Adult Neural Retina Research Articles

Yap haploinsufficiency leads to M\xfcller cell dysfunction and late-onset cone dystrophy

Hippo signalling regulates eye growth during embryogenesis through its effectors YAP and TAZ. Taking advantage of a Yap heterozygous mouse line, we here sought to examine its function in adult neural retina, where YAP expression is restricted to Müller glia. We first discovered an unexpected temporal dynamic of gene compensation. At postnatal stages, Taz upregulation occurs, leading to a gain of function-like phenotype characterised by EGFR signalling potentiation and delayed cell-cycle exit of retinal progenitors. In contrast, Yap+/− adult retinas no longer exhibit TAZ-dependent dosage compensation. In this context, Yap haploinsufficiency in aged individuals results in Müller glia dysfunction, late-onset cone degeneration, and reduced cone-mediated visual response. Alteration of glial homeostasis and altered patterns of cone opsins were also observed in Müller cell-specific conditional Yap-knockout aged mice. Together, this study highlights a novel YAP function in Müller cells for the maintenance of retinal tissue homeostasis and the preservation of cone integrity. It also suggests that YAP haploinsufficiency should be considered and explored as a cause of cone dystrophies in human.

Analysis of Adult Neural Retina Extracellular Vesicle Release, RNA Transport and Proteomic Cargo.

PurposeExtracellular vesicles (EVs) contain RNA and protein cargo reflective of the genotype and phenotype of the releasing cell of origin. Adult neural retina EV release, RNA transfer, and proteomic cargo are the focus of this study.MethodsAdult wild-type mouse retinae were cultured and released EV diameters and concentrations quantified using Nanosight. Immunogold transmission electron microscopy (TEM) was used to image EV ultrastructure and marker protein localization. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze retinal cell transcripts present in EVs. Super-resolution microscopy was used to image fluorescent (green) RNA and (red) lipid membrane labeled EVs, released by adult retina, and internalized by isolated retinal cells. Mass spectrometry was used to characterize the proteomes of adult retina and EVs.ResultsAdult neural retina released EVs at a rate of 1.42 +/− 0.08 × 108/mL over 5 days, with diameters ranging from 30 to 910 nm. The canonical EV markers CD63 and Tsg101 localized to retinal EVs. Adult retinal and neuronal mRNA species present in both retina and EVs included rhodopsin and the neuronal nuclei marker NeuN. Fluorescently labeled RNA in retinal cells was enclosed in EVs, transported to, and uptaken by co-cultured adult retinal cells. Proteomic analysis revealed 1696 protein species detected only in retinal cells, 957 species shared between retina and EVs, and 82 detected only in EVs.ConclusionsThe adult neural retina constitutively releases EVs with molecular cargo capable of intercellular transport and predicted involvement in biological processes including retinal physiology, mRNA processing, and transcription regulation within the retinal microenvironment.

Sostdc1 is expressed in all major compartments of developing and adult mammalian eyes.

This study was conducted in order to study Sostdc1 expression in rat and human developing and adult eyes. Using the yeast signal sequence trap screening method, we identified the Sostdc1 cDNA encoding a protein secreted by the adult rat retinal pigment epithelium. We determined by in situ hybridization, RT-PCR, immunohistochemistry, and western blot analysis Sostdc1 gene and protein expression in developing and postnatal rat ocular tissue sections. We also investigated Sostdc1 immunohistolocalization in developing and adult human ocular tissues. We demonstrated a prominent Sostdc1 gene expression in the developing rat central nervous system (CNS) and eyes at early developmental stages from E10.5days postconception (dpc) to E13dpc. Specific Sostdc1 immunostaining was also detected in most adult cells of rat ocular tissue sections. We also identified the rat ocular embryonic compartments characterized by a specific Sostdc1 immunohistostaining and specific Pax6, Sox2, Otx2, and Vsx2 immunohistostaining from embryonic stages E10.5 to E13dpc. Furthermore, we determined the localization of SOSTDC1 immunoreactivity in ocular tissue sections of developing and adult human eyes. Indeed, we detected SOSTDC1 immunostaining in developing and adult human retinal pigment epithelium (RPE) and neural retina (NR) as well as in several developing and adult human ocular compartments, including the walls of choroidal and scleral vessels. Of utmost importance, we observed a strong SOSTDC1 expression in a pathological ocular specimen of type 2 Peters' anomaly complicated by retinal neovascularization as well in the walls ofother pathological extra-ocular vessels. CONCLUSION: As rat Sostdc1 and human SOSTDC1 are dual antagonists of the Wnt/β-catenin and BMP signaling pathways, these results underscore the potential crucial roles of these pathways and their antagonists, such as Sostdc1 and SOSTDC1, in developing and adult mammalian normal eyes as well as in syndromic and nonsyndromic congenital eye diseases.

