The long noncoding RNA Vax2os1 controls the cell cycle progression of photoreceptor progenitors in the mouse retina

Long noncoding RNAs (lncRNAs) are emerging as regulators of many basic cellular pathways. Several lncRNAs are selectively expressed in the developing retina, although little is known about their functional role in this tissue. Vax2os1 is a retina-specific lncRNA whose expression is restricted to the mouse ventral retina. Here we demonstrate that spatiotemporal misexpression of Vax2os1 determines cell cycle alterations in photoreceptor progenitor cells. In particular, the overexpression of Vax2os1 in the developing early postnatal mouse retina causes an impaired cell cycle progression of photoreceptor progenitors toward their final committed fate and a consequent delay of their differentiation processes. At later developmental stages, this perturbation is accompanied by an increase of apoptotic events in the photoreceptor cell layer, in comparison with control retinas, without affecting the proper cell layering in the adult retina. Similar results are observed in mouse photoreceptor-derived 661W cells in which Vax2os1 overexpression results in an impairment of the cell cycle progression rate and cell differentiation. Based on these results, we conclude that Vax2os1 is involved in the control of cell cycle progression of photoreceptor progenitor cells in the ventral retina. Therefore, we propose Vax2os1 as the first example of lncRNA that acts as a cell cycle regulator in the mammalian retina during development.     

Time course of opsin expression in developing rod photoreceptors

We have investigated the time course of rod photoreceptor determination in the goldfish retina. Rod precursor cells located in the outer nuclear layer of the mature retina continuously generate rod photoreceptors. In this study, we asked when rod precursor cells begin to express opsin, which would signal their commitment to the rod pathway of differentiation. There are three possibilities: a rod precursor could express opsin while still mitotic, at or shortly after the terminal mitosis but before differentiation, or during differentiation. We used immunocytochemistry with antibodies against bromodeoxyuridine, BrdU (a thymidine analogue) and against opsin to determine when during the mitotic history of a cell the expression of opsin first occurred, taking a double labelled cell to be evidence of commitment to the rod cell fate. We found that the first double labelled cells appeared at 4 days after BrdU injection. The number of double labelled cells increased to peak at 10 days, and then fell. These results support the hypothesis that dividing rod precursor cells are probably multipotent stem cells not committed to the rod cell fate.     

Three distinct roles for notch in Drosophila R7 photoreceptor specification

Receptor tyrosine kinases (RTKs) and Notch (N) proteins are different types of transmembrane receptors that transduce extracellular signals and control cell fate. Here we examine cell fate specification in the Drosophila retina and ask how N acts together with the RTKs Sevenless (Sev) and the EGF receptor (DER) to specify the R7 photoreceptor. The retina is composed of many hundred ommatidia, each of which grows by recruiting surrounding, undifferentiated cells and directing them to particular fates. The R7 photoreceptor derives from a cohort of three cells that are incorporated together following specification of the R2-R5 and R8 photoreceptors. Two cells of the cohort are specified as the R1/6 photoreceptor type by DER activation. These cells then activate N in the third cell (the R7 precursor). By manipulation of N and RTK signaling in diverse combinations we establish three roles for N in specifying the R7 fate. The first role is to impose a block to photoreceptor differentiation; a block that DER activation cannot overcome. The second role, paradoxically, is to negate the first; Notch activation up-regulates Sev expression, enabling the presumptive R7 cell to receive an RTK signal from R8 that can override the block. The third role is to specify the cell as an R7 rather than an R1/6 once RTK signaling has specified the cells as a photoreceptor. We speculate why N acts both to block and to facilitate photoreceptor differentiation, and provide a model for how N and RTK signaling act combinatorially to specify the R1/6 and R7 photoreceptors as well as the surrounding non-neuronal cone cells.     

Notch 1 inhibits photoreceptor production in the developing mammalian retina

The transmembrane receptor Notch1 plays a role in development and homeostasis in vertebrates and invertebrates. The mammalian retina is an excellent tissue in which to dissect the precise role of Notch signaling in regulating cell fate and proliferation. However, a systematic analysis has been limited by the early embryonic lethality of Notch1-null mice. Here, Notch1 was conditionally removed from the murine retina either early or late in development. Removal of Notch1 early led to a reduction in the size of the retina as well as aberrant morphology. A decrease in the number of progenitor cells and premature neurogenesis accounted for the reduction in size. Unexpectedly, ablation of Notch1 in early progenitor cells led to enhanced cone photoreceptor production, and ablation of Notch1 at later points led to an almost exclusive production of rod photoreceptor cells. These data suggest that Notch1 not only maintains the progenitor state, but is required to inhibit the photoreceptor fate. These cone enriched mutant mice should prove to be a valuable resource for the study of this relatively rare mammalian photoreceptor cell type.     

