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First published online 18 January 2006
doi: 10.1242/dev.02234
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Departments of Pediatrics and Molecular Biology, Division of Pediatric Hematology-Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75039-9148, USA.
* Author for correspondence (e-mail: scott.cameron{at}utsouthwestern.edu)
Accepted 2 December 2005
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: C. elegans, Hox genes, Programmed cell death
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Clark et al.,
1993; Economides et al.,
2003; Gavalas et al.,
2003; Kenyon,
1986; Lohmann et al.,
2002; Miguel-Aliaga and Thor,
2004; Salser et al.,
1993; Stadler et al.,
2001). Programmed cell death is a widespread and, in most
organisms, essential aspect of normal development; mutations that influence
whether cells survive or undergo programmed cell death contribute to human
disease. Discovering how Hox genes regulate programmed cell death is therefore
important for understanding the roles of Hox genes in development and
disease.
As homeodomain-containing proteins, Hox proteins are likely to act by
regulating transcription. The crucial task is therefore to identify those
targets regulated by Hox proteins that determine programmed cell death or
survival of individual cells or groups of cells. At present, it is not clear
whether, for example, the abnormalities in programmed cell death observed in
Hox mutants represent transformations in cell fate that then indirectly
manifest as abnormal patterns of programmed cell death, or whether Hox genes
directly determine programmed cell death or survival by regulating
transcription of cell death genes. Recent data in Drosophila indicate
that the Hox protein Deformed directly regulates transcription of the
proapoptotic gene reaper to induce programmed cell death and shape a
morphological boundary (Lohmann et al.,
2002). Whether this is a general function of Hox genes during
development needs to be explored.
The genetic pathway for the execution of programmed cell death in C.
elegans is well established and highly conserved across animal species
(Metzstein et al., 1998). In
addition, the essentially invariant pattern of development makes it possible
to study cell fates at the level of individual cells
(Sternberg and Horvitz, 1984).
C. elegans therefore presents an excellent model in which to study
the mechanisms that regulate cell fates and programmed cell death.
The C. elegans genome contains six Hox genes organized as three
gene pairs in a contiguous region of chromosome III. As in other metazoans,
the Hox genes of C. elegans are generally organized along the
chromosome in the order in which they are expressed in the animal, with one
exception (WormBase website,
http://www.wormbase.org,
release WS138, 11 February 2005)
(Brunschwig et al., 1999;
Clark et al., 1993;
Wang et al., 1993). The six
Hox genes include single genes orthologous to labial
(ceh-13), sex combs reduced (lin-39) and
antennapedia (mab-5), and three posterior Hox genes
(egl-5, php-3, nob-1) (Aboobaker
and Blaxter, 2003; Van Auken
et al., 2002; Wang et al.,
1993). Three Hox genes, lin-39, mab-5 and egl-5,
for which mutants bearing null alleles are viable, have been extensively
characterized (Chisholm, 1991;
Clark et al., 1993;
Kenyon, 1986;
Wang et al., 1993). Mutations
affecting these genes result in abnormal patterns of programmed cell death.
Specifically, lin-39 is essential for the survival of six neurons,
the VC neurons, generated in the midregion of the ventral nerve cord
(Clark et al., 1993), and
mab-5 is essential for the programmed cell death of two cells,
P11.aaap and P12.aaap, generated in the posterior ventral nerve cord
(Kenyon, 1986); (Pn.aaap, the
posterior daughter of the anterior daughter of the anterior daughter of the
anterior daughter of any P blast cell). In lin-39 mutants, the six VC
neurons in the midbody of the ventral nerve cord express fates characteristic
of their more anterior and posterior lineal homologs [lineal homologs are
cells arising at corresponding positions of related lineages, and they
typically adopt related fates (Sulston and
Horvitz, 1977)], which undergo programmed cell death, suggesting
lin-39 provides spatial information to the developing animal
(Clark et al., 1993) and
consistent with the functions of Hox genes in other animals
(McGinnis and Krumlauf, 1992).
A similar role has been suggested for mab-5 in the posterior ventral
nerve cord, where the P(11,12).aaap cells of mab-5 mutants survive,
as do their anterior lineal homologs
(Kenyon, 1986).