Activation of Rod Input in a Model of Retinal Degeneration Reverses Retinal Remodeling and Induces Formation of Functional Synapses and Recovery of Visual Signaling in the Adult Retina.

A major cause of human blindness is the death of rod photoreceptors. As rods degenerate, synaptic structures between rod and rod bipolar cells disappear and the rod bipolar cells extend their dendrites and occasionally make aberrant contacts. Such changes are broadly observed in blinding disorders caused by photoreceptor cell death and are thought to occur in response to deafferentation. How the remodeled retinal circuit affects visual processing following rod rescue is not known. To address this question, we generated male and female transgenic mice wherein a disrupted cGMP-gated channel (CNG) gene can be repaired at the endogenous locus and at different stages of degeneration by tamoxifen-inducible cre-mediated recombination. In normal rods, light-induced closure of CNG channels leads to hyperpolarization of the cell, reducing neurotransmitter release at the synapse. Similarly, rods lacking CNG channels exhibit a resting membrane potential that was ~10 mV hyperpolarized compared to WT rods, indicating diminished glutamate release. Retinas from these mice undergo stereotypic retinal remodeling as a consequence of rod malfunction and degeneration. Upon tamoxifen-induced expression of CNG channels, rods recovered their structure and exhibited normal light responses. Moreover, we show that the adult mouse retina displays a surprising degree of plasticity upon activation of rod input. Wayward bipolar cell dendrites establish contact with rods to support normal synaptic transmission, which is propagated to the retinal ganglion cells. These findings demonstrate remarkable plasticity extending beyond the developmental period and support efforts to repair or replace defective rods in patients blinded by rod degeneration.SIGNIFICANCE STATEMENT Current strategies for treatment of neurodegenerative disorders are focused on the repair of the primary affected cell type. However, the defective neurons function within a complex neural circuitry, which also becomes degraded during disease. It is not known whether rescued neurons and the remodeled circuit will establish communication to regain normal function. We show that the adult mammalian neural retina exhibits a surprising degree of plasticity following rescue of rod photoreceptors. The wayward dendrites of rod bipolar cells re-establish contact with rods to support normal synaptic transmission, which is propagated to the retinal ganglion cells. These findings support efforts to repair or replace defective rods in patients blinded by rod cell loss.

New concepts in macrophage ontogeny in the adult neural retina

The number of neurons dedicated to vision itself is thought to be greater than the sum of the four other senses combined. Yet, little attention has been payed to the retina as compared to elsewhere in the central nervous system with respect to microglia, the macrophages of the neural parenchyma. Indeed, major advancements in the understanding of microglial ontogeny and maintenance in brain and spinal cord are now widely appreciated, whereas less notice has been given to the neural retina in this regard. The current Review covers topical concepts on adult microglia and perivascular macrophage ontogenies in the steady state retina, as well as parallels made with these macrophages in other areas of the central nervous system. The subject of recruited monocytes and their descendant monocyte-derived macrophages in degenerative diseases of the retina is also integrated into this Review. Key experiments that have led to the theories covered are highlighted throughout, as are the knowledge gaps that remain unresolved.

Comprehensive interactome of Otx2 in the adult mouse neural retina.