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.     

NeuroD regulates proliferation of photoreceptor progenitors in the retina of the zebrafish

neuroD is a member of the family of proneural genes, which function to regulate the cell cycle, cell fate determination and cellular differentiation. In the retinas of larval and adult teleosts, neuroD is expressed in two populations of post-mitotic cells, a subset of amacrine cells and nascent cone photoreceptors, and proliferating cells in the lineages that give rise exclusively to rod and cone photoreceptors. Based on previous studies of NeuroD function in vitro and the cellular pattern of neuroD expression in the zebrafish retina, we hypothesized that within the mitotic photoreceptor lineages NeuroD selectively regulates aspects of the cell cycle. To test this hypothesis, gain and loss-of-function approaches were employed, relying on the inducible expression of a NeuroD^EGFP fusion protein and morpholino oligonucleotides to inhibit protein translation, respectively. Conditional expression of neuroD causes cells to withdraw from the cell cycle, upregulate the expression of the cell cycle inhibitors, p27 and p57, and downregulate the cell cycle progression factors, Cyclin B1, Cyclin D1, and Cyclin E2. In the absence of NeuroD, cells specific for the rod and cone photoreceptor lineage fail to exit the cell cycle, and the number of cells expressing Cyclin D1 is increased. When expression is ectopically induced in multipotent progenitors, neuroD promotes the genesis of rod photoreceptors and inhibits the genesis of Müller glia. These data show that in the teleost retina NeuroD plays a fundamental role in photoreceptor genesis by regulating mechanisms that promote rod and cone progenitors to withdraw from the cell cycle. This is the first in vivo demonstration in the retina of cell cycle regulation by NeuroD.     

Sequential genesis and determination of cone and rod photoreceptors in Xenopus

In this study, we addressed the temporal sequence of photoreceptor fate determination in Xenopus laevis by examining a number of key events during early cone and rod development. We compared the relative timing and spatial pattern of cone and rod specification using a number of cell type-specific markers, including probes to a long wavelength-sensitive opsin which is expressed by the major cone subtype. Our results show that cones are initially more numerous, and can arise in less mature regions of the retina than rods, although both types of photoreceptors begin to express their respective opsins at about the same time. We applied these markers to an assay of cellular determination to identify the stages of embryonic development at which the earliest photoreceptor fates are induced in vivo. The relative birth order of the major cone and rod subtypes was revealed by simultaneous labeling with markers of cell proliferation and terminal differentiation. Although there is much temporal overlap between the periods of cone and rod genesis and determination in Xenopus, we could discern that the earliest cones are both born and determined before the first rods. Thus, even in the rapidly developing retina of Xenopus, photoreceptors achieve their identities in a sequential fashion, suggesting that the inductive cues which determine specific photoreceptor fates may also arise sequentially during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 227–244, 1998     

A temporally regulated, diffusible activity is required for rod photoreceptor development in vitro.

The retina is a relatively simple and well-characterized CNS structure in which cell-cell interactions have been hypothesized to influence cell type determination. By manipulating cell density in serum-free cultures we show that rat rod photoreceptor development requires a diffusible activity produced by neonatal retinal cells. This effect is not mediated by changes in cell survival or mitosis. Production of the rod promoting activity varies with developmental stage and is temporally correlated with the timing of rod generation in vivo. In low density cultures, which do not support rod development, an increased fraction of cells stain with an antibody specific for another retinal neuron, the bipolar cell. Thus, the diffusible rod promoting activity may influence cell fate determination, and not only terminal differentiation. These results provide an approach for the molecular characterization of developmentally important signals in the vertebrate retina.     

Cell-fate determination in the developing Drosophila eye: role of the rough gene.

The homeobox-gene rough is required in photoreceptor cells R2 and R5 for normal ommatidial assembly in the developing Drosophila eye. We have used several cell-type-specific markers and double mutant combinations to analyze cell-fate determination in rough. We show that the cells that would normally become R2 and/or R5 express a marker, a lacZ insertion in the seven-up (svp) gene, which is indicative of the R1/3/4/6 cell fate. In addition, the analysis of mitotically induced svp,ro double mutant clones in the eye indicates that in rough all outer photoreceptors are under the genetic control of the svp gene. These results show that, in the absence of rough function, R2 and R5 fail to be correctly determined and appear to be transformed into cells of the R3/4/1/6 subtype. This transformation and the subsequent developmental defects do not preclude the recruitment of R7 cells. However, the presence of ommatidia containing more than one R7 and/or R8 cell in rough implies a complex network of cellular interactions underlying cell-fate determination in the Drosophila retina.     

