Neuroblastic Layer Research Articles

Major glycosaminoglycan species in the developing retina: synthesis, tissue distribution and effects upon cell death

Retinal explants mantained in culture medium retain their histotypic structure and develop similarly to the in vivo condition. Extracellular matrix components, particularly the glycosaminoglycans which are not routinely present in dissociated cell cultures are involved in various cellular events. In this work we characterized and determined the localization of sulfated glycosaminoglycans in the extracellular matrix of rat retinal explants at various stages of normal postnatal development and tested whether disruption of the tissue glycosaminoglycan composition may impose either trophic or toxic effects upon distinct retinal cell populations. Our data show that chondroitin sulfate and heparan sulfate glycosaminoglycan chains are synthesized in different proportions during postnatal retinal development. A peak of synthesis of chondroitin sulfates is evident at around P14. Immunohistochemistry showed chondroitin 6-sulfate in the plexiform layers during the earlier stages while later, intense immunoreactivity was found in the outer retina. Heparan sulfate was found in the neuroblastic layer (NBL) at P1, in both nuclear layers from P5 onwards and in the ganglion cell layer (GCL) at all stages. In contrast to chondroitin 6-sulfate, immunoreactivity to heparan sulfate was absent from the outer retina at both P14 and P21. Treatment with heparitinase modulated the rates of cell death in both the GCL and the NBL in P1 retinal explants. Taken together our data show that among the major sulfated glycosaminoglycans, the developing rat retina synthesizes only heparan sulfate and chondroitin sulfates in a spatiotemporally regulated manner, with a peak of chondroitin sulfates at P14, possibly related to photoreceptor differentiation. In addition, our data suggest a role for heparan sulfate as a modulator of sensitivity to cell death in the retina.

Heat shock protein 108 mRNA expression during chicken retina development

In a developmental study on the expression of heat shock protein 108 (HSP108) mRNA in the chicken retina, we found different spatial and temporal expressions of HSP108 mRNA in each retinal layer. While intense HSP108 signals were found in the retina neuroblast layer at embryonic day 5 (E5), the ganglion cell population (GC), inner nuclear layer (IN) and pigment epithelium (PE) showed HSP108 expression at E9. At E14, HSP108 signals were reduced versus the previous stages even though signals were still detected in the GC, the IN, the outer nuclear layer and the PE. HSP108 signals were still detectable at the E21 stage, although each retinal layer showed a much differentiated morphology and diminished signal intensity. These results suggest that HSP108 expression might be developmentally regulated throughout eye organogenesis and that it plays a role in ocular development.

Dlx1, Dlx2, Pax6, Brn3b, and Chx10 homeobox gene expression defines the retinal ganglion and inner nuclear layers of the developing and adult mouse retina.

Distal-less homeobox genes are expressed in the developing forebrain. We assessed Dlx gene expression in the developing and adult mouse retina. Dlx1 and Dlx2 are detected in retinal neuroprogenitors by embryonic day (E) 12.5 (Eisenstat et al. [1999] J. Comp. Neurol. 217-237). At E13.5, the expression of four homeodomain proteins, DLX2, BRN3b, PAX6, and CHX10, define distinct yet overlapping domains in the retinal neuroepithelium. By postnatal day (P) 0, DLX2 is expressed in the neuroblastic layer and the ganglion cell layer (GCL) consisting of ganglion and displaced amacrine cells. DLX1 expression resembles DLX2 to P0 but decreases postnatally. In the adult, DLX2 is localized to ganglion, amacrine, and horizontal cells as determined by coexpression with retinal cell-specific markers. There is coincident expression of DLX2 with gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD)65, and GAD67 in the inner nuclear layer (INL) and GCL. In the adult, DLX2 is coexpressed with BRN3b in ganglion cells; PAX6 in amacrine, horizontal, and ganglion cells; and Chx10 in some bipolar cells. We predict that a combinatorial code of these homeobox genes and others specify retinal cell fate. Our results support a possible role for Dlx1 and Dlx2 in inner retinal development and in the terminal differentiation and/or maintenance of INL interneurons and ganglion cells in the adult. The correlation of DLX2 with GABA expression in the mouse retina closely mirrors the relationship of DLX2 to GABAergic neuronal differentiation in the embryonic forebrain, including neocortex, olfactory bulb and hippocampus, signifying a conservation of function of Dlx genes in the developing central nervous system.

