Vertebrate Eye Research Articles

Roles of LIM homeodomain 2 (Lhx2) in the differentiation of mammalian retinal pigmented epithelium

AbstractNormal vision depends on the retinal pigmented epithelium (RPE), a metabolic cell layer that is vital to the development and function of the adjacent retinal photoreceptors. The aim of this study was to uncover the transcriptional regulatory program and the cis‐regulatory elements required for RPE differentiation and maintenance. Lhx2 is a member of LIM homeodomain (LIM‐HD) protein family; it is a key factor for neural differentiation and is known to be required for early morphogenesis and patterning of the vertebrate eye. To discover the roles of Lhx2 in the differentiation of the mammalian RPE, we conducted functional studies in vivo using conditional mutagenesis in mouse, and knockdown in human RPE derived from embryonic stem cells (hES‐RPE). This was combined with global identification of regulatory regions bound by Lhx2 and a genomic characterization of these sites in the course of RPE differentiation. The results place Lhx2 upstream of a complex regulatory network that controls RPE differentiation, morphology and function in mice and humans.

The structure and oxidation of the eye lens chaperone αA-crystallin.

The small heat shock protein (sHsp) αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the structures of human αA-crystallin oligomers combining cryo-electron microscopy, cross-linking/mass spectrometry, nuclear magnetic resonance spectroscopy and molecular modeling. The different oligomers can be interconverted by the addition or subtraction of tetramers, leading to mainly 12-, 16- and 20-meric assemblies in which interactions between N-terminal regions are important. Cross-dimer domain-swapping of the C-terminal region is a determinant of αA-crystallin heterogeneity. Human αA-crystallin contains two cysteines, which can form an intramolecular disulfide in vivo. Oxidation in vitro requires conformational changes and oligomer dissociation. The oxidized oligomers, which are larger than reduced αA-crystallin and destabilized against unfolding, are active chaperones and can transfer the disulfide to destabilized substrate proteins. This insight into the structure and function of αA-crystallin provides a basis for understanding its role in the eye lens.

Control of size, fate and time by the Hh morphogen in the eyes of flies.

Molecules of the hedgehog (hh) family are involved in the specification and patterning of eyes in vertebrates and invertebrates. These organs, though, are of very different sizes, raising the question of how Hh molecules operate at such different scales. In this paper we discuss the strategies used by Hh to control the development of the two eye types in Drosophila: the large compound eye and the small ocellus. We first describe the distinct ways in which these two eyes develop and the evidence for the key role played by Hh in both; then we consider the potential for variation in the range of action of a "typical" morphogen and measure this range ("characteristic length") for Hh in different organs, including the compound eye and the ocellus. Finally, we describe how different feedback mechanisms are used to extend the Hh range of action to pattern the large and even the small eye. In the ocellus, the basic Hh signaling pathway adds to its dynamics the attenuation of its receptor as cell differentiate. This sole regulatory change can result in the decoding of the Hh gradient by receiving cells as a wave of constant speed. Therefore, in the fly ocellus, the Hh morphogen adds to its spatial patterning role a novel one: patterning along a time axis.

Intermediate filaments in the retinal Müller cells as natural light energy guides

In the current study, we investigated transmission spectrum of the intermediate filaments (IFs) extracted from porcine retinal Müller cells (MC). The recorded transmission spectrum is quite similar to that of the entire guinea pig retina, providing direct proof that the quantum mechanism (QM) of light energy transmission through the inverted retina is in fact the main mechanism determining the high-contrast vision of the vertebrate eyes. The recorded transmission spectrum of the IFs was deconvoluted into four bands, which we assign to different types of IFs. We interpret the differences in the transmission band shape and maxima as differences in the effective electron mass, dependent on the IF structure and composition of its constituting proteins. We analyzed model systems containing IFs and dye molecules, and IFs bridging two adjacent cobalt nanofilms on a substrate. The respective results demonstrate that IFs function as waveguides transferring energy from an energy donor and to an energy acceptor, by way of the exchange mechanism. The presently reported results provide direct experimental confirmation of the earlier proposed quantum mechanism for the high-contrast vision of vertebrate eyes. The mechanism proposed in the current study may be generalized, providing a consistent alternative to Davydov's theory of vibrational solitons in enzymes.

