Developmental study of the expression of B50/GAP-43 in rat retina

B50/GAP-43 has been implicated in neural plasticity, development, and regeneration. Several studies of axonally transported proteins in the optic nerve have shown that this protein is synthesized by developing and regenerating retinal ganglion cells in mammals, amphibians, and fish. However, previous studies using immunohistochemistry to localize B50/GAP-43 in retina have shown that this protein is found in the inner plexiform layer in adults. Since the inner plexiform layer contains the processes of amacrine cells, ganglion cells, and bipolar cells to determine which cells in the retina express B50/GAP-43, we have now used in situ hybridization to localize the mRNA that codes for this protein in the developing rat retina. We have found that B50/GAP-43 is expressed primarily by cells in the retinal ganglion cell layer as early as embryonic day 15, and until 3 weeks postnatal. Some cells in the inner nuclear layer, possibly a subclass of amacrine cells, also express B50/GAP-43 protein and mRNA; however, the other retinal neurons–bipolar cells, photoreceptors, and horizontal cells express little, if any, B50/GAP-43 at any stage in their development. Early in development, the protein appears in the somata and axons of ganglion cells, while later in development, B50/GAP-43 becomes concentrated in the inner plexiform layer, where it continues to be expressed in adult animals. These results are discussed in terms of previous proposals as to the functions of this molecule. © 1993 John Wiley & Sons, Inc.     

In vitro organotypic cultivation of adult newt and rat retinas

Adult rat and newt retinas were studied during long organotypic 3D cultivation. A high proliferation level was discovered in the region of growth by applying DNA synthesis markers and in vitro mitosis registration in newt retina. Aggregates were formed in the retina spheroid cavity because dedifferentiated cells migrated into this region. Small cell populations in nuclear layers also had dividing and migration capacity. Rosette formation has been shown in newt retina. It is a characteristic of fetal retinal development under pathological conditions. The antiGFAP antibody dye demonstrated an increase in the parent Müller cell population and generation of a small cell pool with short GFAP-extensions de novo. Recoverin expression studies detected its translocation from photoreceptor extensions to the cell bodies. Moreover, protein was presented in some cells inside the spheroid. It has been shown for the first time that cell proliferation occurred in the adult rat retinal spheroid developing in vitro; BrdU-positive cells and multiple mitoses were revealed in this fissue. However, the source of proliferation was not in the peripheral retina, and resident macrophages and glial cells located among neurons of the inner nuclear layer had the ability to divide. The antiGFAP antibody showed an increase in GFAP fibers in the rat retina as well as in the newt retina. Recoverin translocated into photoreceptor perikaryons and the outer plexiform layer in cultivated rat retina. Interestingly, some cells with probably de novo expression of recoverin were discovered in rat and newt inner retinas.     

Developmental Alteration of the Expression and Kinase Activity of Cyclin-Dependent Kinase 5 (Cdk5)/p35nck5a in the Rat Retina

Abstract: Neuronal Cdc2-like kinase has been purified from the bovine brain as a proline-directed serine/threonine kinase. This kinase is a heterodimer of Cdk5 and p35^nck5a and influences neuronal maturation or sprouting in the normal brain. In this study, we showed the expression of Cdk5/p35^nck5a kinase in the developing rat retina. The expression of Cdk5 and p35^nck5a increased between 1 week and 3 weeks after birth. These expression levels were most prominent from 2 weeks to 3 weeks after birth and decreased after 4 weeks. The developmental change of Cdk5/p35^nck5a kinase activity coincided with those of the expression of p35^nck5a and Cdk5. An immunohistochemical study showed that Cdk5 was expressed in the ganglion cells and in some cells in the inner nuclear layer at an early stage. With retinal development, Cdk5 was expressed in the inner plexiform layer also. In the adult, the expression of Cdk5 was restricted to the inner plexiform layer and to some cells in the inner nuclear layer. These changes of localization in the developing retina were very close to those of B-50/GAP-43. On the other hand, the expression of p35^nck5a was restricted to the soma of the neuron in the developing retina. This subcellular localization in the developing retina agreed with that in the developing rat brain. The expression levels of Cdk5 and p35^nck5a in retina of rats raised from fetus to 3 weeks after birth in darkness were 36% and 40% respectively, of the baseline for control rats. Moreover, the kinase activity in rats raised in darkness was lower than that in control rats. These data suggest that Cdk5/p35^nck5a may play a role in neuronal plasticity in the developing rat retina.     

