Elastic Instabilities Govern the Morphogenesis of the Optic Cup.

Because the normal operation of the eye depends on sensitive morphogenetic processes for its eventual shape, developmental flaws can lead to wide-ranging ocular defects. However, the physical processes and mechanisms governing ocular morphogenesis are not well understood. Here, using analytical theory and nonlinear shell finite-element simulations, we show, for optic vesicles experiencing matrix-constrained growth, that elastic instabilities govern the optic cup morphogenesis. By capturing the stress amplification owing to mass increase during growth, we show that the morphogenesis is driven by two elastic instabilities analogous to the snap through in spherical shells, where the second instability is sensitive to the optic cup geometry. In particular, if the optic vesicle is too slender, it will buckle and break axisymmetry, thus, preventing normal development. Our results shed light on the morphogenetic mechanisms governing the formation of a functional biological system and the role of elastic instabilities in the shape selection of soft biological structures.

The influences of gender and aging on optic nerve head microcirculation in healthy adults.

Potential differences in the nature of the influences of aging and gender on the optic nerve head (ONH) microcirculation, using laser speckle flowgraphy (LSFG) were evaluated. We studied 908 healthy subjects (men = 701, age: 50.0 ± 9.1 yrs, women = 208, 49.8 ± 9.5 yrs, p = 0.76). The average, maximum (Max), and minimum (Min) mean blur rate (MBR) in a heartbeat were evaluated. The parameters were analyzed separately for the tissue, vessels, and throughout the ONH (All). We investigated which MBR sections are correlated with gender and age by univariate and multivariate regression analyses. The Max MBR-All (r = -0.31) was most strongly correlated with gender (men = 1, women = 0). The Min MBR-All (r = -0.24) was most strongly correlated with age, followed by Min MBR-All (r = -0.20). The factors contributing independently to the Max MBR-All were gender (ß = -0.15), pulse pressure, spherical refraction, ocular perfusion pressure, and red blood cell (RBC) count. The factors contributing independently to the Min MBR-Vessel were gender (ß = -0.09), age (ß = -0.25), body mass index, heart rate, and spherical refraction. The factors contributing independently to the Min-MBR-All were age (ß = -0.22), heart rate, and RBC count. Our results revealed that gender differences influence the Max MBR, and aging influences the Min MBR. These correlations were stronger than that of average MBR.

Retinal Angiogenesis Regulates Astrocytic Differentiation in Neonatal Mouse Retinas by Oxygen Dependent Mechanisms.

In mice, retinal vascular and astrocyte networks begin to develop at birth, expanding radially from the optic nerve head (ONH) towards the retinal periphery. The retinal vasculature grows towards the periphery ahead of differentiated astrocytes, but behind astrocytic progenitor cells (APCs) and immature astrocytes. Endothelial cell specific Vegfr-2 disruption in newborn mice not only blocked retinal vascular development but also suppressed astrocytic differentiation, reducing the abundance of differentiated astrocytes while causing the accumulation of precursors. By contrast, retinal astrocytic differentiation was accelerated by the exposure of wild-type newborn mice to hyperoxia for 24 hours, or by APC specific deficiency in hypoxia inducible factor (HIF)-2α, an oxygen labile transcription factor. These findings reveal a novel function of the retinal vasculature, and imply that in normal neonatal mice, oxygen from the retinal circulation may promote astrocytic differentiation, in part by triggering oxygen dependent HIF-2α degradation in astrocytic precursors.

The gap junction protein Innexin3 is required for eye disc growth in Drosophila.

