tube closure is seen in the hindbrain region at E10.5 of casp9-/- embryos. These
mutants also have enlarged proliferation zones in the fore- and midbrain, resulting
from reduced apoptosis, and causing expansion and protrusion of cranial tissues. casp3
mutant mice show a protrusion of brain tissue consisting of ectopic cell masses with
similarities to the cerebral cortex (Kuida et al., 1996; Woo et al., 1998). Increased
cell number and density of the brain stem in casp3-/- embryos, resulting from reduced
apoptosis, becomes apparent by E12. Protrusions of the neuroepithelium in the retina
are also seen as a result of reduced cell death in this tissue. These phenotypes indicate
that caspase-3 and -9 play nonredundant roles in brain development and that
caspase-3 is also required for normal eye development. In addition, cells from casp9-
and casp3-null mice show similar sensitivities to various apoptotic stimuli. For
example, embryonic stem (ES) cells from casp9 and casp3 mutants are resistant to
apoptosis induced by a range of apoptotic stimuli, including treatment by adriamycin,
etoposide, sorbitol, cisplatin, anisomycin, and UV irradiation (Hakem et al., 1998;
Kuida et al., 1998; Woo et al., 1998). ES cells from casp9 mutants are also resistant
to apoptosis induced by γirradiation whereas casp3-null ES cells are not. MEFs
derived from casp9-/- and casp3-/- mice are resistant to adriamycin and cytotoxic
lymphocyte-mediated apoptosis, and casp3 null MEFs are also resistant to TNF-α-
induced apoptosis while casp9 MEFs are not. A direct role for caspase-9 in activation
of caspase-3 has been demonstrated in casp9-/- mice (Hakem et al., 1998; Kuida et
al., 1998). Caspase-3 processing is deficient in casp9 mutant brain tissue, and lysates
from caspase-9-deficient thymocytes or embryonic brains are unable to process
caspase-3 even in the presence of cytochrome c and dATP. This activity can be
restored by the addition of in vitro translated caspase-9 to the lysates.
casp8 k/o in mice is embryonically lethal (Varfolomeev et al., 1998). casp8-/-embryos
are smaller than normal, display impaired heart muscle development and an
accumulation of erythrocytes in the abdomen and in blood vessels in the trunk area
at E1 1.5 and E12.5. Excessive erythrocytosis is also seen in the liver of mutants,
although the mutants contain reduced hemopoietic stem-cell numbers (Varfolomeev
et al., 1998). casp8-null fibroblasts do not undergo apoptosis in response to CD95
and DR3 ligation, although cell death induced by UV, cytotoxic drugs, vesicular
stomatitis virus, and serum deprivation is unaffected (Varfolomeev et al., 1998).
These results suggest that while caspase-8 plays nonredundant roles in normal
embryonic development and apoptosis mediated by the TNFR family members, it
is dispensable for other apoptotic pathways.
Evolutionarily, caspase-2 is one of the most conserved caspases and is highly related
to CED-3 and DRONC (Kumar et al., 1994; 1997). Caspase-2 is expressed in most
tissues and cell types and undergoes rapid activation in response to a variety of
apoptotic stimuli (Kumar et al., 1994; Harvey et al., 1997; O’Reilly et al., 2002).
Despite this, casp2-/- mice are grossly normal and survive to adulthood (Bergeron et
al., 1998; O’Reilly et al., 2002). casp2-/- females have an apparently increased number
of female germ cells, but these mice show normal fertility (Bergeron et al., 1998).
The thymocytes derived from casp2-null mice are normally sensitive to CD95-
mediated cell death, dexamethasone treatment, and γ-irradiation (Bergeron et al.,
46 GENETICS OF APOPTOSIS