Generation of retinal ganglion cells is modulated by caspase-dependent programmed cell death

Programmed cell death occurs during both early and late neural development. The mechanisms for the regulation and execution of the early cell death as well as its developmental role are still not fully understood. In this work we have studied the early programmed cell death in the retinal neuroepithelium. Apoptotic cells were selectively located around the optic nerve head in the retinal neuroepithelium of 2- to 6-day-old chick embryos. TUNEL-positive cells and cells which were immunostained for activated caspase-3 showed overlapping distributions suggesting that caspase-3 is involved in the early retinal cell death. Caspase-3 involvement in early retinal cell death was also demonstrated by in vivo treatment with caspase inhibitors z-DEVD-fmk and Boc-D-fmk. After 6 h of treatment, the number of TUNEL-positive cells was reduced by 50%. Sustained treatments (20 h) resulted in a slight widening in the central part of the neural retina but the retinal ganglion cell axons maintained their organization and navigation towards the optic fissure. The most prominent result after inhibition of cell death was an increase in the number of retinal ganglion cells which also produced an enlargement of the ganglion cell layer and an increased number of ganglion cell axons. In conclusion, our results show that caspase-dependent programmed cell death occurs in the embryonic chick retina and that it plays a role to modulate the generation of retinal ganglion cells.     

Programmed cell death in the neurulating embryo is prevented by the chaperone heat shock cognate 70

Neuronal cell death is a genuine developmental process, with precise regulation and defined roles. In striking contrast, characterization of cell death that occurs at early stages of neural development is very limited. We previously showed that embryonic proinsulin increases the level of the chaperone heat shock cognate 70 (Hsc70) and reduces the incidence of apoptosis in the neurulating chick embryo [de la Rosa, et al. (1998), Proc. Natl. Acad. Sci. USA, 95, 9950]. We now demonstrate that Hsc70 is directly involved in cell survival during neurulation, as specific downregulation of endogenous Hsc70 by antisense oligodeoxynucleotide interference provoked an increase in apoptosis both in vitro and in ovo. In parallel, activation of caspase-3 was increased after hsc70 antisense oligodeoxynucleotide treatment. Dead cells were located mostly in the developing nervous system, distributed in areas where the incidence of cell death was high. These areas coincided both in vivo and under different death-inducing conditions, including antisense interference and growth factor deprivation. Hsc70 immunostaining was strong in at least some areas of high cell death. Apoptotic cells within these areas presented undetectable Hsc70 levels, however, suggesting that this protein acts as an intrinsic protector of neuroepithelial and neural precursor cells.     

Hypoxia-induced apoptotic cell death is prevented by oestradiol via oestrogen receptors in the developing central nervous system

The neuroprotective effects of oestrogens have been demonstrated against a variety of insults, including excitotoxicity, oxidative stress and cerebral ischemia under certain conditions. However, the molecular mechanisms underlying oestrogen neuroprotection are still unclear. We aimed to determine whether 17beta-oestradiol (E(2)) administration post-hypoxia (p-hx) was neuroprotective and whether these actions were mediated through oestrogen receptors (ER). For this purpose, 12-embyonic day-old chickens were subjected to acute hypoxia [8% (O(2)), 60 min], followed by different reoxygenation periods. To test the neuroprotective effect of E(2) and its mechanism, embryos were injected 30 min after the end of hypoxia with E(2) alone or with ICI 182 780, a competitive antagonist of ER. Cytochrome c (cyt c) release, an indicator of mitochondrial apoptotic pathway, was measured by western blot in optic lobe cytosolic extracts. DNA fragmentation by TUNEL fluorescence and caspase-3 fragmentation by immunofluorescence were detected on optic lobe sections. Acute hypoxia produces a significant increase in cyt c release from mitochondria at 4 h p-hx, followed by an increase in TUNEL positive cells 2 h later (6 h p-hx). Administration of E(2) (0.5 mg/egg) produced a significant decrease in cytosolic cyt c levels at 4 h p-hx, in caspase-3 activation and in TUNEL positive cells at 6 h p-hx compared to vehicle treated embryos. In the E(2)-ICI 182 780 treated embryos, cyt c release, caspase-3 fragmentation and TUNEL positive cells were similar to the hypoxic embryos, thus suggesting the requirement of an E(2)-ER interaction for E(2) mediated neuroprotective effects. In conclusion, E(2) prevents hypoxia-induced cyt c release and posterior cell death and these effects are mediated by oestrogen receptors.     