How mutations in lin-39 and mab-5 determine cell death or
survival is not yet known. Characterization of mab-5 mutants suggests
that mab-5 function is necessary but not sufficient to specify
programmed cell death of the P(11,12).aaap cells
(Salser et al., 1993). For
example, strong loss of function or null mutations in mab-5 result in
survival only of P(11,12).aaap in the ventral nerve cord; programmed cell
death of other cells in the ventral nerve cord occurs normally. In mutants
that ectopically express mab-5, the anteriorly located lineal
homologs of the P(11,12).aaap cells do not undergo programmed cell death
(Salser et al., 1993). These
experiments suggest the existence of factors that prevent programmed cell
death of the P(11,12).aaap anterior lineal homologs or that act with
mab-5 in P(11,12).aaap to ensure their death. Given the similarity in
cell division patterns and fates of cells generated by the P11 and P12
lineages (Sulston and Horvitz,
1977), it was surprising to find that MAB-5 protein is detected in
the P11 blast cell and the five cells ultimately generated by P11 including
P11.aaap, but not in the P12 descendants after the first division
(Salser et al., 1993).
mab-5 might therefore determine cell fates through different
mechanisms in the P11 and P12 lineages, including the P(11,12).aaap programmed
cell deaths.
Hox proteins can bind DNA cooperatively with protein co-factors, including
the PBC family of homeodomain proteins
(Chan et al., 1994). The PBC
family, which includes Drosophila Extradenticle and the mammalian Pbx
proteins (Burglin, 1997), binds
to a specific subset of Hox proteins via an interaction between a conserved
hexapeptide motif found in the Hox protein
(Chang et al., 1995) and a
pocket in the co-factor (Piper et al.,
1999). The C. elegans genome encodes two proteins similar
to Pbx, CEH-20 and CEH-40 (WormBase web site,
http://www.wormbase.org,
release WS138, 11 February 2005), and the Hox proteins LIN-39 and MAB-5 both
contain the hexapeptide motif required for interaction with PBC proteins.
ceh-20 cooperates with lin-39 and mab-5 during
mesoderm differentiation, where a LIN-39/CEH-20 heterodimer directly regulates
the C. elegans homolog of twist
(Liu and Fire, 2000), and
ceh-20 and ceh-40 act partially redundantly during embryonic
development (Van Auken et al.,
2002). ceh-20 mutants have multiple defects in vulval
development and neuronal migration that in part are similar to those of
lin-39 mutants, but also have distinct defects that suggest
lin-39-independent functions
(Yang et al., 2005).
In C. elegans, the BH3 domain-encoding gene egl-1 is
required for programmed cell death of somatic cells, and expression of
egl-1 is sufficient to induce programmed cell death
(Conradt and Horvitz, 1998).
The EGL-1 protein physically interacts with the Bcl2 homolog CED-9 to initiate
programmed cell death (Conradt and
Horvitz, 1998). Two pathways are known that directly regulate
expression of egl-1 and the programmed cell death of specific cells.
In the HSN neurons, the TRA-1 transcription factor binds egl-1
regulatory sequences to prevent programmed cell death
(Conradt and Horvitz, 1999).
egl-1 mutations that prevent TRA-1 binding result in expression of
egl-1 in the HSNs, their programmed cell death and an egg-laying
defect. In a specific subset of pharyngeal neurons, the ces-2 and
ces-1 genes act in a negative regulatory cascade to promote
programmed cell death (Ellis and Horvitz,
1991). The Snail homolog CES-1 directly represses egl-1
in a process opposed by the bHLH proteins HLH-2 and HLH-3
(Thellmann et al., 2003).
These data demonstrate that the fate of programmed cell death of specific
cells can be determined by regulating transcription of egl-1.
We report here that, as part of its function in specifying fates in the P11 and P12 cell lineages, a complex containing the Hox co-factor and Pbx homolog CEH-20 and the Hox protein MAB-5 directly regulates egl-1 expression to induce programmed cell death of P11.aaap.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) at 20°C.
The following mutations were used: LGI, ced-1(e1735); LGII,
muIs16 (Cowing and Kenyon,
1996); LGIII, ceh-20(ay9), ceh-20(ay42) and
unc-36(e251), unc-119(ed3); LGV, egl-1(n1084n3082); LGX,
ceh-40(gk159). The strain NC216 containing the
Pacr-5gfp wdEx75 array and the NC190 strain containing the
Pdel-1gfp wdIs6 integrated array were generously provided
by David Miller (Vanderbilt University, Nashville, TN). ceh-20
alleles were a gift from M. Stern (Yale University, New Haven, CT). The
ceh-40(gk159) allele was isolated and provided by the C.
elegans Knockout Consortium
(http://celeganskoconsortium.omrf.org).
