Growth, Differentiation and Sexuality

tion occur. This meiotic process will provide the
genetic mechanism of random segregation and independent
assortment to create genetic diversity
among the progeny. The completion of meiosis
will result in production of sexual spores that can
be disseminated and can withstand harsh environmental
conditions, an important survival and life
preservation devise. In this regard, any genetic defect
or physiological damage during meiosis will
be detrimental to the survival of the species. Thus,
to overcome such a catastrophe and to prevent defective
vital genes from passing on, the evolution
of meiosis-specific PCD seems plausible.
Meiotic cells are diploid, and they are not as
easy to manipulate as are haploid cells when it
comes to studying mutations that affect meiotic
events. This complication is exacerbated by the
mating-type genes in the tetrapolar sexuality of
some basidiomycetes where selfing is incompatible
(see Casselton and Challen, Chap. 17, this volume).
To overcome this problem, a model system has been
found in a homokaryon AmutBmut of C. cinereus,
wheremutationinboththeA and the B matingtype
loci would eliminate the need of mating to
produce fruiting bodies, and to progress through
meiosis and sporulation (Swamy et al. 1984). In addition,
meiosis in C. cinereus is synchronous and
its progression is regulated by light–dark cycles
(Lu 1967, 2000). Thus, this homokaryon is well
suited for studies in meiotic events, including meiotic
apoptosis (Lu 1996; Celerin et al. 2000; Lu et al.
2003). With this strain, a large number of white-cap
mutants have been created at ETH Zürich, either by
restriction enzyme-mediated integration mutagenesis
(called REMI mutants) or by UV irradiation
(Granado et al. 1997; Kües et al., personal communication).
With a simple hematoxylin staining,
these white-cap mutants were discovered to exhibit
meiotic PCD (Lu and Kües 1999). Further investigation,
using light and electron microscopy, has
revealed that these white-cap mutants can be classified
into four cytologically distinguishable groups
– three show defects in the meiotic prophase I, one
shows defects in sporulation, and all four groups
exhibit basidia-specific PCD, with the hallmarks of
apoptosis (Lu et al. 2003).
Apoptosis in C. cinereus is basidia-specific. The
phenotypes are synchronous chromatin condensation
(Fig. 9.3A), DNA fragmentation as shown by
TUNEL assay (Fig. 9.3B), and cytoplasmic shrinkage
in basidia that are grossly deformed and DAPI
negative, while the neighboring paraphyses are perfectly
healthy, showing a bright nuclear stain with
Programmed Cell Death 181
Fig. 9.3. A–D Meiotic apoptosis in C. cinereus. A Synchronous
chromatin condensation (by DAPI stain)
associated with meiotic arrest at meta-anaphase I. B
TUNEL positive basidia. C Apoptotic (shrunken) basidia
(arrowed) are DAPI negative whereas the neighboring
paraphyses are DAPI positive. Reproduced from Lu et
al. (2003). D The end stage of apoptosis, showing very
shrunken basidia (arrowed) stained with propiono-iron
hematoxylin. Bar = 10 μm
DAPI (Fig. 9.3C). All these are associated with specific
meiotic arrest at metaphase–anaphase I. The
end stage can be demonstrated with a simple hematoxylin
stain (Fig. 9.3D). All meiotic mutants produce
few tetrads that somehow escaped death at the
end stage (see Lu et al. 2003). Some apoptotic phenotypeshavealsobeendocumentedinthespo11-1
mutant, whose identity is based on DNA sequence
similarity to yeast spo11 (Celerin et al. 2000). For
the sporulation mutants, apoptosis is triggered at
the tetrad stage (Lu et al. 2003).
Regardlessofthetimeofdefect,allmeiotic
mutations trigger apoptosis in C. cinereus at a single
entry point (Lu et al. 2003). Only when the arrest
of meiosis is abrogated to enter anaphase I is
apoptosis triggered. The formation of a spindle,
initially well formed and then broken down, has
been demonstrated in the mutant spo11-1 by using
the anti-tubuline antibody (Celerin et al. 2000).
These observations strongly suggest that entry into
themeioticapoptoticpathwayinC. cinereus is under
the metaphase spindle checkpoint control. This
is very different from the multiple checkpoint entries
found in mice (reviewed in Lu et al. 2003).
Thus, in this AmutBmut homokaryon, meiosis can