Ana I. Valenciano

Early neural cell death: numbers and cues from the developing neuroretina

Programmed cell death is a well established key process required for proper development of the nervous system. The regulatory and executor mechanisms controlling survival/death of projection neurons, as well as of other types of differentiated neurons and glial cells, have been studied intensely during neural development. Much less attention has been paid to earlier cell death events affecting neuroepithelial cells and recently born neurons and glial cells. We review here the reports on cell death during vertebrate retina development, our model system for many years, which has provided clear evidence of the importance of early neural cell death. We tentatively categorize the available observations in three death phases, namely morphogenetic cell death, early neural cell death and neurotrophic cell death. The magnitude and the precise regulation of the early phases of cell death are fully comparable to the much better characterized neurotrophic cell death. Therefore, early neural cell death deserves a profound dedicated study; this will help to obtain an integrated understanding of the development of the retina and other parts of the vertebrate nervous system.

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.

Cell death in the nervous system: Lessons from insulin and insulin-like growth factors

Programmed cell death is an essential process for proper neural development. Cell death, with its similar regulatory and executory mechanisms, also contributes to the origin or progression of many or even all neurodegenerative diseases. An understanding of the mechanisms that regulate cell death during neural development may provide new targets and tools to prevent neurodegeneration. Many studies that have focused mainly on insulin-like growth factor-I (IGF-I), have shown that insulin-related growth factors are widely expressed in the developing and adult nervous system, and positively modulate a number of processes during neural development, as well as in adult neuronal and glial physiology. These factors also show neuroprotective effects following neural damage. Although some specific actions have been demonstrated to be anti-apoptotic, we propose that a broad neuroprotective role is the foundation for many of the observed functions of the insulin-related growth factors, whose therapeutical potential for nervous system disorders may be greater than currently accepted.

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.

Balance of pro‐apoptotic transforming growth factor‐β and anti‐apoptotic insulin effects in the control of cell death in the postnatal mouse retina

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.

Programmed cell death in the neurulating embryo is prevented by the chaperone Hsc70

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.

Biotin decreases retinal apoptosis and induces eye malformations in the early chick embryo

Proliferation, cell death and differentiation occur simultaneously in developing retina and are precisely orchestrated. We have studied the effects of biotin (vitamin H) on early retinal development. In vivo administration of biotin to early embryonic chick eyes at moderately elevated levels induced malformations, affecting retina and lens structures. The effects were strictly age dependent and were only found in embryos treated between Hamburger and Hamilton stage 14–17. Biocytin, a biotin analogue, mimicked biotin effects, while avidin could block the effects. At the cellular level, biotin did not affect proliferation but reduced apoptosis. These results suggest that an adequate content of biotin and a precise regulation of retinal cell death are required for the correct morphogenesis of the eye.

The regulated expression of chimeric tyrosine hydroxylase–insulin transcripts during early development

Biological complexity does not appear to be simply correlated with gene number but rather other mechanisms contribute to the morphological and functional diversity across phyla. Such mechanisms regulate different transcriptional, translational and post-translational processes and include the recently identified transcription induced chimerism (TIC). We have found two novel chimeric transcripts in the chick and quail that result from the fusion of tyrosine hydroxylase (TH) and insulin into a single mature transcript. The th and insulin genes are located in tandem and they are generally transcribed independently. However, it appears that two chimeric transcripts containing exons from both the genes can also be produced in a regulated manner. The TH–INS1 and TH–INS2 chimeras differ in their insulin gene content, and they encode two novel isoforms of the TH protein with markedly reduced functionality when compared with the canonical TH. In addition, the TH–INS1 chimeric mRNA generates a small amount of insulin. We propose that TIC is an additional mechanism that can be employed to further regulate TH and insulin expression according to the specific needs of developing vertebrates.