that are involved in the development of the retina. Uncomplicated Leber congenital amaurosis has been associated with mutations of six genes: GUCY2D (encoding RetGC-1) at 17p13.1, RPE65 at 1q31, CRX at 19q13.3, AIPLI at 17p13.1, CRB1 at 1q31-3, and RPGRIP at 14q11. A similar early-onset severe retinal degeneration phenotype has been associated with mutation of TULP1 at 6p21.3. Leber appreciated that the condition he described merged with the phenotypes of early childhood-onset severe retinal degenerations. This insight has been confirmed at the molecular level for mutations of GUCY2D , RPE65 , CRX , AIPL1 , and CRB1 , which cause not only LCA, but also early-childhood and even adult-onset retinal degenerations. This paper reviews the new finding of LCA from mutations of CRB1 and discusses the molecular basis of X-linked blue monochromacy, autosomal recessive congenital achromatopsia from mutations of the genes for ACHM2 ( CNGA3 ) and ACHM3 ( CNGB3 ), X-linked congenital stationary night blindness (CSNB) from mutations of CACNA1F (incomplete CSNB) and NYX (complete CSNB), and the enhanced S-cone syndrome from mutation of the developmental gene, NR2E3 at 15q23, which appears to regulate the development of M- and L-cones from S-cones. These discoveries have opened new areas of cellular and developmental biology for future research into the causes of retinal blindness.

Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk.

The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy (rdy) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.

Folding and subunit assembly of photoreceptor peripherin/rds is mediated by determinants within the extracellular/intradiskal EC2 domain: implications for heterogeneous molecular pathologies.

Peripherin/rds is an integral membrane protein required for the elaboration of rod and cone photoreceptor outer segments in the vertebrate retina; it causes a surprising variety of progressive retinal degenerations in humans and dysmorphic photoreceptors in murine models if defective or absent. (Peripherin/rds is also known as photoreceptor peripherin, peripherin/rds, rds/peripherin, rds, and peripherin-2.) Peripherin/rds appears to act as a structural element in outer segment architecture. However, neither its function at the molecular level nor its role in retinal disease processes are well understood. This report initiates a systematic investigation of protein domain structure and function by examining the molecular and cellular consequences of a series of 14 insertional mutations distributed throughout the polypeptide sequence. Protein expression, disulfide bonding, sedimentation velocity, and subcellular localization of the COS-1 cell-expressed mutant variants were examined to test the hypothesis that protein folding and tetrameric subunit assembly are mediated primarily by EC2, a conserved extracellular/intradiskal domain. Protein folding and tetrameric subunit assembly were not affected by insertion of either an uncharged dipeptide (GA) or a highly charged hendecapeptide (GDYKDDDDKAA) into any one of nine sites residing outside of EC2 as assayed by nonreducing Western blot analysis, sedimentation velocity, and subcellular localization. In contrast, insertions at five positions within the EC2 domain did cause either gross protein misfolding (two sites) or a reduction in protein sedimentation coefficient (two sites) or both (one site). These results indicate that although the vast majority of extramembranous polypeptide sequence makes no measurable contribution to protein folding and tetramerization, discrete regions within the EC2 domain do contain determinants for normal subunit assembly. These findings raise the possibility that multiple classes of structural perturbation are produced by inherited defects in peripherin/rds and contribute to the observed heterogeneity of retinal disease phenotypes.

RotundRacGAP functions with Ras during spermatogenesis and retinal differentiation in Drosophila melanogaster.

