Understanding ERM proteins--the awesome power of genetics finally brought to bear.

In epithelial cells, the Ezrin, Radixin and Moesin (ERM) proteins are involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion and motility, and modulation of signaling pathways. However, discerning the specific cellular roles of ERMs has been complicated by redundancy between these proteins. Recent genetic studies in model organisms have identified unique roles for ERM proteins. These include the regulation of morphogenesis and maintenance of integrity of epithelial cells, stabilization of intercellular junctions, and regulation of the Rho small GTPase. These studies also suggest that ERMs have roles in actomyosin contractility and vesicular trafficking in the apical domain of epithelial cells. Thus, genetic analysis has enhanced our understanding of these widely expressed membrane-associated proteins.

A comprehensive negative regulatory program controlled by Brn3b to ensure ganglion cell specification from multipotential retinal precursors.

The retinal ganglion cells (RGCs) are the sole output neurons in the retina that form the optic nerve and convey light signals detected by photoreceptors to the higher visual system. Their degeneration and damage caused by glaucoma and injury can lead to blindness. During retinogenesis, RGCs are specified from a population of multipotential precursors capable of generating RGC, amacrine, horizontal, and cone cells. How the RGC fate is selected from these multiple neuron fates is unknown at present. Here we show that the previously unsuspected POU domain transcription factor Brn3b (brain-specific homeobox/POU domain protein 3b) plays such a critical role. Loss of Brn3b function in mice leads to misspecification of early RGC precursors as late-born RGC, amacrine, and horizontal cells, whereas misexpressed Brn3b suppresses non-RGC cell fates but promotes the RGC fate. Microarray profiling and other molecular analyses reveal that, in RGC precursors, Brn3b normally represses the expression of a network of retinogenic factor genes involved in fate commitment and differentiation of late-born RGC, amacrine, horizontal, and cone cells. Our data suggest that Brn3b specifies the RGC fate from multipotential precursors not only by promoting RGC differentiation but also by suppressing non-RGC differentiation programs as a safeguard mechanism.

Circadian regulation of iodopsin gene expression in embryonic photoreceptors in retinal cell culture.

A circadian clock regulates a number of diverse physiological functions in the vertebrate eye. In this study, we show that mRNA for the red-sensitive cone pigment, iodopsin, fluctuates with a circadian rhythm in chicken retina. Transcript levels increase in the late afternoon just prior to the time of cone disc shedding. Furthermore, iodopsin mRNA levels are regulated similarly by a circadian oscillator in primary cultures of dispersed embryonic chick retina. Nuclear run-on experiments show that the circadian regulation of iodopsin transcript abundance occurs at the level of gene transcription. Our results provide a demonstration of clock-regulated gene expression in a vertebrate preparation maintained in cell culture.

Rod photoreceptor development in vitro: intrinsic properties of proliferating neuroepithelial cells change as development proceeds in the rat retina.

We describe a reaggregated cell culture system in which retinal neuroepithelial cells from embryonic rats proliferate extensively and give rise to rod photoreceptors on the same schedule in vitro as they do in vivo. Both the proliferative potential of the embryonic neuroepithelial cells and the timing of their differentiation into rods are not changed by the presence of a 50-fold excess of neonatal neural retinal cells, although many more of the embryonic cells develop into rods in these circumstances. In such mixed-age cultures, dividing neonatal cells proliferate much less and give rise to rods much sooner than do dividing embryonic cells, suggesting that the dividing cells at the two ages are intrinsically different. These and other findings suggest that both cell-cell interactions and an intrinsic program in neuroepithelial cells determine cell fate in the developing rat retina.

Two cellular inductions involved in photoreceptor determination in the Xenopus retina.

Cellular determination in the Xenopus retina is not a strict consequence of cell lineage or cell birthdate. This suggests that a retinal cell gets its fate by either local cellular interactions, diffusible factors, or an indeterminate stochastic mechanism. We have performed an in vitro experiment in which cellular contact is controlled to test the first possibility directly. We use these experiments to demonstrate that two cellular inductions are involved in photoreceptor determination in vitro and that these inductions also occur during development in the retina in vivo. The first interaction is responsible for biasing cells toward either a generic photoreceptor or a cone fate, while the second directs cells toward a rod cell fate.

Molecular dynamics of photoreceptor synapse formation in the developing chick retina.

