Ciliary margin-derived BMP4 does not have a major role in ocular development.

Heterozygous Bmp4 mutations in humans and mice cause severe ocular anterior segment dysgenesis (ASD). Abnormalities include pupil displacement, corneal opacity, iridocorneal adhesions, and variable intraocular pressure, as well as some retinal and vascular defects. It is presently not known what source of BMP4 is responsible for these defects, as BMP4 is expressed in several developing ocular and surrounding tissues. In particular, BMP4 is expressed in the ciliary margins of the optic cup which give rise to anterior segment structures such as the ciliary body and iris, making it a good candidate for the required source of BMP4 for anterior segment development. Here, we test whether ciliary margin-derived BMP4 is required for ocular development using two different conditional knockout approaches. In addition, we compared the conditional deletion phenotypes with Bmp4 heterozygous null mice. Morphological, molecular, and functional assays were performed on adult mutant mice, including histology, immunohistochemistry, in vivo imaging, and intraocular pressure measurements. Surprisingly, in contrast to Bmp4 heterozygous mutants, our analyses revealed that the anterior and posterior segments of Bmp4 conditional knockouts developed normally. These results indicate that ciliary margin-derived BMP4 does not have a major role in ocular development, although subtle alterations could not be ruled out. Furthermore, we demonstrated that the anterior and posterior phenotypes observed in Bmp4 heterozygous animals showed a strong propensity to co-occur, suggesting a common, non-cell autonomous source for these defects.

Neonatal and Juvenile Ocular Development in Sprague-Dawley Rats: A Histomorphological and Immunohistochemical Study.

As in many altricial species, rats are born with fused eyelids and markedly underdeveloped eyes. While the normal histology of the eyes of mature rats is known, the histomorphological changes occurring during postnatal eye development in this species remain incompletely characterized. This study was conducted to describe the postnatal development of ocular structures in Sprague-Dawley (SD) rats during the first month of age using histology and immunohistochemistry (IHC). Both eyes were collected from 51 SD rats at 13 time points between postnatal day (PND)1 and PND30. Histologic examination of hematoxylin and eosin-stained sections was performed, as well as IHC for cleaved-caspase-3 and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) to evaluate apoptosis, and IHC for Ki-67 and phospho-histone-H3 to evaluate cell proliferation. Extensive ocular tissue remodeling occurred prior to the eyelid opening around PND14 and reflected the interplay between apoptosis and cell proliferation. Apoptosis was particularly remarkable in the maturing subcapsular anterior epithelium of the lens, the inner nuclear and ganglion cell layers of the developing retina, and the Harderian gland, and was involved in the regression of the hyaloid vasculature. Nuclear degradation in the newly formed secondary lens fibers was noteworthy after birth and was associated with TUNEL-positive nuclear remnants lining the lens organelle-free zone. Cell proliferation was marked in the developing retina, cornea, iris, ciliary body and Harderian gland. The rat eye reached histomorphological maturity at PND21 after a rapid phase of morphological changes characterized by the coexistence of cell death and proliferation.

Improving Urban Stormwater Runoff Quality by Nutrient Removal through Floating Treatment Wetlands and Vegetation Harvest.

Two floating treatment wetlands (FTWs) in experimental tanks were compared in terms of their effectiveness on removing nutrients. The results showed that the FTWs were dominated by emergent wetland plants and were constructed to remove nutrients from simulated urban stormwater. Iris pseudacorus and Thalia dealbata wetland systems were effective in reducing the nutrient. T. dealbata FTWs showed higher nutrient removal performance than I. pseudacorus FTWs. Nitrogen (N) and phosphorous (P) removal rates in water by T. dealbata FTWs were 3.95 ± 0.19 and 0.15 ± 0.01 g/m2/day, respectively. For I. pseudacorus FTWs, the TN and TP removal rates were 3.07 ± 0.15 and 0.14 ± 0.01 g/m2/day, respectively. The maximum absolute growth rate for T. dealbata corresponded directly with the maximum mean nutrient removal efficiency during the 5th stage. At harvest, N and P uptak of T. dealbata was 23.354 ± 1.366 g and 1.489 ± 0.077 g per plant, respectively, approximate twice as high as by I. pseudacorus.

