Eye Formation Research Articles

Physiological effects of KDM5C on neural crest migration and eye formation during vertebrate development

BackgroundLysine-specific histone demethylase 5C (KDM5C) belongs to the jumonji family of demethylases and is specific for the di- and tri-demethylation of lysine 4 residues on histone 3 (H3K4 me2/3). KDM5C is expressed in the brain and skeletal muscles of humans and is associated with various biologically significant processes. KDM5C is known to be associated with X-linked mental retardation and is also involved in the development of cancer. However, the developmental significance of KDM5C has not been explored yet. In the present study, we investigated the physiological roles of KDM5C during Xenopus laevis embryonic development.ResultsLoss-of-function analysis using kdm5c antisense morpholino oligonucleotides indicated that kdm5c knockdown led to small-sized heads, reduced cartilage size, and malformed eyes (i.e., small-sized and deformed eyes). Molecular analyses of KDM5C functional roles using whole-mount in situ hybridization, β-galactosidase staining, and reverse transcription-polymerase chain reaction revealed that loss of kdm5c resulted in reduced expression levels of neural crest specifiers and genes involved in eye development. Furthermore, transcriptome analysis indicated the significance of KDM5C in morphogenesis and organogenesis.ConclusionOur findings indicated that KDM5C is associated with embryonic development and provided additional information regarding the complex and dynamic gene network that regulates neural crest formation and eye development. This study emphasizes the functional significance of KDM5C in Xenopus embryogenesis; however, further analysis is needed to explore the interactions of KDM5C with specific developmental genes.

A Review of Physical and Numerical Approaches for the Study of Gas Stirring in Ladle Metallurgy

This article presents a review of the research into gas stirring in ladle metallurgy carried out over the past few decades. Herein, the physical modeling experiments are divided into four major areas: (1) mixing and homogenization in the ladle; (2) gas bubble formation, transformation, and interactions in the plume zone; (3) inclusion behavior at the steel–slag interface and in the molten steel; and (4) open eye formation. Several industrial trials have also been carried out to optimize gas stirring and open eye formation. Approaches for selecting criteria for scaling to guarantee flow similarity between industrial trials and physical modeling experiments are discussed. To describe the bubble behavior and two-phase plume structure, four main mathematical models have been used in different research fields: (1) the quasi-single-phase model, (2) the volume of fluid (VOF) model, (3) the Eulerian multiphase (E–E) model, and (4) the Eulerian–Lagrangian (E–L) model. In recent years, the E–E model has been used to predict gas stirring conditions in the ladle, and specific models in commercial packages, as well as research codes, have been developed gradually to describe the complex physical and chemical phenomena. Furthermore, the coupling of turbulence models with multiphase models is also discussed. For physical modeling, some general empirical rules have not been analyzed sufficiently. Based on a comparison with the available experimental results, it is found that the mathematical models focusing on the mass transfer phenomenon and inclusion behaviors at the steel-slag interface, vacuum degassing at the gas–liquid interface, dissolution rate of the solid alloy at the liquid–solid interface, and the combination of fluid dynamics and thermodynamics need to be improved further. To describe industrial conditions using mathematical methods and improve numerical modeling, the results of physical modeling experiments and industrial trials must offer satisfactory validations for the improvement of numerical modeling.

Embryonic origin and genetic basis of cave associated phenotypes in the isopod crustacean Asellus aquaticus

Characteristics common to animals living in subterranean environments include the reduction or absence of eyes, lessened pigmentation and enhanced sensory systems. How these characteristics have evolved is poorly understood for the majority of cave dwelling species. In order to understand the evolution of these changes, this study uses an invertebrate model system, the freshwater isopod crustacean, Asellus aquaticus, to examine whether adult differences between cave and surface dwelling individuals first appear during embryonic development. We hypothesized that antennal elaboration, as well as eye reduction and pigment loss, would be apparent during embryonic development. We found that differences in pigmentation, eye formation, and number of segments of antenna II were all present by the end of embryonic development. In addition, we found that cave and surface hatchlings do not significantly differ in the relative size of antenna II and the duration of embryonic development. To investigate whether the regions responsible for eye and pigment differences could be genetically linked to differences in article number, we genotyped F2 hybrids for the four previously mapped genomic regions associated with eye and pigment differences and phenotyped these F2 hybrids for antenna II article number. We found that the region previously known to be responsible for both presence versus absence of pigment and eye size also was significantly associated with article number. Future experiments will address whether pleiotropy and/or genetic linkage play a role in the evolution of cave characteristics in Asellus aquaticus.

