The developing avian retina expresses agrin isoforms during synaptogenesis

The localization, isoform pattern, and mRNA distribution of the synapse-organizing molecule agrin was investigated in the developing avian retina. Injection of anti-agrin Fab fragments into the vitreous humor of chick eyes of embryonic days 3 to 20, a procedure that labels only extracellular agrin, reveals staining in the inner and outer plexiform layers before, during, and after the period of synapse formation. The labeling in these layers changes from a diffuse to a punctate pattern at the time when synapses form. At all stages investigated, the inner limiting membrane (a basal lamina that separates vitreous from neural retina) is intensely labeled, as are the axonal processes of retinal ganglion cells in the optic fiber layer and in the optic nerve, although the staining intensity declines after embryonic day 10 in both retina and optic nerve. In culture, axons of retinal ganglion cells also express agrin-like immunoreactivity on their surfaces. Polymerase chain reaction analysis reveals that several different agrin isoforms are expressed in the developing neural retina. In situ hybridization studies show that agrin isoforms are expressed in the ganglion cell and inner nuclear layers, correlating well with the staining for agrin protein in the optic fiber and plexiform layers. The expression of mRNA coding for several agrin isoforms and the presence of extracellular agrin in the synapse-containing layers during the period of synapse formation is consistent with the idea that agrin isoforms might play a role during synapse formation in the central nervous system. © 1996 John Wiley & Sons, Inc.     

Target cells of vitamin D in the vertebrate retina

Using PAP technique, cellular localization of vitamin D-dependent calcium-binding protein (D-CaBP) was investigated in vertebrate retina with monospecific antisera against chick duodenal D-CaBP. In the chick retina, the receptor cells were positive. In the inner nuclear layer, horizontal cells and some bipolar cells were also positive. Some amacrine cells as well as different levels of the inner plexiform layer were also positive for D-CaBP. A few interspersed ganglion cells were positive but their axons forming the optic tract were negative. Müller's cells were negative. In 1-day-old chicks and 4-week-old rachitic chicks there was paucity and absence, respectively, of D-CaBP staining in horizontal cells. In the mouse, rat, and rabbit the receptors had only trace amounts of reaction product in their outer segment and pedicle. Horizontal cells were densely positive throughout their cellular body and processes. Some amacrine cells in the inner nuclear layer were positive. In the mouse and rat three horizontal levels of the outer plexiform layer were very prominent because of their dense staining for D-CaBP. Many ganglion cells were also positive along with their axons forming the optic nerve. In the rabbit, no positive layers were seen in the inner plexiform layer, and ganglion cells with their fibers were negative. In the frog retina there were smaller amounts of D-CaBP in the receptor cells and horizontal cells than that of the chick retina. Also, the fibers of the ganglionic cells were positive for D-CaBP. In all species studied, some amacrine cells were stained for D-CaBP. Because of its possible roles in membrane calcium transport and intracellular Ca++ regulation, it has perhaps similar functions in these positive cells. The synthesis of D-CaBP is dependent upon vitamin D. These positive cells are thus target cells of vitamin D.     

Spatiotemporal distribution of 1P1 antigen expression in the plexiform layers of developing chick retina

Changes in the distribution of 1P1-antigen in the developing chick retina have been examined by indirect immunofiuorescence staining technique using the novel monoclonal antibody (MAb) 1P1. Expression of the 1P1 antigen was found to be regulated in radial as well as in tangential dimension of the retina, being preferentially or exclusively located in the inner and outer plexiform layers of the neural retina depending on the stages of development. With the onset of the formation of the inner plexiform layer 1P1 antigen becomes expressed in the retina. With progressing differentiation of the inner plexiform layer 1P1 immunofiuorescence revealed 2 subbands at E9 and 6 subbands at E18. At postnatal stages (after P3) immunoreactivity was reduced in an inside-outside sequence leading to the complete absence of the 1P1 antigen in adulthood. 1P1 antigen expression in the outer plexiform layer was also subject to developmental regulation. The spatio-temporal pattern of 1P1 antigen expression was correlated with the time course of histological differentiation of chick retina, namely the synapse rich plexiform layers. Whether the 1P1 antigen was functionally involved in dendrite extension and synapse formation was discussed.     