A mouse model of aniridia reveals the in vivo downstream targets of Pax6 driving iris and ciliary body development in the eye.

The Pax6 transcription factor is essential for development of the brain, eye, olfactory and endocrine systems. Haploinsufficiency of PAX6 in humans and mice causes the congenital condition aniridia, with defects in each of these organs and systems. Identification of the PAX6 transcription networks driving normal development is therefore critical in understanding the pathophysiology observed with loss-of-function defects. Here we have focused on identification of the downstream targets for Pax6 in the developing iris and ciliary body, where we used laser capture microdissection in mouse eyes from E12.5-E16.5, followed by chromatin immunoprecipitation, promoter-reporter assays and immunohistochemistry. We identified 6 differentially expressed genes between wildtype and Pax6 heterozygous mouse tissues and demonstrated that Bmp4, Tgfß2, and Foxc1 were direct downstream targets of Pax6 in developing iris/ciliary body. These results improve our understanding of how mutations in Bmp4, Tgfß2, and Foxc1 result in phenocopies of the aniridic eye disease and provide possible targets for therapeutic intervention.

Lipid globules on the plastid surface in Iris tepal epidermis cells during tepal maturation and senescence.

Epidermis cells in the outer tepals of Iris flowers (Iris×hollandica, cv. Blue Magic) start programmed cell death (PCD) prior to floral opening. The tepals show visible senescence symptoms three days after full opening. Visible senescence coincides with collapse (death) of the upper epidermis cells. In these cells, electron-dense particles (plastoglobuli), membranes, and oil bodies were observed in the plastid interior. Electron-dense globules similar to plastoglobuli, thus apparently mainly consisting of lipids, were found on the plastid surface, from before flower opening until cell death. Such electron-dense globules were also present in the cytosol. The size of some of the globules on the plastid surface increased with time. The globules are likely involved in transfer of lipidic/proteinaceous material from the plastid to the cytosol. As the plastids contained ample oil bodies, up to the time of cell death, cell death was likely not due to lack of reserves. Mitochondrial ultrastructure also remained the same until cell death. The role of mitochondria in PCD is discussed.

Does iris change over time?

Iris as a biometric identifier is assumed to be stable over a period of time. However, some researchers have observed that for long time lapse, the genuine match score distribution shifts towards the impostor score distribution and the performance of iris recognition reduces. The main purpose of this study is to determine if the shift in genuine scores can be attributed to aging or not. The experiments are performed on the two publicly available iris aging databases namely, ND-Iris-Template-Aging-2008-2010 and ND-TimeLapseIris-2012 using a commercial matcher, VeriEye. While existing results are correct about increase in false rejection over time, we observe that it is primarily due to the presence of other covariates such as blur, noise, occlusion, and pupil dilation. This claim is substantiated with quality score comparison of the gallery and probe pairs.

Morphology of the elygium and developing umbraculum in the eye of Amietia vertebralis tadpoles.

The elygium is a darkly pigmented projection over the pupil of the tadpoles of certain species that live mostly at high altitudes. It has been suggested that this structure shades the retina, protecting it from high UV levels. In post-metamorphic individuals, the elygium is replaced by a structure known as an umbraculum. Confusion arose in the past from the inconsistent use of terminology when referring to these two structures. While they may serve the same function, these structures differ fundamentally in structure and origin. Our investigation of the ultrastructure of the eye in Amietia vertebralis tadpoles, using electron and light microscopy, revealed that the elygium consists of an accumulation of melanophores situated within the inner cornea, whereas the umbraculum is a dorsal extension of the iris pigment epithelium, analogous to the ventral pupillary nodule, which is present in most frog species. In A. vertebralis, the umbraculum starts to develop on the iris of older tadpoles (Gosner [1960] stage 37 Herpetologica 16:183-190), medial to the overlying elygium. A smaller, ventral elygium is also present in this species and is similar in structure to the dorsal elygium. The development of the elygium over a six-month period is also described.

Delay of iris flower senescence by cytokinins and jasmonates.