Functional roles of Otx2 transcription factor in postnatal mouse retinal development

We previously reported that Otx2 is essential for photoreceptor cell fate determination; however, the functional role of Otx2 in postnatal retinal development is still unclear although it has been reported to be expressed in retinal bipolar cells and photoreceptors at postnatal stages. In this study, we first examined the roles of Otx2 in the terminal differentiation of photoreceptors by analyzing Otx2; Crx double-knockout mice. In Otx2+/-; Crx-/- retinas, photoreceptor degeneration and downregulation of photoreceptor-specific genes were much more prominent than in Crx-/- retinas, suggesting that Otx2 has a role in the terminal differentiation of the photoreceptors. Moreover, bipolar cells decreased in the Otx2+/-; Crx-/- retina, suggesting that Otx2 is also involved in retinal bipolar-cell development. To further investigate the role of Otx2 in bipolar-cell development, we generated a postnatal bipolar-cell-specific Otx2 conditional-knockout mouse line. Immunohistochemical analysis of this line showed that the expression of protein kinase C, a marker of mature bipolar cells, was significantly downregulated in the retina. Electroretinograms revealed that the electrophysiological function of retinal bipolar cells was impaired as a result of Otx2 ablation. These data suggest that Otx2 plays a functional role in the maturation of retinal photoreceptor and bipolar cells.     

Overexpression of FGF-2 alters cell fate specification in the developing retina of Xenopus laevis

The developing vertebrate retina produces appropriate ratios of seven phenotypically and functionally distinct cell types. Retinal progenitors remain multipotent up until the last cell division, favoring the idea that extrinsic cues direct cell fate. We demonstrated previously that fibroblast growth factor (FGF) receptors are necessary for transduction of signals in the developing Xenopus retina that bias cell fate decisions (S. McFarlane et al., 1998, Development 125, 3967-3975). However, the precise identity of the signal remains unknown. To test whether an FGF signal is sufficient to influence cell fate choices in the developing retina, FGF-2 was overexpressed in Xenopus retinal precursors by injecting, at the embryonic 16-cell stage, a cDNA plasmid encoding FGF-2 into cells fated to form the retina. We found that FGF-2 overexpression in retinal precursors altered the relative numbers of transgene-expressing retinal ganglion cells (RGC) and Müller glia; RGCs were increased by 35% and Müller glia decreased by 50%. In contrast, the proportion of retinal precursors that became photoreceptors was unchanged. Within the photoreceptor population, however, we found a twofold increase in rod photoreceptors at the expense of cone photoreceptors. These data are consistent with an endogenous FGF signal influencing cell fate decisions in the developing vertebrate retina.     

Cellular competence plays a role in photoreceptor differentiation in the developing Xenopus retina

Factors in the environment appear to be responsible for inducing many of the cell fates in the retina, including, for example, photoreceptors. Further, there is a conserved order of histogenesis in the vertebrate retina, suggesting that a temporal mechanism interacts in the control of cellular determination. The temporal mechanism involved could result from different inducing signals being released at different times. Alternatively, the inducing signals might be present at many stages, but an autonomous clock could regulate the competence of cells to respond to them. To differentiate between these mechanisms, cells from young embryonic retinas were dissociated and grown together with those from older embryos, and the timing of photoreceptor determination assayed. Young cells appeared uninfluenced by older cells, expressing photoreceptor markers on the same time schedule as when cultured alone. A similar result was obtained when the heterochronic mixing was done in vivo by grafting a small plug of optic vesicle from younger embryos into older hosts. Even the graft cells at the immediate margin of the transplant failed to express photoreceptor markers earlier than normal, despite their being in contact with older, strongly expressing host cells. We conclude that retinal progenitors intrinsically acquire the ability to respond to photoreceptor-inducing cues by a mechanism that runs on a cell autonomous schedule, and that the conserved order of histogenesis is based in part on this competence clock.     

Effects of alkaline phosphatase inhibitors on chick neural retinal cell differentiation in vitro: ultracytochemical studies

In the matured chick retina, alkaline phosphatase (ALPase) activity is specifically localized in the outer plexiform layer and in horizontal and Müller cells. In the developing chick retina, ALPase activity is first recognized in growing neurites from horizontal cells during the 13th day of incubation, when synaptogenesis begins in the outer plexiform layer. Intraocular administration of ALPase inhibitors to developing chick embryos resulted in developmental disturbances in differentiation of the outer plexiform layer and also of photoreceptor cells. We have now extended these studies to an in vitro system. ALPase activity was studied by ultracytochemistry in cultured retinal cells from chick embryos, and the effects of specific ALPase inhibitor on retinal development were also analyzed. Two cell types showed intense ALPase activity: 1) flat glial cell, which formed a multi-layered epithelial sheet and 2) neuronal cell found within cell aggregates. Some cellular processes forming a neuropil-like structure within these aggregates also showed ALPase activity. When the ALPase inhibitor bromotetramisole was present in the culture medium, there was delay in aggregate formation and the development of neuritic processes was also affected. Moreover, this treatment also caused a considerable reduction in the number of photoreceptor cells present in the culture. The present results indicate that ALPase activity plays a significant role in retinal cell differentiation.     