Clinical Outcomes Using Oversized Back Plates in Type I Boston Keratoprosthesis.

To examine clinical outcomes of oversized titanium back plates in type I Boston keratoprosthesis (KPro) implantation. Retrospective study of 22 sequential eyes (20 patients) undergoing type I KPro implantation with an oversized titanium back plate (larger than trephined wound diameter by 1.0 mm or more), performed by a single surgeon (K.A.C.) from June 2010 to November 2014. Data were collected regarding preoperative eye characteristics, surgical details, and postoperative clinical outcomes. Mean follow-up time per eye was 24.1±14.9 months. All eyes had improved vision after surgery; 13 eyes (59.1%) maintained visual acuity improvement at last follow-up. Initial KPro's were retained in 19 eyes (86.4%); one eye required KPro replacement. Primary retroprosthetic membrane (RPM) developed in three eyes (13.6%), with similar occurrence in aniridic (14.3%) and nonaniridic eyes (13.3%). Secondary RPM's developed in two eyes (9.1%) after vitritis (one eye) and retinal and choroidal detachment (one eye). Glaucoma was a common comorbidity: 2 of 14 eyes (14.3%) with preoperative glaucoma had glaucoma progression, and 4 of 8 eyes (50.0%) without preoperative glaucoma developed glaucoma postoperatively. Other postoperative complications included retinal detachment (5 eyes, 22.7%) and idiopathic vitritis (3 eyes, 13.6%). Oversized titanium KPro back plates are associated with a low rate of primary RPM formation and may have particular utility in reducing primary RPM formation in aniridic eyes. Glaucoma remains a challenge in postoperative KPro management. Complex eyes, at increased risk of postoperative complications, require careful management.

Structural and Functional Analyses of Retinal Ischemia in Eyes with Retinal Vein Occlusion: Relationship with Macular Edema or Microaneurysm Formation

Purpose: To study the structural and functional changes of retinal ischemia and investigate their association with macular edema (ME) or microaneurysm (MA) formation in eyes with retinal vein occlusion (RVO). Methods: Sixty eyes of 30 patients (27 eyes with branch [b]RVO, 3 with central RVO, and 30 fellow eyes) were retrospectively reviewed. Optical coherence tomography (OCT), OCT angiography (OCTA), and microperimetry were performed simultaneously to measure retinal thickness and sensitivity. The presence of ME or MA was also assessed using OCT and fluorescein angiography. Results: The mean retinal sensitivity in the nonperfused areas (NPAs) deteriorated, and this was significantly (r = –0.379, p = 0.0391*) and inversely correlated with duration from disease onset. ME and MA were unlikely to be observed around the area where the retinal sensitivity decreased. In the NPAs, the mean retinal thickness of the superficial capillary plexus (SCP) (p < 0.0001), deep capillary plexus (DCP) (p = 0.0323), and outer retina (p = 0.0008) were significantly thinner than those in the fellow eyes, respectively. Multivariate regression analysis revealed that the thicknesses of the DCP (β: 0.3107, p = 0.0007) and outer retina (β: 0.3482, p = 0.0001) were the independent correlative factors of the retinal sensitivity, but that SCP thickness was not. Conclusion: Deep retinal thinning in NPAs was correlated significantly with a decreased retinal sensitivity, which might be a negative predictor of ME and MA in eyes with RVO.

Fam46a regulates BMP-dependent pre-placodal ectoderm differentiation in Xenopus.

The pre-placodal ectoderm (PPE) is a specialized ectodermal region which gives rise to the sensory organs and other systems. The PPE is induced from the neural plate border during neurulation, but the molecular mechanism of PPE formation is not fully understood. In this study, we examined the role of a newly identified PPE gene, Fam46a, during embryogenesis. Fam46a contains a nucleoside triphosphate transferase domain, but its function in early development was previously unclear. We show that Fam46a is expressed in the PPE in Xenopus embryos, and Fam46a knockdown induces abnormalities in the eye formation and the body color. At the neurula stage, Fam46a upregulates the expression of PPE genes and inhibits neural crest formation. We also show that Fam46a physically interacts with Smad1/Smad4 and positively regulates BMP signaling. From these results, we conclude that Fam46a is required for PPE formation via the positive regulation of BMP signaling. Our study provides a new mechanism of ectodermal patterning via cell-autonomous regulation of BMP signaling in the PPE.