The cellular and molecular mechanisms underlying photoreceptor synaptogenesis are poorly understood. Furthermore, a detailed picture of the molecular composition of photoreceptor synapses, or their subtypes, is not yet available, nor do we know what differences, if any, exist among those subtypes. To address these questions, we investigated temporal and spatial patterns of expression and assembly of photoreceptor presynaptic components during chick embryo retinal development and early posthatched life by using reverse transcriptase polymerase chain reaction (RT-PCR), dissociated retinal cells, laser-capture microdissection (LCM), immunocytochemistry and confocal microscopy. Immunocytochemistry in tissue sections and dissociated cells showed many similarities and few differences in the synaptic composition of rods and cone subtypes, which, however, were found to project to different strata within the outer plexiform layer. A striking finding was the precise timetable of expression of synaptic genes and proteins during synaptogenesis. Although mRNAs for some synaptic molecules appeared as early as embryonic day (ED) 5-8 (the time of inner retina synaptogenesis), others were undetectable before the time of onset of photoreceptor synaptogenesis on ED13, including CAST, rim2, synapsin-2, syntaxin-3, synaptotagmin, glutamate receptors -1, -4, and -5, homer-1 and -2, and tenascin-R. Most synaptic proteins in photoreceptors followed a similar sequence of expression: they were negative or weakly positive before ED13, appeared in inner segments between ED13 and ED15, became subsequently detectable in perinuclear and axonal regions, and by ED18 were assembled into synaptic terminals and became undetectable in the inner segments. The identity of the signals that regulate the coordinated expression of these synaptic components remains to be investigated.

Transient expression of LIM-domain transcription factors is coincident with delayed maturation of photoreceptors in the chicken retina.

In the retina of warm-blooded vertebrates, photoreceptors are specified many days before the onset of synaptogenesis and the expression of photopigments. The factors that regulate the maturation of photoreceptors in the developing retina remain unknown. We report here that photoreceptors transiently express LIM-domain transcription factors during the development of the chicken retina. We examined the differentiation of photoreceptors through the normal course of embryonic development and at the far periphery of the postnatal retina, where the differentiation of photoreceptors is slowed and persists across a spatial gradient. In the embryonic retina, we find visinin-positive photoreceptors that transiently express Islet2 and Lim3 starting at E8 and ending around E15, but persisting in far peripheral regions of the retina through the first 2 weeks of postnatal development. During early stages of photoreceptor maturation, there is coincident and transient expression of the LIM-domain factors with axonin1, a cell surface glycoprotein that is a member of the immunoglobulin superfamily. Coincident with the downregulation of Islet2 and Lim3, we find the upregulation of calbindin, red/green opsin, rhodopsin, and a synaptic marker in the outer plexiform layer (OPL; dystrophin). In the periphery of the postnatal retina, photoreceptors that express Islet2, Lim3, and axonin1 do not overlap with photoreceptors that express calbindin, red/green opsin, rhodopsin, and dystrophin. We propose that Islet2 and Lim3 may promote the expression of genes that are involved in the early stages of differentiation but may suppress the expression of genes that are required in the mature photoreceptors.

Helmsman is expressed in both trachea and photoreceptor development: partial inactivation alters tracheal morphology and visually guided behavior.

We have identified helmsman (hlm), which is expressed in the fruit fly photoreceptor cells during neural network development. Hlm is also expressed in the elongating cells of the embryonic trachea. Both photoreceptor neurons and embryonic trachea cells elongate in precise, targeted growth for cell-to-cell specific recognition. Expression of antisense hlm-interfering RNA during embryogenesis arrests elongation of the developing tracheal cells and blocks maturation. Expression of hlm-interfering RNA during visual system formation results in reduced visual acuity and poor performance in optomotor response, indicative of abnormal neural network development. Hlm is a unique cell surface protein with complement-like protein interaction motifs. We have also cloned hlm from Lucilia cuprina (Australian blowfly), which is approximately 100 million years divergent from Drosophila, and find a remarkable 90% protein identity over the entire 558 amino acid protein. Analysis of the hlm sequence found in other species indicates a significant evolutionary pressure to maintain the hlm protein sequence. Our interpretation is that hlm is involved in cell maturation in both the elongating trachea and elongating photoreceptor cells. Cell adhesion and cell signaling, which are known to use immunoglobulin-like cell adhesion molecules, may use molecular systems analogous to complement to create protein complexes to regulate growth.

Have we achieved a unified model of photoreceptor cell fate specification in vertebrates?

