Optic Cup Formation Research Articles

Pattern of cell death during optic cup formation in the tree shrewTupaia belangeri

Developmental cell death during optic cup formation was investigated in the tree shrew Tupaia belangeri. Twenty-six embryos from days 12 to 16 of prenatal ontogenesis were studied by light microscopy. Prior to the optic vesicle stage, a dorsal area of cell death surrounded the lumen of the V-shaped optic evagination (phase 1). A ventral band of dead cells, found in the optic vesicle (phase 2), preceded a dorsal focus of cell death (phase 3) previously described as a characteristic avian feature. During further invagination (phase 4), a peak of cell death was represented by a ventrodorsal band extending from the diencephalon over the complete optic anlage. The main areas of cell death found in phases 2 to 4 were, topographically, segments of this band. Also, the distinct areas of cell death reported in the literature for the vertebrate species studied so far fit well into this ventrodorsal band found in Tupaia. Thus, most probably, a common spatio-temporal sequence of cell death exists in all of them. In Tupaia, dead cells concentrated at the diencephalic insertion of the optic stalk, the suboptic necrotic center (SONC) reported by several authors, were part of the early ventral band of cell death originating from the median floor of the prosencephalon (phase 2). During optic cup formation, the SONC was part of the ventrodorsal band and, thus, was not secondarily formed by the subdivision of a pre-existing distal ventral area of cell death as reported for several other vertebrates.

Pattern of cell death during optic cup formation in the tree shrew Tupaia belangeri

Pattern of cell death during optic cup formation in the tree shrew Tupaia belangeri

Transient overexpression of the Microphthalmia gene in the eyes of Microphthalmia vitiligo mutant mice.

The murine microphthalmia gene (Mitf) encodes a basic helix-loop-helix transcription factor thought to regulate transcription of genes encoding proteins of the pigmentation pathway. It may promote pigment cell survival and development. The protein encoded by Mitf appears to be critical for eye development, because mutant alleles demonstrate varying degrees of ocular malformation. One of the mildest of these is the Mitf vitiligo (Mitfvit) mutant allele, which exhibits uneven pigmentation of the retinal pigment epithelium (RPE) and slow, progressive photoreceptor cell loss, eventually leading to blindness. In the present study, the expression of Mitf during early eye development in the Mitfvit mutant was compared with that of pigmented wild type mice. Mitf expression quantified by reverse transcriptase-polymerase chain reaction amplification demonstrated a transient elevation of Mitf between embryonic day 10.5 (E10.5) and E13.5 in the Mitfvit mutant compared with wild type mice. In situ hybridization analysis confirmed this elevation and localized Mitf expression to the neuroepithelium during onset of optic vesicle formation (E9.0-E9.5) and, subsequently, to the RPE during optic cup formation (E10-E11.5) in both mutant and wild type eyes. This is the first report of transient elevation of Mitf in any of the Mitf mutants, and the elevation may be relevant to altered levels of pigmentation proteins as well as to the RPE abnormalities observed in the Mitfvit mutant.

Pax-6 gene expression in newt eye development.

pax-6 is thought to be a master control gene of eye development in species ranging from insects to mammals. We have isolated a pax-6 cDNA homolog of the newt, Cynops pyrrhogaster. RT-PCR and sequence analyses predicted four alternatively spliced forms derived from inclusion or exclusion of the region corresponding to exons 5a and 12 in the human pax-6 ortholog. This gene shared extensive sequence identitiy and similar expression patterns with those of mouse and zebrafish. pax-6 signal was first detected at the anterior ridge of the neural plate, and later at the eye and nasal primordium and in the central nervous system - except for the midbrain. The injection of sonic hedgehog (shh) RNA inhibited the expression of pax-6 within the optic vesicle and disturbed eye cup formation. A similar suppressive effect of shh was also observed in the conjugation of the animal caps preloaded with exogenous shh and noggin mRNA, which was used as an inducer of pax-6. In contrast, shh injection had no effect on the expression of pax-6 in the surface ectoderm overlying the optic cup, suggesting that the expression of pax-6 in the surface ectoderm is not regulated by shh in vivo. Moreover, we found transient activation of pax-6 in animal cap explants at the sibling stage of mid-late gastrula. This observation raises the possibility that the ectoderm is competent to the lens-inducing signal at a stage as early as mid gastrula.

Mutations affecting eye morphology in the developing zebrafish (Danio rerio).