Transient expression of adheron molecules during chick retinal development.

Neuritogenesis and synapse formation are transient phenomena mediated in part by filopodial attachments (Tsui, Lankford, and Klein, Proc. Natl. Acad. Sci. 82:8256-8260 1985). The attachments can be labeled by antisera against adherons, adhesive microparticles isolated from cell culture media (Tsui, Schubert, and Klein, J. Cell Biol. 106:2095-2108 1988). Here, two monoclonal antibodies raised against adherons have been found to recognize transiently expressed membrane antigens of developing avian retina. Early in development, monoclonal antibody (mAb) AD1 stained antigens that spanned the entire tissue. With time, immunoreactivity became restricted to optic fiber, ganglion cell, and inner plexiform layers. Immunoblots of embryonic day (E) 13 retina showed a broad band at 66-72 kD for particulate fractions and a fine band at 70 kD for soluble fractions. The particulate forms disappeared as retinas matured, but the soluble form did not. mAb AD2 initially labeled retina antigens of optic fiber, ganglion cell, and inner plexiform layers (IPL). Labeling in the plexiform layer showed discrete lamina. Immunoreactivity first appeared at E9, peaked at E15, and then disappeared shortly after hatching. In isolated cells, AD2 labeled small cell surface aggregates. Cytoarchitectural studies, using whole-mount transmission electron microscopy, showed AD2 antigen in cell surface microfilaments, including some that joined filopodia together. The adheron antigens recognized by mAbs AD1 and AD2 thus were (1) topographically restricted; (2) associated with cell surfaces; and (3) developmentally down-regulated. This pattern suggests a role in developmentally transient cell surface phenomena, such as neurite extension or junction biogenesis.     

Transient expression of adheron molecules during chick retinal development

Neuritogenesis and synapse formation are transient phenomena mediated in part by filopodial attachments (Tsui, Lankford, and Klein, Proc. Natl. Acad. Sci. 82:8256–8260 1985). These attachments can be labeled by antisera against adherons, adhesive microparticles isolated from cell culture media (Tsui, Schubert, and Klein, J. Cell Biol. 106:2095–2108 1988). Here, two monoclonal antibodies raised against adherons have been found to recognize transiently expressed membrane antigens of developing avian retina. Early in development, monoclonal antibody (mAb) AD1 stained antigens that spanned the entire tissue. With time, immunoreactivity became restricted to optic fiber, ganglion cell, and inner plexiform layers. Immunoblots of embryonic day (E) 13 retina showed a broad band at 66–72 kD for particulate fractions and a fine band at 70 kD for suluble fractions. The particulate forms disappeared as retinas matured, but the soluble form did not. mAb AD2 initially labeled retina antigens of optic fiber, ganglion cell, and inner plexiform layers (IPL). Labeling in the plexiform layer showed discrete lamina. Immunoreactivity first appeared at E9, peaked at E15, and then disappeared shortly after hatching. In isolated cells, AD2 labeled small cell surface aggregates. Cytoarchitectural studies, using whole mount transmission electron microscopy, showed AD2 antigen in cell surface microfilaments, including some that joined filopodia together. The adheron antigens recognized by mAbs AD1 and AD2 thus were (1) topographically restricted; (2) associated with cell surfaces; and (3) developmentally down-regulated. This pattern suggests a role in developmentally transient cell surface phenomena, such as neurite extension or junction biogenesis. © 1992 John Wiley & Sons, Inc.     

Immunoreactivity of the septins SEPT4, SEPT5, and SEPT8 in the human eye.

We aimed to examine the distribution of SEPT4, SEPT5, and SEPT8 in the human eye. For each septin, five to six normal human eyes were examined by immunohistochemical staining of paraffin sections using polyclonal antibodies against SEPT4, SEPT5, and SEPT8 and an avidin biotin complex immunodetection system. SEPT4 immunoreactivity (IR) was detected primarily in the epithelium of cornea, lens, and nonpigmented ciliary epithelium; in the endothelium of cornea and vessels of iris and retina; and in the retinal nerve fiber layer, the outer plexiform layer, the outer segments of the photoreceptor cells, the inner limiting membrane of the optic nerve head, and optic nerve axons. SEPT5-IR was present in corneal endothelial cells, iris tissue, nonpigmented ciliary epithelium, and epithelial cells of the lens. SEPT8-IR almost paralleled that of SEPT4, except for a lower SEPT8-IR of the outer photoreceptor segments and a positive staining of the meningothelial cell nests in the subarachnoidal space of the bulbar segment of the orbital optic nerve. In conclusion, SEPT4, SEPT5, and SEPT8 are expressed in various ocular tissues, each revealing a distinct expression pattern. Both physiological and potential pathophysiological role of septins in the human eye deserve further investigation.     