The Otx2 homeodomain transcription factor exerts multiple functions in specific developmental contexts, probably through the regulation of different sets of genes. Protein partners of Otx2 have been shown to modulate its activity. Therefore, the Otx2 interactome may play a key role in selecting a precise target-gene repertoire, hence determining its function in a specific tissue. To address the nature of Otx2 interactome, we generated a new recombinant Otx2(CTAP-tag) mouse line, designed for protein complexes purification. We validated this mouse line by establishing the Otx2 interactome in the adult neural retina. In this tissue, Otx2 is thought to have overlapping function with its paralog Crx. Our analysis revealed that, in contrary to Crx, Otx2 did not develop interactions with proteins that are known to regulate phototransduction genes but showed specific partnership with factors associated with retinal development. The relationship between Otx2 and Crx in the neural retina should therefore be considered as complementarity rather than redundancy. Furthermore, study of the Otx2 interactome revealed strong associations with RNA processing and translation machineries, suggesting unexpected roles for Otx2 in the regulation of selected target genes all along the transcription/translation pathway. The Otx2(CTAP-tag) line, therefore, appears suitable for a systematic approach to Otx2 protein-protein interactions. genesis 53:685-694, 2015. © 2015 Wiley Periodicals, Inc.

Manipulation of the Recipient Retinal Environment by Ectopic Expression of Neurotrophic Growth Factors Can Improve Transplanted Photoreceptor Integration and Survival

Degeneration of the neural retina is the leading cause of untreatable blindness in the developed world. Stem cell replacement therapy offers a novel strategy for retinal repair. Postmitotic photoreceptor precursors derived from the early postnatal (P) retina are able to migrate and integrate into the adult mouse retina following transplantation into the subretinal space, but it is likely that a large number of these cells would be required to restore vision. The adult recipient retina presents a very different environment to that from which photoreceptor precursor donor cells isolated from the developing postnatal retina are derived. Here we considered the possibility that modulation of the recipient environment by ectopic expression of developmentally regulated growth factors, normally present during photoreceptor development, might enhance the migration and integration of transplanted cells into the adult neural retina. Adeno-associated viral (AAV) vectors were used to introduce three growth factors previously reported to play a role in photoreceptor development, IGF1, FGF2, and CNTF, into the adult retina, prior to transplantation of P4 cells derived from the Nrl.GFP(+ve) neural retina. At 3 weeks posttransplantation the number of integrated, differentiated photoreceptor cells present in AAV-mediated neurotrophic factor-treated eyes was assessed and compared to control treated contralateral eyes. We show, firstly, that it is possible to manipulate the recipient retinal microenvironment via rAAV-mediated gene transfer with respect to these developmentally relevant growth factors. Moreover, when combined with cell transplantation, AAV-mediated expression of IGF1 led to significantly increased levels of cell integration, while overexpression of FGF2 had no significant effect on integrated cell number. Conversely, expression of CNTF led to a significant decrease in cell integration and an exacerbated glial response that led to glial scarring. Together, these findings demonstrate the importance of the extrinsic environment of the recipient retina for photoreceptor cell transplantation and show for the first time that it is possible to manipulate this environment using viral vectors to influence photoreceptor transplantation efficiency.

Cell-replacement therapy and neural repair in the retina

Visual impairment severely affects the quality of life of patients and their families and is also associated with a deep economic impact. The most common pathologies responsible for visual impairment and legally defined blindness in developed countries include age-related macular degeneration, glaucoma and diabetic retinopathy. These conditions share common pathophysiological features: dysfunction and loss of retinal neurons. To date, two main approaches are being taken to develop putative therapeutic strategies: neuroprotection and cell replacement. Cell replacement is a novel therapeutic approach to restore visual capabilities to the degenerated adult neural retina and represents an emerging field of regenerative neurotherapy. The discovery of a population of proliferative cells in the mammalian retina has raised the possibility of harnessing endogenous retinal stem cells to elicit retinal repair. Furthermore, the development of suitable protocols for the reprogramming of differentiated somatic cells to a pluripotent state further increases the therapeutic potential of stem-cell-based technologies for the treatment of major retinal diseases. Stem-cell transplantation in animal models has been most effectively used for the replacement of photoreceptors, although this therapeutic approach is also being used for inner retinal pathologies. In this review, we discuss recent advances in the development of cell-replacement approaches for the treatment of currently incurable degenerative retinal diseases.