The Drosophila compound eye develops from a bilayered epithelial sac composed of an upper peripodial epithelium layer and a lower disc proper, the latter giving rise to the eye itself. During larval stages, complex signalling events between the layers contribute to the control of cell proliferation and differentiation in the disc. Previous work in our lab established the gap junction protein Innexin2 (Inx2) as crucial for early larval eye disc growth. By analysing the contribution of other Innexins to eye size control, we have identified Innexin3 (Inx3) as an important growth regulator. Depleting inx3 during larval eye development reduces eye size, while elevating inx3 levels increases eye size, thus phenocopying the inx2 loss- and gain-of-function situation. As demonstrated previously for inx2, inx3 regulates disc cell proliferation and interacts genetically with the Dpp pathway, being required for the proper activation of the Dpp pathway transducer Mad at the furrow and the expression of Dpp receptor Punt in the eye disc. At the developmental timepoint corresponding to eye disc growth, Inx3 colocalises with Inx2 in disc proper and peripodial epithelium cell membranes. In addition, we show that Inx3 protein levels critically depend on inx2 throughout eye development and that inx3 modulates Inx2 protein levels in the larval eye disc. Rescue experiments demonstrate that Inx3 and Inx2 cooperate functionally to enable eye disc growth in Drosophila. Finally, we demonstrate that expression of Inx3 and Inx2 is not only needed in the disc proper but also in the peripodial epithelium to regulate growth of the eye disc. Our data provide a functional demonstration that putative Inx2/Inx3 heteromeric channels regulate organ size.

A wide temporal window for conjunctival papillae development ensures the formation of a complete sclerotic ring.

BACKGROUND: The conjunctival papillae are epithelial thickenings of the conjunctiva that are required for the induction of underlying bones (the scleral ossicles). These transient papillae develop and become inductively active over an extended temporal period (HH 30-36, 6.5-10 dpf). While their inductive capacity was discovered in the mid-1900s, little is known about their development. RESULTS: Through a series of timed surgical ablations followed by in situ hybridization for Bmp2, we show that the ring of conjunctival papillae is not altered if the conjunctival epithelium is ablated either prior to or shortly after papillae induction (i.e., HH 29-30, 6.5-7 dpf). A conjunctival papilla ablated at or prior to HH 34 (8 dpf), when the complete ring is present, regenerates and quickly becomes inductively active, inducing an underlying scleral condensation with only a slight delay. This regenerative capacity extends until HH 35.5, a full 36 hours beyond the normal timeline of papillae induction. As such, the period of epithelial competency for papilla induction is longer than previously identified. CONCLUSIONS: Papilla regeneration is a mechanism that ensures the formation of a complete sclerotic ring and provides another level of redundancy for the induction of a complete sclerotic ring during the normal inductive period. Developmental Dynamics 246:381-391, 2017. © 2017 Wiley Periodicals, Inc.

Imaging Features of Idiopathic Intracranial Hypertension in Children.

Magnetic resonance imaging (MRI) signs of elevated intracranial pressure and idiopathic intracranial hypertension have been well characterized in adults but not in children. The MRIs of 50 children with idiopathic intracranial hypertension and 46 adults with idiopathic intracranial hypertension were reviewed for optic nerve head protrusion, optic nerve head enhancement, posterior scleral flattening, increased perioptic cerebrospinal fluid, optic nerve tortuosity, empty or partially empty sella, tonsillar herniation, enlargement of Meckel's cave meningoceles, and transverse venous sinus stenosis(TSS). Compared to adolescents (11-17 years, n = 40) and adults (>17 years, n = 46), prepubescent children (<11 years, n = 10) had lower frequencies of scleral flattening (50% vs 89% and 85%, P = .02), increased perioptic cerebrospinal fluid (60% vs 84% and 89%, P = .08), optic nerve tortuosity (20% vs 46% and 59%, P = .07), empty or partially empty sella (56% vs 78% and 93%, P = .007), and TSS (67% vs 93% and 96%, P = .04). Children with idiopathic intracranial hypertension have similar MRI findings as adults, but they are less frequent in prepubescent children.

Gene expression analysis during the induction and patterning of the conjunctival papillae in the chick embryonic eye.