Divalent Cations and the Divergence of βγ-Crystallin Function.

The βγ-crystallin superfamily contains both β- and γ-crystallins of the vertebrate eye lens and the microbial calcium-binding proteins, all of which are characterized by a common double-Greek key domain structure. The vertebrate βγ-crystallins are long-lived structural proteins that refract light onto the retina. In contrast, the microbial βγ-crystallins bind calcium ions. The βγ-crystallin from the tunicate Ciona intestinalis (Ci-βγ) provides a potential link between these two functions. It binds calcium with high affinity and is found in a light-sensitive sensory organ that is highly enriched in metal ions. Thus, Ci-βγ is valuable for investigating the evolution of the βγ-crystallin fold away from calcium binding and toward stability in the apo form as part of the vertebrate lens. Here, we investigate the effect of Ca2+ and other divalent cations on the stability and aggregation propensity of Ci-βγ and human γS-crystallin (HγS). Beyond Ca2+, Ci-βγ is capable of coordinating Mg2+, Sr2+, Co2+, Mn2+, Ni2+, and Zn2+, although only Sr2+ is bound with comparable affinity to its preferred metal ion. The extent to which the tested divalent cations stabilize Ci-βγ structure correlates strongly with ionic radius. In contrast, none of the tested divalent cations improved the stability of HγS, and some of them induced aggregation. Zn2+, Ni2+, and Co2+ induce aggregation by interacting with cysteine residues, whereas Cu2+-mediated aggregation proceeds via a different binding site.

Synchrotron X-ray absorption spectroscopy of melanosomes in vertebrates and cephalopods: implications for the affinity of Tullimonstrum

Screening pigments are essential for vision in animals. Vertebrates use melanins bound in melanosomes as screening pigments, whereas cephalopods are assumed to use ommochromes. Preserved eye melanosomes in the controversial fossil Tullimonstrum (Mazon Creek, IL, USA) are partitioned by size and/or shape into distinct layers. These layers resemble tissue-specific melanosome populations considered unique to the vertebrate eye. Here, we show that extant cephalopod eyes also show tissue-specific size- and/or shape-specific partitioning of melanosomes; these differ from vertebrate melanosomes in the relative abundance of trace metals and in the binding environment of copper. Chemical signatures of melanosomes in the eyes of Tullimonstrum more closely resemble those of modern cephalopods than those of vertebrates, suggesting that an invertebrate affinity for Tullimonstrum is plausible. Melanosome chemistry may thus provide insights into the phylogenetic affinities of enigmatic fossils where melanosome size and/or shape are equivocal.

Evolutionary Medicine – A Great Way to Teach Biology

Great teaching is often all about the questions posed to students. When learners get their teeth into an interesting question, they grow as they chew on it. As physicians, we have discovered that disease makes more sense if you ask evolutionary questions, and these questions make a great starting point for science education. One of Darwin's two main questions was how species are related to each other. His answer was that all organisms have common ancestors. This is the foundation for studies of phylogeny. To teach phylogenetic trees, you can use canids or birds, but students are particularly interested if they are tracing the origins of HIV or tuberculosis, or the history of human traits, such as the appendix. Darwin's other question was about why bodies work so well. His answer was natural selection. The characteristics of individuals who have more offspring than others become more common over the generations. This explains why kidneys, hearts, eyes, and brains are so astoundingly well suited to their purposes. As physicians, we constantly confront a very different question. If natural selection is so …

Volt-ampere characteristics of porcine retinal Müller cell intermediate filaments

