Healing modes correlate with visuotectal pattern formation in regenerating embryonic Xenopus retina

After removal of the nasal or the temporal two-thirds of the embryonic (stage 32) eye, the remaining one-third sized fragment undergoes wound healing and then, in most cases, regenerates to form a new eye. Using gross anatomy and histology techniques, we categorized eye fragments into three healing mode categories over the first 24 hr after surgery (stage 37-38). Representative animals were reared through metamorphosis and their visuotectal projections were assayed using standard electrophysiology techniques. In the "rounded-up" healing mode, the cut edges of the fragment pinch to close the wound; retinal cell type layers (pigmented retinal epithelium (pre), photoreceptors, interneurons, ganglion cells) and a lens are present by 24 hr postsurgery. No extraneous or disorganized cells are present either internal or external to the fragments. These fragments regenerated to form normal projections 83% of the time and pattern duplicated projections only 17% of the time. In the "intermediate" healing mode, wound closure is not complete by 24 hr post surgery and groups of disorganized cells are present in the fragment and amassed between the healing cut edges. These fragments formed pattern duplicated projections 72% of the time. In the tongue healing mode, an ectopic mass of cells, contiguous with the main body of the fragment, forms a supernumerary retina in the region of the ablation. At 24 hr post surgery, the cells of the main body fragment form retinal layers; the cells of the tongue, excluding the presence of differentiated pre cells, remain undifferentiated, resembling ciliary margin. The cut edges of the main body fragment eventually fuse with the tongue to form a single eyeball. Tongue fragments formed pattern duplicated projections 100% of the time. In addition, pattern duplicated points derived from nasal fragments appeared most often in the posterior region of the tectum, the normal site of innervation of the nasal retina. This differed significantly from temporal fragment derived duplicated points which appeared more often in the front of the tectum, the normal site of innervation by temporal retina. Thus, the specificity of pattern duplicated innervation is related to the positional values remaining in the fragment after partial retinal ablation. The data indicate that cell movements during healing, whether overt as in the tongue healing mode, or remaining internal to the fragment as in the intermediate healing mode, are intimately correlated with pattern forming mechanisms which underlie pathological visuotectal duplication.     

Embryonic retinal ablation and post-metamorphic optic nerve crush: effects upon the pattern of regenerated retinotectal connections.

We examined relationships between healing observed during embryonic Xenopus retinal and optic nerve regeneration and resultant visuotectal pattern formation. Dorsal (D) and nasoventral (NV) 1/3 sized eye fragments were surgically created in stage 32 Xenopus laevis embryos. Gross anatomical healing modes of these fragments were examined 2 days post-surgery (stage 43). Healing was categorized according to the degree of cell movements observed. Animals were reared through metamorphosis and electrophysiologic mapping techniques were employed on those animals whose eyes regenerated. All D 1/3 fragments showed normal (non-duplicated) projections to the tectum; most (80%) of the healing observed showed little cell movements (the remaining 20% showed substantial cell movements, yet failed to show duplicated projections). Most NV 1/3 fragments (73%) formed two mirror image projections to the contralateral midbrain optic tectum (pattern duplication). Most (88%) of the healing observed among these animals showed massive cell movements in the ventral retinal region (the remaining 12% showed moderate cell movements). The remaining NV 1/3 fragments (27%) showed moderate cell displacement and failed to show duplicated projections). These data are compatible with a cell-movement:intercalary cell division hypothesis in which duplication is dependent upon specific positional confrontation and subsequent cell division. In additional studies, in adult animals, the optic nerves of eyes with duplicated projections were crushed and allowed to regenerate for 1 year. Duplicated projections were restored, indicating that developmental and maturational factors are probably not responsible for duplicative pattern formation; rather, information intrinsic to the eye, possibly created during healing interactions and/or fiber ingrowth to the tectum, underlies duplicate innervation of the tectum.     