Oxygen modulates cell death in the proliferating retina

Many factors probably regulate the process of natural cell death during development. It is present in both the early undifferentiated retina and later following differentiation. Melanin production plays a role in regulating retinal development and when it is absent, cell proliferation and death are enhanced. Here we examine the effects of hyperoxia on this process, as oxygen has been shown to reduce cell death among differentiated photoreceptors late in development. However, in this study we examine its effects much earlier in pigmented and albino pigmentation phenotypes, when most cells are still actively dividing and are not committed to a specific fate. Newborn mice were exposed to high oxygen levels for 24 h and then returned to normal air for varying periods and their retinae examined. Hyperoxia had a dramatic effect on the number of dying cells, reducing them by almost 60% in pigmented animals and by over 80% in albinos. Following the return to normal air there was a gradual increase in their number over 360 min back to normal levels in pigmented mice; however, in albinos there was a complete rebound in levels of cell death within 40 min, reflecting the increased metabolic stress present in albino retinae due to their abnormal levels of proliferation. These results highlight the important role played by oxygen during early natural cell death in the retina and reveal the different developmental conditions present in the retinae of the two pigmentation phenotypes examined.     

Survival and death of mature avian motoneurons in organotypic slice culture: trophic requirements for survival and different types of degeneration

We have developed an organotypic culture technique that uses slices of chick embryo spinal cord, in which trophic requirements for long-term survival of mature motoneurons (MNs) were studied. Slices were obtained from E16 chick embryos and maintained for up to 28 days in vitro (DIV) in a basal medium. Under these conditions, most MNs died. To promote MN survival, 14 different trophic factors were assayed. Among these 14, glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor were the most effective. GDNF was able to promote MN survival for at least 28 DIV. K(+) depolarization or caspase inhibition prevented MN death but also induced degenerative-like changes in rescued MNs. Agents that elevate cAMP levels promoted the survival of a proportion of MNs for at least 7 DIV. Examination of dying MNs revealed that, in addition to cells exhibiting a caspase-3-dependent apoptotic pattern, some MNs died by a caspase-3-independent mechanism and displayed autophagic vacuoles, an extremely convoluted nucleus, and a close association with microglia. This organotypic spinal cord slice culture may provide a convenient model for testing conditions that promote survival of mature-like MNs that are affected in late-onset MN disease such as amyotrophic lateral sclerosis.     

Neurotrophic effects of transferrin on embryonic chick brain and neural retina cell cultures

The viability and differentiation promoting effects of various transferrins [iron-saturated (holo) and iron-depleted (apo) human and chick ovo (conalbumin)-transferrins, and bovine apo-transferrin] were studied, using serum-free, flat-sedimented cell cultures of embryonic chick brain and neural retina. The effects of transferrin (Tf) on the cell cultures depended on the type of Tf used and the parameter measured.Significant differences between brain and neural retina cultures in the effects of apo-ovoTf and iron [supplemented as ammonium-iron (III) citrate] were detected. Maximal levels of mitochondrial activity were observed in the presence of 2 mg/l apo-ovoTf in neural retina cell cultures. In brain cell cultures, 40 mg ovoTf/l were needed to achieve maximal levels. In brain, but not in neural, retina cell cultures ovoTf and optimal concentrations of Fe³⁺ exhibited similar effects on biochemical parameters of cell function and differentiation. Although, in the absence of ovoTf, neuronal outgrowth on areas not covered by glial cells was inhibited in both cell cultures, the differences were more prominent in neural retina cell cultures. Our data strongly suggest that Tf plays a key role in processes not connected directly with its iron transport capability.     

Heterogeneity of muscarinic cholinergic receptors in the developing chick embryo retina

Heterogeneity of muscarinic cholinergic receptors was investigated in chick embryo retina throughout development and in chicks immediately after hatching. The presence of a homogeneous receptor population was evidenced by antagonist binding. The affinity of antagonists increased up to day 14 of incubation, when synaptogenesis occurs. After this stage, it remained substantially unchanged. The number of receptors increased in embryos until hatching. On the contrary, agonists, such as acetylcholine and carbachol, bound to two (high- and low-affinity) binding sites. Through development, the affinity of both significantly increased until day 14, further substantiating the hypothesis of a maturation of the receptor pattern which precedes synapse formation. Muscarinic cholinergic binding seems to identify 3 critical steps in retinal neuronal development. The first is between 7 and 9 days of incubation, the second when synaptogenesis occurs and the third after initiation of function.     

