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1.  The expression of Wnt2b in the optic cup lip requires a border between the pigmented and nonpigmented epithelium 
Molecular Vision  2010;16:2701-2717.
Wnt2b is normally expressed at the optic cup lip and is implicated in ciliary body induction. The lens has often been considered an organizer for the anterior eye, but recent studies demonstrate that the anterior cell fates are correctly specified in the absence of the lens. This study uses Wnt2b as a marker to reveal the mechanism behind the specification of the anterior domain of the optic cup.
Developing chick embryos were used as a model system. Eyes were microsurgically manipulated to assess the role of the lens in the development of the anterior optic cup. Eyes were molecularly manipulated, using fibroblast growth factor expressing replication-incompetent retrovirus, introduced into the retinal pigmented epithelium (RPE) domain. Ectopic fibroblast growth factor transformed the RPE into nonpigmented epithelium (NPE; ciliary body). As the virus does not spread, discrete borders between RPE and NPE were experimentally created. Wnt2b expression was assessed after surgical and molecular manipulation.
Contrary to expectations, we found that the lens is not able to induce Wnt2b expression in optic cup tissue: When the optic cup lip is experimentally misspecified such that it no longer contains the juxtaposition of pigmented and nonpigmented tissue, Wnt2b is not expressed. In addition, if the prelens ectoderm is removed from the optic vesicle before morphogenesis, the resulting lensless optic cup expresses Wnt2b even though it was not in contact with lens tissue. We also show that ectopic lenses do not induce Wnt2b in optic cup tissue. The ciliary body/anterior eye domain is specified at the border of RPE and the NPE of the ciliary body. During development, this border is normally found at the optic cup lip. We can manipulate tissue specification using retroviral-mediated gene transfer, and create ectopic borders between nonpigmented and pigmented tissue. At such borders, Wnt2b is ectopically expressed in the absence of lens contact. Finally, we describe a role for the lens in maintenance of Wnt2b expression and demonstrate support for this in two ways: First, we show that if the lens is removed from the formed optic cup, endogenous Wnt2b expression is specifically lost from the optic cup lip; and second, we show that while ectopic Wnt2b expression is initially found in the majority of ectopic borders, as eye development proceeds ectopic expression is maintained only in those borders that are close to the lens.
Taken together, the results provide support for a model in which the anterior optic cup domain, as described in part by Wnt2b expression, is specified through the elaboration of a border within the optic neuroepithelium rather than through interactions with the surrounding environment.
PMCID: PMC3002962  PMID: 21179237
2.  dlx and sp6-9 Control Optic Cup Regeneration in a Prototypic Eye 
PLoS Genetics  2011;7(8):e1002226.
Optic cups are a structural feature of diverse eyes, from simple pit eyes to camera eyes of vertebrates and cephalopods. We used the planarian prototypic eye as a model to study the genetic control of optic cup formation and regeneration. We identified two genes encoding transcription factors, sp6-9 and dlx, that were expressed in the eye specifically in the optic cup and not the photoreceptor neurons. RNAi of these genes prevented formation of visible optic cups during regeneration. Planarian regeneration requires an adult proliferative cell population with stem cell-like properties called the neoblasts. We found that optic cup formation occurred only after migration of progressively differentiating progenitor cells from the neoblast population. The eye regeneration defect caused by dlx and sp6-9 RNAi can be explained by a failure to generate these early optic cup progenitors. Dlx and Sp6-9 genes function as a module during the development of diverse animal appendages, including vertebrate and insect limbs. Our work reveals a novel function for this gene pair in the development of a fundamental eye component, and it utilizes these genes to demonstrate a mechanism for total organ regeneration in which extensive cell movement separates new cell specification from organ morphogenesis.
Author Summary
Some invertebrates, such as planarians and Hydra, can regenerate fully after amputations that remove large parts of the body. We investigated how cells in the body of planarians provide new cells for eye regeneration after complete head removal. Planarians possess highly potent regenerative cells (neoblasts) in a compartment inside the worm, and these cells must be present in a body fragment for it to regenerate. We identify a pair of transcription factors, sp6-9 and dlx, that are expressed in the optic cup, and use expression of these genes as markers to demonstrate that lineage restriction of eye cells during regeneration begins within the neoblast compartment. dlx and sp6-9 are essential for formation of optic cup progenitors, and inhibition of these genes with RNA interference results in eyes that lack optic cups after regeneration. During eye development in both flies and vertebrates, progenitors form within a patterned epithelium. Interestingly, planarian eye precursors only aggregate once they have stopped cycling and undergone extensive migration. At this stage they already express markers of the terminally differentiated state. Therefore, we identify a mechanism for eye formation during regeneration and a novel function for a conserved gene pair in eye regeneration.
PMCID: PMC3154955  PMID: 21852957
3.  A complex choreography of cell movements shapes the vertebrate eye 
Development (Cambridge, England)  2012;139(2):359-372.
Optic cup morphogenesis (OCM) generates the basic structure of the vertebrate eye. Although it is commonly depicted as a series of epithelial sheet folding events, this does not represent an empirically supported model. Here, we combine four-dimensional imaging with custom cell tracking software and photoactivatable fluorophore labeling to determine the cellular dynamics underlying OCM in zebrafish. Although cell division contributes to growth, we find it dispensable for eye formation. OCM depends instead on a complex set of cell movements coordinated between the prospective neural retina, retinal pigmented epithelium (RPE) and lens. Optic vesicle evagination persists for longer than expected; cells move in a pinwheel pattern during optic vesicle elongation and retinal precursors involute around the rim of the invaginating optic cup. We identify unanticipated movements, particularly of central and peripheral retina, RPE and lens. From cell tracking data, we generate retina, RPE and lens subdomain fate maps, which reveal novel adjacencies that might determine corresponding developmental signaling events. Finally, we find that similar movements also occur during chick eye morphogenesis, suggesting that the underlying choreography is conserved among vertebrates.
PMCID: PMC3243097  PMID: 22186726
Zebrafish; Eye; Morphogenesis; Cell tracking; Retina; Retinal pigmented epithelium (RPE); Lens; Fate map
4.  Lhx1 in the proximal region of the optic vesicle permits neural retina development in the chicken 
Biology Open  2012;1(11):1083-1093.