The effect of NaCl and metabolic profile of propionibacteria on eye formation in experimental Swiss-type cheese

The influence of three brining times (0, 1, and 3 d) and two propionibacteria (PAB) cultures (Prop A and Prop B) on eye formation was investigated in experimental Swiss-type cheeses by comparing PAB counts, biochemical parameters, eye numbers, diameters and volumes. Prop A was strongly inhibited with increasing NaCl content. As a result, eye volume decreased considerably, as well as eye diameter especially towards the cheese surface. Prop B exclusively converted l-lactate and was much less affected by increasing NaCl content, hence, eye formation changed less. The salt sensitivity of the cultures also influenced the spatial distribution of the eyes. The width of the eyeless border zone was mainly affected by three factors: CO2 diffusion to the outside, propionic acid fermentation, and body firmness. The study clearly showed that strain-specific salt sensitivity of PAB is an important feature that strongly influences eye formation in Swiss-type cheeses with different salt content.

Nosip functions during vertebrate eye and cranial cartilage development.

The nitric oxide synthase interacting protein (Nosip) has been associated with diverse human diseases including psychological disorders. In line, early neurogenesis of mouse and Xenopus is impaired upon Nosip deficiency. Nosip knockout mice show craniofacial defects and the down-regulation of Nosip in the mouse and Xenopus leads to microcephaly. Until now, the exact underlying molecular mechanisms of these malformations were still unknown. Here, we show that nosip is expressed in the developing ocular system as well as the anterior neural crest cells of Xenopus laevis. Furthermore, Nosip inhibition causes severe defects in eye formation in the mouse and Xenopus. Retinal lamination as well as dorso-ventral patterning of the retina were affected in Nosip-depleted Xenopus embryos. Marker gene analysis using rax, pax6 and otx2 reveals an interference with the eye field induction and differentiation. A closer look on Nosip-deficient Xenopus embryos furthermore reveals disrupted cranial cartilage structures and an inhibition of anterior neural crest cell induction and migration shown by twist, snai2, and egr2. Moreover, foxc1 as downstream factor of retinoic acid signalling is affected upon Nosip deficiency. Nosip is a crucial factor for the development of anterior neural tissue such the eyes and neural crest cells. Developmental Dynamics 247:1070-1082, 2018. © 2018 Wiley Periodicals, Inc.

Developmentally regulated autophagy is required for eye formation in Drosophila

ABSTRACTThe compound eye of the fruit fly Drosophila melanogaster is one of the most intensively studied and best understood model organs in the field of developmental genetics. Herein we demonstrate that autophagy, an evolutionarily conserved selfdegradation process of eukaryotic cells, is essential for eye development in this organism. Autophagic structures accumulate in a specific pattern in the developing eye disc, predominantly in the morphogenetic furrow (MF) and differentiation zone. Silencing of several autophagy genes (Atg) in the eye primordium severely affects the morphology of the adult eye through triggering ectopic cell death. In Atg mutant genetic backgrounds however genetic compensatory mechanisms largely rescue autophagic activity in, and thereby normal morphogenesis of, this organ. We also show that in the eye disc the expression of a key autophagy gene, Atg8a, is controlled in a complex manner by the anterior Hox paralog Lab (Labial), a master regulator of early development. Atg8a transcription is repressed in front of, while activated along, the MF by Lab. The amount of autophagic structures then remains elevated behind the moving MF. These results indicate that eye development in Drosophila depends on the cell death-suppressing and differentiating effects of the autophagic process. This novel, developmentally regulated function of autophagy in the morphogenesis of the compound eye may shed light on a more fundamental role for cellular self-digestion in differentiation and organ formation than previously thought.Abbreviations: αTub84B, α-Tubulin at 84B; Act5C, Actin5C; AO, acridine orange; Atg, autophagy-related; Ato, Atonal; CASP3, caspase 3; Dcr-2; Dicer-2; Dfd, Deformed; DZ, differentiation zone; eGFP, enhanced green fluorescent protein; EM, electron microscopy; exd, extradenticle; ey, eyeless; FLP, flippase recombinase; FRT, FLP recognition target; Gal4, gene encoding the yeast transcription activator protein GAL4; GFP, green fluorescent protein; GMR, Glass multimer reporter; Hox, homeobox; hth, homothorax; lab, labial; L3F, L3 feeding larval stage; L3W, L3 wandering larval stage; lf, loss-of-function; MAP1LC3, microtubule-associated protein 1 light chain 3; MF, morphogenetic furrow; PE, phosphatidylethanolamine; PBS, phosphate-buffered saline; PI3K/PtdIns3K, class III phosphatidylinositol 3-kinase; PZ, proliferation zone; Ref(2)P, refractory to sigma P, RFP, red fluorescent protein; RNAi, RNA interference; RpL32, Ribosomal protein L32; RT-PCR, reverse transcription-coupled polymerase chain reaction; S.D., standard deviation; SQSTM1, Sequestosome-1, Tor, Target of rapamycin; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay; UAS, upstream activation sequence; qPCR, quantitative real-time polymerase chain reaction; w, white

Early blowing in raw goats' milk cheese: gas production capacity of Enterobacteriaceae species present during manufacturing and ripening.