Our analysis of rotund (rn) null mutations in Drosophila melanogaster revealed that deletion of the rn locus affects both spermatid and retinal differentiation. In the male reproductive system, the absence of RnRacGAP induced small testes, empty seminal vesicles, short testicular cysts, reduced amounts of interspermatid membrane, the absence of individualization complexes, and incomplete mitochondrial condensation. Flagellar growth continued within the short rn null cysts to produce large bulbous terminations of intertwined mature flagella. Organization of the retina was also severely perturbed as evidenced by grossly misshapen ommatidia containing reduced numbers of photoreceptor and pigment cells. These morphological phenotypes were rescued by genomic rnRacGAP transgenes, demonstrating that RnRacGAP function is critical to spermatid and retinal differentiation. The testicular phenotypes were suppressed by heterozygous hypomorphic mutations in the Dras1 and drk genes, indicating cross talk between RacGAP-regulated signaling and that of the Ras pathway. The observed genetic interactions are consistent with a model in which Rac signaling is activated by Ras and negatively regulated by RnRacGAP during spermatid differentiation. RnRacGAP and Ras cross talk also operated during retinal differentiation; however, while the heterozygous hypomorphic drk mutation continued to act as a suppressor of the rn null mutation, the heterozygous hypomorphic Dras1 mutation induced novel retinal phenotypes.

AT 2 , Judgment Day

As reviewed recently in these pages,1 the vasoconstrictor angiotensin II (Ang II) is one of the circulating peptide growth factors, and clinically the most relevant, that fulfill many of Koch’s postulates for having additional effects locally in the myocardium. These criteria include local production of Ang II within the heart, its upregulation there by hypertrophic signals, its release from cardiac cells after mechanical load, the presence of its receptors on the surface of cardiac cells, and the responsiveness of both cardiac myocytes and cardiac fibroblasts to provocation by this peptide.1,2 This local circuit of Ang II production, release, binding, and transduction within the myocardium itself is the most cogent explanation for the effectiveness of ACE inhibitors in heart failure, even at doses that do not alter loading conditions.1,2 See p 346 Three related receptors exist for Ang II: AT1a, AT1b, and AT2, which communicate with a complex cell-wide web of signaling cascades through GTP-binding (G) proteins as the proximal transducer. The operation of a local circuit for Ang II within the myocardium poses two questions, which have attracted much recent experimental effort, and an even larger share of controversy. First, given that multiple receptors exist for Ang II, which type or types are responsible for pathobiology provoked by the local circuit? Second, given that cardiac muscle and nonmuscle cells are both potential targets for Ang II, what is the respective role of each? Genetic models that altogether lack the receptors individually provide an invaluable experimental tool beyond what is achievable by use of pharmacological inhibitors alone. Surprisingly, the Ang II receptor …

Quantitative trait loci affecting the behavior of A/J and CBA/J intercross mice in the elevated plus maze.

How allelic diversity affects neural mechanisms to produce behavioral variation is largely unknown. The elevated plus maze, consisting of open and closed arms, has been used as a model of behavioral variation in rodent exploration. Under dim illumination the nature of the sensory stimuli that influence arm choice is uncertain. Two inbred mouse strains, A/J (Tyrc/Tyrc, the albino phenotype, mutation in tyrosinase) with a strong preference for closed arm entry, and CBA/J (Pdebrdl/Pdebrdl, the retinal degeneration phenotype, mutation in the beta-subunit of rod cGMP phosphodiesterase), with a weak preference for open arm entry, were studied under varying light. Because behavioral differences persist under red light, variation in light perception is not likely to fully account for variation in arm choice. To identify genetic factors influencing arm choice (100 x Open arm entries/Total arm entries) quantitative trait loci analyses (QTL) were performed on (A/J x CBA/J)F2 mice. Two QTLs, one of which includes PDEB, were identified on Chr 5 (LOD > 10) and account for > 30% of the behavioral variation in arm preference. Tyr (Chr 7, 44 cM) was linked to closed arm entries but not arm preference, and is unlikely to be acting through a direct effect on light perception, because A/J arm entries were not affected by red light and there was no interaction with PDEB in the (A/J x CBA/J)F2 mice. Whether the candidate QTLs on Chr 5 affect arm choice through an effect on light perception is unknown, but phenotypic differences between F2 mice with retinal degeneration and CBA/J mice and F2 mice with albinism and A/J mice suggest that factors other than light sensitivity contribute to arm preference in these two strains.

Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D.

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital sensorineural hearing loss, vestibular dysfunction and visual impairment due to early onset retinitis pigmentosa (RP). So far, six loci (USH1A-USH1F) have been mapped, but only two USH1 genes have been identified: MYO7A for USH1B and the gene encoding harmonin for USH1C. We identified a Cuban pedigree linked to the locus for Usher syndrome type 1D (MIM 601067) within the q2 region of chromosome 10). Affected individuals present with congenital deafness and a highly variable degree of retinal degeneration. Using a positional candidate approach, we identified a new member of the cadherin gene superfamily, CDH23. It encodes a protein of 3,354 amino acids with a single transmembrane domain and 27 cadherin repeats. In the Cuban family, we detected two different mutations: a severe course of the retinal disease was observed in individuals homozygous for what is probably a truncating splice-site mutation (c.4488G-->C), whereas mild RP is present in individuals carrying the homozygous missense mutation R1746Q. A variable expression of the retinal phenotype was seen in patients with a combination of both mutations. In addition, we identified two mutations, Delta M1281 and IVS51+5G-->A, in a German USH1 patient. Our data show that different mutations in CDH23 result in USH1D with a variable retinal phenotype. In an accompanying paper, it is shown that mutations in the mouse ortholog cause disorganization of inner ear stereocilia and deafness in the waltzer mouse.

Expression of multiple forms of clusterin during light-induced retinal degeneration

Purpose. Clusterin has been associated with active cell death in several different model systems, including animal models of retinal degeneration. Clusterin is also expressed in normal tissues, a finding that leads to the question of how it could then play a cell death-specific role during tissue regression. To address this paradox, we have examined clusterin expression during light-induced retinal damage in rats. Methods. Normal albino rats were reared in darkness and then exposed to intense visible light to induce retinal degeneration. Clusterin expression was then examined at various times after light treatment. Standard molecular techniques including Northern analysis, immunohistochemistry, and Western analysis were employed. Results. Northern analysis established that the largest increase in clusterin expression occurs after a decrease in interphotoreceptor retinoid binding protein, IRBP, expression (an indication of a photoreceptor cell dysfunction) and after an increase in heme oxygenase 1, HO-1, expression (an oxidative stress inducible gene), suggesting that induction of clusterin expression is an oxidative stress response. Immunohistochemical analysis with two different clusterin-specific antibodies, anti(SGP-2) and anti(301), localized distinct forms of clusterin to Müller cells and degenerating photo-receptor cells. Western analysis demonstrated degeneration associated isoforms of clusterin in light treated retina that are not present in normal retina. Conclusion. Clusterin over-expression is characteristic of a retinal degeneration phenotype and we propose that clusterin action may be defined by the nature in which it is modified. We hypothesize that alternate processing leads to retinal degeneration-specific forms of the protein (65, 61, and 50 kDa) that are not present in normal retina.

Investigating the mechanisms of retinal degenerations with antisense oligonucleotides.

Utilizing antisense oligonucleotides coupled with an intact Xenopus eye rudiment model, we have effectively demonstrated that we are able to downregulate the expression of a photoreceptor-specific protein, rds/peripherin, and generate a loss-of-function model upon which to further study the function of the rds/peripherin gene. The ultrastructure and protein expression patterns very closely resemble those previously documented in both the rds mouse and in human autosomal dominant retinitis pigmentosa due to peripherin/RDS mutations. An identical strategy can be applied to any gene correlated with a degenerative retinal phenotype. As the entire array of genes is revealed through the various genome projects, including human and mouse, it is becoming increasingly critical to evaluate and determine the function of the corresponding gene products. Discovering which gene is responsible for a particular clinical phenotype is only the first of many steps in the development of a treatment or cure for that particular disease. Using our in vitro model, in which the retina is readily accessible to the antisense oligonucleotide yet the normal three-dimensional ultrastructure of the retina is maintained, we can evaluate the function of virtually any gene as the sequence becomes available. A thorough understanding of the function of individual genes will provide insights on the role of gene product in retinal health and pathophysiology. This experimental approach will also allow for specific therapeutic interventions to be evaluated so that targeted treatments can be designed for individuals with specific genetic mutations.