How the eye forms has been one of the fundamental issues in developmental biology. The retinal anlage first appears as the optic vesicle (OV) evaginating from the forebrain. Subsequently, its distal portion invaginates to form the two-walled optic cup, which develops into the outer pigmented and inner neurosensory layers of the retina. Recent work has shown that this optic-cup morphogenesis proceeds as a self-organizing activity without any extrinsic molecules. However, intrinsic factors that regulate this process have not been elucidated. Here we show that a LIM-homeobox gene, Lhx1, normally expressed in the proximal region of the nascent OV, induces a second neurosensory retina formation from the outer pigmented retina when overexpressed in the chicken OV. Lhx2, another LIM-homeobox gene supposed to be involved in early OV formation, could not substitute this function of Lhx1, while Lhx5, closely related to Lhx1, could replace it. Conversely, knockdown of Lhx1 expression by RNA interference resulted in the formation of a small or pigmented vesicle. These results suggest that the proximal region demarcated by Lhx1 expression permits OV development, eventually dividing the two retinal domains.
PMCID: PMC3507191  PMID: 23213388
Lhx1; Lhx5; Optic vesicle; LIM-homeobox; Chicken; Eye development; Neural retina
5.  Eye Morphogenesis and Patterning of the Optic Vesicle 
Organogenesis of the eye is a multi-step process that starts with the formation of optic vesicles followed by invagination of the distal domain of the vesicles and the overlying lens placode resulting in morphogenesis of the optic cup. The late optic vesicle becomes patterned into distinct ocular tissues; the neural retina, retinal pigment epithelium (RPE) and optic stalk. Multiple congenital eye disorders, including anophthalmia or microphthalmia, aniridia, coloboma and retinal dysplasia, stem from disruptions in embryonic eye development. Thus, it is critical to understand the mechanisms that lead to initial specification and differentiation of ocular tissues. An accumulating number of studies demonstrate that a complex interplay between inductive signals provided by tissue-tissue interactions and cell-intrinsic factors is critical to ensure proper specification of ocular tissues as well as maintenance of RPE cell fate. While several of the extrinsic and intrinsic determinants have been identified, we are just at the beginning to understand how these signals are integrated. In addition, we know very little about the actual output of these interactions. In this chapter, we provide an update of the mechanisms controlling early steps of eye development in vertebrates, with emphasis on optic vesicle evagination, specification of neural retina and RPE at the optic vesicle stage, the process of invagination during morphogenesis of the optic cup and maintenance of the RPE cell fate.
PMCID: PMC2958684  PMID: 20959163
6.  Retinal and anterior eye compartments derive from a common progenitor pool in the avian optic cup 
Molecular Vision  2011;17:3347-3363.
The optic cup is created through invagination of the optic vesicle. The morphogenetic rearrangement creates a double-layered cup, with a hinge (the Optic Cup Lip) where the epithelium bends back upon itself. Shortly after the optic cup forms, it is thought to be sub-divided into separate lineages: i) pigmented epithelium in the outer layer; ii) presumptive iris and ciliary body at the most anterior aspect of the inner layer; and iii) presumptive neural retina in the remainder of the inner layer. We test the native developmental potential of the anterior cup to determine if it normally contributes to the retina.
Vital dye and green fluorescent protein (GFP) expressing replication-incompetent retroviral vectors were used to label cells in the nascent optic cup and follow their direct progeny throughout development. Label was applied to either the optic cup lip (n=40), or to the domain just posterior to the lip (n=20). Retroviral labeling is a permanent lineage marker and enabled the analysis of advanced stages of development.
Labeling within the optic cup gave rise to labeled progeny in the posterior optic cup that differentiated as neural retina (20 of 20). In contrast, labeling cells in the optic cup lip gave rise to progeny of labeled cells arrayed in a linear progression, from the lip into the neural retina (36 of 40). Label was retained in cells at the optic cup lip, regardless of age at examination. In older embryos, labeled progeny delaminated from the optic cup lip to differentiate as muscle of the pupillary margin.
The data show that the cells at the optic cup lip are a common progenitor population for pigmented epithelium, anterior eye tissues (ciliary body, iris, and pupillary muscle) and retinal neurons. The findings are supportive of an interpretation where the optic cup lip is a specialized niche containing a multipotent progenitor population.
PMCID: PMC3247166  PMID: 22219630
7.  Laser Scanning Tomography in the EPIC-Norfolk Eye Study: Principal Components and Associations 
To describe Heidelberg Retina Tomograph (HRT) measures, their principal components, and their associations in a British population.
The European Prospective Investigation of Cancer (EPIC)-Norfolk Eye Study is nested within a multicenter cohort study. Measurements were taken with the HRT-2 and the software subsequently updated to yield HRT-3 parameters. Principal components analysis (PCA) was used to identify distinct components of the HRT variables. Generalized estimating equation models were used to examine associations of these components with age, sex, height, body mass index (BMI), blood pressure, social class, education, alcohol intake, smoking status, axial length, IOP, and lens status.
Complete data were available from 10,859 eyes of 6430 participants with a mean age of 68 years. Principal components analysis identified three components with an eigenvalue greater than 1, explaining 79.9% of the variance of all the HRT measures. These were named cup, retinal nerve fiber layer (RNFL), and rim based on the factor loadings they were most correlated with. Older age was significantly associated with a greater cup (P = 0.003), smaller RNFL (P < 0.001), and smaller rim (P < 0.001). Female sex (P = 0.001), higher education (P < 0.001), and shorter axial length (P < 0.001) were associated with a greater RNFL. Lower BMI and higher IOP were associated with a greater cup (both, P < 0.001) and a smaller rim (BMI, P = 0.001; IOP, P < 0.001).
Heidelberg Retina Tomograph measures in this cohort were largely explained by three principal components related to optic disc cup, RNFL, and rim. Associations with cup and rim were distinct to associations with RNFL, suggesting different underlying determinants.
Analysis of Heidelberg Retina Tomograph measurements from a large population-based sample identified three distinct components (optic disc “cup”, “rim” and retinal nerve fiber layer [“RNFL”]). Associations with “cup” and “rim” were distinct to associations with “RNFL”, suggesting different underlying determinants.
