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VOL. 73 (4), 1031-1045, 2007 EARLY NEURAL CELL DEATH: AN OVERLOOKED...

vertebrate development. Genetic inactivation of both proapoptotic
and prosurvival regulatory molecules, as well as apoptotic executor
molecules, causes embryonic or perinatal lethality associated with
severe malformations that mostly affects the nervous system (see 9
for a detailed review). Interestingly, morphological defects were
already visible at early stages of neural development, concomitant
with neurulation and the first waves of neurogenesis.

Embryonic manipulation in the chick is a classic approach that
has been employed to characterize how cell death is regulated (19-24).
We have focused on survival factors as attenuators of early neural cell
death, and in particular proinsulin/insulin (25). In ovo treatment of
chick embryos with insulin-receptor blocking antibodies (20), or with
antisense oligonucleotides (ODNs) (21), augments apoptotic cell
death (Figure 3, A-B). In the retina, the increase in apoptosis parallels
a decrease in the number of retinal ganglion cells (RGCs) (Figure 3,
C-D). Conversely, in ovo treatment with proinsulin reduces naturally
occurring cell death but induces embryonic malformations, including
asymmetric closure of the neuropore, collapse of the optic vesicles,
and bending of the neural tube (Figure 4).

We have further characterized the putative signalling pathway
downstream of proinsulin/insulin at different levels. Upon binding to
its tyrosine-kinase, membrane-bound receptor, proinsulin/insulin
activates both the PI3K/Akt and the Ras/Raf/MAP kinase signaling
cascades. Significantly, RGC survival is regulated by c-Raf in the
embryonic chick neuroretina (26) and retroviral transfection of a
dominant negative form of c-Raf increases PCD while reducing the
density of RGCs correctly situated in their layer (Figure 5).
Furthermore, proinsulin/insulin upregulates the expression of
HSC70, a chaperone involved in survival under conditions of cell
stress. Blocking proinsulin expression with antisense ODNs in
cultured neurulating chick embryos diminishes HSC70 expression
and increases cell death (27). Indeed, antisense ODN interference
experiments established a correlation between proinsulin-induced
cell survival and HSC70 expression, both in cultured chick embryos
and in ovo (23). Thus, HSC70 silencing increases caspase-3 activation
and apoptosis in those embryonic regions where PCD was already
naturally ongoing (Figure 6).

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