Differential expression and cellular localization of doublecortin in the developing rat retina.

Doublecortin is 40 kDa microtubule-associated phosphoprotein required for neuronal migration and differentiation in various regions of the developing central nervous system. We have investigated the expression and cellular localization of doublecortin in the developing rat retina using immunocytochemistry and Western blot analysis. The expression of doublecortin was high from embryonic day 18 (E18) until E20 and was low during the postnatal period. The doublecortin immunoreactivity first appeared in a few radially orientated cells in the mantle zone of the primitive retina at E15. From E16 onward, the immunoreactivity appeared in two different regions: the inner part of the retina and middle of the neuroblastic layer. In the inner part, the somata of cells in the ganglion cell layer, in the distal row of the neuroblastic layer and profiles in the inner plexiform layer showed doublecortin immunoreactivity up to postnatal day 1 (P1). Afterwards, the doublecortin immunoreactivity persisted in the inner plexiform layer until P15, although the intensity decreased gradually with the maturation of the retina. In the middle of the neuroblastic layer, doublecortin immunoreactivity appeared in the radially orientated cells. These cells transformed into horizontal cells. The doublecortin immunoreactivity persisted in these cells up to P21. Given these results, doublecortin may play an important role in the migration and differentiation of specific neuronal populations in developmental stages of the rat retina.

Expression and activation of STAT proteins during mouse retina development

Cytokines and growth factors play important roles in mammalian ocular development and maintenance. Recent studies have indicated that some of these ligands can activate signal transducer and activator of transcription factors (STATs) and modulate gene transcription. The purpose of this study was to investigate the expression and activation of STAT proteins in the developing mouse retina. Anti-STAT and anti-phosphorylated STAT antibodies were used to detect the expression and activation of STATs in embryonic and postnatal neuronal retina, ciliary margin, and retinal pigment epithelium (RPE). In situ hybridization and Western blot were also employed. In embryonic stages, all STAT proteins were expressed in the neuronal retina in distinct cell populations at different embryonic stages. For example, Stat3 expression and activation gradually increased in the inner neuroblast layer and ciliary margin during development. In adult retina, Stat3 was detected in the inner nuclear layer and ganglion cells layers. Stat1 was strongly expressed in both outer and inner plexiform layers. Stat5a was clearly expressed in the outer/inner nuclear layer, the ganglion cell layer, and the inner plexiform layer. Strong expression of Stat3, Stat5a, and Stat6 was observed in the RPE. Activated Stat3 and Stat5a were found in the neural retina and the RPE. Distinct STAT proteins were present in different cell populations in neuronal retina and RPE suggesting multiple functions of STATs in mammalian eye development. Studies of STAT signal pathways in the eye may contribute to the understanding of molecular mechanisms in control of ocular development and pathogenesis.

The Effect of L-Arginine, a Nitric Oxide Synthase Substrate, on Retinal Cell Proliferation in the Postnatal Rat

The effects of L-arginine, a nitric oxide synthase (NOS) substrate, on cell proliferation in the developing postnatal rat retina were studied by immunocytochemistry using anti-bromodeoxyuridine (BrdU) antiserum, and by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Densitometric analysis by immunoblotting confirmed that neuronal NOS expression significantly increased in the L-arginine-treated retinas in comparison with the control retinas at postnatal day (P) 5 to P10. In the retinas of the control and L-arginine-treated rats, BrdU-labeled cells were only seen in the neuroblastic layer of the retinas up to P7. BrdU-labeled cells were significantly more numerous in the retinas of L-arginine-treated rats than in control retinas at P5 in the central retina and at P5 and P7 in the peripheral retina. In addition, TUNEL-positive apoptotic cells were more numerous in the retinas of L-arginine-treated rats at P5 and P7. Our results suggest that NO might play an important role in retinal maturation through regulating proliferative phases in the early stages of rat postnatal development.