PMCID: PMC3803136  PMID: 24030456
diagnostic techniques; epidemiology; axial length; glaucoma; body mass index; optic disk
8.  Eye Development and Retinogenesis 
Three embryonic tissue sources—the neural ectoderm, the surface ectoderm, and the periocular mesenchyme—contribute to the formation of the mammalian eye. For this reason, the developing eye has presented an invaluable system for studying the interactions among cells and, more recently, genes, in specifying cell fate. This article describes how the eye primordium is specified in the anterior neural plate by four eye field transcription factors and how the optic vesicle becomes regionalized into three distinct tissue types. Specific attention is given to how cross talk between the optic vesicle and surface ectoderm contributes to lens and optic cup formation. This article also describes how signaling networks and cell movements set up axes in the optic cup and establish the multiple cell fates important for vision. How multipotent retinal progenitor cells give rise to the six neuronal and one glial cell type in the mature retina is also explained. Finally, the history and progress of cellular therapeutics for the treatment of degenerative eye disease is outlined. Throughout this article, special attention is given to how disruption of gene function causes ocular malformation in humans. Indeed, the accessibility of the eye has contributed much to our understanding of the basic processes involved in mammalian development.
The eye develops from three embryonic tissues. It is an accessible model for studying genetic interactions and signaling switches that coordinate the development and morphogenesis of diverse cell types.
PMCID: PMC3504437  PMID: 23071378
9.  Shaping the eye from embryonic stem cells: Biological and medical implications 
World Journal of Stem Cells  2012;4(8):80-86.
Organogenesis is regulated by a complex network of intrinsic cues, diffusible signals and cell/cell or cell/matrix interactions that drive the cells of a prospective organ to differentiate and collectively organize in three dimensions. Generating organs in vitro from embryonic stem (ES) cells may provide a simplified system to decipher how these processes are orchestrated in time and space within particular and between neighboring tissues. Recently, this field of stem cell research has also gained considerable interest for its potential applications in regenerative medicine. Among human pathologies for which stem cell-based therapy is foreseen as a promising therapeutic strategy are many retinal degenerative diseases, like retinitis pigmentosa and age-related macular degeneration. Over the last decade, progress has been made in producing ES-derived retinal cells in vitro, but engineering entire synthetic retinas was considered beyond reach. Recently however, major breakthroughs have been achieved with pioneer works describing the extraordinary self-organization of murine and human ES cells into a three dimensional structure highly resembling a retina. ES-derived retinal cells indeed assemble to form a cohesive neuroepithelial sheet that is endowed with the intrinsic capacity to recapitulate, outside an embryonic environment, the main steps of retinal morphogenesis as observed in vivo. This represents a tremendous advance that should help resolving fundamental questions related to retinogenesis. Here, we will discuss these studies, and the potential applications of such stem cell-based systems for regenerative medicine.
PMCID: PMC3506970  PMID: 23189212
Retina; Optic cup; Embryonic stem cells; Retinal pigment epithelium; Three dimensional culture
10.  The level of BMP4 signaling is critical for the regulation of distinct T-box gene expression domains and growth along the dorso-ventral axis of the optic cup 
Polarised gene expression is thought to lead to the graded distribution of signaling molecules providing a patterning mechanism across the embryonic eye. Bone morphogenetic protein 4 (Bmp4) is expressed in the dorsal optic vesicle as it transforms into the optic cup. Bmp4 deletions in human and mouse result in failure of eye development, but little attempt has been made to investigate mammalian targets of BMP4 signaling. In chick, retroviral gene overexpression studies indicate that Bmp4 activates the dorsally expressed Tbx5 gene, which represses ventrally expressed cVax. It is not known whether the Tbx5 related genes, Tbx2 and Tbx3, are BMP4 targets in the mammalian retina and whether BMP4 acts at a distance from its site of expression. Although it is established that Drosophila Dpp (homologue of vertebrate Bmp4) acts as a morphogen, there is little evidence that BMP4 gradients are interpreted to create domains of BMP4 target gene expression in the mouse.
Our data show that the level of BMP4 signaling is critical for the regulation of distinct Tbx2, Tbx3, Tbx5 and Vax2 gene expression domains along the dorso-ventral axis of the mouse optic cup. BMP4 signaling gradients were manipulated in whole mouse embryo cultures during optic cup development, by implantation of beads soaked in BMP4, or the BMP antagonist Noggin, to provide a local signaling source. Tbx2, Tbx3 and Tbx5, showed a differential response to alterations in the level of BMP4 along the entire dorso-ventral axis of the optic cup, suggesting that BMP4 acts across a distance. Increased levels of BMP4 caused expansion of Tbx2 and Tbx3, but not Tbx5, into the ventral retina and repression of the ventral marker Vax2. Conversely, Noggin abolished Tbx5 expression but only shifted Tbx2 expression dorsally. Increased levels of BMP4 signaling caused decreased proliferation, reduced retinal volume and altered the shape of the optic cup.
Our findings suggest the existence of a dorsal-high, ventral-low BMP4 signaling gradient across which distinct domains of Tbx2, Tbx3, Tbx5 and Vax2 transcription factor gene expression are set up. Furthermore we show that the correct level of BMP4 signaling is critical for normal growth of the mammalian embryonic eye.
PMCID: PMC1764729  PMID: 17173667
11.  Optic nerve head analyser and Heidelberg retina tomograph: accuracy and reproducibility of topographic measurements in a model eye and in volunteers. 
The accuracy and reproducibility of the optic nerve head analyser (ONHA) and the Heidelberg retina tomograph (HRT) were compared and the performance of the HRT in measuring fundus elevations was evaluated. The coefficient of variation of three repeated measurements in a model eye and in volunteers and the relative error in a model eye was calculated. With ONHA measurements the pooled coefficient of variation in volunteers was 9.3% in measuring cup areas and 8.4% in measuring the cup volume. In a model eye the pooled coefficient of variation was 7.6% for the parameter 'cup area' and 9.9% for the parameter 'cup volume'. The pooled relative error in the model eye was 6.6% for the parameter 'cup area' and 5.1% for the parameter 'cup volume'. With HRT measurements in volunteers the pooled coefficient of variation of both the parameters 'volume below contour' and 'volume below surface' was 6.9%. In the model eye the pooled coefficient of variation was 2.4% for the 'volume below contour' and 4.1% for the parameter 'volume below surface'. The pooled relative error in the model eye was 11.3% for the 'volume below contour' and 11% for the 'volume below surface'. The pooled relative error in measuring retinal elevations in the model eye was 3.8%. The coefficient of variation was 3.5%. The accuracies of the HRT and ONHA were similar. However, as the ONHA 'cup volume' is unreliable in patients because of the design of the ONHA whereas the HRT volume parameters are reliable it seems reasonable to assume that the HRT is superior to the ONHA. Only the HRT is capable of quantifying retinal elevations.