In the current study we reported current-voltage (I/V) characteristics of Müller cell (MC) intermediate filaments (IFs) isolated from porcine retina. It was found that the measured I/V dependences demonstrate behavior of a semiconductor in contact with metal (Au) electrodes. The analysis of the temperature dependence of the experimental I/V curves produced estimates of the parameter values characterizing the electrical conductivity properties of the studied MC IFs. The I/V characteristics and the parameter values allowed to describe the MC IFs as a semiconducting material. The observed properties clarify the mechanism of high-contrast daylight vision of vertebrate eyes. This mechanism was extensively discussed earlier, and is directly dependent on the electric conductivity properties of MC IFs. Significance StatementRetinal cones and rods are physically connected to glial Müller cells by intermediate filaments (IFs). Electric conductivity of IFs allows for a simple quantum mechanical description of light energy transmission through the retina, providing a convincing mechanism that achieves high-contrast vision in vertebrate eyes. Note that classic light transmission is hindered by light scattering on the retinal structures. Electric conductivity of IFs also opens the possibility for energy exchange within and between other cell types by way of IFs and microtubules, and the possibility that such structures may be used for signaling e.g. in the nervous system.

Constant lens fiber cell thickness in fish suggests crystallin transport to denucleated cells

The crystalline lens of the vertebrate eye grows throughout life. This growth may be enormous in fish, while the lens must be functional from larva to adult. During growth, the fiber cells of the lens must increase the concentration of specific proteins (crystallins) in the cytoplasm to increase refractive index. However, the bulk of the fiber cells in a vertebrate lens are denucleated and have no organelles to synthesize proteins. To study how this problem is solved, we first measured lens fiber cell thickness in the Nile tilapia, a teleost fish. In the lenses from 25 fish, in two size groups, fibers were considerably thinner than in other vertebrates. Fiber thickness was about constant along the radius of the lens and the same between the size groups. Since our results provided no evidence for shrinkage of lens fiber cells with growth (expected if protein concentration is increased by expelling water) we included eight additional teleost species to elucidate the mechanism by which the cells increase crystallin concentration. In all species, fiber cell thickness was about constant throughout the lens, with species-specific values. The changes in fiber cell thickness expected from an increase in crystallin concentration by removal of water were modeled. Shrinkage in cell thickness by up to 66% would have been necessary to reach the required crystallin concentration. We conclude that crystallin concentration in denucleated lens fiber cells is increased by transport of proteins from synthetically competent cells in the periphery of the lens.

Upregulation of sine oculis homeobox homolog 3 is associated with proliferation, invasion, migration, as well as poor prognosis of esophageal cancer.

Esophageal cancer (EC) is a common cancer worldwide. Sine oculis homeobox homolog (SIX3) is a human transcription factor that regulates the progression of vertebrate eye and fetal forebrain. However, studies on the function of SIX3 in human tumorigenesis remain rare. In this study, we aim to evaluate the role and the significance of SIX3 in EC. The TCGA database and clinical samples were used to assess the expression of SIX3 in EC patients. The Kaplan-Meier method and Cox's proportional hazards model were performed to analyze the correlations between SIX3 expression and EC clinical outcomes. The expressions of SIX3 in EC cells were measured by quantitative reverse transcription PCR analysis. The cell proliferation was detected using cell counting kit-8 and colony formation assay. The migration and invasion capacity of EC cells were evaluated using wound healing and Transwell methods. Western blot assay was used to measure the alterations in some important protein expression levels in the PI3K/Akt signaling pathway. We found that SIX3 was highly expressed in the EC tissues and cells. In addition, high expression of SIX3 was related to poor survival. The knockdown of SIX3 significantly inhibited the proliferation, migration, and invasion of ECA109 cells. A similar pattern was also found in the proliferation and migration of SKGT-4 cell line. The expression levels of some key proteins in the PI3K/Akt signaling pathway were obviously decreased after cells were transfected with si-SIX3, possibly resulting in PI3K/AKT signaling inactivation. In addition, E-cadherin and N-cadherin showed some change. Collectively, the results shed light on a potentially promoting role of SIX3 in human EC. Thus, SIX3 might be considered a novel prognostic biomarker and therapeutic target for EC patients.