Frizzled 4 Is Required for Retinal Angiogenesis and Maintenance of the Blood-Retina Barrier

PURPOSE. Mice deficient in the secreted protein Norrin or its receptor Frizzled-4 (FZD4) exhibit incomplete vascularization of the neural retina. However, because of early retinal vascular defects in the knockout models, it has not been possible to study FZD4 contribution in ocular neovascular disease. To further understand the role of this signaling pathway in physiological and pathologic angiogenesis, the authors generated a monoclonal antibody that neutralizes FZD4 function in vivo. METHODS. Antibodies were generated by immunizing Fzd4 knockout mice with the cysteine-rich domain of FZD4. A monoclonal antibody (1.99.25) was discovered that antagonizes Norrin- and WNT3A-induced β-catenin accumulation in vitro. 1.99.25 and an isotype-matched negative control antibody were evaluated in models of developmental retinal angiogenesis, oxygen-induced retinopathy, and retinal angiomatous proliferation. The authors also investigated the role of FZD4 in maintaining the blood-retina barrier in normal adult mice. RESULTS. Administration of 1.99.25 inhibited physiological and pathologic sprouting angiogenesis within the retina. Inhibition of FZD4 in developing retinal vascular networks caused the upregulation of PLVAP, a protein normally associated with fenestrated, immature endothelium in the CNS. In the adult neural retina, the administration of 1.99.25 induced PLVAP expression in the deep capillary bed and enabled extravasation of small and large molecules through the blood-retina barrier. CONCLUSIONS. These results demonstrate that FZD4 is required for physiological and pathologic angiogenesis in the retina and for regulation of retinal endothelial cell differentiation. The authors also show that FZD4 is critical for maintaining the integrity of the mature blood-retina barrier.

Long-Term Survival of Photoreceptors Transplanted into the Adult Murine Neural Retina Requires Immune Modulation

Stem cell therapy presents an opportunity to replace photoreceptors that are lost as a result of inherited and age-related degenerative disease. We have previously shown that murine postmitotic rod photoreceptor precursor cells, identified by expression of the rod-specific transcription factor Nrl, are able to migrate into and integrate within the adult murine neural retina. However, their long-term survival has yet to be determined. Here, we found that integrated Nrl.gfp(+ve) photoreceptors were present up to 12 months post-transplantation, albeit in significantly reduced numbers. Surviving cells had rod-like morphology, including inner/outer segments and spherule synapses. In a minority of eyes, we observed an early, marked reduction in integrated photoreceptors within 1 month post-transplantation, which correlated with increased numbers of amoeboid macrophages, indicating acute loss of transplanted cells due to an inflammatory response. In the majority of transplants, similar numbers of integrated cells were observed between 1 and 2 months post-transplantation. By 4 months, however, we observed a significant decrease in integrated cell survival. Macrophages and T cells were present around the transplantation site, indicating a chronic immune response. Immune suppression of recipients significantly increased transplanted photoreceptor survival, indicating that the loss observed in unsuppressed recipients resulted from T cell-mediated host immune responses. Thus, if immune responses are modulated, correctly integrated transplanted photoreceptors can survive for extended periods of time in hosts with partially mismatched H-2 haplotypes. These findings suggest that autologous donor cells are optimal for therapeutic approaches to repair the neural retina, though with immune suppression nonautologous donors may be effective.

