Pax-6 expression during retinal regeneration in the adult newt

The present study examined the expression of Pax-6 during retinal regeneration in adult newts using in situ hybridization. In a normal retina, Pax-6 is expressed in the ciliary marginal zone, the inner part of the inner nuclear layer, and the ganglion cell layer. After surgical removal of the neural retina, retinal pigment epithelial cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, Pax-6 was expressed in all retinal precursor cells. As regeneration proceeded, differentiating cells appeared at the scleral and vitreal margins of the regenerating retina, which had no distinct plexiform layers. In this stage, the expression of Pax-6 was localized in a strip of cells along the vitreal margin of the regenerating retina. In the late stage of regeneration, when the layer structure was completed, the expression pattern of Pax-6 became similar to that of a normal retina. It was found that Pax-6 is expressed in the retinal precursor cells in the early regenerating retina and that the expression pattern of Pax-6 changed as cell differentiation proceeded during retinal regeneration.     

A decay of gap junctions in association with cell differentiation of neural retina in chick embryonic development.

Ultrastructural studies of thin-sectioned and freeze-cleaved materials were performed on developing retinal tissues of 3- to 9-day-old chick embryos to clarify the junctional structures between neural retinal cells and between neural retinal cells and cells of the pigmented epithelium. Frequency, size and position of gap junctions in developing neural retina are different at each stage of development. In 3-day-old embryos, some cells adhere to each other by gap junctions immediately below the outer limiting membrane of neural retinae. The size and number of gap junctions increase remarkably during 5-6 days of incubation. In this period of development, well developed gap junctions consisting of subcompartments of intramembrane particles are found between cell surfaces at both the outer limiting membrane region and the deeper portion of the neural retina. Gap junctions disappear thereafter, and at 7-5 days of incubation, small gap junctions are predominant between cell surfaces at the outer limiting membrane region, while the frequency of gap junctions in the deeper portion is very low. At 9 days of incubation, gap junctions are rarely found. Typical gap junctions are always found between neural retinal cells and those of the pigmented epithelium in embryos up to 7-5 days of incubation. Tight junctions are not found in the neural retina or between neural retina and pigmented epithelium throughout the stages examined.     

Radioautographic study on DNA synthesis of the retina and retinal pigment epithelium of developing mouse embryos.

We report the changes of proliferative activity of the retina and retinal pigment epithelium (RPE) of mouse embryos by detecting cells in the S-phase by light microscopic radioautography using 3H-thymidine. The eyes germs of mouse embryos at the embryonic days 9.5 (E 9.5), E 11.5, E 13.0, E 15.5, E 18.5 of gestational ages, were used for this experiment. Small pieces of the ocular tissues were labelled with 3H-TDR in vitro and light microscopic radioautographs were prepared. The labeling indices of the respective regions of tissues were calculated. Both tissues of retina and RPE showed high percentages of labeling indices from 10% to 50% through the developmental stages. The labeling indices of both tissues in earlier stages were generally higher than those of later stages, and gradually decreased in the later stages. However, the retina and RPE showed different courses of the changes of labeling indices respectively during the embryonic development. In the retina, the labeling indices in the vitreal portions were more than those in the scleral portions during the earlier developmental stages. However, in the later stages, the indices of scleral portions were more than those in the vitreal portions. Comparing the three regions of retina, the labeling indices of the anterior regions were generally higher than those of the equatorial and posterior regions, especially in the vitreal portion. Remarkable differences among three regions were not found in the scleral portion. In the RPE, the labeling indices gradually increased in the anterior region, but decreased in the equatorial and the posterior regions through all the developmental stages. The proliferation of both retina and RPE in the central region occurred earlier than those of the peripheral region.     