Fully differentiated Xenopus eye fragments regenerate to form pattern-duplicated visuo-tectal projections

In order to determine if differentiated Xenopus retina is capable of undergoing regeneration and duplicative pattern formation, we devised a new surgical technique for removal of the temporal two-thirds of the retina. In a series of progressively older larval eyes starting with late tailbud stage embryos (stage 38) and extending to limb-bud stage tadpoles (stage 48), nasal one-third-sized eye fragments successfully regenerated to form nearly normal sized eyes over 75% of the time. Histological preparations showed that early wound healing involved the formation of a neuroepithelium at the ventro-temporal region of the fragment. The pigmented retinal epithelium and associated retinal tissue appeared to be involved in this process. Animals from each stage were reared through metamorphosis and electrophysiologic techniques were employed to determine visuo-tectal projections. Seventy percent of stage 38 animals showed evidence of pattern-duplicated projections. Ninety percent of their responding tectal points showed duplicate innervation from two retinal regions. Older animals (stages 44 to 48) showed less duplication. Only 52% of their responding tectal points duplicated (P less than 0.001). Thus, fully differentiated Xenopus retina can undergo regeneration and duplicative pattern formation similar to that shown by embryonic retinal tissue.     

Experimental induction of microphthalmia in the chick embryo with a single dose of cisplatin

Chick embryos were injected on the fifth day of incubation with 75 ng cis-diamminedichloroplatinum II (cisplatin) and killed at daily intervals. Bilateral microphthalmia appeared in 88% of the surviving embryos; the decrease in eye size was noticeable 2 or 3 days after injection. Coinciding with this, macroscopic, histological, and ultrastructural changes started to appear in the ciliary body: ciliary processes failed to form and the cells in the inner layer of the ciliary epithelium underwent degenerative changes. Changes in the retina appeared somewhat later. Despite the decreased growth rate of the whole eye the neural layer of the retina continued to grow rapidly; as a result, it formed numerous folds and acquired a glandular appearance. In the most severe cases the rapidly growing retina would invade the ciliary region and replace completely the degenerated inner layer of the ciliary epithelium. It has been shown by previous authors that intraocular pressure is a determinant of eye expansion and also that the secretion of water and ions by the ciliary epithelium is important for the maintenance of that intraocular pressure. On this basis, our results are interpreted as indicating that the primary lesion induced by cisplatin was in the ciliary epithelium and that microphthalmia was the consequence of decreased pressure. It is also concluded that the retinal changes were due to the fact that the retina continued to grow despite the lack of expansion of the eye as a whole.     

Nasotemporal asymmetry during teleost retinal growth: preserving an area of specialization.

Teleost fish retinas grow throughout adult life through both cell addition and stretching. Cell division occurs at the periphery of the retina, resulting in annular addition of all cell types except rod photoreceptors, which are added in the central retina. Since many teleosts have a region of high cellular density at the temporal pole of the eye, we analyzed whether and how this specialized region of high visual acuity maintained its relative topographical position through asymmetric circumferential growth. To do this, we measured the pattern of long-term retinal growth in the African cichlid Haplochromis burtoni. We found that the retina expands asymmetrically along the nasotemporal axis, with the nasal retina growing at a higher rate than the temporal, dorsal, or ventral retinae, whose growth rates are equal. This nasotemporal asymmetry is produced via significantly greater expansion of retinal tissue at the nasal pole rather than through differential cell proliferation. The mechanisms responsible for this differential retinal enlargement are unknown; however, such asymmetric expansion very likely minimizes disruption in vision during rapid growth.     