The structure and development of the rat retina: an immunofluorescence microscopical study using antibodies specific for intermediate filament proteins

Rat retina structure was studied between embryonic day 14 and adult with antibodies specific for vimentin, glial fibrillary acidic protein (GFA) and the proteins of the neurofilament triplet. Vimentin could be detected in radial processes throughout the retina at all stages studied. These processes are believed to correspond, in the developing retina, to ventriculocytes, and in the mature retina to Müller cells. They could not normally be stained with any of the other intermediate filament antibodies employed here. We did find, however, that some older albino rats possessed GFA staining in addition to vimentin in these processes. Since we never saw such staining in the retinae of mature non-albino rats, and the retinae of older albino rats often showed signs of degeneration, we concluded that such GFA expression was most likely pathological. Neurofilament protein-positive processes were first detectable at embryonic day 15 1/2 in the inner regions of the retina, and corresponded to the axons of retinal ganglion cells. Such processes were equivalently displayed with antibodies to 68 K and 145 K protein, but were negative with 200 K protein. Some 68 K and 145 K positive fibers could also be decorated with vimentin antibody at this stage, though at later stages this was not the case. At later development stages more 68 K and 145 K neurofilament positive processes appeared, and after the first post-natal week progressively more of such processes became in addition 200 K positive, so that almost all neurofilament positive fibers in the adult stained for all three proteins. Such fibers, in the mature retina corresponded to 68 K and 145 K positive optic nerve fibers, and the processes of neurones in the inner plexiform layer. All fibers in the mature optic nerve fiber layer, but not all of those in the inner plexiform layer were stainable with 200 K antibodies. At 4 days post-natal we were able to detect 68 K and 145 K protein positive profiles in the outer regions of the developing retina, the prospective outer plexiform layer. Such profiles were always in addition vimentin positive, but negative for 200 K protein. During further development such profiles became ordered into a well defined layer and from about post-natal day 13 all of them began to acquire 200 K protein. They could be identified as the processes of horizontal cells. They continued to express vimentin in addition to the three triplet proteins in the adult, a so far unprecedented situation. We were able to detect neurofilament staining in the mature retina only in the above described regions, the inner and outer nuclear layer and the photoreceptor processes being completely free of staining. GFA was first detected in short processes adjacent to the inner limiting membrane which penetrated the optic nerve fiber layer. Such profiles were first detectable in the eye of the newborn animal, and were invariably identically stainable with vimentin at this age. These profiles could be stained with both vimentin and GFA at all later stages examined, although GFA staining became very much stronger than vimentin staining in some profiles in the adult. The results presented here are discussed in terms of development of the different retinal cell types.     

Ageing transition of superoxide dismutase in rat retina

To examine the relationship between retinal ageing and superoxide dismutase, the distribution and expression of the dismutase was studied in the retina of 2-year-old Sprague--Dawley albino rats with immunohistochemistry and immunochemical quantitative analysis. Eight-week-old Sprague--Dawley albino rats were used as controls. In 2-year-old rats, manganese superoxide dismutase (Mn-SOD) immunoreactivities in the photoreceptor inner segments, the outer nuclear layer and the inner plexiform layer were stronger than those in 8-week-old rats. Copper--zinc superoxide dismutase (CuZn-SOD) immunoreactivities in the outer nuclear layer and inner plexiform layer of 2-year-old rats were stronger than those in 8-week-old rats. Faint CuZn-SOD immunoreactivity became visible in the photoreceptor inner segments of 2-year-old rats, whereas no CuZn-SOD immunoreactivity was observed in 8-week-old rats. Our immunochemical quantitative analysis also showed an increase in the immunoreactivities of superoxide dismutases in the sensory retina with age. The transition of the dismutases may have some relationship with retinal ageing. © 1998 Chapman & Hall     

