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Orchestrated proliferation, differentiation, and cell death contribute to the generation of the complex cytoarchitecture of the central nervous system, including that of the neuroretina. However, few studies have comprehensively compared the spatiotemporal patterns of these 3 processes, or their relative magnitudes. We performed a parallel study in embryonic chick and mouse retinas, focusing on the period during which the first neurons, the retinal ganglion cells (RGCs), are generated. The combination of in vivo BrdU incorporation, immunolabeling of retinal whole mounts for BrdU and for the neuronal markers Islet1/2 and β III-tubulin, and TUNEL allowed for precise cell scoring and determination the spatiotemporal patterns of cell proliferation, differentiation, and death. As predicted, proliferation preceded differentiation. Cell death and differentiation overlapped to a considerable extent, although the magnitude of cell death exceeded that of neuronal differentiation. Precise quantification of the population of recently born RGCs, identified by BrdU and β III-tubulin double labeling, combined with cell death inhibition using a pan-caspase inhibitor, revealed that apoptosis decreased this population by half shortly after birth. Taken together, our findings provide important insight into the relevance of cell death in neurogenesis.

Programmed cell death is a genuine developmental process of the nervous system, affecting not only projecting neurons but also proliferative neuroepithelial cells and young neuroblasts. The embryonic chick retina has been employed to correlate in vivo and in vitro studies on cell death regulation. We characterize here the role of two major signaling pathways, PI3K-Akt and MEK-ERK, in controlled retinal organotypic cultures from embryonic day 5 (E5) and E9, when cell death preferentially affects proliferating neuroepithelial cells and ganglion cell neurons, respectively. The relative density of programmed cell death in vivo was much higher in the proliferative and early neurogenic stages of retinal development (E3-E5) than during neuronal maturation and synaptogenesis (E8-E19). In organotypic cultures from E5 and E9 retinas, insulin, as the only growth factor added, was able to completely prevent cell death induced by growth factor deprivation. Insulin activated both the PI3K-Akt and the MEK-ERK pathways. Insulin survival effect, however, was differentially blocked at the two stages. At E5, the effect was blocked by MEK inhibitors, whereas at E9 it was blocked by PI3K inhibitors. The cells which were found to be dependent on insulin activation of the MEK-ERK pathway at E5 were mostly proliferative neuroepithelial cells. These observations support a remarkable specificity in the regulation of early neural cell death., (2007 Wiley Periodicals, Inc)

We analyzed whether the embryonic (E12.5-E14.5) mouse retina possesses genuine neural stem cells and how they respond to defined growth factors and extracellular matrix molecules. Whereas most combinations produced no or limited cell survival and proliferation in culture, FGF-2 plus heparin and laminin stimulated proliferation and the formation of aggregates composed, after two days, of 95.2% nestin-positive cells. However, cells in these aggregates could only be passaged poorly, lost nestin expression and proliferative capacity, and differentiated into neurons. Under the same conditions, olfactory bulb precursor cells divided efficiently and could be expanded. These data suggest that, in addition to FGF-2 and laminin, embryonic retinal neuroepithelial cells need additional extrinsic and/or intrinsic regulators to maintain cell proliferation and self-renewal.

Programmed cell death is an essential, highly regulated process in neural development. Although the role of insulin-like growth factor I in supporting the survival of neural cells has been well characterized, studies on proinsulin/insulin are scarce. Here, we characterize proinsulin/insulin effects on cell death in embryonic day 15.5 mouse retina. Both proinsulin mRNA and proinsulin/insulin immunoreactivity were found in the developing retina. Organotypic embryonic day 15.5 retinas cultured under growth factor deprivation showed an increase in cell death that was reversed by proinsulin, insulin and insulin-like growth factor I, with similar median effective concentration values via phosphatidylinositol-3-kinase activation. Although insulin and insulin-like growth factor I provoked a sustained Akt phosphorylation, proinsulin-induced phosphorylation of Akt was not found. Analysis of the growth factor deprivation-induced cell death mechanisms, using caspase and cathepsin inhibitors, demonstrated that both protease families were required for the effective execution of cell death. The insulin survival effect, which decreased the extent and distribution of cell death to levels similar to those found in vivo, was not enhanced by simultaneous treatment with caspase and cathepsin inhibitors, suggesting that insulin interferes with these protease pathways in the embryonic mouse retina. The mechanisms characterized in this study provide new details on early neural cell death and its genuine regulation by insulin/proinsulin.

