Developmental expression of dynamin in the chick retinotectal system.

Dynamin I, a GTPase involved in the endocytic cycle of synaptic vesicle membranes, is believed to support axonal outgrowth and/or synaptogenesis. To explore the temporal and spatial patterns of dynamin I distribution in neuronal morphogenesis, we compared the developmental expression of dynamin with the expression of presynaptic membrane proteins such as SV2, synaptotagmin, and syntaxin in the chick primary visual pathway. Western blots of retina and tectum revealed a steady increase of synaptotagmin and syntaxin from embryonic Day 7 (E7) to E11, whereas for the same time frame no detectable increase of dynamin was found. Later stages showed increasing amounts of all tested proteins until the first postnatal week. Immunofluorescence revealed that SV2, synaptotagmin, and syntaxin are present in retinal ganglion cell axons from E4 on. In later stages, the staining pattern in the retina and along the visual pathway paralleled the formation and maturation of axons. In contrast, dynamin is not detectable by immunofluorescence in the developing retina and optic tectum before synapse formation. Our data indicate that, in contrast to the early expression of synaptotagmin, SV2, and syntaxin during axonal growth, dynamin is upregulated after synapse formation, suggesting its function predominantly during and after synaptogenesis but not in axonogenesis.(J Histochem Cytochem 47:1297-1306, 1999)     

Calmodulin-dependent protein phosphatase: immunocytochemical localization in chick retina

Calmodulin-dependent protein phosphatase, previously called CaM-BP80 or calcineurin, is present in high concentrations in the central nervous system. The level of the phosphatase has been shown by radioimmunoassay to increase during development in the retinas of embryonic and hatching chicks (Tallant, E.A., and W.Y. Cheung, 1983, Biochemistry, 22:3630-3635). The aims of this study are to immunocytochemically localize the phosphatase in developing and mature retinas and to determine if the phosphatase is present in fractions of retinal synaptic membranes and synaptic junctions. Vibratome slices of fixed chick retina and Western blots of detergent-solubilized retinal fractions are both treated sequentially with rabbit primary antisera and goat anti-rabbit Fab fragments conjugated to peroxidase, and then reacted with hydrogen peroxide and diaminobenzidine. The tissue slices are further processed for electron microscopy. This paper demonstrates the presence of peroxidase reaction product in the retina just before synapse formation. In the outer plexiform layer the product is confined to photoreceptor synaptic terminals, whereas in the inner plexiform layer it is present in synaptic terminals of bipolar cells and in dendrites of ganglion cells. In this latter site the product is present postsynaptically at bipolar and amacrine synapses. The phosphatase is detected in Western blots of both synaptic plasma membrane and synaptic junction fractions.     

Collagen-tailed and globular forms of acetylcholinesterase in the developing chick visual system

We have carried out a comparative study of the developmental profiles of the enzyme acetylcholinesterase, and of its collagen-tailed and globular structural forms, solubilized in the presence of 1 M NaCl, 1% (w/v) sodium cholate and 2 mM EDTA, in the chick retina and optic lobes. The overall acetylcholinesterase activities, both per mg protein and per embryo or chick, are substantially higher in tectum than in retina, from embryonic day 16. The A₁₂ collagen-tailed form of the enzyme is present in similar amounts in the embryonic retina and optic tectum; however, while the A₁₂ activity increases significantly in retina after birth, both by percentage and in absolute terms, the tectal tailed enzyme follows a declining developmental profile, reaching a minimum after 6 months of life. On the other hand, the globular G₄ species shows developmental profiles, both in retina and tectum, rather similar to those obtained for the overall enzyme activity, while the G₂ and G₁ forms are present in comparable concentrations in both tissues. Besides, G₄ is the predominant globular form in the chick optic lobe after hatching, G₂ and G₁ being enriched in the embryonic tectum. In the case of retina, however, all the globular forms contribute more evenly to the total acetylcholinesterase activity, along the developmental period considered.The potential significance of some of the postnatal developmental profiles is discussed in terms of the progressive adjustment of retina and tectum to the requirements of visual function.     

Asymmetrical retinoic acid synthesis in the dorsoventral axis of the retina.

