Ning
Pan, Elizabeth Kimberly, Beryl Hatton, Manisha Shah Dudley, Ute Meisel, and
Ding Xue
CB
347, MCDB, University of Colorado, Boulder, CO 80309
In
C. elegans
131 of the 1090 cells generated during development of the hermaphrodite adult
animal undergo programmed cell death. We are interested in identifying new
genes involved in the life vs. death decisions of cells which normally die.
Specifically, we are interested in studying the signaling pathways that
regulate the deaths of hermaphrodite specific neurons (HSNs). HSNs in C. elegans control egg-laying in
hermaphrodite animals and normally undergo programmed cell death in males where
they are not needed. The life vs.
death decisions of the HSN neurons present an interesting paradigm for studying
sex-specific cell death specification.
Mutations
in several genes (her-1, tra-2,
and egl-1)
have been identified that cause inappropriate death of HSNs in
hermaphrodites. her-1 encodes a novel secreted
molecule, which promotes male differentiation, while tra-2 encodes a putative
transmembrane receptor for the HER-I protein. The egl-1 gene is generally required for
cell-killing in nematodes but is not involved in sex determination. Thus her-1 and tra-2 mediate a novel signal
transduction pathway that integrates into the cell-death pathway through the egl-1 gene to control HSN cell death.
In
order to identify new components in this signaling pathway, we have performed
several genetic screens to isolate suppressors of inappropriate HSN cell death
caused by loss-of-function mutations in the tra-2 gene. We expected to isolate
mutations in genes that control general sex determination, genes that control
general programmed cell death, and genes that are involved in specifying the
death of HSN neurons in males. So far, we have isolated more than forty
mutations. Two of them are alleles of the ced-3 and ced-4 genes, both of which are
general cell-death genes. Fourteen
mutations result in fem phenotypes and are likely to be alleles of fem genes. Five mutations seem to
affect only HSN cell death. Among
them, rsd-1(sm9) (rsd: regulator of sex-specific deaths) is a dominant suppressor
of HSN death in tra-2 hermaphrodites. rsd-4(sm124) is a recessive suppressor of
HSN death in tra-2
hermaphrodites. Both genes were
mapped to LGV, and we are currently doing fine mapping of these two genes to
facilitate their cloning. We are
working on mapping the other three mutations as well. Further genetic and molecular characterization of these rsd genes should help elucidate the
mechanism that controls the life vs. death decision of HSN neurons.
Analysis
of the Sex-Specific Death of the Male-Specific CEM Neurons
Elizabeth
Kimberly, Ning Pan, Beryl Hatton, Ute Meisel, and Ding Xue
CB
347, MCDB, University of Colorado, Boulder, CO 80309
Programmed cell death
(PCD) is an important process during animal development and tissue homeostasis
to clear away unnecessary or abnormal cells. One notable example is the sexually dimorphic process in
which cells or organs that are needed only in one sex are eliminated in the
opposite sex by apoptosis.
In C. elegans two sets of sex-specific neurons, four cephalic
companion neurons (CEMs) and two hermaphrodite specific neurons (HSNs), undergo
sexually dimorphic PCDs. Although
all are born embryonically in both males and hermaphrodites, the CEMs survive
in males and undergo PCD in hermaphrodites. Conversely, the HSNs survive in hermaphrodites but undergo
PCD in males. The sex
determination pathway has been studied extensively in C. elegans but very little is known about what controls the
sex-specific PCDs of these two sets of neurons. We are interested in identifying genes that operate at the
intersection of the sex determination and PCD pathways.
In
C. elegans males
the CEMs are located bilaterally between the two bulbs of the pharynx: two on the dorsal side and two on the
ventral side. We have used a
transgenic strain, pha-1(e2123ts); him-5(e1490);
syEx313 (kindly provided by Paul
Sternberg), in which GFP is specifically expressed only in the CEMs and
a few tail neurons in males, to screen for mutations that cause ectopic PCD of
CEMs in males or survival of CEMs in hermaphrodites. We have screened 8500 haploid genomes and have thus far
isolated four mutations that affect CEMs in males and nine mutations that
affect CEMs in hermaphrodites.