Changes of lysosomal enzymes during hereditary degeneration and histogenesis of retina in mice

Summary In the normal histogenesis of mouse retina localized distribution of acid phosphatase positive granules has been seen around the photoreceptor cell nuclei along the outer limiting membrane. These granules disappear during the development of the rod elements. Temporarily increased activity is also seen along the nuclei of the inner layer adjacent to and in the course of the development of the outer and the inner plexiform layers. Within the inner nuclear layer, the cells at the outer and inner rows develop localized acid phosphatase positive granules which persist in the adult retina. Ganglion cells and the layer of nerve fibres show little change. In the pigment epithelium the enzyme gradually increases. In mice, homozygous for the retinal degeneration gene, degenerating photoreceptor cell nuclei, characterized by perinuclear acid phosphatase staining, can be detected before morphological signs of degeneration. Increased frequency of such nuclei and intensity of staining are recorded with the progress of degeneration. Enzyme activity in the photoreceptor cells, within the inner nuclear layer and in the degenerating photoreceptor cell nuclei is demonstrable using naphthol substrates but not -glycerophosphate. Positive reaction with -glycerophosphate is obtained in these sites in the presence of dimethyl sulphoxide. Existence of differential permeability among the retinal lysosomes is tentatively suggested.     

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.     

Postnatal changes of vascular endothelial growth factor (VEGF) expression in the retinae of normal and hypertensive rats

Vascular endothelial growth factor (VEGF) has been shown to have potent mitotic activity specific to vascular endothelial cells and has been related to vascular permeability, angiogenesis and cell proliferation in both normal and pathological situations. The present study aimed at elucidating the spatio-temporal changes in the postnatal expression pattern of VEGF in the retinae of both normal and hypertensive rats. In situ hybridization with a riboprobe showed that in the pre-hypertensive stage (2 weeks postnatal, prior to the increase of the blood pressure of the hypertensive rat), VEGF expressed strongly in the retinal pigment epithelium (RPE) and inner nuclear layer (INL) but weakly in the ganglion cell layer and nerve fiber layer in both the normal and hypertensive rats. During the early hypertensive stage (6 weeks postnatal, initial increase of the blood pressure of the hypertensive rat), similar expression pattern was maintained but the INL of the hypertensive rat was found to have more positive cells in clusters than that of the normal rat. When a sustained high blood pressure was developed (12 weeks postnatal, sustained hypertensive stage) in the hypertensive rat, the VEGF expression was much reduced in all layers of the retina although weak expression was still observed in the RPE of the normal rat and RPE and INL of the hypertensive rat. Western blot analysis however showed that VEGF protein expression in the retina was much stronger in the hypertensive rat than in the normal rat at 2 and 6 weeks postnatal. At 12 weeks, the VEGF protein returned to a level comparable to that found in the normal rat. It is speculated that the change of the VEGF protein expression pattern during the early phase of the development of hypertension may be related to the subsequent changes in the retinal vasculature of the hypertensive rat.     

Midkine-a Protein Localization in the Developing and Adult Retina of the Zebrafish and Its Function During Photoreceptor Regeneration

Midkine is a heparin binding growth factor with important functions in neuronal development and survival, but little is known about its function in the retina. Previous studies show that in the developing zebrafish, Midkine-a (Mdka) regulates cell cycle kinetics in retinal progenitors, and following injury to the adult zebrafish retina, mdka is strongly upregulated in Müller glia and the injury-induced photoreceptor progenitors. Here we provide the first data describing Mdka protein localization during different stages of retinal development and during the regeneration of photoreceptors in adults. We also experimentally test the role of Mdka during photoreceptor regeneration. The immuno-localization of Mdka reflects the complex spatiotemporal pattern of gene expression and also reveals the apparent secretion and extracellular trafficking of this protein. During embryonic retinal development the Mdka antibodies label all mitotically active cells, but at the onset of neuronal differentiation, immunostaining is also localized to the nascent inner plexiform layer. Starting at five days post fertilization through the juvenile stage, Mdka immunostaining labels the cytoplasm of horizontal cells and the overlying somata of rod photoreceptors. Double immunolabeling shows that in adult horizontal cells, Mdka co-localizes with markers of the Golgi complex. Together, these data are interpreted to show that Mdka is synthesized in horizontal cells and secreted into the outer nuclear layer. In adults, Mdka is also present in the end feet of Müller glia. Similar to mdka gene expression, Mdka in horizontal cells is regulated by circadian rhythms. After the light-induced death of photoreceptors, Mdka immuonolabeling is localized to Müller glia, the intrinsic stem cells of the zebrafish retina, and proliferating photoreceptor progenitors. Knockdown of Mdka during photoreceptor regeneration results in less proliferation and diminished regeneration of rod photoreceptors. These data suggest that during photoreceptor regeneration Mdka regulates aspects of injury-induced cell proliferation.     