PMCID: PMC504930  PMID: 7803352
12.  Self-Organizing Properties of Mouse Pluripotent Cells Initiate Morphogenesis upon Implantation 
Cell  2014;156(5):1032-1044.
Transformation of pluripotent epiblast cells into a cup-shaped epithelium as the mouse blastocyst implants is a poorly understood and yet key developmental step. Studies of morphogenesis in embryoid bodies led to the current belief that it is programmed cell death that shapes the epiblast. However, by following embryos developing in vivo and in vitro, we demonstrate that not cell death but a previously unknown morphogenetic event transforms the amorphous epiblast into a rosette of polarized cells. This transformation requires basal membrane-stimulated integrin signaling that coordinates polarization of epiblast cells and their apical constriction, a prerequisite for lumenogenesis. We show that basal membrane function can be substituted in vitro by extracellular matrix (ECM) proteins and that ES cells can be induced to form similar polarized rosettes that initiate lumenogenesis. Together, these findings lead to a completely revised model for peri-implantation morphogenesis in which ECM triggers the self-organization of the embryo’s stem cells.
Graphical Abstract
•Apoptosis is not essential for the peri-implantation morphogenesis, as believed•Basal membrane proteins create a niche for EPI and drive morphogenesis in ES cells•Polarization and apical constriction reorganize the EPI into a rosette•The proamniotic cavity is formed through hollowing mechanism
The first morphogenetic event by pluripotent stem cells of mouse blastocyst entails epiblast cell polarization and arrangement into a rosette pattern, which parts at its center, leading to cavity formation.
PMCID: PMC3991392  PMID: 24529478
13.  Optic nerve axons and acquired alterations in the appearance of the optic disc. 
The pathophysiologic events in optic nerve axons have recently been recognized as crucial to an understanding of clinically significant acquired alterations in the ophthalmoscopic appearance of the optic disc. Stasis and related abnormalities of axonal transport appear to explain most aspects of optic nerve head swelling, including optic disc drusen and retinal cottonwool spots. Loss of axoplasm and axonal death can be invoked to interpret optic disc pallor, thinning and narrowing of rim tissue, changes in the size and outline of the optic cup, laminar dots, atrophy of the retinal nerve fiber layer, and acquired demyelination and myelination of the retinal nerve fiber layer. It is speculated that the axons may also play a role in the mechanical support of the lamina cribrosa in resisting the pressure gradient across the pars scleralis of the optic nerve head. Axons and their associated glial cells may be involved in those cases where "reversibility" of cupping of the optic disc has been reported. The structure, physiology, and experimental pathologic findings of the optic nerve head have been reviewed. Many aspects concerning the final anatomic appearance of the optic nerve head have been explained. However, many questions remain concerning the intermediate mechanisms by which increased intracranial pressure retards the various components of axonal transport in papilledema and by which increased IOP causes axonal loss in glaucoma. Investigation of the molecular biology of axonal constituents and their responses to abnormalities in their physical and chemical milieu could extend our understanding of the events that result from mechanical compression and local ischemia. Moreover, we have identified a need to further explore the role of axons in the pathophysiology of optic disc cupping.
PMCID: PMC1312472  PMID: 6203209
14.  Cadherin-Mediated Cell Adhesion Is Critical for the Closing of the Mouse Optic Fissure 
PLoS ONE  2012;7(12):e51705.
Coloboma is a congenital disease that contributes significantly to childhood blindness. It results from the failure in closing the optic fissure, a transient opening on the ventral side of the developing eye. Although human and mouse genetic studies have identified a number of genes associated with coloboma, the detailed cellular mechanisms underlying the optic fissure closure and coloboma formation remain largely undefined. N-cadherin-mediated cell adhesion has been shown to be important for the optic fissure closure in zebrafish, but it remains to be determined experimentally how cell-cell adhesions are involved in the mammalian optic fissure closing process. α-catenin is required for cell adhesion mediated by all of the classic cadherin molecules, including N-cadherin. In this study, we used the Cre-mediated conditional knockout technique to specifically delete α-catenin from the developing mouse eye to show that it is required for the successful closing of the optic fissure. In α-catenin conditional mutant optic cups, the major cell fates, including the optic fissure margin, neural retina and retinal pigmented epithelium, are specified normally, and the retinal progenitor cells proliferate normally. However, adherens junctions components, including N-cadherin, β-catenin and filamentous actin, fail to accumulate on the apical side of α-catenin mutant retinal progenitor cells, where adherens junctions are normally abundant, and the organization of the neural retina and the optic fissure margin is disrupted. Finally, the α-catenin mutant retina gradually degenerates in the adult mouse eye. Therefore, our results show that α-catenin-mediated cell adhesion and cell organization are important for the fissure closure in mice, and further suggest that genes that regulate cell adhesion may underlie certain coloboma cases in humans.
PMCID: PMC3519883  PMID: 23240058
15.  AP-2α knockout mice exhibit optic cup patterning defects and failure of optic stalk morphogenesis 
Human Molecular Genetics  2010;19(9):1791-1804.
Appropriate development of the retina and optic nerve requires that the forebrain-derived optic neuroepithelium undergoes a precisely coordinated sequence of patterning and morphogenetic events, processes which are highly influenced by signals from adjacent tissues. Our previous work has suggested that transcription factor activating protein-2 alpha (AP-2α; Tcfap2a) has a non-cell autonomous role in optic cup (OC) development; however, it remained unclear how OC abnormalities in AP-2α knockout (KO) mice arise at the morphological and molecular level. In this study, we show that patterning and morphogenetic defects in the AP-2α KO optic neuroepithelium begin at the optic vesicle stage. During subsequent OC formation, ectopic neural retina and optic stalk-like tissue replaced regions of retinal pigment epithelium. AP-2α KO eyes also displayed coloboma in the ventral retina, and a rare phenotype in which the optic stalk completely failed to extend, causing the OCs to be drawn inward to the midline. We detected evidence of increased sonic hedgehog signaling in the AP-2α KO forebrain neuroepithelium, which likely contributed to multiple aspects of the ocular phenotype, including expansion of PAX2-positive optic stalk-like tissue into the OC. Our data suggest that loss of AP-2α in multiple tissues in the craniofacial region leads to severe OC and optic stalk abnormalities by disturbing the tissue–tissue interactions required for ocular development. In view of recent data showing that mutations in human TFAP2A result in similar eye defects, the current findings demonstrate that AP-2α KO mice provide a valuable model for human ocular disease.