The aim of this study was to identify the main Enterobacteriaceae species responsible for early gas blowing during curdling and the first week of ripening in raw goats' milk cheese. Two batches of raw goats' milk cheese were selected. One of them showed early blowing within the first 24 h of cheese ripening while the other showed no alteration. Although initial levels of Enterobacteriaceae were similar in defective and non-defective cheese, their dynamics (growth and disappearance rates of the species detected) were different. Klebsiella oxytoca and Enterobacter cloacae were the main species in the defective curd, whereas Buttiauxela spp. was predominant in normal curd. Hafnia alvei was the prevailing isolated species for both normal and defective cheese throughout the ripening process. The highest gas production was rendered by K. oxytoca and H. alvei, mainly isolated from curd and cheese. However, other species relevant in milk or curd, like Pantoea ssp. or Buttiauxela spp. were considered as low gas producers. The analysis of digitalized images of cheese showed that most of the cheese eyes were formed before the first week of ripening, although this process continued during maturation.According to the species found in the defective and non-defective cheese, their proportions at different ripening stages, their ability to produce gas and eye formation, K. oxytoca might be considered the most likely responsible for early blowing in raw goats' milk cheeses; while H. alvei increased the eyes number in the later stages of the ripening period.

Silver nanoparticles affect lens rather than retina development in zebrafish embryos

Silver nanoparticles (AgNPs) have been reported to inhibit specification and differentiation of erythroid cells, chromatophores, and myofibrils during zebrafish embryogenesis. However, the knowledge of biological effects of AgNPs on eye development, especially on lens development is scarce. In this study, embryos were exposed to or injected with 0.4 mg/L AgNPs, and the results indicate that no obvious morphological changes in eye formation were observed in the stressed embryos compared to the controls. However, clefts and vacuoles were observed in lens of embryos from AgNPs stressed group. Additionally, the down-regulated expressions of different lens crystallin isoform genes and the normal expression of retinal genes were observed in AgNPs stressed embryos, suggesting AgNPs might inhibit the development of lens rather than the development of retina in zebrafish embryos. Moreover, no obvious cell apoptosis was observed, but normal nuclear DNA and RNA export was observed in lens cells. Together, the data in this study reveal that AgNPs damage the development of lens rather than retina resulting in eye abnormalities via some unknown mechanisms rather than via triggering cells apoptosis or blocking nuclear DNA or RNA export.

Phosphorylation of Arabidopsis SINA2 by CDKG1 affects its ubiquitin ligase activity

BackgroundSEVEN IN ABSENTIA (SINA) is a RING domain-containing ubiquitin ligase involved in Drosophila eye formation. SINA-like proteins in plants are involved in several signaling pathways. Of the 18 SINA-like proteins identified in Arabidopsis, SEVEN IN ABSENTIA 2 (SINA2) lacks a canonical RING domain and is thought to lack ubiquitin ligase activity.ResultsOur results show that SINA2 has E3 ligase activity in vitro, raising the possibility that a modified B-box domain may compensate for its lack of a RING domain. SINA2 physically interacts with the nuclear protein CYCLIN-DEPENDENT KINASE G1 (CDKG1), which acts as a positive regulator of plant responses to abiotic stress. CDKG1 is expressed in multiple tissues and its expression increased in response to abscisic acid (ABA) and osmotic stress. Transgenic Arabidopsis plants that ectopically express CDKG1 exhibit increased tolerance to ABA and osmotic stress treatments during seed germination and cotyledon development, while the loss-of-function cdkg1 mutant plants show reduced tolerance to ABA and osmotic stress treatments. Moreover, CDKG1-dependent phosphorylation of SINA2 positively affects its E3 ubiquitin ligase activity.ConclusionsBased on these results, we propose that CDKG1 modulates SINA2 ubiquitin ligase activity to regulate its effect on plant responses to ABA and osmotic stress.