An aldehyde dehydrogenase present at high levels in the dorsal retina of the embryonic and adult mouse was identified as the isoform AHD-2 known to oxidize retinaldehyde to retinoic acid. Comparative estimates of retinoic acid levels with a reporter cell line placed the retinas among the richest tissues in the entire body of the early embryo; levels in ventral retina, however, exceeded dorsal levels. Retinoic acid synthesis from retinaldehyde in the dorsal pathway was less effective than the ventral pathway at low substrate levels and more effective at high levels. The dorsal pathway was preferentially inhibited by disulfiram, while ventral synthesis was preferentially inhibited by p-hydroxymercuribenzoate. When protein fractions separated by isoelectric focusing were analyzed for retinoic acid synthesizing capacity by a zymography-bioassay, most of the synthesis in dorsal retina was found to be mediated by AHD-2, and ventral synthesis was mediated by dehydrogenase activities distinct in charge from AHD-2. Postnatally, levels of highest retinoic acid synthesis shifted from ventral to dorsal retina. In the adult retina, the dorsal pathway persisted, but the preferential ventral pathway was no longer detectable. Our observations raise the possibility that retinoic acid plays a role in the determination and maintenance of the dorsoventral axis of the retina, and that the morphogenetically significant asymmetry here lies in the spatial arrangement of synthetic pathways.     

Development of NADPH-Diaphorase in the Avian Retina: Regulation by Calcium Ions and Relation to Nitric Oxide Synthase

Abstract: Nitric oxide plays an important role as an intercellular messenger in the CNS. In the present work we measured NADPH-diaphorase activity, which is considered to be a marker of cells producing nitric oxide, in homogenates of the developing chick retina. The enzyme activity can be detected beginning in 8-day-old embryonic retinas with no further quantitative variations throughout development. Arginine analogues inhibit ∼65% of the activity in embryonic retinas and 50% in posthatched retinas. The enzyme is stimulated 50% by 2 m M calcium chloride in retinas from 8 to 14 embryonic days, but this effect decreases to 20% in 17-day embryonic retinas and practically disappears in posthatched animals. The stimulation by calcium is completely blocked by arginine analogues. The decrease in enzyme activity at posthatched retinas is not due to stimulation by endogenous calcium or the presence of insufficient amounts of calmodulin, because addition of EGTA or calmodulin, respectively, did not restore the stimulation to levels observed at embryonic stages. Inhibition of NADPH-diaphorase activity by N ^G-nitro- l -arginine or l - N ^G-(iminoethyl)ornithine is concentration dependent with IC₅₀ values of ∼1 m M at all stages studied. However, in the presence of calcium, the inhibition by both analogues is shifted to the left and is apparently biphasic at all developmental stages, including in posthatched animals, with IC₅₀ values in the low micromolar range. NADPH-diaphorase was also detected by histochemistry in specific groups of cells in the early embryonic retina and in subsets of amacrine and ganglion cells, as well as in photoreceptors, in more developed retinas. The results indicate that different isoforms of nitric oxide synthase are present in the chick retina and that a calcium-dependent isoform is predominant in early periods of development.     

Changes in the glycosylation pattern of prion protein in murine scrapie. Implications for the mechanism of neurodegeneration in prion diseases

In prion diseases, the normal prion protein (PrP(c)) undergoes a conformational change that results in the abnormal form, named scrapie prion protein (PrP(sc)). The visual system of rodents provides a relatively simple neuronal model in which the cell bodies of neurons are confined to the retina and the axons constitute the optic nerve. We investigated by Western blot the profile of PrP(c) in the optic nerve and retina of normal hamsters and mice. We found that in the optic nerve the amount of PrP(c) is significantly higher than in the retina. A less abundant non-glycosylated band was observed in retinas compared with the optic nerve and brain. Similar results were found in the gray and white matter from normal mice and hamsters. After stereotaxic injection of ME7 or 139A in the superior colliculus, a visual target area, the proportion and glycopattern of PrP changed in the retina and optic nerve throughout the course of the disease. Similar results were found in the gray and white matter at terminal stage of scrapie after injection of ME7 and 139A in the dorsal hippocampus. This is the first time that changes in the distribution and glycopattern of PrP have been described in an in vivo model of prion diseases.     