Emigration of neuroepithelial cells from the hindbrain neural tube in the chick embryo

It is generally believed that after the emigration of neural crest, the neuroepithelial cells of the neural tube are committed to differentiate only as neurons and supporting cells of the central nervous system. Neural crest cells arise from the dorsal portion of the developing neural tube and contribute to the formation of the peripheral nervous system and a variety of non-neural structures. In contrast to this view we have recently shown, by focal application of the vital dye Dil in duck embryos, that an additional population of cells emigrates from the neural tube. By using an entirely different technique we confirm and extend these observations in the chick embryo. Replication-deficient retroviral vector LZ12 containing the gene LacZ was utilized to label the neural tube cells. The viral concentrate was microinjected into the lumen of the rostral hind-brain neural tube, considerably after the completion of emigration of neural crest cells. The labeled cells were monitored in whole mounts and histological sections. Initially, the labeled cells were restricted to the neuroepithelium of the hindbrain neural tube. Subsequently, they were seen in the neural tube and in the ganglion of the fifth cranial nerve (trigeminal ganglion). Later, they migrated beyond the trigeminal ganglion, i.e., into the mesenchyme of the first pharyngeal arch. Immunostaining with the neural crest cell marker, HNK-1, indicated that the emigrated neuroepithelial cells were HNK-1 negative. It is concluded that in the chick embryo some neuroepithelial cells emigrate at the site of attachment of the trigeminal nerve, migrate into the ganglion and then into the mesenchyme of the first arch. This cell population differs antigenically from the neural crest cells.     

Induction of cell growth by insulin and insulin-like growth factor-I is associated with jun expression in the otic vesicle

The present report investigates the cellular mechanisms involved in the regulation of cell proliferation by insulin and insulin-like growth factor-I (IGF-I) in the developing inner ear. The results show that insulin and IGF-I stimulate cell proliferation in the otic vesicle. This effect is associated with the induction of the expression of the nuclear proto-oncogene c-jun. The temporal profile of Jun expression coincided with the proliferative period of growth of the otic vesicle. IGF-I promoted the hydrolysis of a membrane glycosyl-phosphatidylinositol, which was characterised as the endogenous precursor for inositol phosphoglycan (IPG). Both purified IPG and a synthetic analogue, 6-O-(2-amino-2-deoxy-α-D-glucopyranosyl)-D-myo-inositol-1,2-cyclic phosphate (C3), were able to mimic the effects of IGF-I on Jun expression. Anti-IPG antibodies blocked the effects of IGF-I, which were rescued by the addition of IPG or its analogue. These results suggest that the sequence involving the hydrolysis of membrane glycolipids and the expression of c-jun and c-fos proto-oncogenes is part of the mechanism that activates cell division in response to insulin and IGF-I during early organogenesis of the avian inner ear. The implications of these observations for otic development and regeneration are briefly discussed. J. Comp. Neurol. 398:323–332, 1997. © 1998 Wiley-Liss, Inc.     

Pathfinding and growth termination of primary trigeminal sensory afferents in the embryonic rat hindbrain

Axons of the trigeminal ganglion convey sensory information from mechanoreceptors, thermoreceptors, and nociceptors in the face and nasal mucosa, then terminate on several groups of neurons including the principal sensory nucleus and the nuclei of the spinal trigeminal tract. To understand guidance mechanisms during the development of trigeminal sensory axons (TA) in the embryonic brain, we first investigated the growth pattern of TA in relation to organization in the hindbrain using flat whole-mount preparation from rat. We found that the primary TA from the trigeminal ganglion entered the brainstem and grew longitudinally within the hindbrain. Whereas descending axons ran just medial to the primary vestibular axons to innervate the spinal nucleus, ascending axons stayed near the entry point. In flat whole-mount culture, the TA extended both ascending and descending branches as they do in vivo. Rostral hindbrain was found to be a less permissive substrate for the TA compared to caudal hindbrain. In addition, the nonpermissive property of the ventral hindbrain substrate restricted the invasion of TA along the entire length of the hindbrain. Thus, cooperation of absolute and relative permissiveness of the substrate plays important roles in the guidance of TA to their targets.     

