Differential effects of bFGF on development of the rat retina

A variety of growth factors can influence the expression of differentiated properties by cell types of the developing retina. One unresolved question has been whether these factors can direct the differentiation pathway of uncommitted precursors or whether they act to help the expression of properties by already committed cells. To address this question we have studied the effects of basic fibroblast growth factor (bFGF) on the differentiation of ganglion cells and rod photoreceptors in explant cultures of embryonic rat retinas. Incubation of retinas in the presence of bFGF accelerated the appearance of differentiated ganglion cells and incubation in the presence of anti-bFGF antibodies delayed the appearance. bFGF had no effect on the appearance of differentiated rod photoreceptors as judged by expression of opsin, although all-trans-retinoic acid did increase the number of cells expressing opsin. bFGF inhibited the formation of rod photoreceptor rosettes suggesting that it does influence some properties of rods or the adjacent Müller glial cells. The results suggest that bFGF can alter the timing of differentiation of retinal ganglion cells but not direct their production from retinal precursors.     

Cell commitment and differentiation in explants of embryonic rat neural retina. Comparison with the developmental potential of dissociated retina

The differentiation of presumptive neural retina following its isolation from rat embryos and growth in explant and monolayer culture has been studied to obtain information regarding the extent to which factors extrinsic and intrinsic to the retina participate in determining molecular and cytological differentiation. Explanted retinal epithelium retained the capacity for mitosis, as shown by [3H]thymidine incorporation, and from the undifferentiated neuroepithelium, retinal cell-types emerged and acquired a laminar organization resembling that in vivo. Characterization of rod photoreceptor cells at both the light and electron microscopic level showed that these cells exhibit differentiated structural features including inner segments, connecting cilia and membranous expansions suggestive of forming outer segments. Immunofluorescent labeling with an antibody to a synaptic vesicle protein, and electron microscopic identification of synaptic elements showed formation of synapses by the photoreceptor cells within the explant. Neurites extending from the explants exhibited growth on laminin, fibronectin and collagen substrates. Since the neurites immunolabeled with antibodies to the 140 kDa subunit of neurofilament and with antibodies to Thy-1, they could be identified as axons of ganglion cells. Antibodies to a variety of cell-type specific antigens showed that the cells expressed molecules associated with the fully differentiated cell. Furthermore, since our approach has been to explant embryonic retina at an age when the antigens are not yet expressed in vivo, the appearance of the antigens in culture represented de novo expression. In contrast, neural retinal cells in dissociated cultures did not exhibit de novo expression of differentiated molecular properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mosaic deletion of Rb arrests rod differentiation and stimulates ectopic synaptogenesis in the mouse retina

The retinoblastoma gene (Rb) regulates neural progenitor cell proliferation and cell fate specification and differentiation. For the developing mouse retina, two distinct functions of Rb have been described: regulation of retinal progenitor cell proliferation and rod photoreceptor development. Cells that would normally become rods fail to mature and remain as immature cells in the outer nuclear layer in the adult. By using Chx10-Cre;Rb(Lox/-) mice, we generated a chimeric retina with alternating apical-basal stripes of wild-type and Rb-deficient tissue. This provides a unique model with which to study synaptogenesis at the outer plexiform layer within regions that lack differentiated rods. In regions where rods failed to differentiate, the outer plexiform layer (OPL) was disrupted. Horizontal cells formed, and their somata were appropriately aligned, but their neurites did not project laterally. Instead many horizonal cell neurites extended apically, forming ectopic synapses with photoreceptors at all levels of the outer nuclear layer. These ectopic photoreceptor terminals contained synaptic ribbons, horizontal cell processes with synaptic vesicles, and a single mitochrondrion characteristic of rod spherules. Rb-deficient bipolar cells differentiated normally, extended dendrites to the OPL, and formed synapses that were indistinguishable from adjacent wild-type cells. In contrast to OPL-positioned synapses, ectopic synapses did not contain bipolar dendrites. This finding suggests that horizontal cells and photoreceptors can form stable synapses that are devoid of bipolar dendrites outside the normal boundaries of the OPL. Finally, analysis of P4, P7, P12, and P15 retinae suggests that the apical horizontal cell processes result from their failure to establish their normal lateral projections during development.     