Spatial and temporal expression patterns of GDNF family receptor alpha4 in the developing chicken retina

GDNF family receptor alpha (GFRalpha) receptors are involved in the regulation of different aspects of embryonic development such as proliferation, migration, differentiation and survival. To determine the possible role of GFRalpha4 in retinal development, we analysed its expression in the developing chicken retina. We found that GFRalpha4 is temporally co-expressed with c-ret. Both, the temporal and spatial expression of GFRalpha4 is developmentally regulated during retinogenesis and is first detected in cells of the ganglion cell layer at E6. As development of the retina proceeds, the expression of GFRalpha4 extends to cells of the inner half of the inner nuclear layer and to cells of the outermost cell row of the inner nuclear layer. Later on, GFRalpha4 expression is also found in additional cells of the outer half of the inner nuclear layer and in a subpopulation of photoreceptors. A central-to-peripheral gradient of retinal differentiation is evident, as the onset of GFRalpha4 expression is first detectable in the central retina, while it is delayed by two days in its periphery.     

Localization of glutamate in the human retina during early prenatal development

In this study we have localized glutamate (GLU) in fetal (14–25 weeks gestation, Wg) human retinas by immunohistochemistry. At 14 Wg, GLU-immunoreactivity (IR) was localized only in the central part of retina, showing a prominently labelled nerve fiber layero A few ganglion cells and displaced amacrine cells were very weakly labelled. At 17 Wg, GLU was localized conspicuously in many ganglion cells, displaced amacrine cells, some amacrine cells and the prospective photoreceptor cell bodies in the neuroepithelial layero With progressive development at 20 and 25 Wg, the IR for GLU was found additionally in the Müller cell endfeet, some bipolar cells as well as in the horizontal cells that were aligned in a row along the outer border of the inner nuclear layer of the central retinao The photoreceptor cell bodies in the outer nuclear layer were also prominently immunopositive for GLU. The developmental distribution of GLU in the human retina tends to indicate that it plays an important role in the differentiation and maturation of retinal neurons.     

Effects of prenatal cocaine exposure in the photoreceptor cells of the rat retina

Despite the increasing evidence of eye abnormalities, the effects of prenatal exposure to cocaine on the visual system are still poorly understood. This study was aimed at analyzing the qualitative and quantitative organization of the retinal photoreceptor cells (PR) and outer nuclear layer (ONL) after prenatal exposure to cocaine in the rat. Pregnant Wistar rats were given sc injections of cocaine hydrochloride (60 mg/kg body wt/d) or saline or were not manipulated; analyses were performed in the 14- and 30-d-old male offspring. Radial semithin and ultrathin sections of epon-embedded flat mounts of the retina showed displaced PR-like cells in the inner nuclear layer (INL), picnotic PR nuclei in INL, and ONL, and retinal PR rosettes and outer-segment debris in the subretinal space. The quantitative study showed an increased density of PR-like nuclei in the INL in PND14 cocaine-treated rats that were within normal values at PND30; no changes were detected in the PR mean nuclear diameter and in the packing density of PR nuclei in the ONL. These data constitute the first morphological demonstration of photoreceptor damage after prenatal cocaine-exposure probably owing to a direct action of the drug and/or to the cocaine-induced ischemia/hypoxia.     