Quantum mechanism of light energy propagation through an avian retina

Taking into account the ultrastructure of the Pied Flycatcher foveal retina reported earlier and the earlier reported properties of Müller cell (MC) intermediate filaments (IFs) isolated from vertebrate retina, we proposed a quantum mechanism (QM) of light energy transfer from the inner limiting membrane level to visual pigments in the photoreceptor cells. This mechanism involves electronic excitation energy transfer in a donor-acceptor system, with the IFs excited by photons acting as energy donors, and visual pigments in the photoreceptor cells acting as energy acceptors. It was shown earlier that IFs with diameter 10 nm and length 117 μm isolated from vertebrate eye retina demonstrate properties of light energy guide, where exciton propagates along such IFs from MC endfeet area to photoreceptor cell area. The energy is mostly transferred via the contact exchange quantum mechanism. Our estimates demonstrate that energy transfer efficiencies in such systems may exceed 80–90%. Thus, the presently developed quantum mechanism of light energy transfer in the inverted retina complements the generally accepted classic optical mechanism and the mechanism whereby Müller cells transmit light like optical fibers. The proposed QM of light energy transfer in the inverted retina explains the high image contrast achieved in photopic conditions by an avian eye, being probably also active in other vertebrates.

Altered ocular parameters from circadian clock gene disruptions.

The pathophysiology of refractive errors is poorly understood. Myopia (nearsightedness) in particular both blurs vision and predisposes the eye to many blinding diseases during adulthood. Based on past findings of diurnal variations in the dimensions of the eyes of humans and other vertebrates, altered diurnal rhythms of these ocular dimensions with experimentally induced myopia, and evolving evidence that ambient light exposures influence refractive development, we assessed whether disturbances in circadian signals might alter the refractive development of the eye. In mice, retinal-specific knockout of the clock gene Bmal1 induces myopia and elongates the vitreous chamber, the optical compartment separating the lens and the retina. These alterations simulate common ocular findings in clinical myopia. In Drosophila melanogaster, knockouts of the clock genes cycle or period lengthen the pseudocone, the optical component of the ommatidium that separates the facet lens from the photoreceptors. Disrupting circadian signaling thus alters optical development of the eye in widely separated species. We propose that mechanisms of myopia include circadian dysregulation, a frequent occurrence in modern societies where myopia also is both highly prevalent and increasing at alarming rates. Addressing circadian dysregulation may improve understanding of the pathogenesis of refractive errors and introduce novel therapeutic approaches to ameliorate myopia development in children.

Investigating the role of body size, ecology, and behavior in anuran eye size evolution

Vertebrate eye size typically scales hypoallemetrically with body size—as animals grow larger their eyes get relatively smaller. Additionally, eye size is highly variable across species, and such variability often reflects functional adaptations to differences in behavior and/or ecology. The selective pressures underlying the evolution of eye size are especially well studied in birds, mammals, and fishes. However, whether similar scaling rules and selective pressures also underlie the evolution of eye size in amphibians remains enigmatic. Variation in eye size is intimately linked with variation in brain anatomy, as the retina is ontogenetically part of the brain. Eye size may therefore coevolve with brain size. Here we use phylogenetic comparative methods to study interspecific variation in eye volume across 44 species of anurans from 8 families from the Hengduan Mountains, China. We relate this variation to key factors known to impact eye size evolution in other vertebrate taxa such as body mass, habitat use, defense strategy and foraging mobility. We found that also in anurans eyes size scaled hypoallometrically with body mass. However, neither of the behavioral or ecological factors explained any variation in relative eye size in our sample. Whether this is representative for other frog species needs to be clarified. We therefore conclude that eye size in frogs is tightly linked to body mass evolution but that, at least in the species investigated here, none of our tested ecological and behavioral factors have a strong influence on eye size evolution.