Photoreceptor-Specific Expression of Platelet-Derived Growth Factor-B Results in Traction Retinal Detachment

Expression of platelet-derived growth factor (PDGF)-A and PDGF-B is increased in patients with proliferative retinopathies in which traction retinal detachments occur. Previous studies have demonstrated that increased expression of PDGF-A in the retina of transgenic mice results in retinal gliosis due to proliferation of astrocytes with different retinal phenotypes based on the time of onset and location of the PDGF-A production. In this study, we investigated the effects of PDGF-B in the retina using gain-of-function transgenic mice that express PDGF-B in photoreceptors. These mice show proliferation of astrocytes, pericytes, and, to a lesser extent, endothelial cells, resulting in ectopic cells on the surface and extending into the retina. The sheets of cells exert traction on the retina resulting in traction retinal detachments similar to those seen in humans with proliferative retinopathies. These studies suggest that PDGF-B has more dramatic effects in the retina than PDGF-A, because it acts on additional cell types, in particular on pericytes, which have a highly developed contractile apparatus. These studies in the retina suggest a means that could be used in other tissues throughout the body to achieve graded PDGF effects. They also provide a new model of traction retinal detachment that can be used to investigate new treatments for patients with proliferative retinopathies.

Genetic heterogeneity of Usher syndrome type II: localisation to chromosome 5q

Usher syndrome is a group of autosomal recessive disorders that includes retinitis pigmentosa (RP) with hearing loss. Usher syndrome type II is defined as moderate to severe hearing loss with...

Isolation and characterization of Drosophila retinal degeneration B suppressors.

The Drosophila retinal degeneration B protein (RdgB) is a novel integral membrane phosphatidylinositol transfer protein required for photoreceptor cell viability and light response. We isolated one intragenic suppressor (rdgBsu100) and four autosomal suppressors of the hypomorphic rdgBKS222 retinal degeneration phenotype. The rdgBsu100 suppressor dramatically slowed rdgBKS222's photoreceptor degeneration without significantly improving the electroretinogram (ERG) light response. One autosomal recessive suppressor [su(rdgB)69] significantly slowed rdgBKS222 retinal degeneration and restored the ERG light response near to that of the wild type. Unlike all the previously characterized rdgB suppressors, the four new autosomal suppressors do not affect the ERG light response in rdgB+ flies. Only Su(rdgB)116 exhibited a mutant phenotype in a rdgB+ background, which was smaller R1-6 rhabdomeres. We also examined the extent to which two previously identified visual transduction mutations suppressed rdgB retinal degeneration. Absence of one of the light-activated calcium channels (trpCM) slowed the onset of rdgB-dependent degeneration. However, loss of protein kinase C (inaC209), which blocks photoreceptor cell deactivation, desensitization, and light adaptation, failed to suppress rdgB degeneration under normal light conditions. This demonstrates that TRP activity, but not INAC, is required for rapid rdgB-dependent degeneration.

Math5 encodes a murine basic helix-loop-helix transcription factor expressed during early stages of retinal neurogenesis.