Inhibition of cell death results in hyperganglionosis: implications for enteric nervous system development

Abstract  The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC) that delaminate from the neural tube and undergo extensive migration and proliferation in order to colonize the entire length of the gut and differentiate into many millions of neurons and glial cells. Although apoptotic programmed cell death is an essential physiological process during development of the majority of the vertebrate nervous system, apoptosis within early ENS development has not been comprehensively investigated. The aim of this study was to determine the presence and extent of apoptosis within the vagal NCC population that gives rise to most of the ENS in the chick embryo. We demonstrated that apoptotic cells, as shown by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and active caspase-3 immunoreactivity, are present within an electroporated green fluorescent protein (GFP) and human natural killer-1 (HNK-1) immunopositive NCC population migrating from the vagal region of the neural tube to the developing foregut. Inhibition of caspase activity in vagal NCC, by electroporation with a dominant-negative form of caspase-9, increased the number of vagal NCC available for ENS formation, as shown by 3-dimensional reconstruction of serial GFP or HNK-1 labelled sections, and resulted in hyperganglionosis within the proximal foregut, as shown by NADPH-diaphorase whole gut staining. These findings suggest that apoptotic cell death may be a normal process within the precursor pool of pre-enteric NCC that migrates to the gut, and as such it may play a role in the control of ENS formation.     

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.     

Neurogenic neuroepithelial and radial glial cells generated from six human embryonic stem cell lines in serum-free suspension and adherent cultures

The great potential of human embryonic stem (hES) cells offers the opportunity both for studying basic developmental processes in vitro as well as for drug screening, modeling diseases, or future cell therapy. Defining protocols for the generation of human neural progenies represents a most important prerequisite. Here, we have used six hES cell lines to evaluate defined conditions for neural differentiation in suspension and adherent culture systems. Our protocol does not require fetal serum, feeder cells, or retinoic acid at any step, to induce neural fate decisions in hES cells. We monitored neurogenesis in differentiating cultures using morphological (including on-line follow up), immunocytochemical, and RT-PCR assays. For each hES cell line, in suspension or adherent culture, the same longitudinal progression of neural differentiation occurs. We showed the dynamic transitions from hES cells to neuroepithelial (NE) cells, to radial glial (RG) cells, and to neurons. Thus, 7 days after neural induction the majority of cells were NE, expressing nestin, Sox1, and Pax6. During neural proliferation and differentiation, NE cells transformed in RG cells, which acquired vimentin, BLBP, GLAST, and GFAP, proliferated and formed radial scaffolds. gamma-Aminobutyric acid (GABA)-positive and glutamate positive neurons, few oligodendrocyte progenitors and astrocytes were formed in our conditions and timing. Our system successfully generates human RG cells and could be an effective source for neuronal replacement, since RG cells predominantly generate neurons and provide them with support and guidance.     

Extracellular potassium and the regulation of acetylcholine receptor synthesis in embryonic chick muscle cells

The effect of elevated extracellular potassium on acetylcholine receptor synthesis was studied in chick embryonic muscle cultures. At physiological ionic strength, potassium chloride, in the 3.3 to 50 mM range, gave rise to a complex dose-response curve whose prominent features are a considerable reduction of receptor appearance rate at 20 mM and a more than 2-fold increase at higher concentrations. The effect of potassium chloride on receptor synthesis appears to be fairly specific: neither was there a duplication of its effect by other electrolytes or solutes, nor did it alter total protein synthesis or receptor stability by more than 30% at any concentration tested; cellular acetylcholinesterase levels actually declined with increasing KCl concentrations. In order to explore the mechanism of the potassium effect, tetrodotoxin (10⁻⁶ M), veratridine (3 × 10⁻⁶ M), D-600 (1.6 × 10⁻⁵ M), and ryanodine (3 × 10⁻⁷ M) were tested in the presence of various concentrations of potassium. Sodium channel toxins as well as calcium effectors modified the potassium response. Based on these findings we propose that the effects of potassium are due to: (a) cessation of spontaneous muscle activity upon raising KCl from 3 to 10 mM; (b) depolarization of the muscle membrane and persistent activation of a calcium channel as concentration is raised from 10 to 20 mM; (c) finally, inactivation or desensitization of the calcium channel, or some other signaling element proximal to the sarcoplasmic reticulum, upon further depolarization.     

人胚中脑神经干细胞增殖能力的研究

目的探讨神经球中细胞的分裂增殖能力。方法自孕13周人胚中脑腹侧分离出未成熟细胞,培养形成神经球,并对其进行扩增。应用单细胞显微操作法对神经球的细胞进行克隆分析;应用流式细胞仪测定神经球细胞的分裂增殖能力。结果大约在接种后7～10d,神经球开始形成;神经球细胞克隆形成率为1.26%±0.33%;流式细胞仪测定神经球中处于有丝分裂期的细胞占细胞总数的10.05%±1.43%。结论自人胚中脑腹侧组织中可以分离出形成神经球的细胞,这些神经球中的细胞具有一定的增殖能力。但并不是所有的细胞都具有增殖能力。     

Development of the human retina: patterns of cell distribution and redistribution in the ganglion cell layer