Trabecular Meshwork TREK-1 Channels Function as Polymodal Integrators of Pressure and pH.

PurposeThe concentration of protons in the aqueous humor (AH) of the vertebrate eye is maintained close to blood pH; however, pathologic conditions and surgery may shift it by orders of magnitude. We investigated whether and how changes in extra- and intracellular pH affect the physiology and function of trabecular meshwork (TM) cells that regulate AH outflow.MethodsElectrophysiology, in conjunction with pharmacology, gene knockdown, and optical recording, was used to track the pH dependence of transmembrane currents and mechanotransduction in primary and immortalized human TM cells.ResultsExtracellular acidification depolarized the resting membrane potential by inhibiting an outward K+-mediated current, whereas alkalinization hyperpolarized the cells and augmented the outward conductance. Intracellular acidification with sodium bicarbonate hyperpolarized TM cells, whereas removal of intracellular protons with ammonium chloride depolarized the membrane potential. The effects of extra- and intracellular acid and alkaline loading were abolished by quinine, a pan-selective inhibitor of two-pore domain potassium (K2P) channels, and suppressed by shRNA-mediated downregulation of the mechanosensitive K2P channel TREK-1. Extracellular acidosis suppressed, whereas alkalosis facilitated, the amplitude of the pressure-evoked TREK-1–mediated outward current.ConclusionsThese results demonstrate that TM mechanotransduction mediated by TREK-1 channels is profoundly sensitive to extra- and intracellular pH shifts. Intracellular acidification might modulate aqueous outflow and IOP by stimulating TREK-1 channels.

Fabrication and characterization of a polymeric curved compound eye

Curved compound eyes, i.e. microlens arrays on curved surfaces, are highly desirable for their unique optical properties including wide field of view, tracking of fast moving objects and so on. However, it is technically challenging to fabricate biomimetic gapless microlens arrays. In this work, we report a simple method for fabricating close-packed microlenses in a kind of stretchable polymeric material PDMS on curved surfaces inspired by the vertebrate eyes. The successfully fabricated polymeric curved compound eye consists of more than 6000 close-packed ommatidia with an average diameter of 600 µm for each ommatidium. The ommatidia are located on a convex surface with a diameter of 40 mm and thus a total field of view of about 180° has been obtained. The optical test on ommatidia shows that the NA for each ommatidium is about 0.21 and the imaging result of the whole compound eye is also given. Furthermore, an optical relay system is introduced to integrate with the compound eye to form a biomimetic compound eye camera. The formed camera is shown to have a great potential for a broad range of optical imaging applications, such as surveillance imaging, target detection and tracking, surveying and mapping, collision-free navigation of terrestrial and aerospace vehicles.

GLI3 repressor but not GLI3 activator is essential for mouse eye patterning and morphogenesis

Since 1967, it is known that the loss of GLI3 causes very severe defects in murine eye development. GLI3 is able to act as a transcriptional activator (GLI3-A) or as a transcriptional repressor (GLI3-R). Soon after the discovery of these GLI3 isoforms, the question arose which of the different isoforms is involved in eye formation – GLI3-A, GLI3-R or even both. For several years, this question remained elusive. By analysing the eye morphogenesis of Gli3XtJ/XtJ mouse embryos that lack GLI3-A and GLI3-R and of Gli3Δ699/Δ699 mouse embryos in which only GLI3-A is missing, we revealed that GLI3-A is dispensable in vertebrate eye formation. Remarkably, our study shows that GLI3-R is sufficient for the creation of morphologically normal eyes although the molecular setup deviates substantially from normality. In depth-investigations elucidated that GLI3-R controls numerous key players in eye development and governs lens and retina development at least partially via regulating WNT/β-CATENIN signalling.