Retinal pigmented epithelium does not transdifferentiate in adult goldfish

The neural retina of adult goldfish can regenerate from an intrinsic source of proliferative neuronal progenitor cells, but it is not known whether the retina can regenerate by transdifferentiation of the retinal pigmented epithelium (RPE), a phenomenon demonstrated in adult newts. In this study, we asked whether following surgical removal of the neural retina in adult goldfish the RPE was capable of autonomously transdifferentiating and generating new neural retina. The retina was prelabeled by injecting the fluorescent dye Fluoro-Gold (FG) into the eye prior to surgical removal; this procedure ensured that residual retina was labeled with FG and could therefore be distinguished from unlabeled, regenerated retina. To examine the time course of retinal regeneration, and to identify regenerated retinal neurons, the thymidine analogue bromodeoxyuridine was injected intraocularly, and retinas were examined up to 2 months later. We found that the RPE did not transdifferentiate; instead, retinas regenerated only when pieces of residual neural retina were left intact. Under these circumstances, newly regenerated cells derived from proliferating cells intrinsic to the residual neural retina. When retinas were completely removed, as was evident from a lack of FG labeling, there was no retinal regeneration. © 1995 John Wiley & Sons, Inc.     

Central and Peripheral Retina Arise through Distinct Developmental Paths

In the mature eye, three distinct tissue fates, retina, ciliary body, and iris, arrange with a strict linear organization along the central (back) to peripheral (front) axis. The establishment of this topographical relationship within the optic vesicle is not well understood. We use a targeted vital labeling strategy to test the derivation of mature eye tissues from the optic vesicle of the chick embryo. Fate mapping uncovers two distinct origins of the neural retina. Contrary to expectations, the central neural retina has a discrete origin within the posterior optic vesicle. The peripheral retina derives from the distal optic vesicle, sharing a common origin with more peripheral tissue fates. This study identifies for the first time two distinct retinal sub-domains, central and peripheral, which arise during embryogenesis. Identification of these discrete retinal compartments provides a framework for understanding functional and disease processes throughout retinal tissue.     

Transdifferentiation Potential of Ciliary and Pigment Epithelial Cells in Lower Vertebrates and Mammals

Cellular composition of the peripheral region of the eye in amphibians and mammals as well as embryonic fissure in amphibians was studied. Different distributions of proliferating cells in retinal pigment epithelium have been revealed in adult amphibians (newt, axolotl, and Xenopus). Single cells incorporated [³H]thymidine in the newt and Xenopus; 0.4% cells, in the axolotl. An embryonic fissure was observed in the eye of the axolotl. Pigment epithelial cells in the embryonic palpebral region actively proliferated: about 20% cells incorporated [³H]thymidine. Proliferating cells were also localized in the ciliary marginal zone of the retina in all studied amphibians, particularly, in the axolotl. In newborn hamsters, [³H]thymidine-labeled cells have been revealed in the pigment epithelium as well as in the outer pigmented and inner unpigmented layers of the ciliary body. Proliferative activity of the peripheral regions of the eye is due to eye growth in adult amphibians and newborn hamsters. After retinectomy, the retina is regenerated from the cells of the growth ciliary marginal zone in all amphibians, pigment epithelial cells in the newt, and pigment epithelial cells of the embryonic fissure in the axolotl. Heterogeneous composition of the pigment epithelium in the newt and axolotl reflects high transdifferentiation potential of these regions. Structural comparison of the peripheral region of the eye in amphibians and mammals demonstrate that the ciliary body of mammals containing stem cells is homologous to the ciliary marginal zone of amphibians containing multipotent cells.     

Nasotemporal asymmetry during teleost retinal growth: preserving an area of specialization

Teleost fish retinas grow throughout adult life through both cell addition and stretching. Cell division occurs at the periphery of the retina, resulting in annular addition of all cell types except rod photoreceptors, which are added in the central retina. Since many teleosts have a region of high cellular density at the temporal pole of the eye, we analyzed whether and how this specialized region of high visual acuity maintained its relative topographical position through asymmetric circumferential growth. To do this, we measured the pattern of long‐term retinal growth in the African cichlid Haplochromis burtoni. We found that the retina expands asymmetrically along the nasotemporal axis, with the nasal retina growing at a higher rate than the temporal, dorsal, or ventral retinae, whose growth rates are equal. This nasotemporal asymmetry is produced via significantly greater expansion of retinal tissue at the nasal pole rather than through differential cell proliferation. The mechanisms responsible for this differential retinal enlargement are unknown; however, such asymmetric expansion very likely minimizes disruption in vision during rapid growth. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 435–442, 1999     

Light microscopic radioautographic study on the DNA synthesis of prenatal and postnatal aging mouse retina after labelled thymidine injection.