Differential CRX and OTX2 expression in human retina and retinoblastoma

The histogenesis of retinoblastoma tumors remains controversial, with the cell-of-origin variably proposed to be an uncommitted retinal progenitor cell, a bipotent committed cell, or a cell committed to a specific lineage. Here, we examine the expression of two members of the orthodenticle family implicated in photoreceptor and bipolar cell differentiation, cone-rod homeobox, CRX, and orthodenticle homeobox 2, OTX2, in normal human retina, retinoblastoma cell lines and retinoblastoma tumors. We show that CRX and OTX2 have distinct expression profiles in the developing human retina, with CRX first expressed in proliferating cells and cells committed to the bipolar lineage, and OTX2 first appearing in the photoreceptor lineage. In the mature retina, CRX levels are highest in photoreceptor cells whereas OTX2 is preferentially found in bipolar cells and in the retinal pigmented epithelium. Both CRX and OTX2 are widely expressed in retinoblastoma cell lines and in retinoblastoma tumors, although CRX is more abundant than OTX2 in the differentiated elements of retinoblastoma tumors such as large rosettes, Flexner-Wintersteiner rosettes and fleurettes. Widespread expression of CRX and OTX2 in retinoblastoma tumors and cell lines suggests a close link between the cell-of-origin of retinoblastoma tumors and cells expressing CRX and OTX2.     

senseless repression of rough is required for R8 photoreceptor differentiation in the developing Drosophila eye.

An outstanding model to study how neurons differentiate from among a field of equipotent undifferentiated cells is the process of R8 photoreceptor differentiation during Drosophila eye development. We show that in senseless mutant tissue, R8 differentiation fails and the presumptive R8 cell adopts the R2/R5 fate. We identify senseless repression of rough in R8 as an essential mechanism of R8 cell fate determination and demonstrate that misexpression of senseless in non-R8 photoreceptors results in repression of rough and induction of the R8 fate. Surprisingly, there is no loss of ommatidial clusters in senseless mutant tissue and all outer photoreceptor subtypes can be recruited, suggesting that other photoreceptors can substitute for R8 to initiate recruitment and that R8-specific signaling is not required for outer photoreceptor subtype assignment. A genetic model of R8 differentiation is presented.     

Distribution of melatonin MT1 receptor immunoreactivity in human retina.

Melatonin is synthesized in the pineal gland and retina during the night. Retinal melatonin is believed to be involved in local cellular modulation and in regulation of light-induced entrainment of circadian rhythms. The present study provides the first immunohistochemical evidence for the localization of melatonin 1a-receptor (MT1) in human retina of aged subjects. Ganglion, amacrine, and photoreceptor cells expressed MT1. In addition, MT1 immunoreactivity was localized to cell processes in the inner plexiform layer and to central vessels of the retina, as well as to retinal vessels but not to ciliary or choroidal vessels. These results support a variety of cellular and vascular effects of melatonin in the human retina. Preliminary evidence from patients with Alzheimer's disease (AD) revealed increased MT1 immunoreactivity in ganglion and amacrine cells, as well as in vessels. In AD cases photoreceptor cells were degenerated and showed low MT1 expression.     

Cellular and ultrastructural features of the adult and the embryonic eye in the marine gastropod,Ilyanassa obsoleta

Light and electron microscopic techniques show that the eye of the marine prosobranch gastropod, Ilyanassa obsoleta, is composed of an optic cavity, lens, cornea, retina, and neuropile, and is surrounded by a connective tissue capsule. The adult retina is a columnar epithelium containing three morphologically distinct cell types: photoreceptor, pigmented, and ciliated cells. The retina is continuous anteriorly with a cuboidal corneal epithelium. The neuropile, located immediately behind the retina, is composed of photoreceptor cell axons, accessory neurons, and their neurites. The embryonic eye is formed from surface ectoderm, which sinks inward as a pigmented cellular mass. At this time, the eye primordium already contains presumptive photoreceptor cells, pigmented retinal cells, and corneal cells. Several days later, just before hatching, the embryonic eye remains in intimate contact with the cerebral ganglion. It has no ciliated retinal cells, neuropile, optic nerve, or connective tissue capsule and its photoreceptor cells lack the electron-lucent vesicles and multivesicular bodies of adult photoreceptor cells. As the eye and the cerebral ganglion grow apart, the optic nerve, neuropile, and connective tissue capsule develop.