胚后64小时龄和35天龄大熊猫视网膜的显微结构

为了解大熊猫眼睛的胚后发育状况,对６４小时龄和３５天龄大熊猫视网膜的组织结构进行了观察,发现胚后６４小时龄大熊猫视网膜的分化程度很低,色素层已形成,但视泡腔明显;神经层由外面数层长梭形,内面数层圆形细胞核及无核的纤维层构成。     

Light- and electron-microscopic study of substance P-immunoreactive neurons in the guinea pig retina

Substance P (SP) immunoreactivity in the guinea pig retina was studied by light and electron microscopy. The morphology and distribution of SP-immunoreactive neurons was defined by light microscopy. The SP-immunoreactive neurons formed one population of amacrine cells whose cell bodies were located in the proximal row of the inner nuclear layer. A single dendrite emerged from each soma and descended through the inner plexiform layer toward the ganglion cell layer. SP-immunoreactive processes ramified mainly in strata 4 and 5 of the inner plexiform layer. SP-immunoreactive amacrine cells were present at a higher density in the central region around the optic nerve head and at a lower density in the peripheral region of the retina. The synaptic connectivity of SP-immunoreactive amacrine cells was identified by electron microscopy. SP-labeled amacrine cell processes received synaptic inputs from other amacrine cell processes in all strata of the inner plexiform layer and from bipolar cell axon terminals in sublamina b of the same layer. The most frequent postsynaptic targets of SP-immunoreactive amacrine cells were the somata of ganglion cells and their dendrites in sublamina b of the inner plexiform layer. Amacrine cell processes were also postsynaptic to SP-immunoreactive neurons in this sublamina. No synaptic outputs onto the bipolar cells were observed.     

Regulated binding of the fibrinogen-like domains of tenascin-R and tenascin-C to the neural EGF family member CALEB

The neural transmembrane protein CALEB was discovered in a screen for novel molecules implicated in neuronal differentiation processes and was found to bind to two proteins of the extracellular matrix, tenascin-C and tenascin-R. The expression of different isoforms of CALEB in axon- and synapse-rich areas in the nervous system is regulated during development. Here we show that an unusual acidic peptide segment of CALEB is sufficient to mediate the binding of CALEB to the fibrinogen-like globes of both tenascin family members as well as to native tenascin-C. We identify a small sequence element within the acidic peptide segment of CALEB as important for this binding. Interestingly, the interactions of CALEB and tenascin-C and -R seem to be regulated during development. We demonstrate that only CALEB-80, the expression of which is up-regulated in the chicken retina during synaptogenesis, but not CALEB-140, expressed later on in development, can bind to the fibrinogen-like domains of tenascin-R or tenascin-C and to native tenascin-C. While both CALEB-80 and CALEB-140 are expressed in the plexiform layers and the optic fiber layer of embryonic chicken retina, CALEB-140 labeling is more intense in the optic fiber layer in comparison to the inner plexiform layer.     

Expression of myelin genes in the developing chick retina

In submammalian animals including chicks, the retina contains oligodendrocytes (OLs), and axons in the optic fiber layer are wrapped with compact myelin within the retina; however, the expression of myelin genes in the chick retina has not been demonstrated yet. In the present study, we examined the expression of three myelin genes (proteolipid protein, PLP; myelin basic protein, MBP; cyclic nucleotide phosphodiesterase, CNP) and PLP in the developing chick retina, in comparison to the localization of Mueller cells. In situ hybridization demonstrated that all three myelin genes began to be expressed at E14 in the chick embryo retina. They are mostly restricted to the ganglion cell layer and the optic fiber layer, with a few exceptions in the inner nuclear layer where Mueller cells reside; however, PLP mRNA+ cells do not express glutamine synthetase, or vice versa. The present results elucidate that myelin genes are expressed only by OLs that are mostly localized in the innermost layer of the developing chick retina.     

