miR‐181a/b control the assembly of visual circuitry by regulating retinal axon specification and growth

Connectivity and function of neuronal circuitry require the correct specification and growth of axons and dendrites. Here, we identify the microRNAs miR‐181a and miR‐181b as key regulators of retinal axon specification and growth. Loss of miR‐181a/b in medaka fish (Oryzias latipes) failed to consolidate amacrine cell processes into axons and delayed the growth of retinal ganglion cell (RGC) axons. These alterations were accompanied by defects in visual connectivity and function. We demonstrated that miR‐181a/b exert these actions through negative modulation of MAPK/ERK signaling that in turn leads to RhoA reduction and proper neuritogenesis in both amacrine cells and RGCs via local cytoskeletal rearrangement. Our results identify a new pathway for axon specification and growth unraveling a crucial role of miR‐181a/b in the proper establishment of visual system connectivity and function. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1252–1267, 2015     

Relationships between complex Delta expression and the specification of retinal cell fates during Drosophila eye development

Analysis of retinal development in Delta (Dl) temperature-sensitive mutants reveals requirements for Delta function in the specification of all retinal cells, including photoreceptors, cone cells, pigment cells and cells that make up interommatidial bristles. In situ hybridization and immunohistochemistry indicate that Delta is expressed dynamically during the specification of different cell types. Comparisons of Delta expression patterns with developmental defects in Dl mutants implies that Delta functions in a cell-nonautonomous manner in the specification of photoreceptors. Delta protein resides predominantly in subcellular vesicles located primarily at the apical ends of developing retinal cells. Localization of Delta protein in Dl and shibire^tsl mutants implies that Delta is targeted to the cell surface, but is efficiently removed via endocytosis, resulting in vesicular accumulation.     

Genetic control of retinal specification and determination in Drosophila

The Drosophila compound eye has long served as an outstanding model system to study many processes, including cell fate specification, cell division, cell growth and cell death. In addition, exploring the molecular basis of eye specification in Drosophila has identified a set of nuclear factors that trigger the conversion of a group of multipotent epithelial cells into eye primordia. These nuclear factors act in complex networks to regulate retinal specification and appear to be conserved throughout phylogeny. Finally, evidence suggests that these nuclear networks have been co-opted to specify cell fates in other tissues. We review the latest developments in the field of retinal specification in Drosophila and discuss several future directions that remain open for investigation.     

Developmental wave of Brn3b expression leading to RGC fate specification is synergistically maintained by miR‐23a and miR‐374

Differential regulation of Brn3b is essential for the Retinal Ganglion Cell (RGC) development in the two phases of retinal histogenesis. This biphasic Brn3b regulation is required first, during early retinal histogenesis for RGC fate specification and secondly, during late histogenesis, where Brn3b is needed for RGC axon guidance and survival. Here, we have looked into how the regulation of Brn3b at these two stages happens. We identified two miRNAs, miR‐23a and miR‐374, as regulators of Brn3b expression, during the early stage of RGC development. Temporal expression pattern of miR‐23a during E10–19, PN1–7, and adult retina revealed an inverse relation with Brn3b expression. Though miR‐374 did not show such a pattern, its co‐expression with miR‐23a evidently inhibited Brn3b. We further substantiated these findings by ex vivo overexpression of these miRNAs in E14 mice retina and found that miR‐23a and miR‐374 together brings about a change in Brn3b expression pattern in ganglion cell layer (GCL) of the developing retina. From our results, it appears that the combined expression of these miRNAs could be regulating the timing of the wave of Brn3b expression required for early ganglion cell fate specification and later for its survival and maturation into RGCs. Taken together, here we provide convincing evidences for the existence of a co‐ordinated mechanism by miRNAs to down regulate Brn3b that will ultimately regulate the development of RGCs from their precursors. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1155–1171, 2014     

Complete reconstruction of the retinal laminar structure from a cultured retinal pigment epithelium is triggered by altered tissue interaction and promoted by overlaid extracellular matrices