维甲酸对神经干细胞的增殖和分化效应

目的 探讨不同浓度维甲酸对神经干细胞的增殖和分化的效应。方法 分离、培养胎龄14d的Wistar孕鼠的神经干细胞，通过与碱性成纤维细胞生长因子（bFGF对比观察维甲酸的促增殖效应；运用神经微丝200（NF-200）和神经胶质酸性纤维蛋白（GFAP）对维甲酸诱导分化的细胞进行组化染色，通过4’，6-二脒基-2-苯基吲哚（DAPI）染胞核，计数各种分化细胞的比例，并对分化的神经元进行胆碱能表型鉴定。结果 维甲酸的增殖效应明显弱于bFGF，但其具有明显的促神经元生成的分化效应，并表达递质，在分化第24天，500nmol／L的浓度使分化的神经元占分化的总细胞数的90．80％。结论 维甲酸具有显著的促神经干细胞分化成神经元的效应。     

Patterns of cell proliferation and cell death in the developing retina and optic tectum of the brown trout

We have analyzed the patterns of cell proliferation and cell death in the retina and optic tectum of the brown trout (Salmo trutta fario) throughout embryonic and postembryonic stages. Cell proliferation was detected by immunohistochemistry with an antibody against the proliferating cell nuclear antigen (PCNA), and apoptosis by means of the TUNEL method. Haematoxylin and DAPI staining were also used to demonstrate apoptotic cells. Photoreceptor cell differentiation was assessed by immunohistochemistry with antibodies against opsins. Throughout embryonic development, PCNA-immunoreactive (PCNA-ir) cells become progressively restricted to the peripheral growth zone of the retina, which appears to be the principal source of new retinal cells from late embryos to adults. However, some PCNA-ir cells are observed secondarily in the differentiated retina, first in the inner nuclear layer of 15-mm alevins and later in the outer nuclear layer of 16-mm alevins, after differentiation of the first rods in the central retina, as demonstrated with opsin immunocytochemistry. Our observations also support the view that the PCNA-ir cells observed secondarily in the INL of the central retina of alevins are photoreceptor precursors. The number and distribution of apoptotic cells in the retina and optic tectum of the trout change throughout development, allowing distinction of several waves of apoptosis. Cell death is detected in proliferating areas at early stages, then in postmitotic or differentiating areas, and later concurring temporal and spatially with the establishment of visual circuits, thus indicating a relationship between apoptosis and proliferation, differentiation and synaptogenesis.     

Synaptogenesis and synaptic protein localization in the postnatal development of rod bipolar cell dendrites in mouse retina

Retinal responses to photons originate in rod photoreceptors and are transmitted to the ganglion cell output of the retina through the primary rod bipolar pathway. At the first synapse of this pathway, input from multiple rods is pooled into individual rod bipolar cells. This architecture is called convergence. Convergence serves to improve sensitivity of rod vision when photons are sparse. Establishment of convergence depends on the development of a proper complement of dendritic tips and transduction proteins in rod bipolar cells. How the dendrites of rod bipolar cells develop and contact the appropriate number of rods is unknown. To answer this question we visualized individual rod bipolar cells in mouse retina during postnatal development and quantified the number of dendritic tips, as well as the expression of transduction proteins within dendrites. Our findings show that the number of dendritic tips in rod bipolar cells increases monotonically during development. The number of tips at P21, P30, and P82 exceeds the previously reported rod convergence ratios, and the majority of these tips are proximal to a presynaptic rod release site, suggesting more rods provide input to a rod bipolar cell. We also show that dendritic transduction cascade members mGluR6 and TRPM1 appear in tips with different timelines. These finding suggest that (a) rod bipolar cell dendrites elaborate without pruning during development, (b) the convergence ratio between rods and rod bipolar cells may be higher than previously reported, and (c) mGluR6 and TRPM1 are trafficked independently during development.     