A requirement for trypsin-sensitive cell-surface components for cell-cell interactions of embryonic neural retina cells

A quantitative assay was used to measure the rate of collection of a population of embryonic neural retina cells to the surface of cell aggregates. The rate of collection of freshly trysinized cells was limited in the initial stages by the rate of replacement of trypsin-sensitive cell- surface components. When cells were preincubated, or "recovered," and then added to cell aggregates, collection occurred at a linear rate and was independent of protein and glycoprotein synthesis. The adhesion of recovered cells was temperature and energy dependent, and was reversibly inhibited by cytochalasin B. Colchicine had little effect on collection of recovered cells. Antiserum directed against recovered cell membranes was shown to bind to recovered cells by indirect immunofluorescence. The antiserum also was shown to inhibit collection of recovered cells to aggregates, suggesting that at least some of the antigens identified might be involved in the adhesion process. The inhibitory effect of the antiserum was dose dependent . Freshly trypsinized cells absorbed neither the immunofluorescence activity nor the adhesion-inhibiting activity. Recovered cells absorbed away both activities. In specificity studies, dorsal neural retina cells adhered to aggregates of ventral optic tectum in preference to aggregates of dorsal optic tectum. The adhesive specificity of the dorsal retina cells was less sensitive to trypsin than the adhesive specificity of ventral retina cells which adhered preferentially to dorsal tectal aggregates only after a period of recovery.     

Cloning and characterization of zRICH, a 2',3'-cyclic-nucleotide 3'-phosphodiesterase induced during zebrafish optic nerve regeneration

We previously reported cloning of cDNAs encoding both components of a protein doublet induced during goldfish optic nerve regeneration. The predicted protein sequences showed significant homology with the mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases (CNPases). CNPases are well-established markers of mammalian myelin; hence, the cDNAs were designated gRICH68 and gRICH70 (for goldfish Regeneration-Induced CNPase Homologues of 68 and 70 kDa). Homologous cDNAs have now been isolated from zebrafish encoding a highly related protein, which we have termed zRICH. RNase protection assays show that zRICH mRNA is induced significantly (fivefold) in optic nerve regenerating zebrafish retinas 7 days following nerve crush. Western blots show a single band in zebrafish brain and retina extracts, with immunoreactivity increasing three-fold in regenerating retinas 21 days postcrush. Immunohistochemical analysis indicated that this increase in zRICH protein expression is localized to the retinal ganglion cell layer in regenerating retina. We have characterized and evaluated the relevance of a conserved beta-ketoacyl synthase motif in zRICH to CNPase activity by means of site-directed mutagenesis. Two residues within the motif, H334 and T336, are critical for enzymatic activity. A cysteine residue within the motif, which corresponds to a critical residue for beta-ketoacyl synthase, does not appear to participate in the phosphodiesterase activity.     

Microtubule-associated proteins characteristic of embryonic brain are found in the adult mammalian retina

We have used monoclonal antibodies against each of the major mammalian brain microtubule-associated proteins (MAPs), MAP1, MAP2, MAP3, MAP5, and tau, to study the timing of appearance and the cytological distribution of these proteins during the development of the rat retina. Western blots of adult rat retina reveal MAPs that are characteristic of embryonic brain, i.e., MAP5 and the low-molecular-weight forms of MAP2 (MAP2c) and tau (juvenile tau). At the onset of neuronal differentiation within the embryonic retina, MAP5, MAP3, MAP2c, and tau are found in the perikarya or extending axons of ganglion cells. High-molecular-weight MAP2, a dendrite marker, does not appear in the retina until the second day of postnatal development, when ganglion cell dendrites ramify within the inner plexiform layer. MAP1, which is characteristic of adult brain, does not appear in the retina until 1 week after birth, and is limited to ganglion cells and their processes. In the adult retina, MAP5 and MAP2c are concentrated within the inner segments and cell bodies of photosensitive cells, whereas tau is found in horizontal cells and more internal cell layers. Since photosensitive cells are unique among retinal neurons in their constant regeneration of their primary processes, the photoreceptive outer segments, both MAP5 and MAP2c appear not only to be involved in events associated with the embryonic differentiation and growth of neurites, but also in process regeneration in adult neurons that maintain some embryonic characteristics.     