Fine structure of the retinal epithelium and tapetum lucidum of the opossum (didelphis virginiana)

The fine structure of the retinal epithelium has been studied by electron microscopy in the opossum (Didelphis virginiana). The retinal epithelium, over most of the retina, is typical of that in other vertebrates and consists of a single layer of heavily pigmented, cuboidal cells. These cells display extensive basal (scleral) infoldings and numerous apical (vitreal) processes which enclose photoreceptor outer segments. A semicircular area of the retinal epithelium in the superior fundus is further specialized as a tapetum lucidum. The reflecting material consists of a large quantity of lipoidal spheres scattered throughout the epithelial cells. Centrally in the tapetal area very few or no melanosomes are found, indicating a non-occlusible tapetum. Peripherally in the tapetum, the epithelial cells contain both reflecting material and melanosomes. As in the non-tapetal area, the epithelial cells of the tapetum display large amounts of smooth endoplasmic reticulum and numerous mitochondria. Bruch's membrane everywhere displays the usual pentalaminate structure described for most vertebrates. The choriocapillaris is also typical, in that numerous fenestrations are present in the endothelium bordering Bruch's membrane.     

A study of the localization and accumulation of S-phase cells in the retina of newt Pleurodeles waltl after experimental pigment epithelial detachment

This work continues the studies of the proliferative ability of cells in the adult newt retina. The model of experimental detachment of the retina from pigment epithelium and two techniques to saturate the ocular tissues in vivo with precursors of DNA synthesis were used: (1) the method of repeated [3H]-thymidine labeling and subsequent autoradiographic analysis of semithin sections and (2) an original method for continuous labeling of thymidine analog bromodeoxyuridine and subsequent immunochemical detection. The data obtained confirm and extend our previous data on the localization of DNA-synthesizing cells in the neural retina and expose the pattern of S-phase cell accumulation after retinal detachment for each proliferation-competent cell population. In addition to cells in the growth zone of the retina, Muller glia, microglia, and minor cell population in the vitreal part of interneurons, DNA-synthesizing cells included astrocytes of the optic nerve and cells of its vascular network. Four weeks after detachment, the number of S-phase cells in the growth zone could reach 15-20%, while the above-mentioned DNA-synthesizing cells in the differentiated retina have low reproductive rate and could produce only one generation within the same period.     

FGF-mediated induction of ciliary body tissue in the chick eye

Upon morphogenesis, the simple neuroepithelium of the optic vesicle gives rise to four basic tissues in the vertebrate optic cup: pigmented epithelium, sensory neural retina, secretory ciliary body and muscular iris. Pigmented epithelium and neural retina are established through interactions with specific environments and signals: periocular mesenchyme/BMP specifies pigmented epithelium and surface ectoderm/FGF specifies neural retina. The anterior portions (iris and ciliary body) are specified through interactions with lens although the molecular mechanisms of induction have not been deciphered. As lens is a source of FGF, we examined whether this factor was involved in inducing ciliary body. We forced the pigmented epithelium of the embryonic chick eye to express FGF4. Infected cells and their immediate neighbors were transformed into neural retina. At a distance from the FGF signal, the tissue transitioned back into pigmented epithelium. Ciliary body tissue was found in the transitioning zone. The ectopic ciliary body was never in contact with the lens tissue. In order to assess the contribution of the lens on the specification of normal ciliary body, we created optic cups in which the lens had been removed while still pre-lens ectoderm. Ciliary body tissue was identified in the anterior portion of lens-less optic cups. We propose that the ciliary body may be specified at optic vesicle stages, at the same developmental stage when the neural retina and pigmented epithelium are specified and we present a model as to how this could be accomplished through overlapping BMP and FGF signals.     

A study of the localization and accumulation of S-phase cells in the retina of newt <Emphasis Type="Italic">Pleurodeles waltl</Emphasis> after experimental pigment epithelial detachment

This work continues the studies of the proliferative ability of cells in the adult newt retina. The model of experimental detachment of the retina from pigment epithelium and two techniques to saturate the ocular tissues in vivo with precursors of DNA synthesis were used: (1) the method of repeated [³H]-thymidine labeling and subsequent autoradiographic analysis of semithin sections and (2) an original method for continuous labeling of thymidine analog bromodeoxyuridine and subsequent immunochemical detection. The data obtained confirm and extend our previous data on the localization of DNA-synthesizing cells in the neural retina and expose the pattern of S-phase cell accumulation after retinal detachment for each proliferation-competent cell population. In addition to cells in the growth zone of the retina, Muller glia, microglia, and minor cell population in the vitreal part of interneurons, DNA-synthesizing cells included astrocytes of the optic nerve and cells of its vascular network. Four weeks after detachment, the number of S-phase cells in the growth zone could reach 15–20%, while the above-mentioned DNA-synthesizing cells in the differentiated retina have low reproductive rate and could produce only one generation within the same period.     