We have identified Math5, a mouse basic helix-loop-helix (bHLH) gene that is closely related to Drosophila atonal and Xenopus Xath5 and is largely restricted to the developing eye. Math5 retinal expression precedes differentiation of the first neurons and persists within progenitor cells until after birth. To position Math5 in a hierarchy of retinal development, we compared Math5 and Hes1 expression in wild-type and Pax6-deficient (Sey) embryos. Math5 expression is downregulated in Sey/+ eyes and abolished in Sey/Sey eye rudiments, whereas the bHLH gene Hes1 is upregulated in a similar dose-dependent manner. These results link Pax6 to the process of retinal neurogenesis and provide the first molecular correlate for the dosage-sensitivity of the Pax6 phenotype. During retinogenesis, Math5 is expressed significantly before NeuroD, Ngn2 or Mash1. To test whether these bHLH genes influence the fates of distinct classes of retinal neurons, we ectopically expressed Math5 and Mash1 in Xenopus retinal progenitors. Unexpectedly, lipofection of either mouse gene into the frog retina caused an increase in differentiated bipolar cells. Directed expression of Math5, but not Xath5, in Xenopus blastomeres produced an expanded retinal phenotype. We propose that Math5 acts as a proneural gene, but has properties different from its most closely related vertebrate family member, Xath5.

Intraocular transplantation of E1A-immortalized retinal precursor cells

The purpose of this study was to examine the effect of the ocular environment on the survival, tumorigenicity, and phenotypic marker expression of immortalized retinal precursor cells transplanted into immunocompetent adult and neonatal Sprague–Dawley rats. E1A-NR.3, a rat immortalized retinal precursor cell culture, was used as an inexhaustible source of experimental graft material. These cells were prelabeled with the fluorescent marker diI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) and transplanted intravitreally (50,000 cells per μL) into 11 adult and 31 neonatal Sprague–Dawley rat eyes. At 1 mo posttransplant, animals were sacrificed and retinal tissue sections examined histologically for the presence of grafted cells, signs of tumor formation, and retinal phenotypic marker expression. No obvious signs of tumor formation or rejection were seen in a total of 42 eyes in the immunocompetent hosts. Our results indicate that E1A-NR.3 cells survive at least 1 month in vivo, and can migrate from the vitreous into neuroretinal cell layers. Subpopulations of surviving grafted cells were seen to express photoreceptor markers rhodopsin and recoverin comparably between in vitro and in vivo conditions. However, the number of cells immunoreactive for vimentin and E1A decreased significantly under in vivo conditions. This report represents the first experimental intravitreal transplantation of E1A-immortalized retinal precursor cells into adult and neonatal rats. The intraocular location and environment appears to affect phenotypic expression of surviving grafted cells, especially with respect to vimentin and E1A expression. The fact that E1A-NR.3 cells survived intraocularly at least 1 mo without tumor formation suggests that the cells may continue to be useful for further in vivo studies of experimental retinal transplantation, and effects of histological location on retinal cell phenotype and histogenesis in immunocompetent hosts.

Intraocular transplantation of E1A-immortalized retinal precursor cells.

The purpose of this study was to examine the effect of the ocular environment on the survival, tumorigenicity, and phenotypic marker expression of immortalized retinal precursor cells transplanted into immunocompetent adult and neonatal Sprague-Dawley rats. EIA-NR.3, a rat immortalized retinal precursor cell culture, was used as an inexhaustible source of experimental graft material. These cells were prelabeled with the fluorescent marker dil (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and transplanted intravitreally (50,000 cells per microL) into 11 adult and 31 neonatal Sprague-Dawley rat eyes. At 1 mo posttransplant, animals were sacrificed and retinal tissue sections examined histologically for the presence of grafted cells, signs of tumor formation, and retinal phenotypic marker expression. No obvious signs of tumor formation or rejection were seen in a total of 42 eyes in the immunocompetent hosts. Our results indicate that EIA-NR.3 cells survive at least 1 month in vivo, and can migrate from the vitreous into neuroretinal cell layers. Subpopulations of surviving grafted cells were seen to express photoreceptor markers rhodopsin and recoverin comparably between in vitro and in vivo conditions. However, the number of cells immunoreactive for vimentin and E1A decreased significantly under in vivo conditions. This report represents the first experimental intravitreal transplantation of E1A-immortalized retinal precursor cells into adult and neonatal rats. The intraocular location and environment appears to affect phenotypic expression of surviving grafted cells, especially with respect to vimentin and E1A expression. The fact that E1A-NR.3 cells survived intraocularly at least 1 mo without tumor formation suggests that the cells may continue to be useful for further in vivo studies of experimental retinal transplantation, and effects of histological location on retinal cell phenotype and histogenesis in immunocompetent hosts.