The DNA synthesis of mouse retina from the 19th prenatal day through 12 months postnatal has been studied by light microscopic radioautography after the injection of tritiated thymidine. A peak of the labeling index after incorporation of tritiated thymidine was found at fetal day 19. The labelled cells decreased gradually with the developing of the eye from the first postnatal day and were completely disappeared in two weeks after birth. The data also indicated obvious regional differences of the incorporation of tritiated thymidine during the periods of the retina development. The labeling index was the greatest in the anterior region compared to the equator region and the posterior region in the same group of age. The average number of the silver grains in labelled nucleus lead to a decrease with the development of the retina after birth, but there was no significant regional differences found in the same group of age. The data shown from this study suggest that the cell differentiation in mouse retina proceed from posterior to anterior region.     

Ciliary margin transdifferentiation from neural retina is controlled by canonical Wnt signaling

The epithelial layers of the ciliary body (CB) and iris are non-neural structures that differentiate from the anterior region of the eyecup, the ciliary margin (CM). We show here that activation of the canonical Wnt signaling pathway is sufficient and necessary for the normal development of anterior eye structures. Pharmacological activation of beta-catenin signaling with lithium (Li(+)) treatment in retinal explants in vitro induced the ectopic expression of the CM markers Otx1 and Msx1. Cre-mediated stabilization of beta-catenin expression in the peripheral retina in vivo induced a cell autonomous upregulation of CM markers at the expense of neural retina (NR) markers and inhibited neurogenesis. Consistent with a cell autonomous conversion to peripheral eye fates, the proliferation index in the region of the retina that expressed stabilized beta-catenin was identical to the wild-type CM and there was an expansion of CB-like structures at later stages. Conversely, Cre-mediated inactivation of beta-catenin reduced CM marker expression as well as the size of the CM and CB/iris. Aberrant CB development in both mouse models was also associated with a reduction in the number of retinal stem cells in vitro. In summary, activation of canonical Wnt signaling is sufficient to promote the development of peripheral eyecup fates at the expense of the NR and is also required for the normal development of anterior eyecup structures.     

A model of retinal ischemia-reperfusion injury in rats by subconjunctival injection of endothelin-1

The retinal ischemia-reperfusion model is used in the study of transient ischemia-related diseases, such as central retinal artery occlusion, angle-closure glaucoma, and others. There are two methods for experimentally producing an ischemia-reperfusion model in the rat retina: (i) the intraocular pressure is greatly raised by increasing the height of the infusion bottle connected with the needle in the anterior chamber; or (ii) the blood vessel that accompanies the optic nerve in retina is ligated. However, each method has some drawbacks. For example, in the first method, the needle must be fixed in the anterior chamber for 1 hr, thus, the technique is not stable and mechanical damage to ocular structures sometimes occurs. In the second method, because of the unavoidable involvement of the optic nerve, damage to the nerve induces retinal changes unrelated to ischemia. In this study, we injected endothelin (ET)-1 under the conjunctiva of the eyeball (subconjunctival injection), and evaluated whether a retinal ischemia-reperfusion model could be generated by this method, simply and noninvasively. We injected 4 x 10(-5) M ET-1 solution into the right eye of the rat and injected a control vehicle (artificial tears) into the left eye. From 5-60 mins after the injection, 50 mg/ml fluorescein isothiocyanate (FITC)-dextran was injected to the left ventricle of heart. Then, the retina was removed and flat mounted. We compared the perfusion conditions of the FITC-dextran to each retina in the right and left eye. There was a complete perfusion of FITC-dextran in the retinal main artery, vein, and the capillary vessels in all of the control eyes. However, perfusion could not be completely observed in the ET-1 injected eye from 5-35 mins after injection; afterwards, the flow was returned. This method of subconjunctival injection of ET-1 is, thus, a feasible technical option for producing a retinal ischemia-reperfusion model in rat.     