PMCID: PMC2850623  PMID: 20150232
16.  Loss of Tbx2 delays optic vesicle invagination leading to small optic cups 
Developmental biology  2009;333(2):360-372.
Tbx2 is a T-box transcription factor gene that is dynamically expressed in the presumptive retina during optic vesicle invagination. Several findings implicate Tbx2 in cell cycle regulation, including its overexpression in tumours and regulation of proliferation during heart development. We investigated the role of Tbx2 in optic cup formation by analysing mice with a targeted homozygous mutation in Tbx2. Loss of Tbx2 caused a reduced presumptive retinal volume due to increased apoptosis, and a delay in ventral optic vesicle invagination leading to the formation of small and abnormally shaped optic cups. Tbx2 is essential for maintenance, but not induction of expression of the dorsal retinal determinant, Tbx5, and acts downstream of Bmp4, a dorsally expressed gene implicated in human microphthalmia. The small retina showed a hypocellular ventral region, loss of Fgf15, normally expressed in proliferating central retinal cells, and increased numbers of mitotic cells in the dorsal region, indicating that Tbx2 is required for normal growth and development across the D-V axis. Dorsal expression of potential regulators of retinal growth, Cyp1b1 and Cx43, and the topographic guidance molecule ephrinb2, was increased, and intra-retinal axons were disorganised resulting in a failure of optic nerve formation. Our data provide evidence that Tbx2 is required for proper optic cup formation and plays a critical early role in regulating regional retinal growth and the acquisition of shape during optic vesicle invagination.
PMCID: PMC2735611  PMID: 19576202
17.  Curvature recognition and force generation in phagocytosis 
BMC Biology  2010;8:154.
The uptake of particles by actin-powered invagination of the plasma membrane is common to protozoa and to phagocytes involved in the immune response of higher organisms. The question addressed here is how a phagocyte may use geometric cues to optimize force generation for the uptake of a particle. We survey mechanisms that enable a phagocyte to remodel actin organization in response to particles of complex shape.
Using particles that consist of two lobes separated by a neck, we found that Dictyostelium cells transmit signals concerning the curvature of a surface to the actin system underlying the plasma membrane. Force applied to a concave region can divide a particle in two, allowing engulfment of the portion first encountered. The phagosome membrane that is bent around the concave region is marked by a protein containing an inverse Bin-Amphiphysin-Rvs (I-BAR) domain in combination with an Src homology (SH3) domain, similar to mammalian insulin receptor tyrosine kinase substrate p53. Regulatory proteins enable the phagocyte to switch activities within seconds in response to particle shape. Ras, an inducer of actin polymerization, is activated along the cup surface. Coronin, which limits the lifetime of actin structures, is reversibly recruited to the cup, reflecting a program of actin depolymerization. The various forms of myosin-I are candidate motor proteins for force generation in particle uptake, whereas myosin-II is engaged only in retracting a phagocytic cup after a switch to particle release. Thus, the constriction of a phagocytic cup differs from the contraction of a cleavage furrow in mitosis.
Phagocytes scan a particle surface for convex and concave regions. By modulating the spatiotemporal pattern of actin organization, they are capable of switching between different modes of interaction with a particle, either arresting at a concave region and applying force in an attempt to sever the particle there, or extending the cup along the particle surface to identify the very end of the object to be ingested. Our data illustrate the flexibility of regulatory mechanisms that are at the phagocyte's disposal in exploring an environment of irregular geometry.
PMCID: PMC3022777  PMID: 21190565
18.  FGF-mediated induction of ciliary body tissue in the chick eye 
Developmental biology  2006;304(1):272-285.
Upon morphogenesis, the simple neuroepithelium of the optic vesicle gives rise to four basic tissues in the vertebrate optic cup: pigmented epithelium, sensory neural retina, secretory ciliary body and muscular iris. Pigmented epithelium and neural retina are established through interactions with specific environments and signals: periocular mesenchyme/BMP specifies pigmented epithelium and surface ectoderm/FGF specifies neural retina. The anterior portions (iris and ciliary body) are specified through interactions with lens although the molecular mechanisms of induction have not been deciphered. As lens is a source of FGF, we examined whether this factor was involved in inducing ciliary body. We forced the pigmented epithelium of the embryonic chick eye to express FGF4. Infected cells and their immediate neighbors were transformed into neural retina. At a distance from the FGF signal, the tissue transitioned back into pigmented epithelium. Ciliary body tissue was found in the transitioning zone. The ectopic ciliary body was never in contact with lens tissue. In order to assess the contribution of the lens on the specification of normal ciliary body, we created optic cups in which the lens had been removed while still pre-lens ectoderm. Ciliary body tissue was identified in the anterior portion of lens-less optic cups. We propose that the ciliary body may be specified at optic vesicle stages, at the same developmental stage when the neural retina and pigmented epithelium are specified and we present a model as to how this could be accomplished through overlapping BMP and FGF signals.
PMCID: PMC1863121  PMID: 17275804
Ciliary body; development; chick; eye; retina; retrovirus; FGF
19.  The retinal pigment epithelium of the eye regulates the development of scleral cartilage 
Developmental Biology  2010;347(1-13):40-52.
The majority of vertebrate species have a layer of hyaline cartilage within the fibrous sclera giving an extra degree of support to the eyeball. In chicks, this is seen as a cuplike structure throughout the scleral layer. However, the mechanisms that control the development of scleral cartilage are largely unknown.
Here we have studied the phases of scleral cartilage development and characterised expression profiles of genes activated during the cartilage differentiation programme. CART1 and SOX9, the earliest markers of pre-committed cartilage, are expressed in the mesenchyme surrounding the optic cup. Later AGGRECAN, a matrix protein expressed during chondrocyte differentiation, is also expressed. The expression of these genes is lost following early removal of the optic cup, suggesting a role for this tissue in inducing scleral cartilage. By grafting young retinal pigment epithelium (RPE) and retina into cranial mesenchyme in vivo, it was found that RPE alone has the ability to induce cartilage formation.