How does a retinal progenitor choose to differentiate as a rod or a cone and, if it becomes a cone, which one of their different subtypes? The mechanisms of photoreceptor cell fate specification and differentiation have been extensively investigated in a variety of animal model systems, including human and non-human primates, rodents (mice and rats), chickens, frogs (Xenopus) and fish. It appears timely to discuss whether it is possible to synthesize the resulting information into a unified model applicable to all vertebrates. In this review we focus on several widely used experimental animal model systems to highlight differences in photoreceptor properties among species, the diversity of developmental strategies and solutions that vertebrates use to create retinas with photoreceptors that are adapted to the visual needs of their species, and the limitations of the methods currently available for the investigation of photoreceptor cell fate specification. Based on these considerations, we conclude that we are not yet ready to construct a unified model of photoreceptor cell fate specification in the developing vertebrate retina.

The developing and evolving retina: using time to organize form.

Evolutionary and other functional accounts of the retina and its normal development highlight different aspects of control of its growth and form than genomic and mechanistic accounts. Discussing examples from opsin expression, developmental regulation of the eye's size and optical quality, regulation of eye size with respect to brain and body size, and the development of the fovea, these different aspects of control are contrasted. Contributions of mouse models, particularly with regard to relative timing of events in different species are reviewed, introducing a Web-based utility for exploration of timing issues (www.translatingtime.net). Variation at the individual level, in early experience, and also across species is an essential source of information to understand normal development and its pathologies.

Molecular mechanisms of vertebrate retina development: implications for ganglion cell and photoreceptor patterning.

Although the neural retina appears as a relatively uniform tissue when viewed from its surface, it is in fact highly patterned along its anterior-posterior and dorso-ventral axes. The question of how and when such patterns arise has been the subject of intensive investigations over several decades. Most studies aimed at understanding retinal pattern formation have used the retinotectal map, the ordered projections of retinal ganglion cells to the brain, as a functional readout of the pattern. However, other cell types are also topographically organized in the retina. The most commonly recognized example of such a topographic cellular organization is the differential distribution of photoreceptor types across the retina. Photoreceptor patterns are highly species-specific and may represent an important adaptation to the visual niche a given species occupies. Nevertheless, few studies have addressed this functional readout of pattern to date and our understanding of its development has remained superficial. Here, we review recent advances in understanding the molecular cascades that control regionalization of the eye anlage, relate these findings to the development of photoreceptor patterns and discuss common and unique strategies involved in both aspects of retinal pattern formation.

Regulation of photoreceptor gene expression by Crx-associated transcription factor network.

Rod and cone photoreceptors in the mammalian retina are special types of neurons that are responsible for phototransduction, the first step of vision. Development and maintenance of photoreceptors require precisely regulated gene expression. This regulation is mediated by a network of photoreceptor transcription factors centered on Crx, an Otx-like homeodomain transcription factor. The cell type (subtype) specificity of this network is governed by factors that are preferentially expressed by rods or cones or both, including the rod-determining factors neural retina leucine zipper protein (Nrl) and the orphan nuclear receptor Nr2e3; and cone-determining factors, mostly nuclear receptor family members. The best-documented of these include thyroid hormone receptor beta2 (Tr beta2), retinoid related orphan receptor Ror beta, and retinoid X receptor Rxr gamma. The appropriate function of this network also depends on general transcription factors and cofactors that are ubiquitously expressed, such as the Sp zinc finger transcription factors and STAGA co-activator complexes. These cell type-specific and general transcription regulators form complex interactomes; mutations that interfere with any of the interactions can cause photoreceptor development defects or degeneration. In this manuscript, we review recent progress on the roles of various photoreceptor transcription factors and interactions in photoreceptor subtype development. We also provide evidence of auto-, para-, and feedback regulation among these factors at the transcriptional level. These protein-protein and protein-promoter interactions provide precision and specificity in controlling photoreceptor subtype-specific gene expression, development, and survival. Understanding these interactions may provide insights to more effective therapeutic interventions for photoreceptor diseases.

Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development.

Understanding the molecular mechanisms by which distinct cell fate is determined during organogenesis is a central issue in development and disease. Here, using conditional gene ablation in mice, we show that the transcription factor Otx2 is essential for retinal photoreceptor cell fate determination and development of the pineal gland. Otx2-deficiency converted differentiating photoreceptor cells to amacrine-like neurons and led to a total lack of pinealocytes in the pineal gland. We also found that Otx2 transactivates the cone-rod homeobox gene Crx, which is required for terminal differentiation and maintenance of photoreceptor cells. Furthermore, retroviral gene transfer of Otx2 steers retinal progenitor cells toward becoming photoreceptors. Thus, Otx2 is a key regulatory gene for the cell fate determination of retinal photoreceptor cells. Our results reveal the key molecular steps required for photoreceptor cell-fate determination and pinealocyte development.

The role of transcription factors in the developing Drosophila eye.