Transgenic Complementation of MARCKS Deficiency with a Nonmyristoylatable, Pseudo-Phosphorylated Form of MARCKS: Evidence for Simultaneous Positive and Dominant-Negative Effects on Central Nervous System Development

MARCKS is a widely expressed protein kinase C substrate that is essential for normal prenatal development of the central nervous system in mice. MARCKS-deficient mice exhibit universal perinatal mortality and numerous developmental abnormalities of the brain and retina. To determine which domains of the protein were important in complementing these neurodevelopmental anomalies, we have interbred MARCKS knockout mice with transgenic mice expressing an epitope-tagged human MARCKS transgene that can completely correct the MARCKS-deficient phenotype. Previous structure–function studies showed that a nonmyristoylatable form of MARCKS could correct all of the neuroanatomical abnormalities, and resulted in approximately 25% viable pups that grew to adulthood and were fertile. The present experiment attempted a similar complementation strategy in which a nonmyristoylatable, “pseudo-phosphorylated” form of the protein was used, which has been shown to be almost completely cytosolic in cell expression studies. Surprisingly, this transgene was able to complement almost all of the cerebral anatomical abnormalities characteristic of the knockout mice. However, these mice also exhibited a universal, novel phenotype: profound retinal ectopia, in which retinal tissue was often found in the vitreous humor as well as extraocularly. Retrospective evaluation of the original MARCKS knockout phenotype revealed that this anomaly was present in about 43% of the knockout mice, and was clearly detectable as early as embryonic day 12.5, before retinal cell differentiation begins. These data suggest that a nonmyristoylatable, pseudo-phosphorylated form of MARCKS can complement most if not all cerebral aspects of the MARCKS-deficient phenotype, but that it appears to worsen a retinal phenotype, perhaps by exerting a dominant-negative effect on a coexpressed MARCKS homologue.

Neutralizing TNF-alpha Activity Modulates T-cell Phenotype and Function in Experimental Autoimmune Uveoretinitis

Inhibiting TNF-α activity prevents tissue destruction without inhibiting retinal T cell infiltration in experimental autoimmune uveoretinitis (EAU) in Lewis rats. To further determine the role of TNF-α in autoimmune uveitis we characterized T cells isolated from retinae after treatment with a TNF-α antagonist. TNF-α activity was neutralized in vivo with a p55 TNF-α receptor-Ig fusion protein (sTNFr-Ig), administered 8 and 10 days after induction of EAU with heterologous retinal antigens. Retinal T-cell phenotype expression was examined by flow cytometry with respect to OX22 status (CD45RBlowor CD45RBhigh), activation (OX40 and CD25 expression) and rate of T-cell apoptosis (Annexin V+PI−expression). Lymphocyte reactivity was assessed by proliferation responses and cytokine production to retinal antigens. Despite greater than 40% of CD4+T cells being activated at the height of disease, the proportion of OX22lowexpression was reduced and T cells exhibited reduced IFN-γ and elevated IL-4 production. Retinal T cells maintained antigen-specific proliferation and demonstrated a low apoptotic rate. Although in both animal groups, comparable numbers of T cells were isolated, neutralizing TNF activity suppressed Th1 effector mechanisms protecting against target organ damage.

FGF1 patterns the optic vesicle by directing the placement of the neural retina domain.