PO2 Profiles in the Retina of the Hemoglobin‐less Icefishes

The retina of vertebrates has one of the highest O2 demands and requires a constant O2 supply. Four general types of retinal circulations have been described for O2 delivery in extant vertebrates: 1) sole reliance on the choriocapillaris; 2) capillaries penetrating the retina; 3) capillarization of the inner side of the retina; and 4) O2 secretion. Oxygen secretion is based on acid secretion into the blood resulting in a potent unloading of O2 from so‐called Root effect hemoglobins (Hb), which is augmented by venous to arterial O2 diffusion in a vascular rete lining the back side of the retina. This choroid rete mirabile (CRM) allows O2 delivery at super‐atmospheric pressure in the avascular retina of some fishes that have the thickest retinae and some of the largest eyes (relative to body size) of all extant vertebrates. The suborder Notothenioidei, which consists of Antarctic and sub‐Antarctic fishes, provides a model system to unravel how the interactions between Hb function and retinal vascularity evolved to produce modern fish eyes. While several phyletically basal notothenioids have been shown to display similar ocular arrangements as in other Perciform species containing a CRM, more phyletically derived notothenioids have highly reduced CRM or have lost it completely. Of particular interest are the species of the family Channichthyidae, icefishes, that are unique among adult vertebrates in possessing pale white blood containing only few vestigial immature erythrocytes and no Hb. Without Hb, O2 secretion is not functional, and icefishes have been demonstrated to possess heavily increased pre‐retinal capillarization most probably to maintain retinal oxygenation and preserve large eyes and thick retinae. However, whether available O2 at different retinal levels actually follows the degree of vascularization has not been tested.Using thin O2 sensors, we measured profiles of partial pressure of O2 (PO2) in the retina of six species of icefishes, two species of related, but red‐blooded dragonfishes (Bathydraconidae), and four species of more distantly related notothens (Nototheniidae). Arterial PO2 was statistically significantly higher in icefishes than in notothens [t‐test; p = 0.0066 (n = 3); PaO2 = 16.64 ± 3.84 kPa in Chaenocephalus aceratus; PaO2 = 4.42 ± 1.40 kPa in Notothenia coriiceps]. In general, pre‐retinal PO2 was high in icefishes (19.20 ± 6.35 kPa in C. aceratus), approaching the theoretical O2 solubility limit without O2 secretion. The PO2 level decreased towards the outer portion of the retina, but with a small peak on the choroid side of the retina, possibly due to a vestigial choriocapillaris. In notothens displaying Root effect, pre‐retinal PO2 was slightly lower (12.07 ± 5.12 kPa in N. coriiceps) but increased to a super‐atmospheric level on the outer portion of the retina (52.25 ± 32.46 kPa in N. coriiceps).Our results demonstrate that retinal PO2 is dictated by vascularization in the Hb‐deprived icefishes, leading to a unique reversal in the retinal oxygen profile.Support or Funding InformationUS National Science Foundation grant PLR‐1444167Carlsberg FoundationVelux FoundationsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cellular and Molecular Dynamics of Vertebrate Eye Morphogenesis: How Neural Crest Shapes the Embryonic Eye