Effects of extracellular matrix and neighboring cells on induction of human embryonic stem cells into retinal or retinal pigment epithelial progenitors

To determine the effects of extracellular matrix and neighboring cells on the differentiation of human embryonic stem cells (hESC) into progenitors of retinal cells and/or retinal pigment epithelium (RPE). HESC were cultured on mouse PA6 stromal cells for approximately 2 weeks to obtain neural progenitors. To induce photoreceptor marker expression, the neural progenitors were cultured on a confluent monolayer of ARPE19 or on laminin-coated dishes. To induce RPE markers, the neural progenitors were seeded onto human Bruch’s membrane or Matrigel. Cells were examined morphologically and stained with different RPE or neural progenitor markers. Microarray techniques were used to compare the gene expression profiles of hESC cultured on mouse fibroblasts or neural progenitors on PA6 cells to the transcriptome of the adult neural retina and RPE. HESC cultured on PA6 cells expressed neural progenitor markers β-tubulin III, PAX6, neural filament, GFAP and vimentin. Culturing these neural progenitors on confluent ARPE19 monolayer induced expression of the photoreceptor progenitor cell marker CRX; culturing neural progenitors on laminin substrates induced a neuronal phenotype with neurite formation. Neural progenitors expressed the RPE marker ZO-1 after culturing on Matrigel-coated dishes and the RPE marker Bestrophin after culturing on human Bruch's membrane explants. Hierarchical clustering analysis of samples suggested that when cultured on PA6 stromal cells hESC exhibited genetic characteristics towards differentiating into neural retina. Microarray analysis showed that after culturing on PA6 cells, stem cells expressed 117 new genes; among these there were 22 genes present in neural retina or RPE cells. The functions of these genes were highly related to cell proliferation, nervous system development and cell adhesion. HESC can be induced to differentiate into neural progenitors after culturing on PA6 cells. These neural progenitors can express RPE markers when cultured on Bruch's membrane or Matrigel, or photoreceptor markers when cultured on confluent ARPE19 or laminin. Additional studies are required to assess the function of hESC induced to express retinal or RPE markers prior to successful intraocular transplantation into animal models of retinal degeneration.

FIZ1 is part of the regulatory protein complex on active photoreceptor-specific gene promoters in vivo

BackgroundFIZ1 (Flt-3 Interacting Zinc-finger) is a broadly expressed protein of unknown function. We reported previously that in the mammalian retina, FIZ1 interacts with NRL (Neural-Retina Leucine-zipper), an essential transcriptional activator of rod photoreceptor-specific genes. The concentration of FIZ1 in the retina increases during photoreceptor terminal maturation, when two key transcription factors NRL and CRX (Cone-Rod Homeobox) become detectable on the promoters of photoreceptor-specific genes (i.e. Rhodopsin, Pde6b). To determine if FIZ1 is involved in regulating CRX-mediated transcriptional activation, we examined FIZ1 subcellular location in mouse neural retina, its ability to interact with CRX, and its association with CRX/NRL target genes.ResultsFIZ1 is present in the nucleus of adult photoreceptors as well as other retinal neurons as shown by transmission electron microscopy with nano-gold labeling. FIZ1 and CRX were co-precipitated from retinal nuclear extracts with antibodies to either protein. Chromatin immunoprecipitation (ChIP) assays revealed that FIZ1 is part of the protein complex on several rod and cone gene promoters, within photoreceptor cells of the mouse retina. FIZ1 complexes with CRX or NRL on known NRL- and CRX-responsive elements, as shown by electrophoretic mobility shift assays with FIZ1 antibody. FIZ1 can directly bind to CRX, as demonstrated using yeast two-hybrid and GST pull-down assays. Co-transfection assays demonstrated that FIZ1 increases CRX-mediated activation of Opsin test promoters. Quantitative ChIP analysis revealed an increased association of FIZ1 with the Rhodopsin promoter in adult (P-25) neural retina versus immature (P-3) neural retina. The quantity of transcriptionally active RNA Polymerase-II within the Rhodopsin gene (Rho) was significantly increased in the adult neural retina, compared to the immature retina.ConclusionFIZ1 directly interacts with CRX to enhance CRX's transactivation activity for target genes. Developmentally, in neural retina tissue, the increased association of FIZ1 with CRX target genes corresponds to an increased association of transcriptionally active Pol-II within the Rho gene. Together with previous findings, our results suggest that FIZ1 may act as a transcriptional co-regulator of photoreceptor-specific genes, recruited by at least two photoreceptor-specific transcription factors, CRX and NRL. Further studies are underway to elucidate the exact role of FIZ1 in photoreceptor gene expression, development and maintenance.