The visual cells of the skate retina: structure, histochemistry, and disc-shedding properties

Earlier studies have shown that visual function in skate is subserved solely by the rod mechanism and that the retina of this elasmobranch contains only rod photoreceptors. Nevertheless, the skate retina is capable of responding to levels of illumination that extend well into the photopic range, and we have detected in histological sections (usually from younger animals) small, proximally displaced, conelike photoreceptors which possibly represent another class of visual cell. However, ultrastructural and histochemical studies showed that the membranous discs of the outer segments of these cells were isolated from the plasma membrane, and that their synaptic terminals appeared immature and unlike those usually associated with cone receptors. In addition, the pattern of incorporation of 3H-fucose, as revealed by radioautography, was similar for both the rods and the smaller visual cells; i.e., the label was concentrated along the basal discs of the outer segment. When we examined the disc-shedding behavior of the visual cells in skates entrained for 2 weeks or longer to a 12-hour light:12-hour dark cycle, enhanced phagocytic activity was seen only following light onset; there was no significant increase following light offset. On the available evidence, it seems reasonable to conclude that the small visual cells are rods that have recently differentiated, and are growing and being incorporated into the photoreceptor layer of the retina.     

Inhibition of alpha(1-6)-linked fucose decreases inner retinal cells and increases photoreceptors in chicken retinal reaggregates

We investigated the developmental role of alpha(1-6)-linked fucose, applying Aleuria aurantia lectin to a specific retinal regeneration system. Thereby, dissociated retinal cells of chicken embryos reaggregate, proliferate, and differentiate in vitro into histotypical spheres, so-called retinospheroids. Under the influence of A. aurantia lectin, processes of proliferation, differentiation and histogenesis of retinospheroids were disturbed. Extending these in vitro studies, we here show that A. aurantia lectin treatment decreases cells of the inner half retina and their processes into inner plexiform layer areas, as revealed by quantitative enzyme histochemistry for butyryl- and acetylcholinesterase, and immunohistochemistry using antibodies to acetylcholinesterase, Pax-6, calbindin-D, and F11. Concomitantly, the number of rod and red/green photoreceptors dramatically increases, using the antibodies rho4D2 and CERN901 (both specific for rods) and CERN906 (specific for red/green cones). These findings show that glycoproteins exhibiting fucose in alpha(1-6)-linkage are involved in processes determining retinal cell fate, strongly shifting the relative ratio of cells of the inner towards cells of the outer retina.     

Activin A promotes progenitor differentiation into photoreceptors in rodent retina

Activins are TGF beta-like proteins that were first discovered for their actions on the reproductive system, but have subsequently been shown to play a role in a variety of developmental processes. Previous studies have demonstrated that activins and their receptors are present in the developing retina, as well as other regions of the embryonic nervous system. We used both in vitro and in vivo approaches to test for functions of activin during retinal development. We found that activin A treatment of embryonic day 18 rat retinal cultures causes the progenitor cells in the cultures to exit the cell cycle and differentiate into rod photoreceptors. This effect is dose-dependent and the promotion of rod photoreceptor differentiation is specific, since the other primary retinal neurons generated in these cultures, the C1+ amacrine cells, are not affected by activin A treatment. Mice with homozygous deletion of the activin betaA gene show a specific decrease in the number of rod photoreceptors compared to wild-type or heterozygous littermates. These data demonstrate that activin A is an important regulator of photoreceptor differentiation in the developing retina.     

Age of differentiation determines rat retinal germinal cell phenotype: induction of differentiation by dissociation

We are interested in the mechanisms that control cell phenotype during the development of the CNS. Since different neuronal types arise at different times during neurogenesis in the retina, we predicted that the factors that determine cell type must be developmentally regulated as well. To test this hypothesis, we induced retinal germinal cells to differentiate at different ages by dissociating the retina into single cells and culturing them on a variety of substrates. Prior to dissociation, the S-phase germinal cells were labeled with 3H-thymidine so that their fate could be specifically followed. We found that our culture conditions promoted the differentiation of the majority of the germinal cells and that these cells differentiated into different neuronal types depending on the age of the animal from which the retina had been taken; embryonic day 14 germinal cells differentiated primarily into ganglion cells, and never produced rods, while germinal cells from postnatal day 1 retina differentiated into rods, but not ganglion cells. These results are consistent with the hypothesis that temporally regulated factors determine cell phenotype during the development of the retina.     