The induction and patterning of the conjunctival papillae (i.e. epithelial thickenings of the conjunctiva required for the induction of the underlying, neural crest-derived scleral ossicles) is complex. It takes place over a period of two days and follows a defined spatiotemporal sequence. In this study, we investigated the spatial and temporal expression pattern of four genes over seven morphological stages of development of these papillae. We show that ß-catenin is expressed during the pre-patterning of the epithelium prior to papilla induction and second that ß-catenin, Ednrb and Inhba are expressed during the induction and patterning of the conjunctival papillae. Furthermore, we identified two genes, ß-catenin and Prox1, that may be involved in the induction of the underlying scleral bones. These data provide an excellent baseline for future studies, setting the stage for functional studies aimed at examining the role of these genes in the patterning of the scleral ossicle system. This study also outlines the similarities between the conjunctival papillae and other placodes and may provide insights into the evolution and development of the conjunctival papillae.

Optic Nerve Head Development in Healthy Infants and Children Using Handheld Spectral-Domain Optical Coherence Tomography.

PURPOSE: To determine feasibility of optic nerve head (ONH) imaging and to characterize ONH development in full-term infants without sedation using handheld spectral-domain optical coherence tomography (SD OCT). DESIGN: Prospective cross-sectional study. PARTICIPANTS: Three hundred fifty-two children aged between 1 day and 13 years. METHODS: All participants were imaged using handheld SD OCT without sedation during a single scan session. The percentage of successful scans was calculated. Interexaminer reproducibility and differences between right and left eyes were assessed using intraclass correlation coefficients (ICCs). Images were analyzed using ImageJ software. The developmental trajectories over time for ONH parameters were calculated using fractional polynomial modelling. MAIN OUTCOME MEASURES: Disc and cup diameter (expressed as distance in micrometers and visual angle in degrees), cup depth, Bruch's membrane opening-minimum rim width (BMO-MRW), retinal thickness, and retinal nerve fiber layer (RNFL; 1700 µm and 6° from the disc center). RESULTS: On average, 70% of participants were imaged successfully. Interexaminer reliability was excellent (ICC, >0.89) for diametric and retinal thickness parameters. Right and left eyes were similar for diametric measurements (ICC, >0.79), but more variable for nasal BMO-MRW, RNFL, and retinal thickness. The mean disc and cup diameter increase by 30% and 40%, respectively, between birth and 13 years of age when expressed as a distance measure, but remained constant (at 5°-5.5° and 2°, respectively) when expressed as a visual angle with reference to the eye nodal point. The peripapillary temporal RNFL demonstrated a marked initial decrease of nearly 35% between birth and approximately 18 months of age. This was followed by a slow increase up to 12 years of age when measured at 1700 µm from the disc center, although there was little change when measured at 6° from the disc center. CONCLUSIONS: We demonstrated feasibility of handheld SD OCT imaging of the ONH in full-term infants and children without anaesthesia or sedation. This is the first in vivo handheld SD OCT study to describe the development of ONH parameters during the critical early years of visual maturation. Our results provide a normative database for use in routine practice and further studies of ONH pathologic features.

Tissue growth constrained by extracellular matrix drives invagination during optic cup morphogenesis.