These include (1) three mutations in two genes required for all
programmed cell deaths, ced-3(sm120, sm144) and egl-1(sm157), (2) mutations (sm117 and sm150) in two genes that affect CEM
cell fate determination, and (3) mutations (sm119 , sm130, and sm146) in three genes that
specifically affect the life vs. death fate of the CEMs or HSNs.
We
have focused on studying the third class of mutants (sm119 , sm130, and sm146). In sm130 mutant animals, the dorsal CEMs but not the ventral
CEMs undergo ectopic PCDs in males, which can be blocked by loss-of-function
mutations in ced-3. sm130 is thus a dorsal CEM-specific
cell death mutant. Both sm119 and sm146 mutations cause partial
reversal of CEM/HSN cell fates: ectopic deaths of HSNs and inappropriate
survival of CEMs in hermaphrodites.
sm119 and
sm146 may
define genes that control the life vs. death fates of both HSNs and CEMs. We are in the process of mapping and
cloning these three genes.
Two
mutations (sm117 and
sm150) in
the second class are also of interest to us. In sm117 mutants, 100% of males lose all four CEMs. This cannot be blocked by mutations in ced-3, indicating that the absence of
the CEMs is not due to their ectopic PCD but rather to abnormal cell fate
transformation. sm117 turns out to be an allele of unc-86, which has been previously
implicated in the cell fate determination of the HSNs but not the CEMs. sm150 is another interesting mutation
that not only causes survival of the CEMs in 58% of the hermaphrodites but also
appears to cause sister cell transformations in several other cell
lineages. Specifically, in sm150 mutants several pharyngeal
cells (e.g. I2 and MC cells) undergo ectopic PCD taking on cell fates that
originally belong to their sister cells.
Consistent with this hypothesis, in sm150; ced-3 double mutants, the deaths of
I2 and MC pharyngeal cells are prevented.
We are in the process of mapping sm150.
In
summary, we have identified several genes that specifically affect the life vs.
death fate of the CEM neurons.
Further characterization of these genes should help reveal how sexually
dimorphic apoptosis is regulated and executed in nematodes and may shed light
on the regulation of sexual dimorphic cell deaths in general.
The egl-41 gene, identified by gf mutations that
cause partial sexual transformation, is identical to sel-10, a negative
regulator of lin-12
Ning Pan, Elizabeth Kimberly, Beryl Hatton, Ute Meisel, Manisha
Dudley, Ding Xue
CB 347,
MCDB, University of Colorado, Boulder, CO 80309
Programmed cell death (PCD) plays an important role in many
biological processes including generating sexual dimorphism. In C.
elegans there are two sets of sex-specific neurons: two
hermaphrodite-specific neurons (HSNs) and four male-specific cephalic companion
neurons (CEMs). The HSNs innervate the vulval muscles and stimulate
egg-laying in the hermaphrodite; the CEMs are believed to play a role in the
chemotaxis of the male to the hermaphrodite for mating. Both sets of
neurons are born during embryogenesis in both sexes, however the unnecessary
set undergoes sex-specific PCD: the HSNs die in males and the CEMs die in
hermaphrodites. The regulation of sex-specific PCD in C. elegans offers an ideal
paradigm for understanding the mechanisms that activate PCD in specific sets of
cells.
The egl-41 gene (egg-laying deficient) was originally
identified by three dominant mutations (n1069, n1074, n1077) that cause
inappropriate HSN cell deaths in hermaphrodites (1,2). In addition, the
CEMs improperly survive, suggesting that egl-41 may play an
important role in regulating sexually dimorphic apoptosis in C. elegans. To determine
the loss-of-function phenotypes of egl-41 and to facilitate its cloning, we
performed an egl-41(n1074) suppressor screen, looking for hermaphrodite
mutants that are non-Egl and without CEMs. We screened 21800 haploid
genomes and identified 12 recessive suppressors (sm122-sm125, sm165-sm168, and sm170-sm173). Among them,
sm171 is the strongest suppressor. Four-factor mapping of sm171
n1074
revealed that sm171 and n1074 are very closely linked, suggesting that sm171 might be an intragenic
suppressor of n1074. Complementation tests between sm171 and the other
suppressors indicate that 9 other suppressor mutations are allelic to sm171.