Genetics of congenital eye malformations: insights from chick experimental embryology.

Embryological manipulations in chick embryos have been pivotal in our understanding of many aspects of vertebrate eye formation. This research was particularly important in uncovering the role of tissue interactions as drivers of eye morphogenesis and to dissect the function of critical genes. Here, we have highlighted a few of these past experiments to endorse their value in searching for hitherto unknown causes of rare congenital eye anomalies, such as microphthalmia, anophthalmia and coloboma. We have also highlighted a number of similarities between the chicken and human eye, which might be exploited to address other eye pathologies, including degenerative ocular diseases.

Investigation of crack initiation mechanisms responsible for the fish eye formation in the Very High Cycle Fatigue regime

The present paper aims to explain the subsurface crack formation occurring in the Very High Cycle Fatigue domain for bcc steels. For this purpose, two high strength steels (a low alloyed steel AISI 5120 and a martensitic stainless steel X40CrMo13) were studied under ultrasonic push-pull fatigue tests. For all tests, a fish eye developed before fracture and, in most cases, the subsurface crack initiation originated from inclusions. Also, during fatigue tests for both materials, the thermal field on specimen surface was recorded by infrared camera and fracture surface observations were performed by Scanning Electron Microscopy (SEM). The SEM pictures reveal the presence of a Fine Granular Area (FGA) surrounding the inclusion in the crack initiation stage. Moreover, earlier in the crack process, slips markings were found in the matrix inside the inclusion dimple indicating that some similarities exist between surface and subsurface crack initiation mechanisms.

The effect of starters with a functional arginine deiminase pathway on cheese ripening and quality

The arginine deiminase (ADI) pathway catalyses the degradation of l-arginine via the intermediate l-citrulline into l-ornithine, ammonia, and CO2. This constitutes an important metabolic feature of some lactic acid bacteria (LAB) in their response to acid stress. We investigated the capacity of four thermophilic starters to degrade arginine via the ADI pathway and studied their influence on the ripening and quality of experimental Gruyere cheeses. One of the starters could not degrade arginine, while another had an impaired ADI pathway and formed mainly citrulline. In cheeses produced with the two starters exhibiting a fully functional ADI pathway, ornithine content, pH-value, and CO2 and eye formation were significantly increased, indicating that the quality and ripening of the cheeses were affected by ADI metabolism. This is the first study investigating thermophilic starter cultures that contain Lactobacillus delbrueckii subsp. lactis with a functional ADI pathway converting arginine into ornithine and CO2 in cheese.

Numerical Simulation of Slag Eye Formation and Slag Entrapment in a Bottom-Blown Argon-Stirred Ladle

A transient mathematical model is developed for simulating the bubble-steel-slag-top gas four-phase flow in a bottom-blown argon-stirred ladle with a 70-ton capacity. The Lagrangian discrete phase model (DPM) is used for describing the moving behavior of bubbles in the steel and slag. To observe the formation process of slag eye, the volume of fluid (VOF) model is used to track the interfaces between three incompressible phases: metal/slag, metal/gas, and slag/gas. The complex multiphase turbulent flow induced by bubble-liquid interactions is solved by a large eddy simulation (LES) model. Slag eye area and slag droplet dispersion are investigated under different gas flow rates. The results show that the movement of bubbles, formation and collapse of slag eye, volatility of steel/slag interface and behavior of slag entrapment can be properly predicted in the current model. When the gas flow rate is 300 L/min, the circulation driven by the bubble plume will stir the entire ladle adequately and form a slag eye of the right size. At the same time, it will not cause strong erosion to the ladle wall, and the fluctuation of the interface is of adequate intensity, which will be helpful for improving the desulfurization efficiency; the slag entrapment behavior can also be decreased. Interestingly, with the motion of liquid steel circulation, the collision and coalescence of dispersed slag droplets occur during the floating process in the vicinity of the wall.