Biochemical investigations of retinotectal adhesive specificity

The preferential adhesion of chick neural retina cells to surfaces of intact optic tecta has been investigated biochemically. The study uses a collection assay in which single cells from either dorsal or ventral halves of neural retain adhere preferentially to ventral or dorsal halves of optic tecta respectively. The data presented support the following conclusions: (a) The adhesion of ventral retina to dorsal tecta seems to depend on proteins located on ventral retina and on terminal β-N-acetylgalactosamine residues on dorsal tecta. (b) The adhesion of dorsal retina to ventral tecta seems to depend on proteins located on ventral tecta and on terminal β- N-acetylgalactosamine residues on dorsal retina. (c) A double gradient model for retinotectal adhesion along the dorsoventral axis is consistent with the data presented. The model utilizes only two complementary molecules. The molecule suggested to be concentrated dorsally in both retina and tectum seems to require terminal β-N-acetylgalactosamine residues for adhesion. Its activity is not affected by protease. A molecule fitting these qualifications, the ganglioside GM(2), could not be detected in a gradient, but lecithin vesicles containing GM(2) adhered preferentially to ventral tectal surfaces. The second molecule, concentrated ventrally in both retina and tectum, is a protein and seems capable of binding terminal β-N- acetylgalactosamine residues. One enzyme, UDP-galactose:GM(2) galactosyltransferase, has been found to be more concentrated in ventral retina than dorsal, but only by 30 percent.     

Expression and involvement of gicerin, a cell adhesion molecule, in the development of chick optic tectum

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It has both a homophilic binding activity and a heterophilic binding activity to neurite outgrowth factor (NOF) a molecule belonging to the laminin family. We have reported many studies on the heterophilic activity of gicerin and NOF, but the function of its homophilic binding activity in vivo had been unclear. In the retina, gicerin is expressed in retinal ganglion cells only when they extend neurites to the optic tectum. In this report we have found that gicerin is also transiently expressed in the optic tectum during this time. First, cell aggregation assays were used to show that gicerin expressed in the optic tectum displays homophilic binding activity. Then, explant cultures of embryonic day 6 chick optic tectum on gicerin-Fc chimeric protein-coated dishes and NOF-coated dishes were carried out. It was found that gicerin-gicerin homophilic interactions promoted cell migration, whereas heterophilic interactions with NOF induced neurite formation. Furthermore, when anti-gicerin antibodies were injected in order to examine the effect of gicerin protein in the formation of the tectal layer in ovo, cell migration was strongly inhibited. These data suggest that homophilic interaction of gicerin participates in the migration of neural cells during the layer formation and plays a crucial role in the organization of the optic tectum.     

Developmental expression of a synaptic vesicle-specific protein in the rat retina

In order to examine the appearance of synaptic vesicles and to correlate it with the formation of the synaptic layers, we have determined the staining pattern of a murine monoclonal antibody (SV 48) to a synaptic vesicle-associated protein in developing rat retina. The antigen was detected by the indirect immunofluorescence technique using cryostat sections of paraformaldehyde-fixed retinas. In the adult retina, the antibody stained both the outer plexiform (OPL) and the inner plexiform layers (IPL). The nuclear layers and the nerve fiber layer (NFL) were devoid of any staining. In prenatal and early postnatal (P) retinas, the antibody stained two bands which corresponded to the respective locations of the NFL and IPL. Staining in the NFL increased until P-4 and began to decline subsequently, and by P-8 little staining was left in this layer. In contrast, in the IPL, the intensity of staining increased gradually and leveled off by P-10. In the outer retina, a band of fluorescence corresponding to the OPL was first observed at P-5 and increased in intensity up to P-10. Immunoblotting studies showed that the major immunoreactive material from adult and embryonic retinas had a Mr approximately 65,000-67,000. As expected from its developmental pattern, all bands appeared initially in the central retina and subsequently in the peripheral retina. Our results show that the synaptic vesicle-protein is present in the nerve fiber layer before synaptogenesis in the central nervous system. Subsequently, the protein is lost from the NFL, possibly as a consequence of synapse formation.     