The zebrafish (Danio rerio) has received considerable attention as a mainstream model for the molecular and genetic study of vertebrate development. In our laboratory, we have conducted a third-generation screen of chemically mutagenized zebrafish for recessive mutations affecting the visual system. This report describes the visible phenotypes and number of morphological mutants so far observed and presents a more detailed histological analysis of six of these mutations. Through analysis of mutant larvae, it was determined that several of the subtle morphological mutations resulted in degeneration of specific cellular layers of the retina. Other mutations resulted in some degeneration distributed diffusely across the entire retina or concentrated at the retinal margin. A single mutation affecting invagination of the optic cup and lens vesicle formation resulted in a failure to develop an anterior chamber. These results demonstrate the utility of a small-scale, highly focused screen for uncovering novel loci involved in retinal and eye development.

Clonal Analysis in the Chicken Retina Reveals Tangential Dispersion of Clonally Related Cells

Development of the chicken retina was investigated through clonal analysis using retroviral vectors. Replication-incompetent retroviral vectors encoding either human placental alkaline phosphatase, β-galactosidase, or a viral core protein were used in paired combinations to infect retinal progenitor cells from Embryonic Days 2.5-7.0. Labeled clones were analyzed late in embryonic development after retinal histogenesis was complete. The early accessibility of the chicken retina, combined with its large final size, resulted in the labeling of much larger clones than had been reported previously in other species. The clones were composed of many cell types, supporting previous conclusions from other vertebrates that the progenitor cells of developing retina are multipotent. The majority of clones derived from early infections consisted of multiple tightly clustered arrays of cells accompanied by dispersed individual cells. Clonal complexity and tangential dispersion were greater in peripheral than central retina and decreased considerably with increasing age of infection. These observations suggest that early during retinal development, shortly after optic cup formation, there is considerable mixing of progenitor cells.

Computer simulation of organogenesis: An approach to the analysis of shape changes in epithelial organs

Embryonic development of epithelial organ primordia often involves changes in several parameters, such as cell height, cell width, cell volume, amount of extracellular space, and cell number. Since these changes often occur simultaneously, it becomes difficult to “separate out” the role that each plays in the developmental process. A computer program has been written that allows the shape of epithelial organs to be reproduced based upon measurements of the primordium. A developmental sequence can be simulated by changing the dimensions of the primordium based upon either measurements of the developmental stages or theoretical projections of changes. The primordium is divided into blocks representing groups of cells, based upon characteristics of the different cell groups. The program allows differences in cell height and circular and spiral curvatures of the primordium to be simulated. Analysis of the optic primordium using this method has allowed recognition of several regional changes during optic cup formation. These are sequential constriction of cell apices at the margin of the optic cup, expansion of the apical surface toward the center of the retinal disc, and spreading of the future pigmented layer. Simulation of other organs permits regions of morphogenetic activity to be identified.

Optic cup formation: a calcium-regulated process.

Invagination of the optic vesicle to form the optic cup is an important event in the formation of the eye in the early embryo. To obtain support for earlier conclusions that a contractile process is involved, calcium dependency of optic cup formation was tested. Heads were excised from chicken embryos at the optic vesicle stage of development (stage 13) and incubated in nutrient medium containing antagonists or agonists of calcium transport. Invagination was reversibly inhibited by the Ca2+ antagonists verapamil and papaverine. It was initiated in a precocious fashion by the Ca2+ ionophore A23187 but only in the presence of external Ca2+. Neither caffeine, theophylline, nor A23187 (in the absence of external Ca2+) were able to initiate precocious optic cup formation. Trifluoperazine and chlorpromazine reversibly inhibited optic cup formation while chlorpromazine sulfoxide had no effect at the concentrations used. The binding of [3H]trifluoperazine to isolated stage 13 heads revealed a class of Ca2+-dependent binding sites having a Kd similar to that of calmodulin. These results indicate a Ca2+-dependence for optic cup formation and that the source of the Ca2+ may be extracellular. This Ca2+ dependence probably is conferred to the system by calmodulin.