Melanin and the Regulation of Mammalian Photoreceptor Topography

Melanin, or products directly associated with it, regulates the maturation of the neural retina because in hypopigmented mammals the central retina fails to develop fully. To determine whether this deficit is reflected in the distribution of photoreceptors, their topography has been studied in the retinae of normally reared pigmented and albino ferrets and animals reared under reduced light conditions. In both strains, the general distribution of rods and cones was similar to that in the cat, cone density peaking in the central retina and rod density peaking in an annulus around the area centralis. The cone population was organized in the form of an orderly mosaic whose regularity was measured at a wide range of retinal eccentricities. No differences were found in cone numbers or their mosaic distribution between pigmented and albino strains, either at the area centralis or at more peripheral regions. In both cases order within the cone mosaic was independent of cell density or retinal eccentricity. In the albinos there was a significant deficit in the number of rods at all retinal locations when compared with rod numbers in the pigmented animals. There were no differences between normally reared and dark-reared animals in this respect either within or between the strains. Therefore, the albino gene must have a selective and specific effect on the development of this cell type in the outer retina. Ganglion cells and rods are both affected by the albino gene, while cones are not. Because cones and ganglion cells are generated during the same period and rods are generated later, the albino gene cannot be acting during a particular developmental time window. Because the cone mosaic was normal in the albinos, in spite of a large rod deficit, the factors that regulate the spacing of cones cannot depend in any significant manner upon the later generation and subsequent addition of rods to the outer retina.     

Immunolocalization of basic fibroblast growth factor and its receptor in adult goldfish retina.

The neural retina of teleost fish can regenerate following surgical or neurotoxic lesions. As a first attempt to uncover the factors important for the regenerative response, we used immunocytochemistry to demonstrate the presence of basic fibroblast growth factor (bFGF) and its receptor in the goldfish retina. The bFGF-immunoreactivity was present throughout the retina, but was most intense in photoreceptor cells, especially cones, and Müller glia. Immunoreactivity for the bFGF receptor was strongest in the axon terminals of photoreceptors, both rods and cones. This pattern of immunolocalization is especially interesting since the proliferating cells that are thought to be responsible for generating the neural regenerate are located among the photoreceptor axon terminals. These proliferating cells have been identified as rod precursors because in the intact retina they give rise only to rod photoreceptors. When the neural retina is damaged, however, rod precursors are thought to be the source of proliferating neuroepithelial cells responsible for generating the retinal regenerate. The role played by bFGF in normal neurogenesis, cell differentiation, and/or neuronal regeneration in the fish retina has yet to be determined.     

Rod bipolar cells in the macaque monkey retina: immunoreactivity and connectivity.

Rod bipolar cells in the macaque monkey retina were labeled by three antibodies: an antibody against the alpha- and beta-subspecies of protein kinase C (PKC), a polyclonal antiserum against the L7 protein from mouse cerebellum, and a monoclonal antibody against rabbit olfactory bulb (MAb 115A 10). The MAb 115A10 antibody also labeled some cone bipolar and some amacrine cells. The antibody against PKC was used to study the synaptic connectivity of rod bipolar cells. Reconstructions of 28 rod spherules showed that usually two and up to four rod bipolar processes invaginate each rod spherule. Six rod bipolar axons in the inner plexiform layer were reconstructed; they all showed the same pattern of connectivity. Synaptic output at rod bipolar dyads usually was onto two amacrine cell profiles: one that resembled the All amacrine cell and another that frequently made a reciprocal synapse. Rod bipolar cells did not contact ganglion cells. Synaptic input to rod bipolar cells came from reciprocal amacrine cells at dyads and other amacrine cells. In these respects, the rod pathway in the monkey is very similar to that described in cat and rabbit. The density of rod bipolar cells was determined and compared with the density of rods. There is a maximum of 15,000-20,000 rod bipolar cells/mm2 at 1-3 mm eccentricity, close to where rod density is maximum. Rod density is 10 times higher than rod bipolar cell density within 2 mm of the fovea, and 30 times higher at 15 mm eccentricity. This change in relative density is compensated by an increase in the number of rods contacted by individual rod bipolar cells (seen in Golgi-stained whole-mount retina) so that the number of rod bipolar terminal boutons in each rod photoreceptor remains relatively constant with changing eccentricity. We estimate that each rod bipolar cell is contacted by about 20 rods at 2-4 mm eccentricity and about 60 rods at 6-7 mm eccentricity.     