Immunohistochemical localization of metallothionein in the eye of rats

In order to elucidate possible physiological roles of metallothionein (MT), we have studied immunohistological localization of MT in the eye of the rat, using an avidin-biotin peroxidase complex method. As a result, strong MT immunostaining was observed in the epithelium of the lens and cornea. In the retina, considerably strong MT immunostaining was observed in the pigment cell layer while the nerve fiber layer and inner plexiform layer showed weak MT staining. Glial cells in the optic nerve were found to have marked MT staining. The present result is consistent with the hypothesis that MT may be involved not only in activation of zinc enzymes and cell proliferation through supply of zinc ions, but also in a protective mechanism in the blood-retina barrier.     

Immunohistochemical localization of metallothionein in the eye of rats

Summary In order to elucidate possible physiological roles of metallothionein (MT), we have studied immunohistological localization of MT in the eye of the rat, using an avidin-biotin peroxidase complex method. As a result, strong MT immunostaining was observed in the epithelium of the lens and cornea. In the retina, considerably strong MT immunostaining was observed in the pigment cell layer while the nerve fiber layer and inner plexiform layer showed weak MT staining. Glial cells in the optic nerve were found to have marked MT staining. The present result is consistent with the hypothesis that MT may be involved not only in activation of zinc enzymes and cell proliferation through supply of zinc ions, but also in a protective mechanism in the blood-retina barrier.     

Neuropeptide Y (NPY)-like immunoreactive amacrine cells in retinas of frog and goldfish

Summary The distribution of neuropeptide Y (NPY)-like immunoreactivity in rat, rabbit, chick, frog and goldfish retinas was investigated by immunohistochemistry. Positive results were observed only in the frog and goldfish retinas. NPY immunoreactivity was associated with a small population of amacrine cell bodies in the inner nuclear layer and cell processes in the inner plexiform layer of both retinas. In the frog retina, three distinct layers containing immunoreactivity were observed in the inner plexiform layer. In contrast, the immunoreactivity in the same area of the goldfish retina was more or less separated into two layers. Convincing evidence could not be found for the co-existence of NPY-like material with other putative transmitter-like substances in the two retinas.Radioimmunoassay revealed the presence of small amounts of NPY-like immunoreactivity in the rabbit retina; the goldfish and frog retinas contained significantly more immunoreactive material. High performance liquid chromatography of the immunoreactive material in frog and goldfish retinas showed each retina containing different molecular forms of NPY-like proteins, neither of which resembled porcine NPY or PYY.The endogenous NPY-like material of the frog retina can be released by potassium depolarisation in a calciumdependent way. In view of all these data an NPY-like protein must now be considered a potential retinal transmitter.     

Nitric oxide synthase immunoreactivity in the developing and adult human retina

Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide (NO) from L-arginine. In this study, the cellular localization of neuronal NOS (nNOS) activity in the human retina since fetal development was examined by immunohistochemistry. No detectable staining in the fetal retina was present at 14 weeks of gestation (wg), the earliest age group examined. A centro-peripheral gradient of development of nNOS immunoreactivity was evident at 16–17 wg, with the midperipheral retina showing nNOS immunoreactivity in most of the cell types and the inner plexiform layer while the peripheral part demonstrated moderate immunoreactivity only in the ganglion cell layer and photoreceptor precursors. A transient increase in nNOS immunoreactivity in the ganglion cells and Müller cell endfeet between 18–19 and 24–25 wg was observed at the time when programmed cell death in the ganglion cell layer, loss of optic nerve fibres as well as increase in glutamate immunoreactivity and parvalbumin (a calcium binding protein) immunoreactivity in the ganglion cells was reported. These observations indicate that programmed cell death of ganglion cells in the retina may be linked to glutamate toxicity and NO activity, as also suggested by others in the retina and cerebral cortex. The presence of nNOS immunoreactivity in the photoreceptors from 16–17 weeks of fetal life to adulthood indicates other functions, besides their involvement in photoreceptor function of transduction and information processing.     

