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.     

Changing sensitivity to cell death during development of retinal photoreceptors

Photoreceptor cell death occurs during both normal and pathological retinal development. We tested for selective induction and blockade of cell death in either retinal photoreceptors or their precursors. Organotypical retinal explants from rats at postnatal days 3-11 were treated in vitro for 24 hr with thapsigargin, okadaic acid, etoposide, anisomycin, or forskolin. Explant sections were examined for cell death, and identification of either photoreceptors or proliferating/immediate postmitotic cells followed imunohistochemistry for either rhodopsin or bromodeoxyuridine and proliferating cell nuclear antigen, respectively. Photoreceptor cell death was selectively induced by either thapsigargin or okadaic acid, whereas death of proliferating/immediate postmitotic cells was induced by etoposide. Prelabeling of proliferating precursors allowed direct demonstration of changing sensitivity of photoreceptors to various chemicals. Degeneration of both photoreceptors and proliferating/immediate postmitotic cells depended on protein synthesis. Increase of intracellular cyclic AMP blocked degeneration of postmitotic, but not of proliferating, photoreceptor precursors. The selective induction and blockade of cell death show that developing photoreceptors undergo progressive changes in mechanisms of programmed cell death associated with phenotypic differentiation.     

Differentiation-dependent sensitivity to cell death induced in the developing retina by inhibitors of the ubiquitin-proteasome proteolytic pathway

The effects of inhibitors of proteasome function were studied in the retina of developing rats. Explants from the retina of neonatal rats at postnatal day (P) 3 or P6 were incubated with various combinations of the proteasome inhibitor carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132), the protein synthesis inhibitor anisomycin, or the adenylyl cyclase activator forskolin. MG132 induced cell death in a subset of cells within the neuroblastic (proliferative) layer of the retinal tissue. The cells sensitive to degeneration induced by either MG132 or anisomycin, were birthdated by bromodeoxyuridine injections. This showed that the MG132-sensitive population includes both proliferating cells most likely in their last round of cell division, and postmitotic undifferentiated cells, at a slightly earlier stage than the population, sensitive to anisomycin-induced cell death. The results show that sensitivity to cell death induced by proteasome inhibitors defines a window of development in the transition from the cell cycle to the differentiated state in retinal cells.     

Expression pattern of a newt Notch homologue in regenerating newt retina

We isolated part of a newt Notch homologue, N-Notch, from regenerating newt retina. The spatio-temporal pattern of N-Notch expression was studied by in situ hybridization at different stages of newt retinal regeneration. Proliferating cells were confirmed by the injection of bromodeoxyuridine (BrdU). In the early stage of regeneration, when the retina was one to two cells thick, all proliferating retinal progenitors expressed N-Notch. As the thickness of the retina increased with regeneration, N-Notch expression decreased in BrdU-positive cells on the vitreal side of the retina. Subsequently, presumptive retinal ganglion cells that were BrdU-negative cells appeared at the vitreal edge of the regenerating retina. These differentiating cells did not express N-Notch. Later, N-Notch expression decreased in the BrdU-positive cells on the scleral surface of the retina. Subsequently, presumptive photoreceptor cells that were BrdU-negative cells appeared in this region. These differentiating cells also did not express N-Notch. The proliferating retinal progenitors ceased expressing N-Notch and then stopped dividing during the differentiation of ganglion cells and photoreceptor cells. It was found that retinal regeneration involves the expression of an important developmental signaling molecule, Notch, in retinal progenitors and the expression of Notch ceased as cell differentiation proceeded during retinal regeneration.     

Apoptotic cell death of proliferating neuroepithelial cells in the embryonic retina is prevented by insulin

The role of programmed cell death is well established for connecting neurons. Conversely, much less is known about apoptosis affecting proliferating neuroepithelial cells. Chick retina from day 4 to day 6 of embryonic development (E), essentially proliferative, presented a defined distribution of apoptotic cells during normal in vivo development, as visualized by TdT-mediated dUTP nick end labelling (TUNEL). Insulin, expressed in the early chick embryonic retina as proinsulin, attenuated apoptosis in growth factor-deprived organotypic culture of E5 retina. This effect was demonstrated both by TUNEL and by staining of pyknotic nuclei, as well as by release of nucleosomes. Application of a 1 h [methyl-3H]thymidine pulse in ovo at E5, followed by organotypic culture in the presence or absence of insulin, showed that this factor alone decreased the degradation of labelled DNA to nucleosomes by 40%, as well as the proportion of labelled pyknotic nuclei. Both features are a consequence of apoptosis affecting neuroepithelial cells, which were in S-phase or shortly after. In addition, when the E5 embryos were maintained in ovo after the application of [methyl-3H]thymidine, 70% of the apoptotic retinal cells were labelled, indicating the in vivo prevalence of cell death among actively proliferating neuroepithelial cells. Apoptotic cell death is thus temporally and spatially regulated during proliferative stages of retinal neurogenesis, and embryonic proinsulin is presumably an endogenous protective factor.     

