Genetics of Apoptosis

Using this assay, genetic screens were undertaken for mutations that perturbed the
overall level of apoptosis in the embryo (White et al., 1994). A collection of genomic
deletions representing about half of the genome was screened for defects in apoptosis.
In these screens, large numbers of embryos were collected from parents heterozygous
for a genomic deletion, and defects in apoptosis were analyzed in the 25% of the
progeny homozygous for the deletion. Although most of these deletions are large,
deleting many functions that are required for normal embryonic development, the
large supply of maternal gene products is generally sufficient to allow embryonic
development to proceed long enough to assess apoptosis.
The majority of deletion strains examined in this screen produced no quantitative
defects in apoptosis in homozygous embryos. Embryos homozygous for a number of
genomic deletions were found to have a significant increase in the overall level of
apoptosis. This may reflect the loss of antiapoptotic functions, such as DIAP1 (see
below). In addition, general defects in development can also result in widespread
apoptosis (see, for example, Pazdera et al., 1998).
Three overlapping genomic deletions completely eliminated apoptosis in
homozygous embryos (White et al., 1994). These homozygous embryos died at the
end of embryogenesis, and contained more cells than wild-type embryos. It is very
likely that the presence of extra cells in these embryos is a direct result of the lack of
apoptosis. However, since the deletions are relatively large and eliminate a number
of genes, embryonic lethality cannot be attributed directly to the lack of apoptosis.
Attempts were made to generate single-gene mutations that showed the same cell
death-defective phenotype as the deletion. This proved unsuccessful, as the loss of
more than one gene in the interval is necessary to see the cell death-defective
phenotype. Four genes, reaper (rpr) (White et al., 1994), head involution defective
(hid) (Grether et al., 1995), grim (Chen et al., 1996), and the newly identified sickle
(skl) (Christich et al., 2002; Srinivasula et al., 2002; Wing et al., 2002), have now
been identified in this genomic region. These genes lie clustered together, within
about 250 kb. The protein products of all four genes share a short stretch of homology
at the N-terminus, which is important for their function. Otherwise the genes are
fairly dissimilar. Deletions that remove only one of these genes have no or very weak
effects on embryonic apoptosis, while the removal of two has stronger effects (Grether
et al., 1995; Chen et al., 1996; Peterson et al., 2002). Deletion of the genes hid, rpr,
and grim together results in a complete block in embryonic apoptosis. A role for skl
in developmental apoptosis remains to be demonstrated.

3.

How Rpr, Hid, Grim, and SkI initiate apoptosis

Overexpression of Rpr, Hid, Grim, or SkI is sufficient to induce ectopic apoptosis
in some tissues of the developing fly and in mammalian and insect cultured cells
(Grether et al., 1995; Chen et al., 1996; Pronk et al., 1996; White et al., 1996; Evans
et al., 1997; Claveria et al., 1998; McCarthy and Dixit, 1998; Haining et al., 1999).
Apoptosis is induced on overexpression of Rpr, Hid, or Grim in embryos homozygous

186 GENETICS OF APOPTOSIS