We have conducted several consecutive SNP mappings of egl-41(n1074),narrowing down its
position to a very small interval of 4 cosmids on LG V. We found that
injection of these four cosmids can counteract the Egl and CEM survival
phenotypes of the egl-41(n1074) mutation, suggesting that the normal
activity of egl-41 antagonizes the likely altered function of the EGL-41 mutant
product. Using this characteristic, we performed further rescuing
experiments and found egl-41 to be identical to the previously
characterized gene sel-10(suppressor/enhancer of lin-12). sel-10 is a negative
regulator of lin-12 (lineage-abnormal) and encodes an F-box/WD40
repeat-containing protein (3,4). The sel-10(ar41) mutant displays no
obvious defects in either HSNs or CEMs, similar to what we have observed in our
egl-41(n1074) intragenic suppressors. In addition, injection of a 6.9 kb
PCR product derived from the sel-10 region of the n1074animals into either
wild-type or sel-10 animals mimicked the egl-41(n1074) mutant phenotypes.
We have identified the molecular lesions corresponding to two egl-41alleles, n1074and n1077(both are a G567E
missense mutation in SEL-10), and four n1074 intragenic
suppressors. sm122 and sm170 are missense
mutations (R472Q and S467F, respectively) in the sixth WD40 repeat of SEL-10; sm170 is a splice site
mutation before exon 10; sm123 is a missense mutation in the first WD40
repeat of SEL-10.
Currently we are sequencing other suppressor mutations to
determine if and how they affect the sel-10 ORF. In
addition we are doing experiments to determine what proteins interact with
SEL-10 and how these interactions are affected by the gain-of-function and
loss-of-function mutations in sel-10. These experiments should provide
crucial insight into how sel-10 and its interacting partners affect the
life vs. death fates of the HSNs and CEMs in C. elegans.
1) Desai, C., Garriga, J., McIntire, S.L.,
Horvitz, H.R. (1988) Nature 336, 638-646.
2) Desai, C. and Horvitz, H.R. (1989)
Genetics 121, 703-721.
3) Sundaram, M. and Greenwald, I. (1993)
Genetics 135, 765-783.
4)
Hubbard, E.J., Wu, G., Kitajewski, J., and Greenwald, I. (1997)
Genes and Development 11, 3182-3193.
The egl-41 gene is identical to sel-10, a negative
regulator of lin-12.
Ning Pan, Elizabeth Kimberly, Beryl Hatton, Ute Meisel, Ding Xue. MCD
Biology, University of Colorado, Boulder, CO.
Programmed cell death (PCD) plays an important role in many
biological processes including generating sexual dimorphism. In C. elegans, two
hermaphrodite-specific neurons (HSNs) and four male-specific cephalic companion
neurons (CEMs) are born during embryogenesis in both sexes but undergo PCD in
the sex where they are not needed. The egl-41 gene (egg
laying-defective) was originally identified by three dominant mutations (n1069,
n1074, n1077) that cause inappropriate HSN deaths and improper CEM survivals
in hermaphrodite animals (Desai and Horvitz, 1989), suggesting that egl-41 plays an important
role in regulating sex-specific deaths. To clone egl-41 and to understand
how it works, we performed an egl-41(n1074) suppressor screen
and have isolated 10 intragenic and 4 extragenic suppressors of n1074. Using SNP mapping,
we mapped egl-41(n1074) to a small interval of 4 cosmids on LG V.
Interestingly, injection of these 4 cosmids can counteract the Egl and CEM survival
phenotypes of the egl-41(n1074) hermaphrodites, suggesting that the normal
activity of egl-41 antagonizes the likely "altered" function of the EGL-41
mutant product. Using this characteristic, we performed further
"rescuing" experiments and found egl-41 to be identical to sel-10
(suppressor/enhancer of lin-12; Sundaram and Greenwald, 1993), which
encodes an F-box/WD40 repeat-containing protein and is likely a component of
the SCF E3 ubiquitin ligase complex (Hubbard et al., 1997), exerting its functions
by promoting degradation of its target proteins. We have identified molecular
lesions corresponding to two egl-41 alleles, n1074 and n1077 (both G567E
substitutions in SEL-10), and all ten n1074 intragenic
suppressors (all but one are missense mutations).