Interleukin-4 blocks proliferation of retinal progenitor cells and increases rod photoreceptor differentiation through distinct signaling pathways

Interleukin-4 (IL-4), an anti-inflammatory cytokine, has been related to the differentiation of the rodent retina in vitro, but constitutive presence of either IL-4 or of IL-4 receptor in the retina has not been reported. In this work we examined the expression of IL-4 and its specific receptor alpha subunit (IL-4Ralpha). IL-4Ralpha is expressed both in neural retina and non-neural ocular tissue, while IL-4 was found mainly in non-neural tissue. We characterized a novel trophic effect of IL-4 upon the retina. We showed that IL-4 can inhibit the proliferation of retinal cells (approximately 40%) through the cAMP-PKA pathway and associated with a reduction of cyclin D1 and increase of p27(kip1). IL-4 also promotes the differentiation of rod photoreceptors. Activation of tyrosine kinases, protein kinase C, and mitogen-activated kinases of the Erk family were required for IL-4-induced rod photoreceptor differentiation, independent of the release of other trophic factors in culture. Taken together, our results show, for the first time, that IL-4 directly modulates proliferation of retinal cells and rod photoreceptor differentiation, through distinct signaling pathways.     

Role of nitric oxide in photoreceptor survival in embryonic chick retinal cell culture

The presence of nitric oxide synthase (NOS) in chick retina during development has allowed us to study the role of nitric oxide (NO) during retinal differentiation in dissociated chick retinal cell culture from embryonic day 6. We have demonstrated the presence of nicotinamide adenine dinucleotide phosphate diaphorase staining in these cultures after 3 days in vitro (Div), with a maximal intensity after 8 Div, corresponding to embryonic day 14. Immunohistochemistry studies confirmed the presence of the two isoforms of NOS, NOS-I and -III, in dissociated retinal cell cultures at 8 Div. Addition of NG-monomethyl-L-arginine, a NOS inhibitor, to retinal cell cultures prevented NO production but did not modify the appearance and the survival of ganglion and amacrine cells. However, immunohistochemical analysis with distinct markers for photoreceptor cells (rods and cones) showed that inhibition of endogenous NOS in retinal cell cultures prevented the developmental decrease of rod number between 5 and 8 Div, thus supporting the hypothesis that NO may be involved in the cell death of rods during the development of the retina.     

Neuronal addition and retinal expansion during growth of the crucian carp eye

Using standard paraffin technique the addition of new cells in crucian carp retinas was examined. Between eye diameters 4.4 and 10.0 mm the number of ganglion cells increases from 103,000 to 205,000, INL cells from 1.5 to 3 million, cones from 250,000 to 900,000, and rods from 2 to 9 million. Concomitantly retinal area increases fivefold and the cell densities decrease by 37% for the cones, 57% for th e INL cells, and 58% for the ganglion cells, while the rod density remains stable. In relation to the rods the cell ratios at different retinal loci undergo marked changes during growth. The contributions to retinal growth by addition of new neurons and by expansion of the retina have been determined for the different retinal layers. The layer of rods grows exclusively by addition of new rod mosaic. In the cone layer 81% of growth is due to addition of new cone mosaic. In the inner nuclear layer (INL) 56% of growth is due to addition of new cells and in the ganglion cell layer 52% is due to cell addition. In each case retinal expansion accounts for the remainder of increase in retinal area. On morphological grounds six cone types can be found in the crucian carp retina. Their ratios are constant during retinal growth and at different retinal loci.     