In the early embryo, the eyes form initially as relatively spherical optic vesicles (OVs) that protrude from both sides of the brain tube. Each OV grows until it contacts and adheres to the overlying surface ectoderm (SE) via an extracellular matrix (ECM) that is secreted by the SE and OV. The OV and SE then thicken and bend inward (invaginate) to create the optic cup (OC) and lens vesicle, respectively. While constriction of cell apices likely plays a role in SE invagination, the mechanisms that drive OV invagination are poorly understood. Here, we used experiments and computational modeling to explore the hypothesis that the ECM locally constrains the growing OV, forcing it to invaginate. In chick embryos, we examined the need for the ECM by (1) removing SE at different developmental stages and (2) exposing the embryo to collagenase. At relatively early stages of invagination (Hamburger-Hamilton stage HH14[Formula: see text]), removing the SE caused the curvature of the OV to reverse as it 'popped out' and became convex, but the OV remained concave at later stages (HH15) and invaginated further during subsequent culture. Disrupting the ECM had a similar effect, with the OV popping out at early to mid-stages of invagination (HH14[Formula: see text] to HH14[Formula: see text]). These results suggest that the ECM is required for the early stages but not the late stages of OV invagination. Microindentation tests indicate that the matrix is considerably stiffer than the cellular OV, and a finite-element model consisting of a growing spherical OV attached to a relatively stiff layer of ECM reproduced the observed behavior, as well as measured temporal changes in OV curvature, wall thickness, and invagination depth reasonably well. Results from our study also suggest that the OV grows relatively uniformly, while the ECM is stiffer toward the center of the optic vesicle. These results are consistent with our matrix-constraint hypothesis, providing new insight into the mechanics of OC (early retina) morphogenesis.

A quantitative analysis of growth control in the Drosophila eye disc.

The size and shape of organs is species specific, and even in species in which organ size is strongly influenced by environmental cues, such as nutrition or temperature, it follows defined rules. Therefore, mechanisms must exist to ensure a tight control of organ size within a given species, while being flexible enough to allow for the evolution of different organ sizes in different species. We combined computational modeling and quantitative measurements to analyze growth control in the Drosophila eye disc. We find that the area growth rate declines inversely proportional to the increasing total eye disc area. We identify two growth laws that are consistent with the growth data and that would explain the extraordinary robustness and evolutionary plasticity of the growth process and thus of the final adult eye size. These two growth laws correspond to very different control mechanisms and we discuss how each of these laws constrains the set of candidate biological mechanisms for growth control in the Drosophila eye disc.

Regulation of Drosophila eye development by the transcription factor Sine oculis.

Homeodomain transcription factors of the Sine oculis (SIX) family direct multiple regulatory processes throughout the metazoans. Sine oculis (So) was first characterized in the fruit fly Drosophila melanogaster, where it is both necessary and sufficient for eye development, regulating cell survival, proliferation, and differentiation. Despite its key role in development, only a few direct targets of So have been described previously. In the current study, we aim to expand our knowledge of So-mediated transcriptional regulation in the developing Drosophila eye using ChIP-seq to map So binding regions throughout the genome. We find 7,566 So enriched regions (peaks), estimated to map to 5,952 genes. Using overlap between the So ChIP-seq peak set and genes that are differentially regulated in response to loss or gain of so, we identify putative direct targets of So. We find So binding enrichment in genes not previously known to be regulated by So, including genes that encode cell junction proteins and signaling pathway components. In addition, we analyze a subset of So-bound novel genes in the eye, and find eight genes that have previously uncharacterized eye phenotypes and may be novel direct targets of So. Our study presents a greatly expanded list of candidate So targets and serves as basis for future studies of So-mediated gene regulation in the eye.

Cadherin-mediated cell adhesion is critical for the closing of the mouse optic fissure.

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, ß-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.

Is the ataxia of Charlevoix-Saguenay a developmental disease?

The autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is considered a neurodegenerative disease caused by mutations in the SACS gene, located on chromosome 13q12.12. It is a syndrome that comprises skeletal, retinal and neurological manifestations, among which feature spasticity, cerebellar ataxia and peripheral neuropathy. Five patients with a molecular diagnosis of ARSACS underwent clinical, radiological, and ophthalmologic examinations. Every one of the identified causal mutations was novel. Spastic ataxia, peripheral neuropathy, pes cavus, and hammertoes were found in every case. T2 and T2-fluid attenuation inversion recovery-weighted MRI sequences demonstrated cerebellar atrophy and a hypointense linear striation at the pons. Tensor diffusion sequences revealed that the hypointense striation corresponded with hyperplasia of the pontocerebellar fibres, which gave place to abnormally thick middle cerebellar peduncles. Stereophotographs of the optic discs showed an increased number of retinal fibres, and ocular coherence tomography, increased thickness of the retinal nerve fibre layer. The authors suggest that the hyperplasic pontocerebellar fibres compress the pyramidal tracts at the pons since a very early stage of central nervous system development, causing spasticity, and may also cause cerebellar atrophy by means of glutamate-induced excitotoxicity. The abnormal amount of retinal fibres traversing the optic discs could have caused the detected mild peripheral visual field defects. Taken together, these facts point to a developmental cause in ARSACS, as it does not exhibit the tissue atrophy characteristic of degenerative diseases. Clinical deterioration in ARSACS seems to be mediated by phenomena (compression of the pyramidal tracts and cerebellar glutamate-mediated excitotoxicity) derived from the developmental anomalies referred to, while the neuromuscular symptoms are caused by a peripheral neuropathy with pathologic features suggestive of a similar origin. These observations should be taken into account when research about the origin of ARSACS is undertaken.

Birth weight deviation and early postnatal growth are related to optic nerve morphology at school age in children born preterm.

The aim of this study was to evaluate the influence of early and later postnatal growth variables on optic disc morphology in children (n = 53) born at gestational age <32 wk. On fundus photographs taken at a median age of 5.4 y, the optic discs were evaluated using digital image analysis and compared with those of a control group (n = 203). The results were analyzed in relation to gestational age, birth weight (BW) SD score (SDS), IGF-1 weight at postmenstrual age 32 wk (SDS), and weight, length, and head circumference (SDS) at follow-up. The preterm children's optic disc and neuronal rim areas were smaller than in the control group. Low BW (SDS) and weight at wk 32 (SDS) were associated with larger area of the optic cup and reduced neuronal rim area. Preterm children with known brain lesions (n = 6) had significantly larger cups than preterm children without known brain lesions. The association found between both low BW and poor early growth and later reduced neuronal tissue of the optic nerve indicate that early weight gain is important for neural development in preterm children.

Knockout of GARPs and the ß-subunit of the rod cGMP-gated channel disrupts disk morphogenesis and rod outer segment structural integrity.

Ion flow into the rod photoreceptor outer segment (ROS) is regulated by a member of the cyclic-nucleotide-gated cation-channel family; this channel consists of two subunit types, alpha and beta. In the rod cells, the Cngb1 locus encodes the channel beta-subunit and two related glutamic-acid-rich proteins (GARPs). Despite intensive research, it is still unclear why the beta-subunit and GARPs are coexpressed and what function these proteins serve. We hypothesized a role for the proteins in the maintenance of ROS structural integrity. To test this hypothesis, we created a Cngb1 5'-knockout photoreceptor null (Cngb1-X1). Morphologically, ROSs were shorter and, in most rods that were examined, some disks were misaligned, misshapen and abnormally elongated at periods when stratification was still apparent and degeneration was limited. Additionally, a marked reduction in the level of channel alpha-subunit, guanylate cyclase I (GC1) and ATP-binding cassette transporter (ABCA4) was observed without affecting levels of other ROS proteins, consistent with a requirement for the beta-subunit in channel assembly or targeting of select proteins to ROS. Remarkably, phototransduction still occurred when only trace levels of homomeric alpha-subunit channels were present, although rod sensitivity and response amplitude were both substantially reduced. Our results demonstrate that the beta-subunit and GARPs are necessary not only to maintain ROS structural integrity but also for normal disk morphogenesis, and that the beta-subunit is required for normal light sensitivity of the rods.

Pax2 in the optic nerve of the goldfish, a model of continuous growth.