MIO-M1 Cells and Similar Müller Glial Cell Lines Derived from Adult Human Retina Exhibit Neural Stem Cell Characteristics

Growing evidence suggests that glial cells may have a role as neural precursors in the adult central nervous system. Although it has been shown that Müller cells exhibit progenitor characteristics in the postnatal chick and rat retinae, their progenitor-like role in developed human retina is unknown. We first reported the Müller glial characteristics of the spontaneously immortalized human cell line MIO-M1, but recently we have derived similar cell lines from the neural retina of several adult eye donors. Since immortalization is one of the main properties of stem cells, we investigated whether these cells expressed stem cell markers. Cells were grown as adherent monolayers, responded to epidermal growth factor, and could be expanded indefinitely without growth factors under normal culture conditions. They could be frozen and thawed without losing their characteristics. In the presence of extracellular matrix and fibroblast growth factor-2 or retinoic acid, they acquired neural morphology, formed neurospheres, and expressed neural stem cell markers including betaIII tubulin, Sox2, Pax6, Chx10, and Notch 1. They also expressed markers of postmitotic retinal neurons, including peripherin, recoverin, calretinin, S-opsin, and Brn3. When grafted into the subretinal space of dystrophic Royal College of Surgeons rats or neonatal Lister hooded rats, immortalized cells migrated into the retina, where they expressed various markers of retinal neurons. These observations indicate that adult human neural retina harbors a population of cells that express both Müller glial and stem cell markers and suggest that these cells may have potential use for cell-based therapies to restore retinal function. Disclosure of potential conflicts of interest is found at the end of this article.

Development and pathology of the hyaloid, choroidal and retinal vasculature

During embryogenesis, the development and differentiation of the eye requires the concomitant formation of the neural/glial elements along with a dense vascular network. The adult neural retina is supported by two distinct vascular systems, the proper retinal vessels and the choroidal vessels. The two beds differ not only in their pattern of embryonic differentiation, but also in their function in the adult organism. The retinal vasculature has barrier properties similar to those observed in the brain, whereas the choroidal vessels display a highly fenestrated phenotype. The hyaloid vasculature is a transient embryonic vascular bed which is complete at birth in mammals and regresses contemporaneously with the formation of the retinal vasculature. The dependence of the retina on its blood supply makes it highly vulnerable to any vascular changes and indeed ocular diseases, such as proliferative retinopathy, age-related macular degeneration and the hyperplastic primary vitreous, which are associated with abnormalities of the different vascular beds of the eye. A number of factors have been implicated in developmental and pathological changes in vessel formation and regression, including fibroblast growth factors, platelet-derived endothelial growth factor and vascular endothelial growth factor, among others. The purpose of this review is to describe and discuss new insights into the mechanisms and molecular cues involved in the development of the normal and pathological vascular systems of the eye. The characterization of the molecules and cell-cell interactions involved in the formation, stabilization and regression of new vessels has led to the identification of potential control points for therapeutic intervention.

Calmodulin gene expression in the neural retina of the adult rat

Calmodulin (CaM) mRNAs are expressed with low abundancy in the adult rat neural retina. However, when digoxigenin (DIG)-labeled cRNA probes specific for each CaM mRNA population were hybridized at slightly alkaline pH (pH 8.0), the widespread distribution of CaM mRNA-expressing cells was revealed, with similar abundance for all three CaM genes. The CaM genes displayed a uniquely similar, layer-specific expression throughout the retina, and no significant differences were found in the distribution patterns of the CaM mRNA populations or the labeled cell types. The strongest signal for all CaM mRNAs was demonstrated in the ganglion cell layer and the inner nuclear layer, where the highest signal intensity was found within the inner sublamina. Similarly intermediate signal intensities for all CaM genes were detected in the inner and outer plexiform layers, within the vicinity of the outer limiting membrane and in the retinal pigment epithelium. A very low specific signal was characteristic in the outer nuclear layer and the photoreceptor inner segment layer, while no specific hybridization signal was observed in the photoreceptor outer segment layer. In summary, all CaM genes exhibited a similar and a characteristically layer-specific expression pattern in the adult rat retina.