It is not known whether tepal senescence in Iris flowers is regulated by hormones. We applied hormones and hormone inhibitors to cut flowers and isolated tepals of Iris × hollandica cv. Blue Magic. Treatments with ethylene or ethylene antagonists indicated lack of ethylene involvement. Auxins or auxin inhibitors also did not change the time to senescence. Abscisic acid (ABA) hastened senescence, but an inhibitor of ABA synthesis (norflurazon) had no effect. Gibberellic acid (GA3 ) slightly delayed senescence in some experiments, but in other experiments it was without effect, and gibberellin inhibitors [ancymidol or 4-hydroxy-5-isopropyl-2-methylphenyltrimethyl ammonium chloride-1-piperidine carboxylate (AMO-1618)] were ineffective as well. Salicylic acid (SA) also had no effect. Ethylene, auxins, GA3 and SA affected flower opening, therefore did reach the flower cells. Jasmonates delayed senescence by about 2.0 days. Similarly, cytokinins delayed senescence by about 1.5-2.0 days. Antagonists of the phosphatidylinositol signal transduction pathway (lithium), calcium channels (niguldipine and verapamil), calmodulin action [fluphenazine, trifluoroperazine, phenoxybenzamide and N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide hydrochloride (W-7)] or protein kinase activity [1-(5-isoquinolinesulfonyl)-2-methylpiperazine hydrochloride (H-7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-8) and N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride (H-9)] had no effect on senescence, indicating no role of a few common signal transduction pathways relating to hormone effects on senescence. The results indicate that tepal senescence in Iris cv. Blue Magic is not regulated by endogenous ethylene, auxin, gibberellins or SA. A role of ABA can at present not be excluded. The data suggest the hypothesis that cytokinins and jasmonates are among the natural regulators.

[Study on the nitrogen and phosphorus uptake ability of four plants cultivated on floating-bed].

Plant floating-bed tested engineering was constructed for eutrophication control in Dian-shan Lake, the characteristics and nutrient uptake abilities of Canna indica, Iris pseudacorus, Thalia dealbata and Lythrum salicaria were compared. It shows that using upper and lower nylon nets to fix the plants on the floating-bed is beneficial for them to grow and reproduce rapidly. Survival rates of Canna indica, lris pseudacorus, Thalia dealbata and Lythrum salicaria are 83.33%, 83.33%, 76.67% and 53.33% respectively. Ramets of Canna indica and Thalia dealbata are 64 and 78 respectively in November, and the biomass (fresh weight) of these two plants are 32.0 and 38.6 kg per individual plant. Nitrogen (N) and phosphorus (P) content in stems/leaves of Canna indica and Thalia dealbata are greater than those in roots. The ratio between stems/leaves and roots of N, P content in Canna indica are 1.40 and 1.21 respectively, while 1.59 and 1.08 in Thalia dealbata. The difference of cumulative N, P content in plants is mostly on account of different plant biomass. N uptake ability of Thalia dealbata is the highest, which is 457.11 g per square; Canna indica has the highest P uptake ability, which is 41.29 g per square. N, P uptake ability of stems/leaves in Canna indica are 2.17 and 1.86 times higher than that of roots, while 1.73 and 1.17 times higher respectively in Thalia dealbata. Thus, Canna indica and Thalia dealbata are recommended as the floating-bed plants to control the eutrophication in Dian-shan Lake.

[Clonal plasticity of Iris japonica under different soil and canopy conditions in subtropocal evergreen broad-leaved forest].

An investigation was made on the growth indices of Iris japonica growing on the yellow soil and Karst soil within a subtropical evergreen broad-leaved forest and in the forest gaps, aimed to approach the clonal plasticity of I. japonica in heterogeneous habitats. The results showed that the ramet height and diameter and the rhizome diameter of I. japonica were significantly higher in yellow soil habitat than in Karst soil habitat, while the individual density was in adverse. Light had significant effects on rhizome diameter, but soil condition had lesser effects under the same light environment. Within the forest of Karst soil habitat, the bud number and flower number of I. japonica were zero, possibly due to the double pressure from soil quality and light environment for the balance between resources uptake and energy assignment and for the sustaining of growth. In a certain extent, I. japonica in yellow soil habitat tended to K strategy via decreasing the individual number for improving competition to adapt environment, while I. japonica in Karst soil habitat tended to r strategy via increasing individual number for sustaining competition capability.