The ceh-40(gk159) allele deletes 968 bp of genomic sequence including
all of predicted exons 2, 3, 4 and 5. If transcribed and translated, the
deletion would be predicted to generate a truncated protein that includes the
initial 31 amino acids of CEH-40 followed by missense amino acids and
premature termination. Some nematode strains used in this work were provided
by the Caenorhabditis Genetics Center, which is funded by the NIH
National Center for Research Resources (NCRR). Lineage analysis was performed
as described by Sulston and Horvitz
(Sulston and Horvitz, 1977).
Biolistic bombardment was performed as described
(Praitis et al., 2001).
Plasmid constructs
The wild-type 7.6 kb egl-1 genomic region is contained in plasmid
pBC08 (Conradt and Horvitz,
1998). Site-specific mutagenesis was performed using the method of
Quikchange (Stratagene) and all DNA segments exposed to PCR or mutagenesis
were fully sequenced. For the egl-1 transgene containing mutations in
sites 1 and 2 and a deletion in the F23B12.1 predicted phosphatase, a 370
nucleotide PflF1 fragment was removed from the F23B12.1-coding
sequences and the plasmid religated. To construct the pMP0017
Pceh-20ceh-20:cfp translational fusion plasmid, a 3.6 kb
SacI PflMI fragment of the ceh-20 genomic region,
including 
1 kb of 5' sequence and 1 kb of 3' sequence was
amplified by long PCR and cloned into pBSKSII+. Site-specific
mutagenesis was used to change the stop codon to an AscI restriction
site, into which the cfp-coding region from pPD134.96 (kindly
provided by Andy Fire, Stanford University, Palo Alto, CA) was cloned. This
plasmid was injected at a concentration of 50 ng/µl into
ceh-20(ay42), where it rescued the egg-laying defect, and together
with a lin-15-rescuing plasmid into ced-3(n717);
lin-15(n765) mutants. To construct the
Pegl-1histone:gfp reporter, overlap PCR was used to
replace the open reading frame of egl-1 by an AgeI
restriction site. A fusion of the R08C7.3 histone 2A gene to
gfp was inserted into the AgeI site. This plasmid was
microinjected into ced-3(n717); lin-15(n765) mutants at a
concentration of 10 ng/µl and integrated after gamma irradiation. The
mxIs14 integrated array was mapped to LG X.
Fusion proteins and electrophoretic mobility shift assays
Full length ceh-20 and mab-5 cDNAs amplified from
wild-type C. elegans total RNA were cloned as MscI
XhoI fragments generated by PCR into a derivative of pSP73 that
contains the Xenopus ß-globin 5' untranslated region
(Swift et al., 1998).
Oligonucleotides encoding the myc or FLAG epitopes were then ligated into
MscI-digested mab-5 or ceh-20 expression plasmids.
Those regions of constructs generated by PCR or changed after ligation of the
oligonucleotides were completely sequenced. Proteins were generated by coupled
transcription and translation in vitro using SP6 polymerase and TnT wheat-germ
extract (Promega) and efficiency of protein generation was estimated by
synthesis of an aliquot including 35S-methionine followed by
autoradiography (for native proteins) or after western blot (for
epitope-tagged proteins). For EMSAs, proteins were preincubated for 15 minutes
at 37°C in a 10 µl reaction including 20 mM HEPES (pH 7.9), 15%
glycerol and 0.2 mM EDTA. Probe (50,000 cpm) in 10 µl with 100 mM KCl, 5 mM
DTT, 5 mM MgCl2, 50 ng polydI-dC, 50 ng E. coli genomic
DNA and 1% NP40 was also preincubated for 15 minutes at 30°C. Probe and
proteins were combined and incubated on ice for 30 minutes, then separated on
an 8% polyacrylamide gel in 0.5xTBE buffer run at 25 mA at 4°C.
Probe sequences are available on request.