Characterization of Wnt signaling components and activation of the Wnt canonical pathway in the murine retina.

The neuroepithelial layer of the developing eyecup contains multipotential precursor cells that give rise to all of the neurons and the one glial cell type present in the adult retina. Patterning within the retinal neuroepithelium is regulated by cell intrinsic as well as cell extrinsic mechanisms. Although the identity of some of the signaling molecules that regulate retinal development is known, the function of many others, especially members of the Wnt family, has yet to be characterized in the context of retinal development. We undertook a comprehensive in situ hybridization analysis to examine the expression of Wnt pathway components in the developing and adult mouse neural retina. Our findings confirm and extend previous expression studies in mice and other vertebrates, as we show that Wnt-3, -5a, -5b, and -7b are expressed in the neural retina and that there is a dynamic pattern of Wnt receptor (Mouse frizzled [Mfz]) and Wnt antagonist (Secreted-frizzled-related protein [Sfrp]) gene expression in the embryonic and perinatal neural retina. Moreover, we show that Wnt-13 is expressed in the pigment epithelium overlying the distal part of the eyecup and the ciliary margin and that Mfz-4, -6, and -7 are expressed in different regions within the ciliary margin. To determine where activation of canonical Wnt signaling is occurring in the retina, we examined reporter gene expression in TCF/Lef-LacZ mice and we demonstrate that the highest levels of beta-gal activity are found in the ciliary margin, adjacent to and within the Wnt-13 expression domain, implicating Wnt-13 signaling in the development of the ciliary margin and its derivatives.

Retinal remodeling in inherited photoreceptor degenerations.

Photoreceptor degenerations initiated in rods or the retinal pigmented epithelium usually evoke secondary cone death and sensory deafferentation of the surviving neural retina. In the mature central nervous system, deafferentation evokes atrophy and connective re-patterning. It has been assumed that the neural retina does not remodel, and that it is a passive survivor. Screening of advanced stages of human and rodent retinal degenerations with computational molecular phenotyping has exposed a prolonged period of aggressive negative remodeling in which neurons migrate along aberrant glial columns and seals, restructuring the adult neural retina (1). Many neurons die, but survivors rewire the remnant inner plexiform layer (IPL), forming thousands of novel ectopic microneuromas in the remnant inner nuclear layer (INL). Bipolar and amacrine cells engage in new circuits that are most likely corruptive. Remodeling in human and rodent retinas emerges regardless of the molecular defects that initially trigger retinal degenerations. Although remodeling may constrain therapeutic intervals for molecular, cellular, or bionic rescue, the exposure of intrinsic retinal remodeling by the removal of sensory control in retinal degenerations suggests that neuronal organization in the normal retina may be more plastic than previously believed.

Distribution of Lutein and Zeaxanthin Stereoisomers in the Human Retina

The distribution of macular pigment stereoisomers in the human retina has been mapped and a pathway to account for the presence of the non-dietary carotenoid,meso-zeaxanthin, is proposed. Adult neural retinas were cut into three concentric areas centered on the fovea, and the extracted carotenoids were analysed and purified by high-performance liquid chromatography. The dicarbamate or dibenzoate derivatives of the collected zeaxanthin fractions for each tissue sample were further analysed by HPLC to determine their stereoisomer composition. Whole retinas from infant eyes were similarly analysed. The results show that, relative to zeaxanthin, the concentration of lutein in the adult neural retina increases with radial distance from the fovea while that ofmeso-zeaxanthin decreases. Infant retinas were found to have more lutein and lessmeso-zeaxanthin, relative to zeaxanthin, than adult retinas. Small quantities of (3S, 3′S)-zeaxanthin were also found in the adult retina, particularly in the macula. It is proposed that lutein and zeaxanthin are transported into an individual's retina in the same proportions found in his or her blood serum. Some of the lutein is then converted intomeso-zeaxanthin, primarily in the macula, by a mechanism which is less developed in infants than adults.