Interestingly, animals harboring the loss-of-function sel-10(ar41) mutation or the sel-10(sm171
n1074) mutation, the strongest n1074 suppressor, display
no obvious defects in either HSNs or CEMs. However, both mutations can suppress
hermaphrodite HSN death and CEM survival phenotypes caused by a weak
loss-of-function mutation in tra-2 (sexual transformer). In addition, we found
that loss-of-function mutations in both fem-1 (feminization) and fem-2 are epistatic to
the sel-10(n1074) mutation.
We are in the process of cloning n1074 extragenic
suppressors to identify potential SEL-10 targets or interacting proteins. We
are also carrying out biochemical experiments to look for SEL-10 targets or
co-factors and have identified one candidate SEL-10 substrate. These
experiments should help provide crucial insight into how sel-10 affects the life
vs. death fates of the HSNs and CEMs in C. elegans and how it may
affect cell death in general.
Molecular genetic characterization of a putative inhibitor of
CEM cell death.
Elizabeth Kimberly, Erin Peden, David Kokel, Ning Pan, Beryl
Hatton, Ute Meisel, Ding Xue. MCDB, University of Colorado, Boulder, CO.
Programmed cell death (PCD) generates sexual dimorphism by
eliminating sex-specific cells or organs that are not needed in a specific sex
of a species. We are interested in identifying genes that regulate sexually
dimorphic PCD in C. elegans. Four male-specific cephalic companion
neurons (CEMs) and two hermaphrodite-specific neurons (HSNs) undergo
sex-specific PCDs in C. elegans. Although all are born embryonically in
both males and hermaphrodites, the CEMs survive only in males and the HSNs
survive only in hermaphrodites. The regulation of sex-specific PCDs in C.
elegans offers an ideal paradigm for understanding the mechanisms that
activate PCD in specific sets of cells.
In C. elegans males, the CEMs are located between the two
bulbs of the pharynx: two on the dorsal side and two on the ventral side. We
have used a transgenic strain, pha-1(e2123ts); him-5(e1490); syEx313 (kindly provided by
Maureen Barr and Paul Sternberg), in which GFP is specifically expressed only
in CEMs and a few tail neurons in males, to screen for mutations that cause
abnormal CEM PCD patterns. From a screen of 8500 haploid genomes, we isolated
four mutations that affect CEM PCDs in males and nine mutations that inhibit
CEM PCDs in hermaphrodites. We are most interested in the mutant rsd-6(sm130) (regulator of
sex-specific death). In sm130 mutant males, both the dorsal CEMs and
ventral CEMs undergo ced-3-dependent ectopic PCD. However, the dorsal
CEMs are more strongly affected: only 8% of dorsal CEMs are present compared
with 47% of ventral CEMs that survive. In sm130 hermaphrodites,
CEMs undergo PCD normally. Since sm130 is a recessive mutation, it may
affect a cell death inhibitor that regulates sex-specific CEM death. Genetic
epistasis analysis suggests that rsd-6 acts downstream of tra-1, the terminal
regulator of the sex determination pathway, and sel-10, a regulator of
both CEM and HSN PCDs (see Ning Pan’s abstract), to regulate sex-specific CEM
deaths. Three-factor mappings and single nucleotide polymorphism (SNP) mappings
placed rsd-6 in a two cosmid interval on the left arm of LG X. We found that
one of the cosmids robustly rescues the sm130 phenotype. We have
localized the rescuing activity to a region of the cosmid that encodes a
homeobox protein, CEH-30. Further molecular and biochemical characterization of
the rsd-6 gene should help reveal how sexually dimorphic apoptosis is
regulated and executed in nematodes and may shed light on the regulation of
sexually dimorphic cell deaths in general.