Transforming growth factor (TGF)-beta and insulin display opposite effects in regulating programmed cell death during vertebrate retina development; the former induces apoptosis while the latter prevents it. In the present study we investigated coordinated actions of TGF-beta and insulin in an organotypic culture system of early postnatal mouse retina. Addition of exogenous TGF-beta resulted in a significant increase in cell death whereas exogenous insulin attenuated apoptosis and was capable of blocking TGF-beta-induced apoptosis. This effect appeared to be modulated via insulin-induced transcriptional down-regulation of TGF-beta receptor II levels. The analysis of downstream signalling molecules also revealed opposite effects of both factors; insulin provided survival signalling by increasing the level of anti-apoptotic Bcl-2 protein expression and phosphorylation and down-regulating caspase 3 activity whereas pro-apoptotic TGF-beta signalling reduced Bcl-2 mRNA levels and Bcl-2 phosphorylation and induced the expression of TGF-induced immediate-early gene (TIEG), a Krüppel-like zinc-finger transcription factor, mimicking TGF-beta activity.

The signaling cascade Ras/Raf/mitogen-activated protein kinases modulates cell proliferation, differentiation, and survival, all key cellular processes during neural development. To better define the in vivo role of Raf during chick retinal neurogenesis, we interfered with Raf-dependent signaling during days 4.5 to 7.5 of embryonic development by expressing a dominant negative mutant of c-Raf (DeltaRaf), which blocks Ras-dependent Raf activation, and by overexpressing wild-type c-Raf. DeltaRaf expression induced an increase in cell death by apoptosis, whereas it did not affect overall cell proliferation and differentiation. In parallel, the number of Islet-1/2-positive and TUJ1-positive retinal ganglion cells were diminished in their definitive layer, whereas there was an increase in the number of mislocated Islet-1/2-positive cells. This disturbed morphogenesis correlated with a disruption of the optic fiber layer. Conversely, c-Raf overexpression caused moderate opposite effects on apoptosis. These results frame in vivo early neurogenesis processes in which c-Raf is essential.

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.

Regulated preproinsulin gene expression in nonpancreatic tissues during development has been demonstrated in rodents, Xenopus and chicken. Little is known, however, about the synthesis and processing of the primary protein product, proinsulin, in comparison with these events in pancreas. Using specific antisera and immunocytochemistry, immunoblot and HPLC criteria, we characterize the differential processing of proinsulin in developing neuroretina, liver and pancreas. The chick embryo pancreas expresses the convertase PC2, and largely processes proinsulin to insulin. In contrast, little or no mature PC2 is present in embryonic liver and neuroretina and the (pro)insulin immunoactivity identified is predominantly proinsulin.

While the role of heat shock proteins under experimental stress conditions is clearly characterized, their expression in unstressed cells and tissues and their functions in normal cell physiology, besides their chaperone action, remain largely undetermined. We report here the identification in chicken of the antigen recognized by the monoclonal antibody PM1 [Hernández-Sánchez et al. (1994) Eur. J. Neurosci., 6,1801-1810] as the noninducible chaperone heat-shock cognate 70 (Hsc70). Its identity was determined by partial peptide sequencing, immuno-crossreactivity and two-dimensional gel-electrophoresis. In addition, we examined its expression during chick embryo retinal neurogenesis. The early widespread Hsc70 immunostaining corresponding to most, if not all, of the neuroepithelial cells becomes restricted to a subpopulation of these cells in the peripheral retina as development proceeds. On the other hand, retinal ganglion cells, differentiating in the opposite central-to-peripheral gradient, retained Hsc70 immunostaining. Other molecular chaperones, the heat-shock proteins Hsp40, Hsp60 and Hsp90, did not seem to compensate the loss of Hsc70. They also showed decreasing immunostaining patterns as neurogenesis proceeds, although distinctive from that of Hsc70, whereas Hsp70 was not detected in the embryonic retina. This precise cellular and developmental regulation of Hsc70, a generally considered constitutive molecular chaperone, in unstressed embryos, together with the expression of other chaperones, provides new tools and a further insight on neural precursor heterogeneity, and suggests possible specific cellular roles of chaperone function during vertebrate neurogenesis.