Retinal neural progenitors express topographic map markers

Transplantation of neural stem cells for replacing neurons after neurodegeneration requires that the transplanted stem cells accurately reestablish the lost neural circuits in order to restore function. Retinal ganglion cell axons project to visual centers of the brain forming circuits in precise topographic order. In chick, dorsal retinal neurons project to ventral optic tectum, ventral neurons to dorsal tectum, anterior neurons to posterior tectum and posterior neurons to anterior tectum; forming a continuous point-to-point map of retinal cell position in the tectal projection. We found that when stem cells derived from ventral retina were implanted in dorsal host retina, the stem cells that became ganglion cells projected to dorsal tectum, appropriate for their site of origin in retina but not appropriate for their site of implant in retina. This led us to ask if retinal progenitors exhibit topographic markers of cell position in retina. Indeed, retinal neural progenitors express topographic markers: dorsal stem cells expressed more Ephrin B2 than ventral stem cells and, conversely, ventral stem cells expressed more Pax-2 and Ventroptin than dorsal stem cells. The fact that neural progenitors express topographic markers has pertinent implications in using neural stem cells in cell replacement therapy for replacing projecting neurons that express topographic order, e.g., analogous neurons of the visual, auditory, somatosensory and motor systems.     

Norepinephrine acts as D1-dopaminergic agonist in the embryonic avian retina: late expression of beta1-adrenergic receptor shifts norepinephrine specificity in the adult tissue

Dopamine is the main catecholamine found in the chick retina whereas norepinephrine is only found in trace amounts. We compared the effectiveness of dopamine and norepinephrine in promoting cyclic AMP accumulation in retinas at embryonic day 13 (E13) and from post-hatched chicken (P15). Dopamine (EC(50)=10microM) and norepinephrine (EC(50)=30microM), but not the beta(1)-adrenergic agonist isoproterenol, stimulated over seven-fold the production of cyclic AMP in E13 retina. The cyclic AMP accumulation induced by both catecholamines in embryonic tissue was entirely blocked by 2microM SCH23390, a D(1) receptor antagonist, but not by alprenolol (beta-adrenoceptor antagonist). In P15 retinas, 100microM isoproterenol stimulated five-fold the accumulation of cAMP. This effect was blocked by propanolol (10microM), but not by 2microM SCH23390. Embryonic and adult retina display beta(1) adrenergic receptor mRNA as detected by RT-PCR, but the beta(1) adrenergic receptor protein was detected only in post-hatched tissue. We conclude that norepinephrine cross-reacts with D(1) dopaminergic receptor with affinity similar to that of dopamine in the embryonic retina. In the mature retina, however, D(1) receptors become restricted to activation by dopamine. Moreover, as opposed to the embryonic tissue, norepinephrine seems to stimulate cAMP accumulation via beta(1)-like adrenergic receptors in the mature tissue.     

Expression of membrane-bound and soluble guanylyl cyclase mRNAs in embryonic and adult retina of the medaka fish Oryzias latipes

Localization of mRNAs for four membrane-bound guanylyl cyclases (membrane GCs; OlGC3, OlGC4, OlGC5, and OlGC-R2), three soluble guanylyl cyclase subunits (soluble GC; OlGCS-alpha(1), OlGCS-alpha(2), and OlGCS-beta(1)), neuronal nitric oxide synthase (nNOS), and cGMP-dependent protein kinase I (cGK I) was examined in the embryonic and adult retinas of the medaka fish Oryzias latipes by in situ hybridization. All of the membrane GC mRNAs were detected in the photoreceptor cells of the adult and embryonic retinas, but in different parts; the OlGC3 and OlGC5 mRNAs were expressed in the proximal part and the OlGC4 and OlGC-R2 mRNAs were expressed in the outer nuclear layer. The mRNA for nNOS was expressed in a scattered fashion on the inner side of the inner nuclear layer in the adult and embryonic retinas. The mRNAs (OlGCS-alpha(2) and OlGCS- beta(1)) of two soluble GC subunits (alpha(2) and beta(1)) were expressed mainly in the inner nuclear layer and the ganglion cell layer of the embryonic retina while the mRNAs of the soluble GC alpha(1) subunit and cGK I were not detected in either the adult or embryonic retina. These results suggest that NO itself and/or the cGMP generated by soluble GC (alpha(2)/beta(1) heterodimer) play a novel role in the neuronal signaling and neuronal development in the medaka fish embryonic retina in addition to the role played by phototransduction through membrane GCs in the adult and embryonic retinas.     