The occurrence of apoptosis during retinal regeneration in adult newts

The present study examined the occurrence of apoptosis, identified by an in situ technique for detecting DNA fragmentation, in the regenerating retina of adult newts following ablation of the retina. Apoptosis occurs in the initial phase of regeneration when retinal precursor cells are actively proliferating. In the late stage of regeneration, when two synaptic layers are forming, apoptosis occurs mainly in the ganglion cell layer and inner nuclear layer. We found that apoptosis occurred with proliferation, differentiation, formation of retinal layers and retinotectal projections during retinal regeneration. Our findings suggest that apoptosis is closely related to these phenomena.     

DNA repair in the degenerating mouse retina

In light of different recent results suggesting that the adult mammalian central nervous system can produce new neurons, possibly as an endogenous repair mechanism, we investigated whether neurogenesis occurs in response to photoreceptor degeneration in the rd1 mouse, a model of human-inherited retinal dystrophy. Bromodeoxy-Uridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression experiments detected cell proliferation in the extreme peripheral retina, in both wt and rd1 retina, independent of degeneration. BrdU incorporation and PCNA expression also occurred in rd1 photoreceptors. Our results strongly suggest that these photoreceptors undergo DNA repair: p53, PCNA, and DNA ligase IV are expressed before photoreceptor death, consistent with a model where photoreceptors expressing the rd1 mutation activate a process of DNA repair but which is overwhelmed by the disease mutation leading to apoptotic death. The existence of such a balance offers potential new targets for neuroprotective approaches.     

Embryonic stem cells are capable of generating a neuronal network in the adult mouse retina

The integration of embryonic stem (ES) cells as well-differentiated neuronal cells into the retinas of adult mice was investigated. ES cells were transplanted in adult mouse retinas by intravitreal injections. Neuronal differentiation of the ES cells was investigated by morphological and immunohistochemical examinations on post-operative days 5 and 30. ES cell apoptosis was examined by in situ terminal dUTP-biotin nick end labeling of DNA fragments. Differentiated ES cells growing along the retinal surface developed fine neuronal cell processes around cell nuclei and generated neuronal networks into the retinal inner plexiform layer (IPL) 30 days after transplantation. The differentiated ES cells expressed retinal and neuronal markers. Many apoptotic cells were recognized in transplanted ES cells at day 5 but not at day 30 after transplantation. ES cells may be useful for neural tissue regeneration in the adult mammalian retina.     

Early histogenesis of the teleostean retina: studies using a novel immunochemical marker, proliferating cell nuclear antigen (PCNA/cyclin)

Immunoreactive proliferating cell nuclear antigen or cyclin (ir-PCNA), detected by immunofluorescence using a human autoantibody, was used as a marker for proliferating neuroblasts during early development of the teleostean retina. In a killifish (Medaka) and a cyprinid (goldfish), ir-PCNA was present in all retinal cells at the monolayer stage (embryonic day 2 in Medaka, days 3-4 in goldfish). Subsequently ir-PCNA was lost, as neuroblasts became postmitotic in a centre-to-periphery and proximal-to-distal (ganglion cells first, photoreceptors last) sequence. In late larvae and adults, neuronal ir-PCNA was confined to the ring of proliferating neuroblasts at the retinal margin and scattered rod precursors in the outer nuclear layer. The changes in localization of ir-PCNA during development parallel closely the changes in localization of [3H]thymidine incorporation, reported previously.     

Adenovirus-mediated gene transfer of Bcl-xL impedes neurite regeneration in vitro

Mouse retinal explants were transfected with recombinant adenovirus vector carrying the green fluorescent protein (GFP) gene and the rat bcl-xL gene (Adeno-Bcl-xL) to determine its ability to protect retinal ganglion cells against apoptotic cell death and to promote retinal ganglion cell neurite regeneration. Adeno-Bcl-xL-incubated retinas had reduced apoptosis compared to controls. However, neurite regeneration in adeno-treated retinas was less than that of vector-free retina. These results suggest that the usefulness of adenovirus vectors for gene therapy for retinal ganglion cells may be limited.     