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.

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 μM) 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.

Pituitary Adenylyl Cyclase-activating Polypeptide Prevents Induced Cell Death in Retinal Tissue through Activation of Cyclic AMP-dependent Protein Kinase

Multiple neuroactive substances are secreted by neurons and/or glial cells and modulate the sensitivity to cell death. In the developing retina, it has been shown that increased intracellular levels of cAMP protect cells from degeneration. We tested the hypothesis that the neuroactive peptide pituitary adenylyl cyclase-activating polypeptide (PACAP) has neuroprotective effects upon the developing rat retina. PACAP38 prevented anisomycin-induced cell death in the neuroblastic layer (NBL) of retinal explants, and complete inhibition of induced cell death was obtained with 1 nm. A similar protective effect was observed with PACAP27 and with the specific PAC1 receptor agonist maxadilan but not with glucagon. Photoreceptor cell death induced by thapsigargin was also prevented by PACAP38. The neuroprotective effect of PACAP38 upon the NBL could be reverted by the competitive PACAP receptor antagonist PACAP6-38 and by the specific PAC1 receptor antagonist Maxd.4. Molecular and immunohistochemical analysis demonstrated PAC1 receptors, and treatment with PACAP38 induced phospho-cAMP-response element-binding protein immunoreactivity in the anisomycin-sensitive undifferentiated postmitotic cells within the NBL. PACAP38 produced an increase in cAMP but not inositol triphosphate, and treatment with the cAMP-dependent protein kinase inhibitor R(p)-cAMPS blocked the protective effect of PACAP38. The results indicate that activation of PAC1 receptors by PACAP38 modulates cell death in the developing retina through the intracellular cAMP/cAMP-dependent protein kinase pathway.

Mucin-Like Glycoprotein Associated with Photoreceptor Cells in the Postnatal Developing Rat Retina

Purpose: To examine the developmental expression of mucin-like glycoprotein associated with photoreceptor cells (MLGAPC), and to confirm that MLGAPC with a relative mass of 120 and 135 kD correspond to the cone and rod types, respectively. Methods: We isolated the cDNA of the core protein of rat MLGAPC, prepared an anti-rat MLGAPC core protein antibody and then examined the expression of the MLGAPC core protein in postnatal developing rat retinas by means of Western blotting and immunohistochemistry. Results: On postnatal early days when cone photoreceptor cells were thought to be developing, MLGAPC was detected as a single band of 120 kD and was found to be positive in the apical portion of the neuroblastic layer. After postnatal day 7, when rod photoreceptor cells were developing, MLGAPC was detected as a minor band of 120 kD and an additional major band of 135 kD and was present throughout the differentiated photoreceptor and outer plexiform layers. Conclusions: The present findings revealed the developmental expression of MLGAPC and confirmed that MLGAPC of 120 and of 135 kD correspond to the cone and rod types, respectively. This demonstration is advantageous for studying the physiological roles of MLGAPC in the photoreceptor and outer plexiform layers, since the cone- and rod-associated glycoproteins should be investigated separately.

Two phases of increased cell death in the inner retina following early elimination of the ganglion cell population.