Ultrastructure of the lens anterior capsule in secondary cataract associated with uveitis under an electron microscope

Objective: To observe the ultrastructure of lens epithelium cells in cataract associated with uveitis. Methods: A cross-sectional study. Seven patients(7 eyes) received phacoemulsification in Peking University First Hospital from August 2016 to March 2017 due to complicated cataract associated with uveitis[2 males and 5 females,with an average age of (49±20) years] and 3 patients of age-related cataract (3 eyes, females, aged 54, 71, 74 years) were enrolled. Anterior capsule samples were collected during surgery. Transmission electron microscopy was performed. The cell morphology, the proportion of cell nuclear and organelle changes and the proportion of apoptosis in cataract associated with uveitis were described statistically by x±s or median (range). Results: Spindle-shaped epithelial cells were observed in cataract associated with uveitis (100%), while 28.00%,16.67%,16.67% of spindle-shaped epithelial cells were observed in age-related cataract. The organelle changes included mitochondria swelling and damage to the tight cell junction in cataract associated with uveitis. The tight junction between two cells was damaged in different degrees, only 8.33% (0-16.67%)of the cell nuclei appeared normal, and increased chromatin density(47.07%±22.28%), nuclear pyknosis(38.02%±19.61%) and nuclear fragmentation (9.96%±8.10%) were observed in cataract secondary to uveitis. The apoptosis rate was (48.16%±26.66%) in cataract associated with uveitis and correlated to the duration of intraocular inflammation. While the apoptosis rate were 0, 8.33%, 0 in age-related cataract patients. No autophagosome was observed in cataract associated with uveitis. Conclusions: Ultrastructure changes existed in lens epithelium cells in cataract associated with uveitis. The increased rate of apoptosis and inhibition of autophagy could be the possible mechanism of cataract formation in uveitic eyes. (Chin J Ophthalmol, 2018, 54: 357-362).

Endosulfan reduces fertilization success and causes abnormal embryo development to zebrafish

Reproductive and teratogenic effects of endosulfan were investigated by exposure of mature breeding zebrafish (Danio rerio) to environmentally relevant concentrations of 0.01–1.2 μg L−1 and monitoring their spawning, fertilization success, and gonad histology. Fertilization success was negatively correlated to duration of exposure at endosulfan concentration of 1.2 μg L−1. No apparent effect was observed on fish gonads after histological examination. Teratogenic effects were assayed by exposing newly spawned embryos to endosulfan at 1.0–200 μg L−1 and determining coagulation, tail development, eye formation, and spontaneous movements 24 h after fertilization; pigmentation, heart rate, edema, and mortality after 48 h and hatch time, mortality, and spinal deformations after 144 h. At 50–100 μg L−1, endosulfan caused impaired tail development and differentiation, and at 10–200 μg L−1 spinal deformation and increased mortality, depending upon the timing of exposure.

Polycomb group (PcG) proteins and Pax6 cooperate to inhibit in vivo reprogramming of the developing Drosophila eye.

How different cells and tissues commit to and determine their fates has been a central question in developmental biology since the seminal embryological experiments conducted by Wilhelm Roux and Hans Driesch in sea urchins and frogs. Here, we demonstrate that Polycomb group (PcG) proteins maintain Drosophila eye specification by suppressing the activation of alternative fate choices. The loss of PcG in the developing eye results in a cellular reprogramming event in which the eye is redirected to a wing fate. This fate transformation occurs with either the individual loss of Polycomb proteins or the simultaneous reduction of the Pleiohomeotic repressive complex and Pax6. Interestingly, the requirement for retinal selector genes is limited to Pax6, as the removal of more downstream members does not lead to the eye-wing transformation. We also show that distinct PcG complexes are required during different developmental windows throughout eye formation. These findings build on earlier observations that the eye can be reprogrammed to initiate head epidermis, antennal and leg development.

Phosphorylation states change Otx2 activity for cell proliferation and patterning in the Xenopus embryo.

The homeodomain transcription factor Otx2 has essential roles in head and eye formation via the negative and positive regulation of its target genes, but it remains elusive how this dual activity of Otx2 affects cellular functions. In the current study, we first demonstrated that both exogenous and endogenous Otx2 are phosphorylated at multiple sites. Using Xenopus embryos, we identified three possible cyclin-dependent kinase (Cdk) sites and one Akt site, and analyzed the biological activities of phosphomimetic (4E) and nonphosphorylatable (4A) mutants for those sites. In the neuroectoderm, the 4E but not the 4A mutant downregulated the Cdk inhibitor gene p27xic1 (cdknx) and posterior genes, and promoted cell proliferation, possibly forming a positive-feedback loop consisting of Cdk, Otx2 and p27xic1 for cell proliferation, together with anteriorization. Conversely, the 4A mutant functioned as an activator on its own and upregulated the expression of eye marker genes, resulting in enlarged eyes. Consistent with these results, the interaction of Otx2 with the corepressor Tle1 is suggested to be phosphorylation dependent. These data suggest that Otx2 orchestrates cell proliferation, anteroposterior patterning and eye formation via its phosphorylation state.