Sequence comparisons
Family Relations and SeqComp software
(Brown et al., 2002) was used
to compare sequences in the egl-1 genes of C. elegans and
C. briggsae.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), which express the predicted acetylcholine receptor subunit
acr-5 and the sodium channel del-1
(Esmaeili et al., 2002;
Winnier et al., 1999). We used
gfp reporter constructs to examine expression of these markers in
ced-3 mutants, in which the death of the P(11,12).aaap cells was
prevented by a block in programmed cell death, and compared the results with
those where death of the cells was prevented by mutation of mab-5.
Thirty out of 30 animals of genotype Pdel-1gfp;
ced-3(n717) expressed gfp in P11.aaap, while only one out of
30 animals of genotype Pdel-1gfp; mab-5(n1384)
did so. Similarly, 24 of 30 animals of genotype Pacr-5gfp;
ced-3(n717) expressed gfp in P11.aaap, while only two out of
30 animals of genotype Pdel-1gfp; mab-5(n1384)
did so. These data suggest that mab-5 may determine aspects of
P11.aaap identity in addition to inducing programmed cell death of the cell,
or that it directly promotes expression of the acr-5 and
del-1 transgenes.
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). ceh-20 and ceh-40 each encode homologs of
the extradenticle/Pbx TALE class of homeodomain proteins
(http://www.wormbase.org,
release WS138, 11 February 2005). ceh-20 acts with lin-39
and mab-5 to pattern mesoderm
(Liu and Fire, 2000), and
redundantly with ceh-40 during embryonic development
(Van Auken et al., 2002).
ceh-20 mutants also have defects in neuronal migration that are in
part similar to those in lin-39 mutants
(Yang et al., 2005). In the
ventral nerve cord, ceh-20 mutants have defects in programmed cell
death consistent with loss of lin-39 function in the midbody and of
mab-5 function in the posterior
(Table 1). In mutants carrying
the strong loss-of-function allele ceh-20(ay42) (M. Stern, personal
communication; null alleles are lethal), the defects include programmed cell
death of six cells in the midregion of the ventral nerve cord and survival of
two cells in the posterior ventral nerve cord
(Fig. 1). We followed cell
lineages in ceh-20(ay42) mutants and directly observed programmed
cell death of the VC neurons in the midbody, as in lin-39 mutants,
and survival of the P(11,12).aaap cells, as in mab-5 mutants
(Fig. 1).
ceh-40(gk159) mutants have a normal pattern of cell deaths in the
ventral nerve cord, and the defects in cell death in mutants carrying a weak
allele of ceh-20, ay9 are not enhanced by the ceh-40(gk159)
deletion, suggesting that ceh-40 is dispensable for determining the
pattern of programmed cell death in the ventral nerve cord
(Table 1). In contrast to the
highly penetrant defects in lin-39 and mab-5 function, we
did not observe any transformation of the P12 lineage to the P11 fate, as is
observed in egl-5 mutants
(Chisholm, 1991), suggesting
that ceh-20 may not act as the co-factor for egl-5 in this
lineage.
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).
Hox genes act upstream of or parallel to the cell death gene egl-1
To address where in the genetic pathway for programmed cell death the Hox
genes might act, we determined whether loss-of-function mutations in
egl-1 could prevent programmed cell deaths in lin-39
mutants. The Plin-11gfp reporter construct is expressed in
the six VC motoneurons of wild-type animals
(Cameron et al., 2002).
lin-39(n1760); Plin-11gfp mutants do not express
the reporter in the midbody (Fig.
3), consistent with programmed cell death of the VC neurons in
these mutants (Clark et al.,
1993). In lin-39(n1760); egl-1(n1084n3082);
Plin-11gfp mutants the VC neurons survive and express the
Plin-11gfp reporter, suggesting that lin-39 acts
upstream of or parallel to egl-1. We performed similar experiments
with ceh-20 mutants (Fig.
3). Mutants carrying the weak ay9 allele of
ceh-20 express the reporter in few cells in the midbody, consistent
with programmed cell death of many of the VC neurons in these mutants. The VC
neurons survive and express the Plin-11gfp reporter in
ceh-20(ay9); egl-1(n1084n3082); Plin-11gfp
mutants, but expression of the reporter was weak in some VC neurons,
suggesting ceh-20 may determine other aspects of VC identity in
addition to being required for their survival. This suggestion is supported by
the phenotype of mutants carrying the strong ay42 allele of
ceh-20. In ceh-20(ay42); egl-1(n1084n3082);
Plin-11gfp mutants the VC neurons survive but do not
express Plin-11gfp
(Fig. 3). Expression of
Plin-11gfp in lin-39 null alleles but not in
ceh-20 mutants also suggests that ceh-20 has
lin-39-independent functions. These data are consistent with the
model that lin-39 and ceh-20 act through egl-1 to
prevent programmed cell death of the VC neurons.