Morphofunctional interactions of peripheral nerve fibers of the iris with neurons developing in the anterior chamber of the eye in rats.

Electron microscopic studies were performed on intraocular transplants of embryonic septal and hippocampal tissue developing in the anterior chamber of the eye in rats for 3-4 months. The aim of the study was to seek ultrastructural identification of peripheral nerve fibers entering transplants from the iris, and to assess their ability to establish true synaptic contacts with transplanted CNS neurons. Bundles of myelinated and unmyelinated axons surrounded by Schwann cell cytoplasm were seen within the perivascular spaces of ingrowing blood vessels. Both types of peripheral fiber were also identified in the neuropil areas of transplants. At the ultrastructural level, unmyelinated axons were found to be free of glial Schwann cell sheaths and to form typical asymmetrical synapses with the dendrites and dendritic spines of transplant neurons. These results provide evidence of the high morphofunctional plasticity of both parts (central, peripheral) of the nervous system.

[Morpho-functional interactions of the iris peripheral nervous fibers with the neurons developing in the rat anterior eye chamber].

The intraocular grafts of the septal or hippocampal embryonic tissues developing in the rat anterior eye chamber for three to four months were investigated by electron microscopy. The aim of this study was both the ultrastructural identification of the peripheral nervous fibers entering the grafts from host iris and the estimation of their capacity to establish true synaptic contacts with the central nervous system neurons of the grafts. The bundles of myelinated and unmyelinated axons, surrounded by the Schwann cell cytoplasm, were observed within the perivascular spaces of the ingrowing blood vessels. In the neuropil areas of the grafts, both types of the peripheral nervous fibers were also identified. It was demonstrated on the ultrastructural level that the unmyelinated axons lost their glial envelope of the Schwann cell and formed the typical asymmetric synapses with the dendrites and dendritic spines of the grafted neurons. The results are indicative of the high morpho-functional plasticity of both parts of the nervous system.

Pax6 dosage requirements in iris and ciliary body differentiation.

Pax6 is a highly conserved transcription factor that controls the morphogenesis of various organs. Changes in Pax6 dosage have been shown to affect the formation of multiple tissues. PAX6 haploinsufficiency leads to aniridia, a pan-ocular disease primarily characterized by iris hypoplasia. Herein, we employ a modular system that includes null and overexpressed conditional alleles of Pax6. The use of the Tyrp2-Cre line, active in iris and ciliary body (CB) primordium, enabled us to investigate the effect of varying dosages of Pax6 on the development of these ocular sub-organs. Our findings show that a lack of Pax6 in these regions leads to dysgenesis of the iris and CB, while heterozygosity impedes growth of the iris and maturation of the iris sphincter. Overexpression of the canonical, but not the alternative splice variant of Pax6 results in severe structural aberrations of the CB and hyperplasia of the iris sphincter. A splice variant-specific rescue experiment revealed that both splice variants are able to correct iris hypoplasia, while only the canonical form rescues the sphincter. Overall, these findings demonstrate the dosage-sensitive roles of Pax6 in the formation of both the CB and the iris.

Deletion of G protein-coupled receptor 48 leads to ocular anterior segment dysgenesis (ASD) through down-regulation of Pitx2.

The development of the anterior segment of the mammalian eye is critical for normal ocular function, whereas abnormal development can cause glaucoma, a leading cause of blindness in the world. We report that orphan G protein-coupled receptor 48 (Gpr48/LGR4) plays an important role in the development of the anterior segment structure. Disruption of Gpr48 causes a wide spectrum of anterior segment dysgenesis (ASD), including microphthalmia, iris hypoplasia, irdiocorneal angle malformation, cornea dysgenesis, and cataract. Detailed analyses reveal that defective iris myogenesis and ocular extracellular matrix homeostasis are detected at early postnatal stages of eye development, whereas ganglion cell loss, inner nuclear layer thinness, and early onset of glaucoma were detected in 6-month-old Gpr48(-/-) mice. To determine the molecular mechanism of ASD caused by the deletion of Gpr48, we performed gene expression analyses and revealed dramatic down-regulation of Pitx2 in homozygous knockout mice. In vitro studies with the constitutively active Gpr48 mutant receptor demonstrate that Pitx2 is a direct target of the Gpr48-mediated cAMP-CREB signaling pathway in regulating anterior segment development, suggesting a role of Gpr48 as a potential therapeutic target of ASD.