Neurons in the inner nuclear layer (INL) of the vertebrate retina undergo considerable programmed cell death during development, but the determinants of this cell death remain largely unknown. The present study examines the role of retinal ganglion cells in support of INL neurons in the developing ferret retina. The retinal ganglion cell population was eliminated by optic nerve transection at postnatal day (P) 2, and the incidence of cell death was examined using terminal deoxytransferase dUTP nick-end labelling (TUNEL) at various ages during the first 3 postnatal weeks. Significant increases in TUNEL-positive cells were observed in the neuroblast layer (NBL) as early as P3, prior to synapse formation within the inner plexiform layer (IPL), and again in the INL at P22, the normal peak of naturally occurring cell death within the ferret's INL. A decrease in TUNEL-positive cells was found in the NBL at P8. These results show three phases of response to the loss of retinal ganglion cells and suggest that cells in the NBL/INL are normally dependent on retinal ganglion cells for their survival. Recent studies have shown that certain populations of retinal neurons are reduced in adult animals that had lost the population of ganglion cells during early development, so the present study also examined when this reduction could first be detected. The number of parvalbumin-immunoreactive amacrine cells was decreased significantly in the NBL of the manipulated eye as early as P8, when we could first label this population, and this difference persisted through adulthood. The fact that cell death in the NBL has already increased within 24 hours of ganglion cell elimination, coupled with the specificity of this effect on the adult complement of INL cell types, shows that cell-cell interactions controlling survival are already highly specific for particular types of retinal neuron early in development

Transient expression of somatostatin mRNA in developing ganglion cell layers of rat retina

Somatostatin (SOM) mRNA in developing ganglion cell layer (GCL) detected by in situ hybridization histochemistry and SOM peptide in developing optic chiasma and optic tract detected by immunocytochemistry were monitored to explore whether ganglion cells expressing SOM project to the visual center. Most of these cells in the developing GCL expressed SOM transiently from embryonic day 13 (E13) to E21. The cells expressing SOM mRNA initially followed a central-to-peripheral pattern of development. The cells expressing SOM mRNA in the retinas of fetuses became detectable at E13. From E14 to E17 the number of cells expressing SOM mRNA increased rapidly. At E17 most of the cells in the developing GCL expressed SOM mRNA. From E18 to postnatal days the positive cells became sparse except at the postnatal day 0 (PND0) the positive cells decreased dramatically in comparison with that at the E21. At PND15, the positive cells only can be found in the inner neuroblastic layer and in the ganglion cell layer. At PND20 the distribution pattern and the number of the positive cells were essentially the same as that in adult rat. SOM immunoreactivity was detectable at E16 in the developing optic chiasma and optic tract; the majority of the fibers in these area were SOM positive. From E16 to E18 the density of the immunostaining increased rapidly, whereas from E19 to E21 the density decreased. At PND0 no positive fibers were seen. The transient presence of SOM in most of the ganglion cells in the developing ganglion cell layer has prompted us to study the role of SOM in generation and differentiation of the retinal ganglion cells, and formation of the retina-visual center projections.

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.

Paracrine neuroprotective effect of nitric oxide in the developing retina.

The retina of newborn rats consists of the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL) containing amacrine cells and the neuroblastic layer (NBL). In retinal explants, the GCL enters cell death after sectioning of the optic nerve, whereas there is almost no cell death in the NBL. When protein synthesis is inhibited with anisomycin, cell death is blocked in the GCL and induced in the NBL. We tested the roles of nitric oxide (NO) on cell death in the retina in vitro. Either L-arginine, the substrate for NO synthase or the NO donor S:-nitroso-acetylpenicillamine (SNAP) blocked cell death induced by anisomycin in the NBL, but had no effect in the GCL. Sepiapterin, a precursor of the nitric oxide synthase (NOS)-cofactor tetrahydrobiopterin also had a protective effect against anisomycin. The use of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble form of guanylyl cyclase, showed that anti-apoptotic effect of SNAP is partially mediated by cGMP generated by activation of guanylyl cyclase. NADPH-diaphorase histochemistry stained cells only in the GCL and INL. Thus, the degenerative effect of anisomycin is observed within the NBL, whereas the localization of NOS is restricted to the GCL and INL. The protective effect of both the NO substrate and cofactor upon cell death induced by anisomycin in the NBL, indicates that NO produced by amacrine and ganglion cells is a paracrine modulator of cell death within the retinal tissue.