Multipotent and Stem Cells in the Developing, Definitive, and Regenerating Vertebrate Eye

This is a review of the experimental studies on the vertebrate retina neurogenesis. Data are provided on the distribution and localization of multipotent and stem cells in the developing, definitive, and regenerating eye. At the early stages of retina development, the neuroepithelial cells divide synchronously, thus leading to the accumulation of a certain number of the retinal rudiment cells. Synchronous divisions precede the asynchronous ones, when the differentiation of the retinal cells is initiated. The neuroepithelial cells are multipotent: the neuroblast is a source of the cells of different types, for example, neurons and glial cells. The proliferating multipotent cells are preserved in the ciliary-terminal zone of the retina of amphibians, fish, and chickens during their entire life. The differentiated pigment epithelium cells also proliferate in this area of the eye. The multipotent cells of the retinal ciliary-terminal zone and cells of the pigment epithelium in the eye periphery provide for the growth of amphibian and fish eyes during the entire life of these animals. In adult mammals, clonable and self-renewable cells were found among the pigmented differentiated cells in the ciliary folds. In a culture, the stem cells form spheroids consisting of depigmented and proliferating cells. Upon transdifferentiation, the cells of spheroids form rods, bipolar cells, and ganglion and glial cells, thus suggesting the possible regenerative potencies of the stem cells in the ciliary body of the mammalian eye. The main event of retinal regeneration in newts is the transdifferentiation of the pigment epithelium cells. The results of comparative analysis suggest that the stem cells of the ciliary body in the mammalian eye and pigment epithelium cells in lower vertebrates exhibit similar potencies and use similar mechanisms during the formation of the cells of the neural series.     

Expression of monocarboxylate transporters in rat ocular tissues

The aim of the present study was to determine the distribution of monocarboxylate transporter (MCT) subtypes 1-4 in the various structures of the rat eye by using a combination of conventional and real-time RT-PCR, immunoblotting, and immunohistochemistry. Retinal samples expressed mRNAs encoding all four MCTs. MCT1 immunoreactivity was observed in photoreceptor inner segments, Müller cells, retinal capillaries, and the two plexiform layers. MCT2 labeling was concentrated in the inner and outer plexiform layers. MCT4 immunolabeling was present only in the inner retina, particularly in putative Müller cells, and the plexiform layers. No MCT3 labeling could be observed. The retinal pigment epithelium (RPE)/choroid expressed high levels of MCT1 and MCT3 mRNAs but lower levels of MCT2 and MCT4 mRNAs. MCT1 was localized to the apical and MCT3 to the basal membrane of the RPE, whereas MCT2 staining was faint. Although MCT1-MCT4 mRNAs were all detectable in iris and ciliary body samples, only MCT1 and MCT2 proteins were expressed. These were present in the iris epithelium and the nonpigmented epithelium of the ciliary processes. MCT4 was localized to the smooth muscle lining of large vessels in the iris-ciliary body and choroid. In the cornea, MCT1 and MCT2 mRNAs and proteins were detectable in the epithelium and endothelium, whereas evidence was found for the presence of MCT4 and, to a lesser extent, MCT1 in the lens epithelium. The unique distribution of MCT subtypes in the eye is indicative of the pivotal role that these transporters play in the maintenance of ocular function. retina; eye; immunohistochemistry; polymerase chain reaction     

The rat retina is a useful in vivo model to study membrane lipid synthesis: Rates of biosynthesis of neutral glycerides and phospholipids