Caspase regulation of neuronal progenitor cell apoptosis

Programmed cell death (apoptosis) of both proliferating neuroblasts and postmitotic neurons is essential for normal nervous system development. To study the molecular regulation of apoptosis in neuronal progenitor cells, we developed a flow cytometric assay capable of distinguishing between viable, apoptotic, and necrotic cell populations. Incubation of freshly dissociated telencephalic cells from gestational day 12-13 mouse embryos with either cytosine arabinoside (AraC) or staurosporine caused a marked increase in the percentage of apoptotic cells. Both drugs induced caspase-3 activation, as determined by in vitro cleavage of a caspase-3 substrate and immunocytochemical detection of activated caspase-3. Treatment of telencephalic cells with the broad caspase inhibitor BAF, blocked caspase-3 activation and protected cells against both AraC and staurosporine-induced apoptotic death. These results indicate that neuronal progenitors possess a caspase-dependent apoptotic pathway, the activation of which may regulate neuronal progenitor cell numbers in vivo.     

Timing and topography of cell genesis in the rat retina

To understand the mechanisms of cell fate determination in the vertebrate retina, the time course of the generation of the major cell types needs to be established. This will help define and interpret patterns of gene expression, waves of differentiation, timing and extent of competence, and many of the other developmental processes involved in fate acquisition. A thorough retinal cell "birthdating" study has not been performed for the laboratory rat, even though it is the species of choice for many contemporary developmental studies of the vertebrate retina. We investigated the timing and spatial pattern of cell genesis using 3H-thymidine (3H-TdR). A single injection of 3H-TdR was administered to pregnant rats or rat pups between embryonic day (E) 8 and postnatal day (P) 13. The offspring of prenatally injected rats were delivered and all animals survived to maturity. Labeled cells were visualized by autoradiography of retinal sections. Rat retinal cell genesis commenced around E10, 50% of cells were born by approximately P1, and retinogenesis was complete near P12. The first postmitotic cells were found in the retinal ganglion cell layer and were 9-15 microm in diameter. This range includes small to medium diameter retinal ganglion cells and large displaced amacrine cells. The sequence of cell genesis was established by determining the age at which 5, 50, and 95% of the total population of cells of each phenotype became postmitotic. With few exceptions, the cell types reached these developmental landmarks in the following order: retinal ganglion cells, horizontal cells, cones, amacrine cells, rods, bipolar cells, and Müller glia. For each type, the first cells generated were located in the central retina and the last cells in the peripheral retina. Within the sequence of cell genesis, two or three phases could be detected based on differences in timing, kinetics, and topographic gradients of cell production. Our results show that retinal cells in the rat are generated in a sequence similar to that of the primate retina, in which retinogenesis spans more than 100 days. To the extent that sequences reflect underlying mechanisms of cell fate determination, they appear to be conserved.     

Cell birthdays and rate of differentiation of ganglion and horizontal cells of the developing cat's retina

Tritiated thymidine autoradiography experiments demonstrated that three cell classes are produced by ventricular cells during the first phase of neurogenesis: retinal ganglion cells, A-type horizontal cells, and cone photoreceptors. Light microscopy and scanning electron microscopy were used to study the migration and morphological differentiation of these three cell classes. The patterns of postmitotic migration are of interest because these three classes of cells are found in three different layers of the adult retina. Cones retain their position at the outer limiting membrane (OLM) throughout life and do not migrate. Ganglion cells migrate immediately to the proximal (vitread) layer of the retina and begin to differentiate. In contrast, A-type horizontal cells migrated away from the OLM within 10-14 days after their final mitosis but were morphologically relatively undifferentiated at that time. Subsequent differentiation of the A-type horizontal cell is also protracted; dendrites are not observed until approximately 3 weeks after the final mitosis. These observations suggest that there are several interacting mechanisms involved in neurogenesis: a sequence that produces a specific cohort of committed cells at a specific time, the subsequent migration of postmitotic neuroblasts to an appropriate position in the retina, and a spatial gradient of differentiation increasing from distal to proximal layers. While this distribution of differentiated cells early in fetal development is striking, the existence of underlying time-dependent processes that might cause this apparent spatial phenomenon cannot be eliminated.     