Neurogenesis in the ventricular layer and the development of cell topography in the ganglion cell layer have been studied in whole-mounts of human fetal retinae. At the end of the embryonic period mitotic figures were seen over the entire outer surface of the retina. By about 14 weeks gestation mitosis had ceased in central retina and differentiation of photoreceptor nuclei was evident within a well-defined area which constituted about 2% of total retina area. This area was approximately centered on the site of the putative fovea, identified by the exclusive development of cone nuclei at that location. The area of retina in which mitosis had ceased increased as gestation progressed. By mid-gestation mitosis in the ventricular layer occupied about 77% of the outer surface of the retina and by about 30 weeks gestation mitosis in the ventricular layer had ceased. Cell density distributions in the ganglion cell layer were nonuniform at all stages studied (14-40 weeks). Densities were highest at about 17 weeks gestation, and by mid-gestation the adult pattern of cell topography was present with maps showing elevated cell densities in posterior retina and along the horizontal meridian. Cell densities generally declined throughout the remainder of the gestation period, except in the posterior retina, where densities in the perifoveal ganglion cell layer remained high during the second half of gestation. There is a rapid decline in cell density in the foveal ganglion cell layer toward the end of gestation, and it is suggested that the persistence of high densities in the perifoveal region may be related to migration of cells away from the developing fovea. The total population of cells in the ganglion cell layer was highest (2.2-2.5 million cells) between about weeks 18 and 30 of gestation. After this the cell population declined rapidly to 1.5-1.7 million cells. It is suggested that naturally occurring neuronal death is largely responsible for this decline.     

Radial migration in the cerebral cortex is enhanced by signals from thalamus

The six layered cerebral cortex derives from cells that divide in the ventricular zone and migrate to their final destination in the cortical plate (future cortex). In the mouse, cortical layer III and IV neurons undergo their final mitotic division at around E16, at which time thalamic axons are beginning to enter the cortex. We used bromodeoxyuridine-birth dating of cells in cortical slice cultures to show that the thalamus enhances the migration out of the ventricular zone of future layer III/IV cells. When cortical slices were cultured alone, less than 35% of cells born in vitro on E16 were present in the pial half of the slice after 48 h in culture. In contrast, when cortical slices were cocultured with thalamus, 69% of these cells were found in the pial half of the slice. Explants of other developing tissues did not mimic the effect of the thalamus. The thalamus had no obvious effect on cortical radial glial cells, cortical cell viability or maintenance of cortical slice structure. We found that most precursors born at a similar age but in vivo, shortly before cortical slices were isolated, migrated to the pial half of the cultured slices in the absence of a cocultured thalamic explant. Thus, E16 cortical slices cultured without thalamus permit migration of cells born in vivo and therefore already exposed to the thalamus. Our results indicate that the thalamus provides factors to E16-born cortical precursors that enhance their directed migration out of the ventricular zone to the cortical plate.     

Multiple-site optical recording reveals embryonic organization of synaptic networks in the chick spinal cord

We examined embryonic expression of postsynaptic potentials in stages 26-31 (E5 to E7) chick spinal cord slices. Slow optical signals related to the postsynaptic potentials which were evoked by electrical stimulation of afferent fibers were identified in the dorsal grey matter and the ventral motoneuronal area. In cervical spinal cord (C13) preparations, the dorsal slow signal appeared from stage 28 (E6), whilst the ventral slow signal was recognized from stage 29. At stages 26 and 27 (E5), no slow signal was observed in either the dorsal or ventral regions. On the other hand, in lumbosacral spinal cord (LS5) preparations, the dorsal, as well as ventral, slow signals appeared from stage 29; at stage 28 no slow signal was detected in the dorsal or ventral regions. These results suggest that there are differences in the ontogenetic expression of synaptic functions between the dorsal and ventral regions, and between the cervical and lumbosacral spinal cords. In embryos older than stage 29, removal of Mg2+ from the bathing solution markedly enhanced the amplitude and incidence of the ventral slow signal. In addition, in C13 preparations at stage 28, removal of Mg2+ elicited small slow signals in the ventral region in which no synaptic response was evoked in normal Ringer's solution. The slow signals induced in the Mg2+-free solution were blocked by 2-amino-5-phosphonovaleric acid (APV), showing that they are attributable to N-methyl- D-aspartate (NMDA) receptors. These results suggest that functional synaptic connections via polysynaptic pathways are already generated on motoneurons, but are suppressed by a Mg2+ block on the NMDA receptors at developmental stages when synaptic transmission from the primary afferents to the dorsal interneurons is initially expressed in the dorsal region.