There are some exceptions within the vertebrates where scleral cartilage is not present; one such example is the placental mammals. However, we found that the cartilage differentiation pathway is initiated in mice as seen by the expression of Cart1 and Sox9, but expression of the later cartilage marker Aggrecan is weak. Furthermore, cartilage forms in mouse peri-ocular mesenchyme micromass culture. This suggests that the process halts in vivo before full differentiation into cartilage, but that murine scleral mesenchyme has retained the potential to make cartilage in vitro.
RA, Wnts and Bmps have been linked to the cartilage development process and are expressed within the developing RPE. We find that RA may have a role in early scleral cartilage development but is not likely to be the main factor involved.
These data reveal the course of scleral cartilage formation and highlight the key role that the optic cup plays in this process. The driving element within the optic cup is almost certainly the retinal pigmented epithelium.
PMCID: PMC2977850  PMID: 20707994
Eye; Development; Sclera; Cartilage; RPE; Neural crest
20.  The Actomyosin Machinery Is Required for Drosophila Retinal Lumen Formation 
PLoS Genetics  2014;10(9):e1004608.
Multicellular tubes consist of polarized cells wrapped around a central lumen and are essential structures underlying many developmental and physiological functions. In Drosophila compound eyes, each ommatidium forms a luminal matrix, the inter-rhabdomeral space, to shape and separate the key phototransduction organelles, the rhabdomeres, for proper visual perception. In an enhancer screen to define mechanisms of retina lumen formation, we identified Actin5C as a key molecule. Our results demonstrate that the disruption of lumen formation upon the reduction of Actin5C is not linked to any discernible defect in microvillus formation, the rhabdomere terminal web (RTW), or the overall morphogenesis and basal extension of the rhabdomere. Second, the failure of proper lumen formation is not the result of previously identified processes of retinal lumen formation: Prominin localization, expansion of the apical membrane, or secretion of the luminal matrix. Rather, the phenotype observed with Actin5C is phenocopied upon the decrease of the individual components of non-muscle myosin II (MyoII) and its upstream activators. In photoreceptor cells MyoII localizes to the base of the rhabdomeres, overlapping with the actin filaments of the RTW. Consistent with the well-established roll of actomyosin-mediated cellular contraction, reduction of MyoII results in reduced distance between apical membranes as measured by a decrease in lumen diameter. Together, our results indicate the actomyosin machinery coordinates with the localization of apical membrane components and the secretion of an extracellular matrix to overcome apical membrane adhesion to initiate and expand the retinal lumen.
Author Summary
Biological tubes are integral units of tissues and organs such as lung, kidney, and the cardiovascular system. The fundamental design of tubes involves a central lumen wrapped by a sheet of cells. To function properly, the tubes require a precise genetic control over their creation, the diametric growth and maintenance of the lumen during development. In the fruit fly, Drosophila melanogaster, the photoreceptor cells of the eye form a tubular structure. The formation of the retinal lumen is critical for separating and positioning the light sensing organelles of each photoreceptor cell to achieve visual sensitivity. In an effort to investigate the mechanisms of Drosophila retinal lumen formation, we identified a contractile machinery that was present at the apical portion of photoreceptor cells. Our data is consistent with the idea that a contractile force contributes to the initial separation of the juxtaposed apical membranes and subsequent enlargement of the luminal space. Our work suggests that building a biological tube requires not only an extrinsic pushing force provided by the growing central lumen, but also a cell intrinsic pulling force powered by contraction of cells lining the lumen. Our findings expand and demonstrate the coordination of several molecular mechanisms to generate a tube.
PMCID: PMC4168998  PMID: 25233220
21.  Canine and Human Visual Cortex Intact and Responsive Despite Early Retinal Blindness from RPE65 Mutation 
PLoS Medicine  2007;4(6):e230.
RPE65 is an essential molecule in the retinoid-visual cycle, and RPE65 gene mutations cause the congenital human blindness known as Leber congenital amaurosis (LCA). Somatic gene therapy delivered to the retina of blind dogs with an RPE65 mutation dramatically restores retinal physiology and has sparked international interest in human treatment trials for this incurable disease. An unanswered question is how the visual cortex responds after prolonged sensory deprivation from retinal dysfunction. We therefore studied the cortex of RPE65-mutant dogs before and after retinal gene therapy. Then, we inquired whether there is visual pathway integrity and responsivity in adult humans with LCA due to RPE65 mutations (RPE65-LCA).
Methods and Findings
RPE65-mutant dogs were studied with fMRI. Prior to therapy, retinal and subcortical responses to light were markedly diminished, and there were minimal cortical responses within the primary visual areas of the lateral gyrus (activation amplitude mean ± standard deviation [SD] = 0.07% ± 0.06% and volume = 1.3 ± 0.6 cm3). Following therapy, retinal and subcortical response restoration was accompanied by increased amplitude (0.18% ± 0.06%) and volume (8.2 ± 0.8 cm3) of activation within the lateral gyrus (p < 0.005 for both). Cortical recovery occurred rapidly (within a month of treatment) and was persistent (as long as 2.5 y after treatment). Recovery was present even when treatment was provided as late as 1–4 y of age. Human RPE65-LCA patients (ages 18–23 y) were studied with structural magnetic resonance imaging. Optic nerve diameter (3.2 ± 0.5 mm) was within the normal range (3.2 ± 0.3 mm), and occipital cortical white matter density as judged by voxel-based morphometry was slightly but significantly altered (1.3 SD below control average, p = 0.005). Functional magnetic resonance imaging in human RPE65-LCA patients revealed cortical responses with a markedly diminished activation volume (8.8 ± 1.2 cm3) compared to controls (29.7 ± 8.3 cm3, p < 0.001) when stimulated with lower intensity light. Unexpectedly, cortical response volume (41.2 ± 11.1 cm3) was comparable to normal (48.8 ± 3.1 cm3, p = 0.2) with higher intensity light stimulation.
Visual cortical responses dramatically improve after retinal gene therapy in the canine model of RPE65-LCA. Human RPE65-LCA patients have preserved visual pathway anatomy and detectable cortical activation despite limited visual experience. Taken together, the results support the potential for human visual benefit from retinal therapies currently being aimed at restoring vision to the congenitally blind with genetic retinal disease.