Iris development in vertebrates; genetic and molecular considerations.

The iris plays a key role in visual function. It regulates the amount of light entering the eye and falling on the retina and also operates in focal adjustment of closer objects. The iris is involved in circulation of the aqueous humor and hence functions in regulation of intraocular pressure. Intriguingly, iris pigmented cells possess the ability to transdifferentiate into different ocular cell types of retinal pigmented epithelium, photoreceptors and lens cells. Thus, the iris is considered a potential source for cell-replacement therapies. During embryogenesis, the iris arises from both the optic cup and the periocular mesenchyme. Its interesting mode of development includes specification of the peripheral optic cup to a non-neuronal fate, migration of cells from the surrounding periocular mesenchyme and an atypical formation of smooth muscles from the neuroectoderm. This manner of development raises some interesting general topics concerning the early patterning of the neuroectoderm, the specification and differentiation of diverse cell types and the interactions between intrinsic and extrinsic factors in the process of organogenesis. In this review, we discuss iris anatomy and development, describe major pathologies of the iris and their molecular etiology and finally summarize the recent findings on genes and signaling pathways that are involved in iris development.

[Effects of Zn on the growth, Cd accumulation and physiological resistance of Iris lactea var. chinensis under Cd stress].

A solution culture experiment was conducted to examine the effects of Zn (1-100 mg x L(-1)) on the growth, Cd accumulation, and antioxidant enzyme activities of Iris lactea var. chinensis under Cd stress (10 mg x L(-1)). The results showed that after the addition of Zn, the Cd content changed a little in shoots, but increased significantly in roots, with a trend of rising first and dropping then. When treated with 1 mg Zn x L(-1), the Cd content in roots was the highest, being 51.4% higher than the control. When the Zn concentration was higher than 1 mg x L(-1), the Cd content in roots decreased to some extent, but was still higher than the control. Low concentration (1-10 mg x L(-1)) Zn increased the shoot biomass and leaf chlorophyll content, but decreased the malondialdehyde (MDA) content. At 10 mg Zn x L(-1), the chlorophyll a and b contents went up to the maximum, being 5.21% and 22.27% higher than the control, respectively, while the MDA content decreased by 25.46%, indicating that low concentration Zn could alleviate Cd damage. With increasing Zn concentration, the alleviation effect of Zn on Cd damage decreased gradually. When the Zn concentration was 100 mg x L(-1), I. lactea var. chinensis was damaged most seriously, with the biomass and chlorophyll content reduced and MDA content increased significantly. Within the ranges of test Cd and Zn concentrations, the activities of superoxide dismutase (SOD) and peroxidase (POD) had an increasing trend, suggesting that SOD and POD played an important antioxidant protective role for Cd or Zn-Cd stressed I. lactea var. chinensis.

Determinative role of Wnt signals in dorsal iris-derived lens regeneration in newt eye.

We have previously shown that lens regeneration from the pigmented epithelium of the dorsal iris in the adult newt eye proceeds in two steps after lens removal or intraocular FGF2 injection. The FGF2-dependent proliferation of iris pigmented epithelium and activation of early lens genes that occur over the entire circumference of the iris comprise the first step, while subsequent dorsally confined lens development marks the second step. Here, we investigated the expression of Wnt and Wnt receptor Frizzled genes in lens-regenerating iris tissues. Wnt2b and Frizzled4 were activated only in the dorsal half of the iris in synchrony with the occurrence of the second step, whereas Wnt5a and Frizzled2 were activated in both halves throughout the period of the first and second steps. Cultured explants of the iris-derived pigmented epithelium in the presence of FGF2 underwent dorsal-specific lens development fully recapitulating the in vivo lens regeneration process. Under these conditions, Wnt inhibitors Dkk1, which specifically inhibits the canonical signal pathway, and/or sFRP1 repressed the lens development, while exogenous Wnt3a, which generally activates the canonical pathway like Wnt2b, stimulated lens development from the dorsal iris epithelium and even caused lens development from the ventral iris epithelium, albeit at a reduced rate. Wnt5a did not elicit lens development from the ventral epithelium. These observations indicate that dorsal-specific activation of Wnt2b determines the dorsally limited development of lens from the iris pigmented epithelium.