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;
Mann and Affolter, 1998). Four
such matches were identified (Fig.
4). Transgenic animals carrying an egl-1 genomic
construct in which all four sites had been mutated from TGATNNAT to TCCATGGT
had defects in programmed cell death in the ventral nerve cord
(Fig. 4). Specifically, one
cell in the P11 lineage survived in virtually all transgenic animals, and one
cell in the P12 lineage often survived. Mutation of only the candidate Hox
co-factor/Hox site at position +5995 relative to the egl-1 ATG was
completely sufficient for this phenotype
(Fig. 4; see Fig. S1 in the
supplementary material). Mutation of this site in the
Pegl-1histone:gfp reporter resulted in a failure to
express gfp in P11.aaap, indicating that this site regulates
transcription of egl-1 in this cell
(Fig. 4). This site is also
conserved in the egl-1 gene of Caenorhabditis remanei (data
not shown). Transgenic animals with mutations of two nucleotides in the Hox
half site or three nucleotides in the Hox co-factor half site were
indistinguishable from the TCCATGGT mutant, suggesting binding by both a Hox
and its co-factor is required for regulation
(Fig. 4).
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ceh-20 is expressed in P(11,12).aaap and many other cells
To examine in which cells the ceh-20 gene is expressed, and
specifically to test whether ceh-20 is expressed in P11.aaap where
CEH-20 could interact with MAB-5 to regulate egl-1, we generated a
rescuing cfp reporter construct. We generated transgenic animals that
expressed a full-length CEH-20 protein with a fusion of CFP to the C terminus
of the protein. Introduction of this construct into ceh-20(ay42)
mutants rescued the egg-laying defect (data not shown). We introduced this
construct into ced-3 mutants, in which P11.aaap and the other cells
that undergo programmed cell death in the ventral nerve cord survive, and
examined expression of the reporter construct. Consistent with a recent report
of the expression pattern of a similar construct
(Yang et al., 2005), we
identified broad expression in many nuclei, including most ventral nerve cord
neurons. We specifically identified expression in P11.aaap, which undergoes
programmed cell death in a ceh-20-dependent fashion
(Fig. 6). To address the
possibility that ceh-20 is required for expression of mab-5,
we examined expression of a Pmab-5gfp reporter
(Cowing and Kenyon, 1996) in
ceh-20(ay42) mutants and identified no significant difference
(Fig. 6), suggesting that
ceh-20 is not required for expression of mab-5 in the P11
lineage. However, as ceh-20(ay42) is a strong loss of function but
not null allele, this does not rule out regulation of Hox genes by
ceh-20.
egl-1 is regulated differently in P11.aaap and P12.aaap
To determine precisely which cells are affected by mutation of the site at
+5995, we followed P11 and P12 cell lineages in transgenic animals carrying
the TCCATGGT mutation at position +5995 of an integrated egl-1
transgene (Fig. 7). In six out
of seven animals, mutation of the site resulted in survival of P11.aaap, which
survives in ceh-20 and mab-5 mutants. By contrast, in seven
of seven animals P12.aaap, which survives in ceh-20 and
mab-5 mutants, underwent programmed cell death. In three out of seven
animals, we observed survival of P12.pp, a cell that in wild-type animals
undergoes programmed cell death (Sulston
and Horvitz, 1977). MAB-5 protein is present in P11.aaap when the
cell is generated (Salser et al.,
1993), consistent with binding by a CEH-20/MAB-5 complex to the
site at +5995 and direct activation of egl-1 transcription in this
cell. By contrast, in P12.aaap, MAB-5 is likely to act indirectly, as MAB-5
protein is undetectable after the first division in this lineage
(Salser et al., 1993), 4 hours
prior to the programmed cell death of P12.aaap. Survival of some P12.pp cells
in animals with a mutation in the site at +5995 suggests that this site may
mediate activation of egl-1 transcription to induce death of this
cell. As P12.pp cell death occurs normally in mab-5 and
ceh-20 mutants, perhaps a more posterior Hox gene and Hox co-factor
[such as EGL-5 and the MEIS homolog UNC-62
(Van Auken et al., 2002)] act
upon this site.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and with indirect regulation of egl-1 by
CEH-20/MAB-5 in P12.aaap.