Establishment of the scleral cartilage in the chick.

This study was undertaken to investigate the establishment of the scleral cartilage in the chick embryo. Johnston et al. (1974) has demonstrated that most of the cells of the scleral cartilage originate in the cranial neural crest. By means of a series of chorioallantoic grafts of pigmented retina, and its adherent periocular mesenchyme from stage 11 to 25, the present experiments show that the cranial neural crest cells arrive at the eye in sufficient numbers to form cartilage by stage 14. Pigmented retina, denuded of mesenchyme, from stage 16 embryos implanted into the head of stage 13 embryos induces cartilage formation in head mesenchyme. However, neither pigmented retina nor spinal cord could induce cartilage formation in chorioallantoic mesenchyme. Combination grafts of cranial neural crest and presumptive optic vesicle developed neural tissue, pigmented retina, and in some cases sclera-like cartilage. Thus, periorbital mesenchyme, derived largely from cranial neural crest, at about stage 14 develops the scleral cartilage in response to induction by the pigmented retina.     

Electron microscopic observations on the retinal pigment epithelium and choroid in the domestic pig (Sus scrofa)

Summary The morphology of the retinal pigment epithelium (RPE) and adjacent choroid has been investigated by electron microscopy in the domestic pig. The RPE consists of a single layer of cells which are columnar posteriorly but become cuboidal and even squamous moving peripherally in the fundus. The cells of the RPE layer regardless of location display basal (scleral) infoldings and apical (vitreal) processes and are joined laterally by junctional complexes. Throughout the retina the epithelial cells are rich in smooth endoplasmic reticulum and mitochondria but less so in rough endoplasmic reticulum and polysomes. The epithelial nucleus is vesicular and basally located. In the superior fundus an area of the RPE is very lightly pigmented and richer in lysosomes than is this layer in the inferior and peripheral fundus. The choroid overlying this area is also lightly pigmented and contains much collagen in a lamellar arrangement. This region may represent a vestigial tapetum fibrosum. Bruch's membrane is slightly thicker posteriorly but is everywhere seen to have a pentalaminate substructure. The choriocapillaris is a single layer of large capillaries which show numerous fenestrations facing the RPE. In the superior fundus the choriocapillaris is also highly fenestrated facing the choroid.     

Retinal fate and ganglion cell differentiation are potentiated by acidic FGF in an in vitro assay of early retinal development.

One of the earliest events in vertebrate eye development is the establishment of the pigmented epithelium and neural retina. These fundamentally different tissues derive from the invaginated optic vesicle, or optic cup. Even after achieving a fairly advanced state of differentiation, the pigmented epithelium exhibits the same potential as the optic cup in that it can "transdifferentiate" into neural retina. C. M. Park and M. J. Hollenberg (Dev. Biol. 134, 201-205, 1989) discovered that administration of basic fibroblast growth factor, coupled with retinal removal, could trigger this transformation in vivo. We have developed a quantitative in vitro assay to study the role(s) of the fibroblast growth factor (FGF) family in this phenomenon and more generally in early retinal development. We found that several aspects of the process, including inhibition of pigmented epithelium differentiation, proliferation, and conversion to a retinal fate, were not strictly correlated. Both acidic and basic FGFs were found to potentiate all aspects of the process, with acidic FGF being 4 to 20 times more potent than basic FGF for inhibition of pigmentation and induction of retinal antigens. Depending upon its concentration, acidic FGF induced from 40% to 80% of the cells in the explants to produce antigens normally expressed by retinal ganglion cells, the first cell type to be generated in retinal development. Expression of such a ganglion cell marker could be directly stimulated in non-dividing cells as well as in dividing cells, indicating that conversion from the pigmented epithelial to retinal fate did not require cell division. These data suggest that acidic FGF, or a related molecule, may function in establishment of retinal fate from the optic cup. This effect may be directly or indirectly mediated by induction of retinal ganglion cell fate among multipotent progenitor cells.     