The effect of inhibitors of glycoconjugate synthesis on optic cup formation in the chick embryo

Invagination of the optic primordium in the chick embryo coincides with the appearance of material at the apical cell surface that precipitates cetylpyridinium chloride. Since this precipitate is soluble only at high salt concentrations, it is suspected of being an acidic glycoconjugate. In order to test whether glycoconjugates play a role in optic cup formation, embryos were treated with drugs that interfere with glycoconjugate synthesis. 6-Diazo-5-oxo- l-norleucine was selected as a general inhibitor of glycoconjugates and tunicamycin as a specific inhibitor of asparagine-linked oligosaccharides. Both inhibitors prevented invagination of the optic vesicle and resulted in absence of a cetylpyridinium chloride precipitate at the apical cell surfaces. In addition to the effect within the cavity of the optic vesicle, both inhibitors also affected the attachment between the lens epithelium and the retinal disc. During normal development, the basal laminae of the lateral surface of the optic vesicle and the overlying ectoderm fuse to form a thickened basement membrane at the margin of the future retinal disc. As the retinal disc and lens epithelium invaginate, additional attachments form toward the center of the two folding layers. These attachments are perturbed by both diazo-oxo-norleucine and tunicamycin. However, the lens epithelium forms a vesicle even if the optic cup does not form. Since both inhibitors have been reported to affect processes in addition to glycoconjugate synthesis, the effect of tunicamycin on sugar, amino acid, and sulfate incorporation was studied. The inhibitor had a significant effect only on glucosamine incorporation and appeared to selectively inhibit an increase in incorporation that began in control heads at the time of invagination.

Microfilament patterns in the developing chick eye: Their role in invaginations

The function of microfilaments in early eye morphogenesis in the chick was investigated using an ATP-containing contraction medium to induce contraction of intracellular actomyosin systems. Invagination of the optic vesicle broadly similar to that seen in normal optic cup formation was seen when the treated eyes were at early stages, before the normal onset of optic cup invagination or lens placode formation. In older embryos where optic cup formation had begun the cupping process was actually reversed. In contrast neither the presumptive lens epithelium of the earliest embryos nor the fully formed lens placodes invaginated in response to the medium, although almost completely closed lens vesicles completed their closure. These results suggest that while optic cup formation is at least partly the result of active microfilament contraction the initial invagination of the presumptive lens is brought about by some other mechanism.

Studies on an anophthalmic strain of mice: VI. Lens and cup interaction

In the embryology of the eye region in the anophthalmic strain of mice ( ZRDCT Ch ), development proceeds normally until Day 10 (26 somites). At this time a lens is induced, but it is smaller in size and may be improperly centered in the optic cup. Where the lens is centered in relation to the optic cup determines whether microphthalmia or anophthalmia will occur. Also, we observed that optic cup formation is different in normal control strains.

The distribution of intercellular gap junctions in the developing retina and pigment epithelium of Xenopus laevis.

The distribution of gap junctions in the developing retina and pigment epithelium of Xenopus has been examined from optic vesicle to photoreceptor outer segment stages. In the retina and pigment epithelium walls of the optic vesicle gap junctions are found between ventricular cells in the apical contact zone and close to the outside wall; a neural tube type of distribution. Optic cup formation brings the retina and pigment epithelium into contact and gap junctions form between them. After the stage of retinal specification gap junctions, previously more numerous dorsally, are more often found in the ventral retina and pigment epithelium. They continue to be found in the retinal centre between ventricular cell processes in the apical contact zone and near the vitreal basement lamella above the ganglion cell layer until stage 35. Gap junctions are occasionally made by ganglion cells and their axons, providing possible pathways initiating cell differentiation. After stage 35 gap junctions seem to be confined to the peripheral rim of the eye cup where new cells are formed throughout larval life. The observed distribution suggests that gap junctions may provide intercytoplasmic pathways for retinal specification intrinsic to the retina and pigment epithelium.

Effects of chloramphenicol on the development of the zebrafish, Brachydanio rerio.

Fertilized eggs of the zebrafish, Brachydanio rerio, were subjected at 26 °C to three concentrations of chloramphenicol, 0.125, 0.250, and 0.500 mg/ml, at seven developmental stages ranging from cleavage to optic cup formation for periods of 12, 24, and 36 h. Subsequently, they were transferred to aquarial water to complete 48 h of development. Most extreme anomalies were induced by chloramphenicol in eggs initially subjected during cleavage and blastulation and less extreme anomalies in eggs exposed during or after gastrulation. In addition to general retardation of development, anomalies of the nervous, muscular, and vascular systems were induced. A unique type of spina bifida was of frequent occurrence. The teratogenic responses of the zebrafish egg are discussed in relation to the present concept of the mode of action of this antibiotic.