Topologically restricted appearance in the developing chick retinotectal system of Bravo, a neural surface protein: experimental modulation by environmental cues [published erratum appears in J Cell Biol 1991 Mar;112(5):1049]

A novel neural surface protein, Bravo, shows a pattern of topological restriction in the embryonic chick retinotectal system. Bravo is present on the developing optic fibers in the retina; however, retinal axons in the tectum do not display Bravo. The appearance of Bravo in vitro is modulated by environmental cues. Axons growing out from retinal explants on retinal basal lamina, their natural substrate, express Bravo, whereas such axons growing on collagen do not. Retinal explants provide a valuable system to characterize the mechanism of Bravo restriction, as well as the cellular signals controlling it. Bravo was identified with monoclonal antibodies from a collection generated against exposed molecules isolated by using a selective cell surface biotinylation procedure. The NH2-terminal sequence of Bravo shows similarity with L1, a neural surface molecule which is a member of the immunoglobulin superfamily. This possible relationship to L1, together with its restricted appearance, suggests an involvement of Bravo in axonal growth and guidance.     

Evidence for accumulation of the β-subunit of β-conglycinin in soybean [Glycine max (L) Merr.] embryonic axes

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total proteins from soybean cotyledons and embryonic axes revealed the presence of similar storage proteins in both organs. Results from Western blot analysis conducted with antibodies raised against the purified #-subunit of #-conglycinin demonstrated accumulation of the #-subunit in embryonic axes. This accumulation followed a temporal pattern similar to that shown by the cotyledons. Axis #-conglycinin was broken down during the initial stage(s) of seed germination and was completely mobilized within 3 days after imbibition. Subcellular fractions were isolated from developing embryonic axes using metrizamide density gradients and analyzed by Western blots. Storage proteins were enriched in the lighter fractions of the gradient as well as with immunoglobulin heavy-chain binding protein. Electron microscopy of the storage-protein-enriched gradient fraction revealed small vesicles and protein aggregates. Protein A-gold immunocytochemistry was used to localize the occurrence of the #-subunit of #-conglycinin within the protein aggregates present in the metrizamide gradient and in the protein bodies present in mature embryonic axes.     

Immunologic detection of retina cognin on the surface of embryonic cells

The retina cell-aggregating glycoprotein, referred to as the retina cognin, has been demonstrated to be located at the surface of embryonic neural retina cells. The term cognin is used to indicate its postulated role in the mechanism of mutual recognition and morphogenetic association of embryonic cells. Antiserum was prepared to the highly purified retina cognin derived from isolated cell membranes of chick embryo retina, and it was used to detect the cognin on cells from chick embryos by means of complement-mediated cell lysis. Retina cells (from 10-day embryos) freshly dissociated with trypsin showed little—if any—lysis by the cognin antiserum; this is consistent with the sensitivity of the cognin to trypsin. However, the cells became susceptible to immunolysis after a period of incubation at 37 °C, which indicates regeneration of the cognin at the cell surface during the recovery period. This regeneration required protein synthesis. Immunofluorescence tests showed binding of the antiserum to the surface of the recovered cells, thereby further demonstrating the surface location of the cognin. The presence, availability or ability to regenerate the cognin, as assayed here, declined sharply with the embryonic age of the cells. Addition of exogenous cognin to freshly trypsin-dissociated retina cells (from 10-day embryos) markedly increased their susceptibility to immunolysis by the cognin antiserum, which indicates that the added cognin becomes associated with the surface of these cells. In contrast, addition of retina cognin to cells freshly trypsinized from 10-day embryo optic tectum and cerebrum, or from 14-day retina did not increase their susceptibility to immunolysis by the cognin antiserum. These results are consistent with earlier findings that enhancement of cell aggregation by the retina cognin is tissue-specific and stage-specific. Cells from non-neural tissues of the chick embryo were not lysed by the retina cognin antiserum. However, neural tissues, such as optic tectum, were found to contain cells which showed surface cross-reaction with the retina cognin antiserum.     

A study of ocular morphogenesis in the rat using (3H)thymidine autoradiography: evidence for thymidine recycling in the developing retina.

With the use of light microscopic autoradiography, the incorporation and subsequent distribution of [³H]thymidine within the retinas of sequentially staged rat embryos was studied during the period when large numbers of cells become necrotic and were resorbed within the optic rudiment. In the 48 hr following injection of isotope on embryonic Day 11, the number of grains overlying the dorsal, nondegenerate portion of the retina decreased to a low level, whereas ventrally, where the retina becomes intensely necrotic, the grain counts remained at much greater levels. The results indicate that thymidine may be recycled within normally degenerate regions of the embryonic retina.