The immunocytochemical localization of neuronal nitric oxide synthase in the developing rat retina.

The localization of nitric oxide synthase (NOS) was investigated in the developing rat retina by immunocytochemistry and western blot analysis, using an antiserum directed against neuronal NOS. NOS-labeled cells were first detected at postnatal day 5 (P5) in the inner row of the neuroblastic layer. These cells were considered to correspond to the type 1 cell of the adult rat retina. Type 2 cells, characterized by a small soma and weak immunoreactivity, and a class of displaced amacrine cells were detected at P9 and P7, respectively. By P14 or P15, the time of eye opening, NOS immunoreactivity appeared in some bipolar cells. NOS was first expressed at the protein level at P9. Thereafter, quantitative evaluation by immunoblotting confirmed that the intensity of the immunoreactive bands increased abruptly, reaching the same value as is found in the adult retina at P21. Our results demonstrate that differentiation of NOS-labeled cells follows a discrete developmental pattern and is most active during the 2nd postnatal period in the rat retina.

Regional and cellular specificity of the expression of TPRD, the tetratricopeptide Down syndrome gene, during human embryonic development

The TPRD gene (tetratricopeptide (TPR) containing Down syndrome gene) is one of the candidate genes in the Down syndrome chromosomal region-1. Duplication of this gene may be the cause of major phenotypic features of Down syndrome. Here we show that the TPRD expression is developmentally regulated during human embryogenesis. At the earliest stages of development (Carnegie 8–12) TPRD expression is ubiquitous. At later developmental stages (Carnegie stages 14, 16 and 18), it becomes restricted to the nervous system, as is the case for the mtprd gene during mouse development. We extended our analysis of TPRD expression during fetal development of the human nervous system (13, 22 and 24 weeks). A new oblique illumination technique was used to compare signal intensity and cell density. Some regions of the nervous system such as the external cortical layers of the brain, and the inner neuroblastic layer of the eye, strongly express the TPRD gene.

Ontogeny of a photic response in the retina and suprachiasmatic nucleus in the mouse

The ontogeny of photic responsiveness in the retina and the suprachiasmatic nucleus (SCN) of C57BL/6J mouse was studied using the enhanced expression of the immediate early gene c- fos as a marker of neuronal activation. c- fos expression was assessed by immunocytochemical localisation of its protein product. Light induction of Fos-like protein in the retina and SCN occurred first at postnatal day four (PD 4). At this stage of development, some cells in the inner part of the neuroblastic layer and in the ganglion cell layer showed positive immunoreaction; the number of Fos-like positive cells increased with age until it reached adult levels by PD 15. Induction of Fos-like expression at PD 4 in the SCN mainly occurred in the ventrolateral region, the region that receives the greatest density of retinal innervation. These results indicate that retinal input can activate cells in the SCN even before eyelids open, and the SCN can be stimulated by photic inputs as early as day 4 after birth.

Choline acetyltransferase-immunoreactive neurons in the developing rat retina.

The development of cholinergic cells in the rat retina has been examined with immunocytochemistry by using antisera against choline acetyltransferase (ChAT). ChAT-immunoreactive (IR) cells were first detected at embryonic day 17 (E17) in the transitional zone between the neuroblastic layer (NBL) and ganglion cell layer (GCL). At E20, ChAT-IR cells are located exclusively in the GCL. At postnatal day 0 (P0), ChAT immunoreactivity appeared for the first time in cells at the distal margin of the NBL. Two prominent bands of labeled processes were first visible at P3, and by P15, these two bands resembled those of the adult retina. In addition, ChAT immunoreactivity appeared transiently in horizontal cells from P5 to P10. The number of ChAT-IR cells increased steadily up to P15. This resulted in a 93.8-fold increase between E17 and P15 (680-63,800 cells). However, after P15, the number declined by 19% from 63,800 cells at P15 to 51,800 in the adult. At all ages, the spatial density of each ChAT-IR cell population in the central retina was higher than in the periphery. In both central and peripheral regions, the peak density of ChAT-IR cells in the GCL was attained at E20. However, in the INL, the peak densities occurred at P3 in the central region and at P5 in the peripheral region. Up to P15, the soma diameter of ChAT-IR cells in the INL and GCL in each region increased continuously, reaching peak values at P15. Our results demonstrate that ChAT immunoreactivity is expressed in early developmental stages in the rat retina, as in other mammals, and that acetylcholine released from ChAT-IR cells may have neurotrophic functions in retinal maturation.