四指马鲅视网膜早期发育的组织学研究

本文采用石蜡连续切片技术、H.E染色和显微测量法,对四指马鲅(Eleutheronema tetradactylum)早期发育过程中视网膜的结构、分化和形成过程以及视觉特性进行了研究。结果显示,受精后8 h54 min,视杯已经形成。初孵仔鱼视网膜没有分化。2日龄仔鱼可以清晰的辨认出色素上皮层、外核层、内核层和神经节细胞层。3日龄仔鱼内核层已经分化出水平细胞、双极细胞和无长突细胞。4日龄仔鱼视网膜10层结构完整。9日龄至14日龄,外核层胞核数目与神经节细胞数目的比值增大,视网膜会聚程度升高,是该鱼视觉特性发生变化的过渡期,这与其从浮游到浅海中下层和泥沙质海底活动的生态迁移相适应。在生长发育的早期阶段,其视网膜内核层水平细胞仅有1到2层,属于感光系统不甚发达的类型。该鱼在仔鱼浮游生活阶段,视敏度较高,视觉对其行为和摄食活动具有重要作用,适应生活于光照较充足的环境中,转入浅海中下层和泥沙质海底后,光敏度和视敏度均较差,视觉在其行为和摄食活动中不具有主要作用。     

Molecular cloning and characterization of a new neuron-specific homologue of rat polypyrimidine tract binding protein

Cancer-associated retinopathy (CAR) is a rare form of retinal degeneration and also one of the paraneoplastic neurologic disorders. Sera of CAR patients usually contain high titers of antibodies against retinal proteins, and CAR is believed to be an autoimmune disease. Using serum from a CAR patient as a molecular probe, a homologue of the polypyrimidine tract-binding protein (PTB) was isolated from a cDNA library of rat neonatal retina. This homologue, named PTB-like protein (PTBLP), encodes a 532 amino acid residue protein and has 73.5 and 68.8% homology with PTB and with a regulator of differentiation 1, respectively. Functional domains in the PTB, such as nuclear localization signals and four RNA recognition motifs (RRMs), were highly conserved. The expression of PTBLP mRNA was observed in the retina and brain but not in liver, kidney, spleen, or lung. The expression of PTBLP protein in rat retina was distributed in most of the cells in the ganglion cell layer and some cells in the inner nuclear layer. The PTBLP protein was localized in the nuclei of these cells. These results suggest that PTBLP is a new member of the PTB gene family and a neuron-specific homologue.     

Localization of basic fibroblast growth factor to the developing capillaries of the bovine retina

The basic fibroblast growth factor (FGF) is a potent mitogen that has vascular endothelium as one of its principle target cells. Recent work has provided both the complete amino acid sequence of basic FGF and the nucleotide sequence of the genes for both human and bovine basic FGF. Although capillary endothelial cells have been shown to produce basic FGF in vitro and to deposit basic FGF in their extracellular matrix in vitro as well, no direct evidence yet exists for the distribution of basic FGF in vivo. Antipeptide antibodies were prepared against a 15-amino-acid sequence from the amino terminus of basic FGF in order to avoid cross-reactivity with acidic FGF, a protein with 55% overall homology to basic FGF. After affinity purification, these antisera were used to localize the basic fibroblast growth factor in the fetal and adult bovine retina. Immunoreactive material was found in capillaries of the inner nuclear layer, a capillary network undergoing development during the third trimester in the fetal bovine eye. Although the resolution of the technique does not permit a unique assignment of cellular localization, the presence of stain immediately adjacent to the lumen of capillaries suggests that capillary endothelial cells may produce the basic fibroblast growth factor in vivo during vascular development.     

Fgf signaling is required for photoreceptor maintenance in the adult zebrafish retina

Fibroblast growth factors (Fgf) are secreted signaling molecules that have mitogenic, patterning, neurotrophic and angiogenic properties. Their importance during embryonic development in patterning and morphogenesis of the vertebrate eye is well known, but less is known about the role of Fgfs in the adult vertebrate retina. To address Fgf function in adult retina, we determined the spatial distribution of components of the Fgf signaling pathway in the adult zebrafish retina. We detected differential expression of Fgf receptors, ligands and downstream Fgf targets within specific retinal layers. Furthermore, we blocked Fgf signaling in the retina, by expressing a dominant negative variant of Fgf receptor 1 conditionally in transgenic animals. After blocking Fgf signaling we observe a fast and progressive photoreceptor degeneration and disorganization of retinal tissue, coupled with cell death in the outer nuclear layer. Following the degeneration of photoreceptors, a profound regeneration response is triggered that starts with proliferation in the inner nuclear layer. Ultimately, rod and cone photoreceptors are regenerated completely. Our study reveals the requirement of Fgf signaling to maintain photoreceptors and for proliferation during regeneration in the adult zebrafish retina.