胭脂鱼眼早期形态发生研究

采用组织学方法观察了胭脂鱼(Myxocyprinus asiaticus) 眼的发生过程, 结果显示: 胭脂鱼眼的发育经历了眼原基形成期、眼囊形成期、视杯形成期、晶体板形成期、晶体囊形成期、角膜原基形成期、角膜上皮形成期、视网膜细胞增殖期、晶状体成熟期、眼色素形成期以及眼成型期等11个时期。视网膜发育最早, 起始于眼原基的形成, 直至眼成型期分化完成, 形成了厚度不一的8层细胞, 由内向外依次为神经纤维层、神经细胞层、内网层、内核层、外网层、外核层、视杆视锥层和色素上皮层, 且发育历时最长, 约264h。晶状体的发育在视网膜之后, 始于晶体板的形成, 于出膜前期成熟, 发育历时最短, 约74h。角膜发育最晚, 始于角膜原基的形成, 出膜1 d分化为透明的成熟角膜, 发育历时约96h。出膜4 d仔鱼眼色素沉积明显, 视网膜各层分化明显, 晶状体内部完全纤维化, 眼的形态结构基本发育完全。     

Effects of alkaline phosphatase inhibitors on chick neural retinal cell differentiation in vitro: ultracytochemical studies

In the matured chick retina, alkaline phosphatase (ALPase) activity is specifically localized in the outer plexiform layer and in horizontal and Müller cells. In the developing chick retina, ALPase activity is first recognized in growing neurites from horizontal cells during the 13th day of incubation, when synaptogenesis begins in the outer plexiform layer. Intraocular administration of ALPase inhibitors to developing chick embryos resulted in developmental disturbances in differentiation of the outer plexiform layer and also of photoreceptor cells. We have now extended these studies to an in vitro system. ALPase activity was studied by ultracytochemistry in cultured retinal cells from chick embryos, and the effects of specific ALPase inhibitor on retinal development were also analyzed. Two cell types showed intense ALPase activity: 1) flat glial cell, which formed a multi-layered epithelial sheet and 2) neuronal cell found within cell aggregates. Some cellular processes forming a neuropil-like structure within these aggregates also showed ALPase activity. When the ALPase inhibitor bromotetramisole was present in the culture medium, there was delay in aggregate formation and the development of neuritic processes was also affected. Moreover, this treatment also caused a considerable reduction in the number of photoreceptor cells present in the culture. The present results indicate that ALPase activity plays a significant role in retinal cell differentiation.     

The presence of dopamine-β-hydroxylase-like enzyme in the vertebrate retina

An analysis of the dopamine-β-hydroxylase activity in various ocular tissues revealed low enzymatic activity in all the retinas analysed. Bovine and monkey retinas had the highest enzyme activity. The enzyme is also present in bovine optic nerve and pigment epithelium/choroid complex, and low levels are present in the vitreous. Immunohistochemical analysis of various retinas with an antiserum raised to bovine adrenal dopamine-β-hydroxylase-showed positive staining only in monkey and bovine retinas. In both instances dopamine-β-hydroxylase immunoreactivity was associated with cells in the ganglion cell layer. These cells are probably ganglion cells since some positive staining was also observed in the bovine optic nerve. Autoradiographical analysis of the uptake of [³H]noradrenaline showed that grains were associated with some structures in the ganglion cell layer. A single prominent layer of terminals at the junction of the inner nuclear and inner plexiform layer of the bovine retina was also apparent. This was considered to be due to [³H]noradrenaline accumulation by dopaminergic amacrine cells. The results suggest that a small number of noradrenergic and/or adrenergic ganglion cells may exist in certain retinas.     

Calretinin and calbindin in the retina of the developing chick

Summary Calretinin and calbindin-D28k are two calcium-binding proteins that are present in largely different sets of nerve cells in the central nervous system. Their appearance during development of the chick retina was studied by immunohistochemistry and Western blots. The patterns are mature one day before hatching. Each cell type acquires its characteristic calcium-binding protein several days after its differentiation has started, but in most cases before morphological maturation is complete. There is also an early phase of calbindin immunoreactivity in many immature amacrine cells, and of calretinin immunoreactivity in the presumptive photoreceptor layer, suggesting that these proteins may have distinct functions in differentiating cells.Abbreviations CR+ Immunoreactive for calretinin only - CB+ immunoreactive for calbindin only - CR+CB+ immunoreactive for both antisera - IPL inner plexiform layer - OPL outer plexiform layer