Retinal dysplasia and degeneration in RARbeta2/RARgamma2 compound mutant mice.

The eye is the organ whose development is the most frequently altered in response to maternal vitamin A deficiency [VAD; Warkany, J. and Schraffenberger, S. (1946). Archs Ophthalmol. 35, 150-169]. With the exception of prenatal retinal dysplasia, all the ocular abnormalities of the fetal VAD syndrome are recapitulated in mouse mutants lacking either RARalpha and RARbeta2, RARalpha and RARgamma, RARgamma and RARbeta2, or RXRalpha [Lohnes, D., Mark, M., Mendelsohn, C., Dolle, P., Dierich, A., Gorry, P., Gansmuller, A. and Chambon, P. (1994) Development 120, 2723-2748; Mendelsohn, C., Lohnes, D., Décimo, D., Lufkin, T., LeMeur, M., Chambon, P. and Mark, M. (1994) Development 120, 2749-2771; Kastner, P., Grondona, J. Mark, M., Gansmuller, A., LeMeur, M., Décimo, D., Vonesch, J.L., Dollé, P. and Chambon, P. (1994) Cell 78, 987-1003], thus demonstrating that retinoic acid (RA) is the active vitamin A metabolite during prenatal eye morphogenesis. Whether retinoids are also involved in postnatal eye development could not be investigated, as VAD newborns are not viable and the above RAR double null mutants and RXRalpha null mutants died in utero or at birth. We report here the generation of viable RARbeta2/RARgamma2 double null mutant mice, which exhibit several eye defects. The neural retina of newborn RARbeta2gamma2 mutants is thinner than normal due to a reduced rate of cell proliferation, and from day 4 shows multiple foci of disorganization of its layers. These RARbeta2gamma2 mutants represent the first genetically characterized model of retinal dysplasia and their phenotype demonstrates that RARs, and therefore RA, are required for retinal histogenesis. The RARbeta2gamma2 retinal pigment epithelium (RPE) cells display histological and/or ultrastructural alterations and/or fail to express cellular retinol binding protein I (CRBPI). Taken altogether, the early onset of the RPE histological defects and their striking colocalisation with areas of the neural retina displaying a faulty laminar organization, a reduced neuroblastic proliferation, and a lack of photoreceptor differentiation and/or increased apoptosis, make the RPE a likely target tissue of the RARbeta2gamma2 double null mutation. A degeneration of the adult neural retina, which may similarly be secondary to a defective RPE, is also observed in these mutants, thus demonstrating an essential role of RA in the survival of retinal cells. Moreover, all RARbeta2gamma2 mutants display defects in structures derived from the periocular mesenchyme including local agenesis of the choroid and of the sclera, small eyelids, and a persistence of the primary mesenchymal vitreous body. A majority of the RARbeta2 single null mutants also exhibit this latter defect, thus demonstrating that the RARbeta2 isoform plays a unique role in the formation of the definitive vitreous body.

Diffusible rod-promoting signals in the developing rat retina.

We previously developed a reaggregate cell culture system in which embryonic rat retinal neuroepithelial cells proliferate and give rise to opsin-expressing rod photoreceptor cells (rods) on the same schedule in vitro as they do in vivo. We showed that the proportion of neuroepithelial cells in the embryonic day 15 (E15) retina that differentiated into opsin+ rods after 5-6 days in such cultures increased by approximately 40-fold when the E15 cells were cultured in the presence of an excess of postnatal day 1 (P1) neural retinal cells. In the present study, we have further analyzed this rod-promoting activity of neonatal neural retinal cells. We show that the activity is mediated by a diffusible signal(s) that seems to act over a relatively short distance. Whereas neonatal (P1-P3) neural retina has rod-promoting activity, E15 and adult neural retina, neonatal thymus, cerebrum and cerebellum do not. Finally, we show that neonatal neural retina promotes rod but not amacrine cell development.