Adenovirus-mediated gene transfer of Bcl-xL impedes neurite regeneration in vitro

Mouse retinal explants were transfected with recombinant adenovirus vector carrying the green fluorescent protein (GFP) gene and the rat bcl-x(L) gene (Adeno-Bcl-xL) to determine its ability to protect retinal ganglion cells against apoptotic cell death and to promote retinal ganglion cell neurite regeneration. Adeno-Bcl-xL-incubated retinas had reduced apoptosis compared with controls. However, neurite regeneration in adeno-treated retinas was less than that of vector-free retina. These results suggest that the usefulness of adenovirus vectors for gene therapy for retinal ganglion cells may be limited.     

Voltage‐gated K+ current: a marker for apoptosis in differentiating neuronal progenitor cells?

We investigated apoptosis during early stages of in vitro differentiation of neuronal precursors generated by embryonic day 14 (E14) mouse striata stem cells. Differentiation was in conditions of suboptimal growth factor supply. Apoptosis reached 10-15% of cells and affected proliferating as well as postmitotic cells, including TUJ1-positive cells. Inhibition of apoptosis led to an increased proportion of TUJ1-positive cells generated by stem cells. K(+) current was reported to be related to apoptosis. Outward K(+) currents were present in differentiating neuronal precursors that were consistent with delayed rectifier and transient A-type currents. The amplitude of the delayed rectifier current varied during the first 4 days of stem cell differentiation. Current amplitude was greatly increased in the presence of staurosporine but reduced at elevated extracellular K(+) concentration. In addition, the amplitude of the current was significantly diminished by inhibiting several caspases, but not caspase 8. In Bax knock-out transgenic neuronal precursors, K(+) current was not decreased after the first day but at later stages of cell differentiation. At this early stage, apoptosis of proliferating cells and of TUJ1-positive cells was not reduced by the absence of Bax, but was by caspase 9 inhibition. Thus, activation of a delayed rectifier K(+) current in differentiating stem cells is related to apoptosis. Recordings of this current revealed that apoptosis at early stages of neuronal differentiation occurred in two phases that did not exhibit similar dependence on the proapoptotic protein Bax and that probably used different pathways.     

The RB protein family in retinal development and retinoblastoma: new insights from new mouse models

The Rb gene was isolated almost 20 years ago, but fundamental questions regarding its role in retinal development and retinoblastoma remain. What is the normal function of RB protein in retinogenesis? What is the cell-of-origin of retinoblastoma? Why do retinoblastoma tumors have recurrent genetic lesions other than Rb inactivation? Why is retinoblastoma not induced by defects in cell cycle regulators other than Rb? Why is the retina so sensitive to Rb loss? Recently developed conditional Rb knockout models provide new insight into some of these issues. The data suggest that RB protein may not control the rate of progenitor division, but is critical for cell cycle exit when dividing retinal progenitors differentiate into postmitotic transition cells. This finding focuses attention on the ectopically dividing transition cell, rather than the progenitor, as the cell-of-origin. Cell-specific analyses in the RB-deficient retina reveal that ectopically dividing photoreceptors, bipolar and ganglion cells die, but amacrine, horizontal and Muller cells survive and stop dividing when they terminally differentiate. Rare amacrine transition cells escape cell cycle exit and generate tumors. These data suggest that post-Rb mutations are required to overcome growth arrest associated with terminal differentiation, rather than apoptosis as previously suggested. To explain why perturbing cell cycle regulators other than RB does not initiate retinoblastoma, we speculate that mutations in other components of the RB pathway perturb cell cycle arrest, but only RB loss triggers genome instability in retinal transition cells, which may be critical to facilitate post-Rb mutations necessary for transformation. Cell-specific differences in the effect of Rb loss on genome stability may contribute to the tremendous sensitivity of retinal transition cells to tumorigenesis. The new mouse models of retinoblastoma will be invaluable for testing these possibilities.     

In vitro isolation and expansion of human retinal progenitor cells

Human retinal development proceeds with temporal and spatial precision. Although differentiation starts around the beginning of the third month of gestation, the majority of cells in the outer neuroblastic layer of human neural retina are still proliferating, as evidenced by their Ki-67 immunoreactivity. In the present study, the proliferating human retinal progenitor cells (HRPCs) were isolated and expanded in culture. They were capable of dividing for multiple generations (with passage 8, the latest tested) and differentiating to several retinal cell phenotypes. These findings indicate that human retina at the 10th-13th week of gestation harbors progenitor cells that can be maintained and expanded in vitro for multiple generations. The availability of such cells may have important implications with respect to human degenerative retinal diseases, as these HRPCs have the potential to be used therapeutically to replace damaged retinal neurons.     

Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death.

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein synthesized and secreted by retinal pigment epithelial (RPE) cells in early embryogenesis and has been shown to be present in the extracellular matrix between the RPE cells and the neural retina. It induces neuronal differentiation and promotes survival of neurons of the central nervous system from degeneration caused by serum withdrawal or glutamate cytotoxicity. Because the role of PEDF in the retina is still unknown, we examined its ability to protect cultured retinal neurons against hydrogen peroxide (H(2)O(2))-induced cell death. Retinas of 0-2-day-old Sprague-Dawley rats were isolated and dissociated, and the neurons were maintained for 2 weeks in a synthetic serum-free medium. Immunocytochemical labeling showed that 50-60% of the cultured cells were rod photoreceptors. Treatment with H(2)O(2) induced significant death of retinal neurons in a dose- and time-dependent manner. Pretreatment with PEDF prior to insult greatly attenuated H(2)O(2)-induced cytotoxicity, and its effect was shown to be dose dependent. Cytotoxicity was determined by 3,(4,5-dimethylthiazol-2-yl)2, 5-diphenyl-tetrazolium bromide and lactate dehydrogenase assays, and apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling assay. The present study also showed that H(2)O(2)-induced retinal neuron death was by apoptosis that could be inhibited by PEDF. Combination of PEDF with basic fibroblast growth factor, brain-derived neurotrophic factor, or ciliary neurotrophic factor improves the protection. These data strongly suggest that PEDF is a potential neuroprotective agent in the treatment of retinal degeneration.     

Differential effects of cyclin-dependent kinase blockers upon cell death in the developing retina

Pharmacological blockers of cyclin-dependent kinases (CDKs) can inhibit cell cycle progression. Deferoxamine (DFO) and mimosine (MIMO) arrest cells reversibly at the G1/S transition and olomoucine (OLO) inhibits the cell cycle at both G1/S and G2/M. We investigated the effect of these drugs upon cell death in histotypical explants taken from the retina of neonatal rats. Degeneration of retinal ganglions cells (RGC) induced by axotomy was inhibited by OLO (100 microM) but not by DFO (up to 2 mM) or MIMO (up to 1 mM). On the other hand, after 1 day in vitro, all cell cycle inhibitors induced cell death in the neuroblastic layer (NBL) of the explants. DFO and MIMO induced cell death only of proliferating cells, identified either by their incorporation of bromodeoxyuridine or by immunolabeling the proliferating cell nuclear antigen. In turn, OLO induced cell death of both proliferating and post-mitotic cells. However, the post-mitotic cells were unlabeled with markers of retinal differentiation. Our results indicate that cyclin-dependent kinases are involved in the control of sensitivity to cell death in the retina, and that retinal cells present differentiation-dependent responses to modulation of CDK activity.     

Spatiotemporal gradients of differentiation of chick retina types I and II cholinergic cells: identification of a common postmitotic cell population.

The chick retina has three types of cholinergic amacrine cells. We have found that Types I and II differentiate from a common population of postmitotic cells temporarily located in the inner plexiform layer (IPL cells). Golgi staining and immunocytochemistry for choline acetyltransferase (ChAT) and gamma-aminobutyric acid (GABA) were used to trace the development and fate of IPL cells. Transformation of the shape of IPL cells into those typical of both conventional amacrine cells and those displaced to the ganglion cell layer are seen. All IPL cells are doubly immunoreactive, for ChAT and GABA, from the time they appear as a cell population within the inner plexiform layer (IPL) until their separation into the two amacrine cell populations. Polarization and early stages of shape differentiation of both types occur while they are in the IPL, starting in the dorsocentral area in the temporal retina and spreading to the rest of the retina. Three spatial gradients of differentiation are observed: from central-to-peripheral, dorsal-to-ventral, and temporal-to-nasal retina. Our findings suggest that the fate of both types of cells in the chick is determined locally, whereas their postmitotic precursors are within the IPL. The presence of GABA and acetylcholine in both types of amacrine cells at early stages of their morphogenesis, well before they have synaptic interactions, suggests a morphogenetic role for these molecules in inner retinal differentiation.