Early neurogenesis progresses by an initial massive proliferation of neuroepithelial cells followed by a sequential differentiation of the various mature neural cell types. The regulation of these processes by growth factors is poorly understood. We intend to understand, in a well-defined biological system, the embryonic chicken retina, the role of the insulin-related growth factors in neurogenesis. We demonstrate the local presence of signaling elements together with a biological response to the factors. Neuroretina at days 6-8 of embryonic development (E6-E8) expressed proinsulin/insulin and insulin-like growth factor I (IGF-I) mRNAs as well as insulin receptor and IGF type I receptor mRNAs. In parallel with this in vivo gene expression, E5 cultured neuroretinas synthesized and released to the medium a metabolically radiolabeled immunoprecipitable insulin-related peptide. Furthermore, insulin-related immunoreactive material with a HPLC mobility close to that of proinsulin was found in the E6-E8 vitreous humor. Exogenous chicken IGF-I, human insulin, and human proinsulin added to E6 cultured neuroretinas showed relatively close potencies stimulating proliferation, as determined by [methyl-3H]thymidine incorporation, with a plateau reached at 10(-8) M. These factors also stimulated neuronal differentiation, indicated by the expression of the neuron-specific antigen G4. Thus, insulin-related growth factors, interestingly including proinsulin, are present in the developing chicken retina and appear to play an autocrine/paracrine stimulatory role in the progression of neurogenesis.

To better understand the role of insulin-related growth factors in neural development, we have characterized by in situ hybridization in chicken embryonic retina the patterns of gene expression for insulin, insulin-like growth factor I (IGF-I), their respective receptors and the IGF binding protein 5 (IGFBP5) from early stages (E6) until late stages (E18)--an analysis not performed yet in any species. In addition, we studied the effect of insulin and IGF-I on cultured neuroepithelial cells. Insulin receptor mRNA and IGF-I receptor mRNA were both present and showed a similar, widespread pattern throughout retina development. Insulin mRNA could be detected only by reverse transcription coupled to polymerase chain reaction. IGF-I mRNA was concentrated in the ciliary processes and extraocular muscles early in development (embryonic day 6; E6) and in maturing retinal ganglion cells subsequently (E9-15). IGFBP5 mRNA was preferentially localized in the more differentiated central retinal zone and was maximally concentrated in the inner nuclear and ganglion cell layers at E9. These findings suggest a near constitutive expression of insulin receptor and IGF-I receptor genes, while IGF-I and IGFBP5 showed a highly focal spatiotemporal regulation of gene expression. Insulin and IGF-I, already at 10(-8) M, increased the proportion of PM1-positive neuroepithelial cells found in E5 retinal cultures without affecting significantly the total number of proliferating cells. Together, these data support the finding that, during early neurogenesis in chicken retina, insulin and IGF-I have a specific paracrine/autocrine action. This action, as well as possible effects elicited subsequently, may be dictated by restricted-local synthesis of the ligands and limited access to the factors contained in the vitreous humour. In the case of IGF's role, local IGFBPs expression can contribute to the fine modulation.

Transforming growth factor (TGF)-β and insulin display opposite effects in regulating programmed cell death during vertebrate retina development; the former induces apoptosis while the latter prevents it. In the present study we investigated coordinated actions of TGF-β and insulin in an organotypic culture system of early postnatal mouse retina. Addition of exogenous TGF-β resulted in a significant increase in cell death whereas exogenous insulin attenuated apoptosis and was capable of blocking TGF-β-induced apoptosis. This effect appeared to be modulated via insulin-induced transcriptional down-regulation of TGF-β receptor II levels. The analysis of downstream signalling molecules also revealed opposite effects of both factors; insulin provided survival signalling by increasing the level of anti-apoptotic Bcl-2 protein expression and phosphorylation and down-regulating caspase 3 activity whereas pro-apoptotic TGF-β signalling reduced Bcl-2 mRNA levels and Bcl-2 phosphorylation and induced the expression of TGF-induced immediate-early gene (TIEG), a Krüppel-like zinc-finger transcription factor, mimicking TGF-β activity. [ABSTRACT FROM AUTHOR]