In the developing Drosophila compound eye, multipotent precursor cells are induced to develop into particular cell types through sequential induction. In the target cells, transcription factors may be modulated by the inductive signals to execute their instructions. Four recently isolated genes may encode such developmentally modulated transcription factors.

Effects of cadmium on photoreceptors and ganglionic cells of retinal layer in mice embryo--an ultrastructural study.

Cadmium (Cd) is one of the environmental contaminant and because of its non-decomposable character, it can damage nature. In this study, TEM was used in order to assess the ultrastructural effects of Cd on photorececptor and ganglionic cells of mouse retinal layer. Apoptotic nuclei, heterochromatic nuclei, deletion of nucleus membrane, invisible nucleolus, and apoptotic cells with mitochondrial changes were observed in mice embryo (days 15 of gestation) following CdCl2 injection to mothers on day 9 of gestation. Cadmium exposure caused apoptotic changes both in photoreceptors and ganglionic cells.

Exploring RPE as a source of photoreceptors: differentiation and integration of transdifferentiating cells grafted into embryonic chick eyes.

PURPOSE: To study the possibility of generating photoreceptors through programming RPE transdifferentiation by examining cell differentiation after transplantation into the developing chick eye. METHODS: RPE was isolated, and the cells were dissociated, cultured, and guided to transdifferentiate by infection with retrovirus expressing neuroD (RCAS-neuroD), using RCAS-green fluorescence protein (GFP) as a control. The cells were then harvested and microinjected into the developing eyes of day 5 to day 7 chick embryos, and their development and integration were analyzed. RESULTS: Cells from the control culture integrated into the host RPE. When grafted cells were present in large number, multilayered RPE-like tissues were formed, and the extra tissues consisted of grafted cells and host cells. None of the cells from the control culture expressed photoreceptor-specific genes. In contrast, most cells from RCAS-neuroD-infected culture remained depigmented. A large number of them expressed photoreceptor-specific genes, such as visinin and opsins. Antibodies against red opsin decorated the apical tips and the cell bodies of the grafted, transdifferentiating cells. In the subretinal space, visinin(+) cells aligned along the RPE or an RPE-like structure. When integrated into the host outer nuclear layer, grafted cells emanated elaborate, axonal arborization into the outer plexiform layer of the host retina. CONCLUSIONS: Cultured RPE cells retained their remarkable regenerative capabilities. Cells guided to transdifferentiate along the photoreceptor pathway by neuroD developed a highly ordered cellular structure and could integrate into the outer nuclear layer. These data suggest that, through genetic programming, RPE cells could be a potential source of photoreceptor cells.

The sevenless signaling pathway: variations of a common theme.

Many developmental processes are regulated by intercellular signaling mechanisms that employ the activation of receptor tyrosine kinases. One model system that has been particular useful in determining the role of receptor tyrosine kinase-mediated signaling processes in cell fate determination is the developing Drosophila eye. The specification of the R7 photoreceptor cell in each ommatidium of the developing Drosophila eye is dependent on activation of the Sevenless receptor tyrosine kinase. This review will focus on the genetic and biochemical approaches that have identified signaling molecules acting downstream of the Sevenless receptor tyrosine kinase which ultimately trigger differentiation of the R7 photoreceptor cell.

Identification and functional characterization of conserved promoter elements from glass: a retinal development gene of Drosophila.

The Drosophila melanogaster glass transcription factor acts in photoreceptor cell development, glass transcription in the developing eye begins in the morphogenetic furrow. We report a deletion analysis which shows that a 5147 bp fragment can confer near wild-type expression in the developing eye. We completed the sequence of a similar Drosophila virilis fragment and comparison of these two genomic sequences reveals seven perfectly conserved sequence elements of 15 bp (or longer). We placed oligomers of each element 5' to an hsp70 promoter-lacZ fusion construct, and examined the patterns of beta-galactosidase expression produced in transgenic files. Three of these elements drive beta-galactosidase expression in the developing eye, and one is inhibitory.

Overlapping and specific patterns of GDNF, c-ret and GFR alpha mRNA expression in the developing chicken retina.

GDNF and the GDNF receptors, c-Ret, GFR alpha 1 and 2 mRNA is expressed in the developing chicken retina. GDNF labelling was mainly found in embryonic day 4-5 retina but weak labelling could also be found over scattered retinal cells at later stages. c-ret labelling was found over ganglion cells, amacrine and horizontal cells; the preferred GDNF receptor (GFR alpha 1) over amacrine and horizontal cells; and the less preferred GDNF receptor (GFR alpha 2) over ganglion cells, amacrine cells and photoreceptors.