Patterning of the bipotential retinal primordia (the optic vesicles) into neural retina and retinal pigmented epithelium depends on its interaction with overlaying surface ectoderm. The surface ectoderm expresses FGFs and the optic vesicles express FGF receptors. Previous FGF-expression data and in vitro analyses support the hypothesis that FGF signaling plays a significant role in patterning the optic vesicle. To test this hypothesis in vivo we removed surface ectoderm, a rich source of FGFs. This ablation generated retinas in which neural and pigmented cell phenotypes were co-mingled. Two in vivo protocols were used to replace FGF secretion by surface ectoderm: (1) implantation of FGF-secreting fibroblasts, and (2) injection of replication-incompetent FGF retroviral expression vectors. The retinas in such embryos exhibited segregated neural and pigmented epithelial domains. The neural retina domains were always close to a source of FGF secretion. These results indicate that, in the absense of surface ectoderm, cells of the optic vesicles display both neural and pigmented retinal phenotypes, and that positional cues provided by FGF organize the bipotential optic vesicle into specific neural retina and pigmented epithelium domains. We conclude that FGF can mimic one of the earliest functions of surface ectoderm during eye development, namely the demarcation of neural retina from pigmented epithelium.

Differences in the retinal GABA system among control, spastic mutant and retinal degeneration mutant mice

Immunocytochemical methods were used to compare the GABA system in control mice and two mutant strains: spastic which has reduced glycine receptors and retinal degeneration mutant in which the photoreceptors degenerate and reportedly have increased GABA and GAD levels. We found that the spastic mutant retina had reduced GABA-immunoreactivity (IR) in the proximal retina, reduced staining for GAD-1440 in the OPL, and reduced GABAA receptor staining in the OPL, compared to control. The retinal degeneration mutant retinas had enhanced GABA-IR throughout the retina, particularly in Müller cells, bipolar cells and IPL, and enhancement of GABAA receptor staining in the OPL, compared to control. The distributions of GABA-IR, GAD-1440-IR and GABAA receptor-IR in retinas of spastic mutant mice that also expressed the retinal degeneration phenotype resembled those found in retinas of mice that expressed only the retinal degeneration phenotype rather than those that expressed only the spastic mutation. No differences were observed among the conditions for GAD-65, GAD-67 or GABA-T. Our results with the spastic and retinal degeneration mutant mice demonstrate that attenuation in the glycinergic system and photoreceptor degeneration, respectively, is accompanied by alterations in different aspects of the GABA system, giving impetus for caution in the interpretation of experiments involving genetic manipulation of complex phenotypes.

The phosphatidylinositol transfer protein domain of Drosophila retinal degeneration B protein is essential for photoreceptor cell survival and recovery from light stimulation.

The Drosophila retinal degeneration B (rdgB) gene encodes an integral membrane protein involved in phototransduction and prevention of retinal degeneration. RdgB represents a nonclassical phosphatidylinositol transfer protein (PITP) as all other known PITPs are soluble polypeptides. Our data demonstrate roles for RdgB in proper termination of the phototransduction light response and dark recovery of the photoreceptor cells. Expression of RdgB's PITP domain as a soluble protein (RdgB-PITP) in rdgB2 mutant flies is sufficient to completely restore the wild-type electrophysiological light response and prevent the degeneration. However, introduction of the T59E mutation, which does not affect RdgB-PITP's phosphatidylinositol (PI) and phosphatidycholine (PC) transfer in vitro, into the soluble (RdgB-PITP-T59E) or full-length (RdgB-T59E) proteins eliminated rescue of retinal degeneration in rdgB2 flies, while the light response was partially maintained. Substitution of the rat brain PITPalpha, a classical PI transfer protein, for RdgB's PITP domain (PITPalpha or PITPalpha-RdgB chimeric protein) neither restored the light response nor maintained retinal integrity when expressed in rdgB2 flies. Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo. Expression of either RdgB-T59E or PITPalpha-RdgB in rdgB+ flies produced a dominant retinal degeneration phenotype. Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPalpha-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism. This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.