Retinal remodeling in inherited photoreceptor degenerations

Photoreceptor degenerations initiated in rods or the retinal pigmented epithelium usually evoke secondary cone death and sensory deafferentation of the surviving neural retina. In the mature central nervous system, deafferentation evokes atrophy and connective re-patterning. It has been assumed that the neural retina does not remodel, and that it is a passive survivor. Screening of advanced stages of human and rodent retinal degenerations with computational molecular phenotyping has exposed a prolonged period of aggressive negative remodeling in which neurons migrate along aberrant glial columns and seals, restructuring the adult neural retina (1). Many neurons die, but survivors rewire the remnant inner plexiform layer (IPL), forming thousands of novel ectopic microneuromas in the remnant inner nuclear layer (INL). Bipolar and amacrine cells engage in new circuits that are most likely corruptive. Remodeling in human and rodent retinas emerges regardless of the molecular defects that initially trigger retinal degenerations. Although remodeling may constrain therapeutic intervals for molecular, cellular, or bionic rescue, the exposure of intrinsic retinal remodeling by the removal of sensory control in retinal degenerations suggests that neuronal organization in the normal retina may be more plastic than previously believed.     

Retina and lens regeneration in anuran amphibians

Anuran amphibians can regenerate the retina through differentiation of stem cells in the ciliary marginal zone and through transdifferentiation of the retinal pigmented epithelium. By contrast, the regeneration of the lens has been demonstrated only in larvae of species belonging to the Xenopus genus, where the lens regenerates through transdifferentiation of the outer cornea. Retinal pigmented epithelium to neural retina and outer cornea to lens transdifferentiation processes are triggered and sustained by signaling molecules belonging to the family of the fibroblast growth factor. Both during retina and lens regeneration there is a re-activation of many of the genes which are activated during development of the eye, even though the spatial and temporal pattern of gene expression is not a simple repetition of that found in development.     

Proliferative activity of the pigment epithelium and regenerating retinal cells in Ambystoma mexicanum

Cellular sources of retinal regeneration and proliferative activity of the cells taking part in retina restoration have been studied in axolotls using 3H-thymidine. The cells of ciliary-terminal zone proved to be the main source of retinal restoration. Besides these cells, the pigmented cells of the iris inner and outer layers and pigment epithelium cells can take part in this process. Morphological stages of retinal regeneration have been established and regular changes in the level of proliferation in different zones of regenerating retina have been found with respect to the stage of retina restoration. The high level of proliferative activity of the pigment epithelium cells found soon after the operation favoured the restoration of disturbed integrity of the pigment epithelium layer, the increase of cell density in it, the elongation of the pigment epithelium layer, the formation of processes, and, sometimes, the replenishment of regenerating retina.     

Retinal stem cells and regeneration

The optic vesicle gives rise to several very different epithelial tissues, including the neural retina, the pigmented epithelium, the iris, the ciliary epithelium of the ciliary body and the optic stalk. Retinal regeneration can arise from several different cellular sources; in some species, the process involves interconversion, or transdifferentiation, among cells of the different tissue types. Therefore, prior to a discussion of retinal regeneration, we will briefly discuss current knowledge about the influence of signaling molecules in cell fate determination in ocular tissues. Next, we will detail the evidence for neurogenesis in the mature retina. Lastly, we will describe various types of regenerative phenomena that occur in the retina, from complete regeneration of functional retina in fish and amphibians, to the more limited neuronal production that occurs in avian and mammalian retinas.     

A novel function for Hedgehog signalling in retinal pigment epithelium differentiation