Cognin distribution during differentiation of embryonic chick retinal cells in vitro

Differentiation of individual retina neurons is closely linked to development of retina function. This differentiation may be intrinsic to the cell or determined by the position of the cell within the developing tissue. Retina cognin, a cell-cell recognition protein, which may itself mediate position-dependent cell interactions in vivo exhibits a characteristic change in distribution during embryonic chick development. Cognin is progressively lost from the outer retina in a manner which appears position-dependent. We asked if this change in cognin distribution was actually position-dependent or intrinsic to the retina cells. Neural retina cells from 8-day-old chick embryos were cultured in vitro. Continued differentiation of the cultured cells was demonstrated by neurite outgrowth and characteristic increases in choline acetyltransferase and glutamic acid decarboxylase activity. In such cultures, the characteristic developmentally related disappearance of retina cognin occurred as in vivo. This indicated that this aspect of retina neuronal differentiation was independent of position within the tissue and likely intrinsic to individual cells after 8 days of embryonic development.     

Photoreceptor mosaic: number and distribution of rods and cones in the rhesus monkey retina

Video-enhanced differential interference contrast optics was used to determine the number and distribution of photoreceptors across the entire retinal surface of 9 eyes obtained from 7 adult rhesus monkeys. We found that the retina of this primate contains an average of 3,100,000 cones (+/- 130,000) and 61,000,000 rods (+/- 7,500,000). Variation among animals in the number of rods and cones cannot be accounted for by differences in sex, age, or retinal surface area, nor is there a correlation between the number of rods and cones (a retina with a high number of rods does not typically have a high number of cones). Cone density peaks at 141,000 cones/mm2 in the foveola and decreases about 100-fold toward the periphery. Rod density in a central annulus around the fovea is 130,000/mm2 and decreases 6-8-fold toward the periphery. In all 9 retinae, we found that an area 4-5 mm dorsal to the fovea had the highest rod density at 184,000 rods/mm2. The functional significance of this area, which we term the dorsal rod peak (DRP), may be related to high sensitivity vision under scotopic conditions. Outside of the DRP, rod density is symmetrical around the major axes of the retina, whereas cone density is elevated in nasal retina. Among animals, both rods and cones display a 2-fold individual difference in receptor density at any given eccentricity. Although rods and cones differ in absolute number, the location and magnitude of their peak densities, and their central to peripheral density gradients, the ratio of the density of rods to cones (15-30:1) is remarkably stable from 3 mm to 15 mm eccentricity. The relative consistency in the proportion of rods and cones in extrafoveal retina may be related to mechanisms of retinal development and functional interactions between scotopic and photopic systems.     

Survival and differentiation of adult rat-derived neural progenitor cells transplanted to the striatum of hemiparkinsonian rats

We investigated the survival, distribution and differentiation capabilities of adult rat hippocampus-derived progenitor cells (AHPs) by grafting them into either the intact or dopamine (DA)-denervated adult rat striatum (ST). Furthermore, we tested the effects of the in vivo administration of retinoic acid (RA) on the differentiation of the grafted cells. AHPs, prelabeled in vitro with bromodeoxyuridine (BrdU) and primed with RA, were transplanted bilaterally into the ST of hemiparkinsonian rats. Twenty animals were divided in four groups: three groups received i.p. injections of RA (1.5 mg/kg/day) for 1, 2 or 4 weeks and one group received vehicle injections for 4 weeks. Approximately 60% of the implanted BrdU-immunoreactive (BrdU+) cells were present in either intact or lesioned ST after 5 weeks of transplantation, with a striking widespread radial distribution from the implantation site. The cells became morphologically integrated with the surrounding host tissue, with no evidence of tumor formation. Approximately 18% of the BrdU+ cells were immunoreactive for the glial precursor marker NG2 and occasionally BrdU+ cells co-expressed the neuronal marker TuJ1. This differentiation pattern was similar in the intact and DA-denervated ST. Although further research is needed to find more adequate methods to drive the differentiation of these cells toward the desired phenotypes, the survival, differentiation potential and widespread distribution throughout the ST observed in this study suggest that AHPs may be useful in treatment of degenerative disorders affecting the nervous system.