Retinal damage induced by cisplatin in neonatal rats and mice

Purpose. The morphologic response of the retina at different neonatal ages to various doses of cis -platinum(II)diamminedichloride (cisplatin) was examined in rats and mice. Methods. Cisplatin was given to rats at a dose of 1, 3 or 5 mg/kg at 0 days or 5 mg/kg at 7 or 14 days of age, and to mice at 0.5, 1.5, 3 or 6 mg/kg at 0 days or 6 mg/kg at 7 or 14 days of age, and the animals examined 12 and 24 hrs, and 3 and 7 days after the treatment. Results. In both species, regardless of gender, with = 3 mg/kg cisplatin treatment (lethal dose) at day 0, retinal damage characterized by the appearance of aggregations of TUNEL-positive cells scattered in the undifferentiated neuroblastic layer was seen at 24 hrs, and led to rosette formation at day 3 and 7 (retinal dysplasia). At the ultrastructural level, neuroblastic cells showed condensation of chromatin and shrinkage of the cytoplasm, and rosettes encircled by an outer limiting membrane. Cell debris phagocytosed by pigment epithelial cells was seen. However, cisplatin at < 3 mg/kg in 0-day-old animals or at high dose in = 7-day-old animals caused no damage to the retina. Conclusions. A critical period (day 0) for the administration and a threshold dose (= 3 mg/kg) of cisplatin in the development of retinal damage in rats and mice was seen. Although the cisplatin dose necessary to damage the retina caused a high incidence of mortality, it was below the human therapeutic dose.

Gamma irradiation leads to two waves of apoptosis in distinct cell populations of the retina of newborn rats.

Gamma radiation induces apoptosis in the proliferative zone (neuroblastic layer) of the developing rat retina. We asked whether sensitivity to apoptosis might be related to distinct phases of the cell cycle. Explants of newborn rat retina or newborn pups were gamma-irradiated and apoptosis was detected by chromatin condensation, DNA fragmentation in situ and DNA electrophoresis. After 6 hours, early appearing apoptotic bodies were located mainly towards the outer tier of the neuroblastic layer. In contrast, after 24 hours, late-appearing apoptotic cells were located towards the inner margin of the neuroblastic layer, a region associated with the S phase of the cell cycle. Labeling of a cohort of cells with the nucleotide analog bromo-deoxyuridine (BrdU) at the time of irradiation, showed that these cells die in the late wave of apoptosis. BrdU given 3 hours before fixation labeled a large number of late apoptotic cells, but no early apoptotic cells. After labeling of all cycling cells with BrdU, 40% of the early apoptotic profiles were unlabeled, and thus post-mitotic. The same schedules of cell death were identified after gamma irradiation in vivo. The results show that irradiation leads to two waves of apoptosis in distinct cell populations. An early wave comprises both post-mitotic cells and proliferating cells out of the S phase. The late wave comprises cells in S phase, which pass through this phase again to die. The antioxidant pyrrolidinedithiocarbamate prevented the early but not the late wave of apoptosis following irradiation, and blocked lipid peroxidation at 6 hours after the insult, suggesting that the two waves of apoptosis are indeed mediated by distinct mechanisms.