Pax2 is a well known transcription factor which participates in optic nerve development. It assures the correct arrival and package of the newly formed retinal axons and the adequate differentiation of the newly formed glial cells. Pax2 protein expression is continuous throughout adult life in the goldfish optic nerve. We have found two populations of astrocytes in the optic nerve: Pax2(+) and Pax2(-). Moreover, we have observed that the Pax2(+) astrocytes from the optic nerve head present differences in number and organization to those of the rest of the optic nerve. In the optic nerve head some Pax2(+) astrocytes, principally localized in the glia limitans, have thin GFAP(+) processes and weak cytokeratin and ZO1 immunolabeling. Several Pax2(+) astrocytes are in close association with the GFAP(+)/GS(+) Müller cell vitreal processes and with the growing Zn8(+) retinal ganglion cell axons. However, in the intraorbital segment, Pax2(+) astrocytes are more numerous and they have strongly cytokeratin(+)/ZO1(+) processes and form part of the reticular astrocytes and the glia limitans. We also found Pax2(-) astrocytes in both the optic nerve head and the intraorbital segment. In the intraorbital segment there are GS(+)/Pax2(-) cells which are absent from the optic nerve head. We propose that the maintenance of Pax2 protein expression in adult goldfish optic nerve could be related to the continuous addition of new ganglion cell axons and new glial cells.

Fifty years later: the disk goes to the prom.

Although age-related macular degeneration is the most prevalent macular disease in the world, numerous discoveries regarding the molecular bases of vision have been made through genetic association studies of rare inherited maculopathies. In this issue of the JCI, Yang et al. present a functional genetics study that identifies a role for prominin 1 (PROM1), best known as a stem cell and/or progenitor cell marker, in the biogenesis of retinal photoreceptor disk arrays (see the related article beginning on page 2908). This study supports an established model in which disk morphogenesis occurs through membrane evagination and extends other recent studies assigning PROM1 important functions outside of the stem cell niche.

Visual phenotype in Williams-Beuren syndrome challenges magnocellular theories explaining human neurodevelopmental visual cortical disorders.

Williams-Beuren syndrome (WBS), a neurodevelopmental genetic disorder whose manifestations include visuospatial impairment, provides a unique model to link genetically determined loss of neural cell populations at different levels of the nervous system with neural circuits and visual behavior. Given that several of the genes deleted in WBS are also involved in eye development and the differentiation of retinal layers, we examined the retinal phenotype in WBS patients and its functional relation to global motion perception. We discovered a low-level visual phenotype characterized by decreased retinal thickness, abnormal optic disk concavity, and impaired visual responses in WBS patients compared with age-matched controls by using electrophysiology, confocal and coherence in vivo imaging with cellular resolution, and psychophysics. These mechanisms of impairment are related to the magnocellular pathway, which is involved in the detection of temporal changes in the visual scene. Low-level magnocellular performance did not predict high-level deficits in the integration of motion and 3D information at higher levels, thereby demonstrating independent mechanisms of dysfunction in WBS that will require remediation strategies different from those used in other visuospatial disorders. These findings challenge neurodevelopmental theories that explain cortical deficits based on low-level magnocellular impairment, such as regarding dyslexia.

Sensory deficits in mice hypomorphic for a mammalian homologue of unc-53.

The migration of cells and the extension of cellular processes along pathways to their defined destinations are crucial in the development of higher organisms. Caenorhabditis elegans unc-53 plays an important role in cell migration and the outgrowth of cellular processes such as axons. To gain further insight into the biological function of unc53H2, a recently identified mammalian homologue of unc-53, we have generated mice carrying a mutation of unc53H2 and provide evidence that unc53H2 is involved in neuronal development and, more specifically, the development of different sensory systems. The unc53H2 hypomorphic mouse showed a general impaired acuity of several sensory systems (olfactory, auditory, visual and pain sensation) which in case of the visual system was corroborated by the morphological observation of hypoplasia of the optic nerve. We hypothesize that in analogy with its C. elegans homologue, unc53H2 may play a role in the processes of cellular outgrowth and migration.