Sonic hedgehog is involved in eye field separation along the proximodistal axis. We show that Hh signalling continues to be important in defining aspects of the proximodistal axis as the optic vesicle and optic cup mature. We show that two other Hedgehog proteins, Banded hedgehog and Cephalic hedgehog, related to the mouse Indian hedgehog and Desert hedgehog, respectively, are strongly expressed in the central retinal pigment epithelium but excluded from the peripheral pigment epithelium surrounding the ciliary marginal zone. By contrast, downstream components of the Hedgehog signalling pathway, Gli2, Gli3 and X-Smoothened, are expressed in this narrow peripheral epithelium. We show that this zone contains cells that are in the proliferative state. This equivalent region in the adult mammalian eye, the pigmented ciliary epithelium, has been identified as a zone in which retinal stem cells reside. These data, combined with double labelling and the use of other retinal pigment epithelium markers, show that the retinal pigment epithelium of tadpole embryos has a molecularly distinct peripheral to central axis. In addition, Gli2, Gli3 and X-Smoothened are also expressed in the neural retina, in the most peripheral region of the ciliary marginal zone, where retinal stem cells are found in Xenopus, suggesting that they are good markers for retinal stem cells. To test the role of the Hedgehog pathway at different stages of retinogenesis, we activated the pathway by injecting a dominant-negative form of PKA or blocking it by treating embryos with cyclopamine. Embryos injected or treated at early stages display clear proximodistal defects in the retina. Interestingly, the main phenotype of embryos treated with cyclopamine at late stages is a severe defect in RPE differentiation. This study thus provides new insights into the role of Hedgehog signalling in the formation of the proximodistal axis of the eye and the differentiation of retinal pigment epithelium.     

Differential effect of dopamine on mitosis in early postnatal albino and pigmented rat retinae

Insufficient levels of L-DOPA, released from the retinal pigment epithelium (RPE), in albino animals are considered responsible for the abnormal development of the underlying neural retina. L-DOPA normalizes retinal neurogenesis by reducing levels of cell proliferation either by acting on the cells directly or by being converted into dopamine. Here we report the effects of dopamine on mitosis in early postnatal neural retinae from albino and pigmented rats, using 4D (x, y, z and time) confocal microscopy. Exogenous dopamine significantly prolongs mitosis in retinae from albino, but not pigmented, animals. As fewer cells move into and divide in the ventricular zone (VZ) in the presence of dopamine, we conclude that the overall cell cycle is affected. The D1 receptor blocker, SCH 23390, inhibits these effects. Thus, the differential effects of dopamine on neural retinae from pigmented and albino rats in vitro must result from the activation of D1 receptors, which are present in the retina from birth. Immunohistochemical labeling of D1 receptors shows that the pattern of their distribution is similar between pigmentation phenotypes, but levels of expression may be elevated in albinos. Labeling is most intense in the inner plexiform layer but is present throughout the neuroblastic layer. These findings are discussed in light of previous reports of reduced catecholamine levels in the albino retina.     

In vitro stimulation of cartilage in embryonic chick neural crest cells by products of rentinal pigmented epithelium.

The retinal pigmented epithelium of the chick embryo influences head neural crest mesenchymal cells to form the scleral cartilage of the eye. The possible role of extracellular matrix in this interaction was studied. Extracellular matrix was deposited on Millipore filters in vitro by pigmented epithelial cells which were then killed by distilled water lysis. When grown on the Millipore filters which had carried pigmented epithelium, clonal neural crest and periocular mesenchyme “target” cells formed cartilage in 61 of 155 experiments. Cartilage was not formed when the cells were grown on naked filters nor did gels of purified Type I and Type II collagen promote chondrogenesis. It is concluded that extracellular matrix deposited by the pigmented epithelium in vitro is a potent stimulus for the induction of chondrogenesis in competent mesenchyme, and that living pigmented epithelial cells need not be present for such induction.     

The association between prion proteins and Aβ₁₋₄₂ oligomers in cytotoxicity and apoptosis

Microglia are cells from non-neuronal lineages that reside in the central nervous system. In zebrafish, early macrophages migrate from the yolk sac to the brain and retina at 26-30 hour post fertilization (hpf) and transform into microglia at 55-60 hpf. The migration of macrophages into the central nervous system requires signaling by macrophage colony stimulating factor-1 receptor (csf-1r), which is encoded by the gene fms. In this study, we show that the targeted knockdown of csf-1r with morpholino oligonucleotides delays migration of macrophages from the yolk sac to the retina, and this delay in macrophage migration results in microphthalmia, delay in cell cycle withdrawal among retinal progenitors and the absence of neuronal differentiation. When embryos were allowed to survive beyond the time when morpholino-dependent translation inhibition is lost, microglia re-occupy the retina and neuronal differentiation partially recovers. Our data demonstrate that microglia are required for normal retinal growth and neurogenesis. This study provides new insight into the neurogenic role of microglia during retinal development in zebrafish.