The retina regenerates from retinal pigment epithelial (RPE) cells by transdifferentiation in the adult newt and Xenopus laevis when it is surgically removed. This was studied under a novel culture condition, and we succeeded, for the first time, in developing a complete retinal laminar structure from a single epithelial sheet of RPE. We cultured a Xenopus RPE monolayer sheet isolated from the choroid on a filter cup with gels overlaid and found that the retinal tissue structure differentiated with all retinal layers present. In the culture, RPE cells isolated themselves from the culture substratum (filter membrane), migrated, and reattached to the overlaid gel, on which they initiated transdifferentiation. This was exactly the same as observed during in vivo retina regeneration of X. laevis. In contrast, when RPE monolayers were cultured similarly without isolation from the choroid, RPE cells proliferated, but remained pigmented instead of transdifferentiating, indicating that alteration in tissue interaction triggers transdifferentiation. We then examined under the conventional tissue culture condition whether altered RPE‐choroid interaction induces Pax6 expression. Pax6 was upregulated in RPE cells soon after they were removed from the choroid, and this expression was not dependent of FGF2. FGF2 administration was needed for RPE cells to maintain Pax6 expression. From the present results, in addition to our previous ones, we propose a two‐step mechanism of transdifferentiation: the first step is a reversible process and is initiated by the alteration of the cell‐extracellular matrix and/or cell–cell interaction followed by Pax6 upregulation. FGF2 plays a key role in driving RPE cells into the second step, during which they differentiate into retinal stem cells. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Stem‐cell‐based therapies for retinal degenerative diseases: Current challenges in the establishment of new treatment strategies

Various advances have been made in the treatment of retinal diseases, including new treatment strategies and innovations in surgical devices. However, the treatment of degenerative retinal diseases, such as retinitis pigmentosa (RP) and age‐related macular degeneration (AMD), continues to pose a significant challenge. In this review, we focus on the use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to treat retinal diseases by harnessing the ability of stem cells to differentiate into different body tissues. The retina is a tissue specialized for light sensing, and its degradation leads to vision loss. As part of the central nervous system, the retina has very low regenerative capability, and therefore, treatment options are limited once it degenerates. Nevertheless, innovations in methods to induce the generation of retinal cells and tissues from ESCs/iPSCs enable the development of novel approaches for these irreversible diseases. Here we review some historical background and current clinical trials involving the use of stem‐cell‐derived retinal pigment epithelial cells for AMD treatment and stem cell‐derived retinal cells/tissues for RP therapy. Finally, we discuss our future vision of regenerative treatment for retinal diseases with a partial focus on our studies and introduce other interesting approaches for restoring vision.     

Lgr5+ amacrine cells possess regenerative potential in the retina of adult mice

Current knowledge indicates that the adult mammalian retina lacks regenerative capacity. Here, we show that the adult stem cell marker, leucine‐rich repeat‐containing G‐protein‐coupled receptor 5 (Lgr5), is expressed in the retina of adult mice. Lgr5⁺ cells are generated at late stages of retinal development and exhibit properties of differentiated amacrine interneurons (amacrine cells). Nevertheless, Lgr5⁺ amacrine cells contribute to regeneration of new retinal cells in the adult stage. The generation of new retinal cells, including retinal neurons and Müller glia from Lgr5⁺ amacrine cells, begins in early adulthood and continues as the animal ages. Together, these findings suggest that the mammalian retina is not devoid of regeneration as previously thought. It is rather dynamic, and Lgr5⁺ amacrine cells function as an endogenous regenerative source. The identification of such cells in the mammalian retina may provide new insights into neuronal regeneration and point to therapeutic opportunities for age‐related retinal degenerative diseases.     

The zebrafish young mutation acts non-cell-autonomously to uncouple differentiation from specification for all retinal cells

Embryos from mutagenized zebrafish were screened for disruptions in retinal lamination to identify factors involved in vertebrate retinal cell specification and differentiation. Two alleles of a recessive mutation, young, were isolated in which final differentiation and normal lamination of retinal cells were blocked. Early aspects of retinogenesis including the specification of cells along the inner optic cup as retinal tissue, polarity of the retinal neuroepithelium, and confinement of cell divisions to the apical pigmented epithelial boarder were normal in young mutants. BrdU incorporation experiments showed that the initiation and pattern of cell cycle withdrawal across the retina was comparable to wild-type siblings; however, this process took longer in the mutant. Analysis of early markers for cell type differentiation revealed that each of the major classes of retinal neurons, as well as non-neural Müller glial cells, are specified in young embryos. However, the retinal cells fail to elaborate morphological specializations, and analysis of late cell-type-specific markers suggests that the retinal cells were inhibited from fully differentiating. Other regions of the nervous system showed no obvious defects in young mutants. Mosaic analysis demonstrated that the young mutation acts non-cell-autonomously within the retina, as final morphological and molecular differentiation was rescued when genetically mutant cells were transplanted into wild-type hosts. Conversely, differentiation was prevented in wild-type cells when placed in young mutant retinas. Mosaic experiments also suggest that young functions at or near the cell surface and is not freely diffusible. We conclude that the young mutation disrupts the post-specification development of all retinal neurons and glia cells.