Wnt2b/beta-catenin-mediated canonical Wnt signaling determines the peripheral fates of the chick eye.

Wnt signaling orchestrates multiple aspects of central nervous system development, including cell proliferation and cell fate choices. In this study, we used gene transfer to activate or inhibit canonical Wnt signaling in vivo in the developing eye. We found that the expression of Wnt2b or constitutively active (CA) beta-catenin inhibited retinal progenitor gene (RPG) expression and the differentiation of retinal neurons. In addition, Wnt signal activation in the central retina was sufficient to induce the expression of markers of the ciliary body and iris, two tissues derived from the peripheral optic cup (OC). The expression of a dominant-negative (DN) allele of Lef1, or of a Lef1-engrailed fusion protein, led to the inhibition of expression of peripheral genes and iris hypoplasia, suggesting that canonical Wnt signaling is required for peripheral eye development. We propose that canonical Wnt signaling in the developing optic vesicle (OV) and OC plays a crucial role in determining the identity of the ciliary body and iris. Because wingless (wg) plays a similar role in the induction of peripheral eye tissues of Drosophila, these findings indicate a possible conservation of the process that patterns the photoreceptive and support structures of the eye.

Growth of the cornea from infancy to adolescence.

The intraocular distance and iris diameter of children and young adolescents were measured, with the aid of a measuring microscope, from photographs of their faces. True intraocular distance was measured with an intraocular caliper at the same time that the photographs were taken. These data were then compiled and horizontal visible iris diameters (HVIDs) were calculated. An equation was derived from the optics of the Gullstrand model eye to calculate horizontal corneal diameter (HCD) from HVID. Comparisons of HVIDs revealed no significant correlation with age in either a regression plot of cross-sectional data for subjects aged 1 month to 1 year, or all subjects whose ages ranged from 1 month to adolescence. Additional longitudinal data for 13 individuals, who had been photographed as both an infant (mean = 3.4 months) and as an older child or adolescent (mean = 8.6 years), were then compiled and HVIDs for these subjects at two different ages were compared. A Wilcoxon signed rank test revealed a small but significant amount of growth, 0.318 mm (p-value = 0.013), in the HVIDs over a mean age difference of 8.3 years for individuals measured twice during their lifetimes. The regression equation for this growth was: HVID = 10.52 (+/-0.095 S.E.) + 0.0305 (+/-0.014) x Age (years). From a comparison of data from earlier literature and our own measurements, we conclude that, after birth, the fastest growth of the cornea must occur during the first few months of life.

BMP inhibition-driven regulation of six-3 underlies induction of newt lens regeneration.

Lens regeneration in adult newts is a classic example of how cells can faithfully regenerate a complete organ through the process of transdifferentiation. After lens removal, the pigment epithelial cells of the dorsal, but not the ventral, iris dedifferentiate and then differentiate to form a new lens. Understanding how this process is regulated might provide clues about why lens regeneration does not occur in higher vertebrates. The genes six-3 and pax-6 are known to induce ectopic lenses during embryogenesis. Here we tested these genes, as well as members of the bone morphogenetic protein (BMP) pathway that regulate establishment of the dorsal-ventral axis in embryos, for their ability to induce lens regeneration. We show that the lens can be regenerated from the ventral iris when the BMP pathway is inhibited and when the iris is transfected with six-3 and treated with retinoic acid. In intact irises, six-3 is expressed at higher levels in the ventral than in the dorsal iris. During regeneration, however, only expression in the dorsal iris is significantly increased. Such an increase is seen in ventral irises only when they are induced to transdifferentiate by six-3 and retinoic acid or by BMP inhibitors. These data suggest that lens regeneration can be achieved in noncompetent adult tissues and that this regeneration occurs through a gene regulatory mechanism that is more complex than the dorsal expression of lens regeneration-specific genes.