The phospholipid composition was studied in the whole rat retina, as well as in its subcellular fractions. A relative enrichment of phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine was observed in rod outer segments (ROS) in comparison with entire retina: nuclear-photoreceptor inner segmentssynaptic bodies (P₁) and synaptosomal-mitochondrial (P₂) fractions. Phosphatidylcholine was the predominant phospholipid class found in all subcellular fractions analyzed. The microsomal fraction was relatively enriched in phosphatidic acid and in phosphatidylinositol. In addition, the rat eye has been used as an in vivo system to study membrane lipid synthesis. After intravitreal injections of [2-³H]glycerol a rapid labeling of retinal glycerolipids took place. Up to 120 min after injection only the glycerol backbone of lipids was labeled. Phosphatidic acid and diacylglycerol displayed rapid rates of synthesis and breakdown. Fastest rates of labeling were attained by phosphatidylcholine followed by phosphatidylinositol. Differences were found when in vitro labeling by [2-³H]glycerol was compared with intravitreal injections. Labeling of phospholipids of subcellular fractions by intravitreally injected [2-³H]glycerol showed that most of the label accumulated in microsomal phosphatidylcholine and phosphatidylinositol. Diacylglycerols and phosphatidylethanolamine also took up 10 and 20% respectively of the precursor. It is concluded that the rat eye is a useful experimental model to study synthesis and metabolism of membrane lipids in the retina.     

Early onset of phenotype and cell patterning in the embryonic zebrafish retina

The regular arrangement of retinal cone cells in a mosaic pattern is a common feature of teleosts. In the zebrafish, Brachydanio rerio, the retinal cone mosaic comprises parallel rows consisting of a repeating motif of four cone types. In order to elucidate the temporal and spatial aspects of the genesis of the cone mosaic in the developing retina, we generated a monoclonal antibody that specifically binds to the double cone photoreceptor of the adult. We first saw staining in the developing retina with this antibody, FRet 43, at 48 hours postfertilization, the time at which the first photoreceptor cells undergo their final mitotic division. We then injected embryonic fish with the thymidine analog, 5-bromo-2'-deoxyuridine (BrdU), confirming with a double-labeling experiment that the onset of FRet 43 antigenicity occurs within three hours of the cellular division that generates the double cone photoreceptors. Then we stained tangential sections of the 54-hour embryonic retina with FRet 43, further showing that cells devoid of staining alternate with stained pairs of cells in a pattern that is consistent with the arrangement of photoreceptors in the adult cone mosaic. These results indicate that a marker of the double cone phenotype is expressed at approximately the same time as cellular birthday and that the mosaic patterning is present within 6 hours of this expression.     

The temporal requirement for vitamin A in the developing eye: mechanism of action in optic fissure closure and new roles for the vitamin in regulating cell proliferation and adhesion in the embryonic retina

Mammalian eye development requires vitamin A (retinol, ROL). The role of vitamin A at specific times during eye development was studied in rat fetuses made vitamin A deficient (VAD) after embryonic day (E) 10.5 (late VAD). The optic fissure does not close in late VAD embryos, and severe folding and collapse of the retina is observed at E18.5. Pitx2, a gene required for normal optic fissure closure, is dramatically downregulated in the periocular mesenchyme in late VAD embryos, and dissolution of the basal lamina does not occur at the optic fissure margin. The addition of ROL to late VAD embryos by E12.5 restores Pitx2 expression, supports dissolution of the basal lamina, and prevents coloboma, whereas supplementation at E13.5 does not. Surprisingly, ROL given as late as E13.5 completely prevents folding of the retina despite the presence of an open fetal fissure, showing that coloboma and retinal folding represent distinct VAD-dependent defects. Retinal folding due to VAD is preceded by an overall reduction in the percentage of cyclin D1 positive cells in the developing retina, (initially resulting in retinal thinning), as well as a dramatic reduction in the cell adhesion-related molecules, N-cadherin and β-catenin. Reduction of retinal cell number combined with a loss of the normal cell-cell adhesion proteins may contribute to the collapse and folding of the retina that occurs in late VAD fetuses.