The study by Samuel Jacobson and colleagues suggests that retinal gene therapy can improve retinal, visual pathway, and visual cortex responses to light stimulation, even after prolonged periods of blindness and in congenitally blind patients.
Editors' Summary
The eye captures light but the brain is where vision is experienced. Treatments for childhood blindness at the eye level are ready, but it is unknown whether the brain will be receptive to an improved neural message. Normal vision begins as photoreceptor cells in the retina (the light-sensitive tissue lining the inside of the eye) convert visual images into electrical impulses. These impulses are sent along the optic nerve to the visual cortex, the brain region where they are interpreted. The conversion of light into electrical impulses requires the activation of a molecule called retinal, which is subsequently recycled by retinal pigment epithelium (RPE) cells neighboring the retina. One of the key enzymes of the recycling reactions is encoded by a gene called RPE65. Genetic changes (mutations) in RPE65 cause an inherited form of blindness called Leber congenital amaurosis (LCA). In this disease, retinal is not recycled and as a result, the photoreceptor cells cannot work properly and affected individuals have poor or nonexistent vision from birth. Previous studies in dog and mouse models of the human disease have demonstrated that the introduction of a functional copy of RPE65 into the RPE cells using a harmless virus (gene therapy) dramatically restores retinal activity. Very recently, a pioneering gene therapy operation took place in London (UK) where surgeons injected a functional copy of RPE65 into the retina of a man with LCA. Whether this operation results in improved vision is not known at this time.
Why Was This Study Done?
Gene therapy corrects the retinal defects in animal models of LCA but whether the visual pathway from the retina to the visual cortex of the brain can respond normally to the signals sent by the restored retina is not known. Early visual experience is thought to be necessary for the development of a functional visual cortex, so replacing the defective RPE65 gene might not improve the vision of people with LCA. In this study, the researchers have studied the visual cortex of RPE65-deficient dogs before and after gene therapy to see whether the therapy affects the activity of the visual cortex. They have also investigated visual pathway integrity and responsiveness in adults with LCA caused by RPE65 mutations. If the visual pathway is disrupted in these patients, they reasoned, gene therapy might not restore their vision.
What Did the Researchers Do and Find?
The researchers used a technique called functional magnetic resonance imaging (fMRI) to measure light-induced brain activity in RPE65-deficient dogs before and after gene therapy. They also examined the reactions of the dogs' pupils to light (in LCA, the pupils do not contract normally in response to light because there is reduced signal transmission along the visual pathway). Finally, they measured the electrical activity of the dogs' retinas in response to light flashes—the retinas of patients with LCA do not react to light. Gene therapy corrected the defective retinal and visual pathway responses to light in the RPE65-deficient dogs and, whereas before treatment there was no response in the visual cortex to light stimulation in these dogs, after treatment, its activity approached that seen in normal dogs. The recovery of cortical responses was permanent and occurred soon after treatment, even in animals that were 4 years old when treated. Next, using structural MRI, the researchers studied human patients with LCA and found that the optic nerve diameter in young adults was within the normal range and that the structure of the visual cortex was very similar to that of normal individuals. Finally, using fMRI, they found that, although the visual cortex of patients with LCA did not respond to dim light, its reaction to bright light was comparable to that of normal individuals.
What Do These Findings Mean?
The findings from the dog study indicate that retinal gene therapy rapidly improves retinal, visual pathway, and visual cortex responses to light stimulation, even in animals that have been blind for years. In other words, in the dog model of LCA at least, all the components of the visual system remain receptive to visual inputs even after long periods of visual deprivation. The findings from the human study also indicate that the visual pathway remains anatomically intact despite years of disuse and that the visual cortex can be activated in patients with LCA even though these people have very limited visual experience. Taken together, these findings suggest that successful gene therapy of the retina might restore some functional vision to people with LCA but proof will have to await the outcomes of several clinical trials ongoing or being planned in Europe and the USA.
Additional Information.
Please access these Web sites via the online version of this summary at
General information on gene therapy is available from the Oak Ridge National Laboratory
Information is provided by the BBC about gene therapy for Leber congenital amaurosis (includes an audio clip from a doctor about the operation)
The National Institutes of Health/National Eye Institute (US) provides information about an ongoing gene therapy trial of RPE65-Leber congenital amaurosis gives details on treatment trials for Leber congenital amaurosis
The Foundation Fighting Blindness has a fact sheet on Leber congenital amaurosis (site includes Microsoft Webspeak links that read some content aloud)
The Foundation for Retinal Research has a fact sheet on Leber congenital amaurosis
Find more detailed information on Leber congenital amaurosis and the gene mutations that cause it from GeneReviews
WonderBaby, information for parents of babies with Leber congenital amaurosis
PMCID: PMC1896221  PMID: 17594175
22.  SOX2 hypomorphism disrupts development of the prechordal floor and optic cup 
Mechanisms of development  2012;129(0):1-12.
Haploinsufficiency for the HMG-box transcription factor SOX2 results in abnormalities of the human ventral forebrain and its derivative structures. These defects include anophthalmia (absence of eye), microphthalmia (small eye) and hypothalamic hamartoma (HH), an overgrowth of the ventral hypothalamus. To determine how Sox2 deficiency affects the morphogenesis of the ventral diencephalon and eye, we generated a Sox2 allelic series (Sox2IR, Sox2LP, and Sox2EGFP), allowing for the generation of mice that express germline hypomorphic levels (<40%) of SOX2 protein and that faithfully recapitulate SOX2 haploinsufficient human phenotypes. We find that Sox2 hypomorphism significantly disrupts the development of the posterior hypothalamus, resulting in an ectopic protuberance of the prechordal floor, an upregulation of Shh signaling, and abnormal hypothalamic patterning. In the anterior diencephalon, both the optic stalks and optic cups (OC) of Sox2 hypomorphic (Sox2HYP) embryos are malformed. Furthermore, Sox2HYP eyes exhibit a loss of neural potential and coloboma, a common phenotype in SOX2 haploinsufficient humans that has not been described in a mouse model of SOX2 deficiency. These results establish for the first time that germline Sox2 hypomorphism disrupts the morphogenesis and patterning of the hypothalamus, optic stalk, and the early OC, establishing a model of the development of the abnormalities that are observed in SOX2 haploinsufficient humans.