Precise and stereotyped 3‐dimensional tissue structure, established during the process of morphogenesis, is essential for proper organ function. In the visual system, aberrant structural development of the embryonic eye can lead to visual impairment and blindness. Despite increasing knowledge of pathways controlling the differentiation of many cell types in the eye, we still lack a basic understanding of the mechanisms controlling its morphogenesis. Using a combination of 4‐dimensional timelapse imaging, computational approaches, and zebrafish genetics, we seek to unravel the cellular and molecular basis, including both intrinsic and extrinsic cues, for the morphogenesis of the initial embryonic eye structure, the optic cup. One particular extrinsic cell population of interest is the neural crest: migratory mesenchymal cells are essential for the development and morphogenesis of many epithelial organs. In mouse, eye defects have been described in a neural crest mutant, but the mechanisms underlying aberrant optic cup development are unknown. Using zebrafish, we asked when and how neural crest contacts the developing eye, and how optic cup defects arise.We find that at the earliest stages of optic vesicle formation, neural crest cells are in contact with the optic vesicle; by the end of optic cup morphogenesis, these neural crest derived cells encapsulate the optic stalk and optic cup, except for its distal, lens‐facing side. To test the role of neural crest, we utilize the zebrafish tfap2ats213;foxd3zdf10 double mutant: in the absence of neural crest, optic cup invagination is defective, and using 4D cell tracking, we pinpointed the cell movements disrupted by loss of neural crest.What molecule is neural crest providing to control optic cup morphogenesis? Strikingly, using electron microscopy, we directly observe loss of basement membrane assembly in the neural crest mutant. Nidogen, a laminin‐collagen crosslinking protein expressed by neural crest and essential for optic cup morphogenesis in embryonic stem cells, appears to be a crucial factor: dominant negative nidogen disrupts wild type optic cup morphogenesis without affecting neural crest migration, and overexpression of wild type nidogen partially rescues optic cup morphogenesis in neural crest mutants. There are four nidogen genes in zebrafish (nid1a, nid1b, nid2a, and nid2b), and we are now generating quadruple mutants to examine loss‐of‐function phenotypes.Our data suggest that neural crest provides crucial extracellular matrix factors to regulate optic cup morphogenesis, and highlight diverse roles mesenchyme can play in epithelial organ development and morphogenesis.Support or Funding InformationNIH/NEI R01EY025378, R01EY025780 to KMK. CDB was supported by the University of Utah T32 Developmental Biology Training Grant (NIH T32HD007491).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis.

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid fissure closure. Recently, the identification of individuals with coloboma in the superior aspect of the iris, retina, and lens led to the discovery of a novel structure, referred to as the superior fissure or superior ocular sulcus (SOS), that is transiently present on the dorsal aspect of the optic cup during vertebrate eye development. Although this structure is conserved across mice, chick, fish, and newt, our current understanding of the SOS is limited. In order to elucidate factors that contribute to its formation and closure, it is imperative to be able to observe it and identify abnormalities, such as delay in the closure of the SOS. Here, we set out to create a standardized series of protocols that can be used to efficiently visualize the SOS by combining widely available microscopy techniques with common molecular biology techniques such as immunofluorescent staining and mRNA overexpression. While this set of protocols focuses on the ability to observe SOS closure delay, it is adaptable to the experimenter's needs and can be easily modified. Overall, we hope to create an approachable method through which our understanding of the SOS can be advanced to expand the current knowledge of vertebrate eye development.

Homozygous frameshift mutations in FAT1 cause a syndrome characterized by colobomatous-microphthalmia, ptosis, nephropathy and syndactyly

A failure in optic fissure fusion during development can lead to blinding malformations of the eye. Here, we report a syndrome characterized by facial dysmorphism, colobomatous microphthalmia, ptosis and syndactyly with or without nephropathy, associated with homozygous frameshift mutations in FAT1. We show that Fat1 knockout mice and zebrafish embryos homozygous for truncating fat1a mutations exhibit completely penetrant coloboma, recapitulating the most consistent developmental defect observed in affected individuals. In human retinal pigment epithelium (RPE) cells, the primary site for the fusion of optic fissure margins, FAT1 is localized at earliest cell-cell junctions, consistent with a role in facilitating optic fissure fusion during vertebrate eye development. Our findings establish FAT1 as a gene with pleiotropic effects in human, in that frameshift mutations cause a severe multi-system disorder whereas recessive missense mutations had been previously associated with isolated glomerulotubular nephropathy.