PMCID: PMC4077342  PMID: 22522080
Sox2; Prechordal floor; Optic cup; Optic stalk; Shh; Patterning
23.  Coupling Mechanical Deformations and Planar Cell Polarity to Create Regular Patterns in the Zebrafish Retina 
PLoS Computational Biology  2012;8(8):e1002618.
The orderly packing and precise arrangement of epithelial cells is essential to the functioning of many tissues, and refinement of this packing during development is a central theme in animal morphogenesis. The mechanisms that determine epithelial cell shape and position, however, remain incompletely understood. Here, we investigate these mechanisms in a striking example of planar order in a vertebrate epithelium: The periodic, almost crystalline distribution of cone photoreceptors in the adult teleost fish retina. Based on observations of the emergence of photoreceptor packing near the retinal margin, we propose a mathematical model in which ordered columns of cells form as a result of coupling between planar cell polarity (PCP) and anisotropic tissue-scale mechanical stresses. This model recapitulates many observed features of cone photoreceptor organization during retinal growth and regeneration. Consistent with the model's predictions, we report a planar-polarized distribution of Crumbs2a protein in cone photoreceptors in both unperturbed and regenerated tissue. We further show that the pattern perturbations predicted by the model to occur if the imposed stresses become isotropic closely resemble defects in the cone pattern in zebrafish lrp2 mutants, in which intraocular pressure is increased, resulting in altered mechanical stress and ocular enlargement. Evidence of interactions linking PCP, cell shape, and mechanical stresses has recently emerged in a number of systems, several of which show signs of columnar cell packing akin to that described here. Our results may hence have broader relevance for the organization of cells in epithelia. Whereas earlier models have allowed only for unidirectional influences between PCP and cell mechanics, the simple, phenomenological framework that we introduce here can encompass a broad range of bidirectional feedback interactions among planar polarity, shape, and stresses; our model thus represents a conceptual framework that can address many questions of importance to morphogenesis.
Author Summary
Many tissues and organs, including sensory organs like the vertebrate retina and inner ear, are built from sheets of connected cells called epithelia. The precise arrangement of different types of cells within these epithelia can be essential to their function. (For example, photoreceptor cells in eyes must be properly spaced to collect an optimal, undistorted signal.) We combine experimental observations with computational modeling to understand how a particular example of such epithelial organization—the planar crystalline packing of cone photoreceptor cells in the fish retina—is created. Specifically, we introduce a model where the strength of cell-cell adhesion along an interface depends on the orientation of that interface. When a global mechanical compression is applied along one direction, this model can recapitulate observed features of the cone packing and gives qualitatively correct predictions of the cone photoreceptor pattern observed in regenerated and mutant retinas. Our analysis shows that simple local interactions can direct the creation of regular, long-ranged order among epithelial cells, and it also clarifies the mechanical interactions needed to establish and maintain the integrity of the retinal epithelium. Our model may thus ultimately provide a foundation for insights into diseases in which epithelial integrity is lost.
PMCID: PMC3426565  PMID: 22936893
24.  Confocal Scanning Laser Tomography of the Optic Nerve Head of Glaucoma Patients: Inter-Correlation of Disc Parameters 
Ghana Medical Journal  2009;43(4):150-156.
To evaluate the scanning laser tomography characteristics of the optic nerve head in patients with primary open angle (POAG) glaucoma using the Heidelberg retina tomography (HRT II).
A clinic-based retrospective study.
A total of 84 eyes of 42 POAG patients with good quality HRT II Images were studied at Charles Nicolle Hospital University department of Ophthalmology out-patient clinic, Tunis.
Characteristics of optic disc pattern of glaucoma patients were documented using the HRT II. Association of disc area with the other HRT parameters and inter-eye difference in the HRT parameters were assessed using simple and multiple regression analysis.
Main out-come measures
Disc area, cup area, rim area, cup-to-disc area ratio, cup volume, rim volume, mean cup depth, maximum cup depth and mean retinal nerve fibre layer thickness.
Twenty-seven males and 15 females were studied. The mean age of glaucoma patients was 48.9±2.7 years. The mean disc area, cup area, cup-to-disc area ratio and rim area were 2.19±0.46 (range, 1.23 – 3.16mm2), 0.95±0.94 (range 0.08 – 2.15), 0.42±0.21(range 0.004–0.92), 1.25± 0.46 (range 0.18–2.64) respectively. Disc area was positively correlated to the cup area (p=0.001), rim area (p=0.001), cup to disc area ratio (p=0.03), and mean cup depth (p=0.02).The glaucoma diagnosis score was strongly correlated with the rim area (p< 0.001), cup area (p< 0.001), mean cup depth (p< 0.001) and cup disc area ratio (p< 0.001). Absolute inter eye parameter between the two eyes were positively correlated with disc area (p< 0.05).
There was a significant correlation of the parameters between the two eyes and between the disc area and some other HRT parameters.
PMCID: PMC2956369  PMID: 21326994
Diagnosis; disk area; disk cup; glaucoma; Scanning Laser Tomography
25.  Three-dimensional stress analysis of threaded cups – a finite element analysis 
International Orthopaedics  2007;32(2):195-201.
A three-dimensional model of the left acetabulum with inserted threaded cup has been generated, based on the finite element method, to calculate stress patterns in the standing phase during walking. In this study, a hemispherical cup with sharp threads, a parabolic cup with flat threads and a conical cup with sharp threads were analysed and compared. Stress patterns in both implant components and adjacent bony structures were calculated in a directly postoperative situation. The different cups were found to induce different stress patterns, deformations and shifting tendencies. The inlays deform notably and show characteristic rotational movement patterns together with the shell. The inclination angle increases in the hemispherical cup and decreases in the parabolic cup. The conical cup levers outward almost parallel to the bone stock by approximately 0.05 mm. The pole surfaces of the various cups – especially the very convex area next to the threads – induce increased compressive stress in the superior section of the acetabular base. This is increased by a factor of three in the conical cup in comparison to the hemispherical cup and less so in comparison to the parabolic cup. This study illustrates that three-dimensional stress calculations are suitable for procuring additional biomechanical information to augment clinical studies, for evaluating implants and for establishing stability prognoses, especially for newly developed prototypes.
PMCID: PMC2269023  PMID: 17318551

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