Despite the similar patterns of cell division and cell fates in P11, P12
and their descendants, different mechanisms determine these lineages. In first
larval stage animals, the P11 and P12 blast cells migrate into the ventral
nerve cord from lateral positions, and laser ablation studies indicate that
both cells are initially each capable of adopting the P12 fate
(Sulston and White, 1980).
Multiple intercellular signaling pathways, including EGF/EGFR-like
(LIN-3/LET-23) and Wnt/Wnt receptor (LIN-44/LIN-17) pathways specify the P12
fate, in part by acting through the Hox protein EGL-5
(Jiang and Sternberg, 1998).
egl-5 is a homolog of Abd-B
(Aboobaker and Blaxter, 2003;
Chisholm, 1991;
Wang et al., 1993). In
egl-5-null mutants P12 is transformed to the P11 fate
(Chisholm, 1991), and
egl-5 represses expression of mab-5 in the P12 lineage after
the first division (Salser et al.,
1993). Less is known about how the P11 fate is specified. Direct
activation of egl-1 transcription by CEH-20/MAB-5 in P11.aaap is one
molecular mechanism operative in the P11 lineage that is inactive in the P12
lineage.
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).
Partial reconstruction of the posterior ventral nerve cord of a mab-5
mutant indicated that the `undead' P11.aaap cell extended an axon like that of
a VB motoneuron (Kenyon,
1986), consistent with a homeotic transformation. We find that if
the programmed cell death of P11.aaap is prevented by a mutation in
ced-3, it expresses acr-5 and del-1, markers of the
VB motoneuron fate (Esmaeili et al.,
2002; Winnier et al.,
1999). In mab-5 mutants, however, P11.aaap generally does
not express these markers, suggesting that the transformation may be aberrant.
Survival of P11.aaap in mab-5 mutants probably reflects a specific
defect in activation of egl-1 transcription and programmed cell
death. In this capacity, egl-1 acts as a realizator gene
(Garcia-Bellido, 1977)
regulated by mab-5 and ceh-20 to effect morphogenesis, in
this case by deleting a single cell from the developing animal. Previous
target genes directly regulated by Hox proteins in C. elegans include
only transcription factors (Koh et al.,
2002; Liu and Fire,
2000). Identification of egl-1 as a realizator gene
activated by a Hox co-factor/Hox complex to induce programmed cell death in
C. elegans supports the suggestion that non-homeotic targets of Hox
proteins are common and evolutionarily ancient
(Hombria and Lovegrove,
2003).
Mutations in the Hox gene lin-39 also result in defects in
programmed cell death; specifically, the six VC neurons of the midregion of
the ventral nerve cord require lin-39 for survival. We find that
ceh-20 is also required for survival of the VCs. Our data also
suggest that ceh-20 may determine one aspect of VC identity,
expression of lin-11, through a mechanism that is lin-39
independent. This suggestion is consistent with lin-39-independent
functions for ceh-20 in vulval development and neuronal migration
(Yang et al., 2005). The
mechanism through which lin-39 and ceh-20 regulate VC
survival is not clear at present. Analogous to P11.aaap, a LIN-39/CEH-20
complex could directly repress transcription of egl-1 in the VC
neurons to ensure survival of the VC neurons. To confirm this model,
regulatory sites in egl-1 through which a LIN-39/CEH-20 complex acts
must be identified. An alternative model is that LIN-39 and CEH-20 regulate
egl-1 indirectly, as MAB-5 apparently does in P12.aaap.
In Drosophila, the Hox genes deformed and abd-B
activate transcription of reaper to induce programmed cell death and
establish segment boundaries (Lohmann et
al., 2002). reaper and other functionally related genes
initiate programmed cell death in Drosophila primarily by inhibiting
the action of DIAP1, a RING domain-containing protein that ubiquitinates the
caspase DRONC and promotes its degradation
(Ditzel et al., 2003;
Goyal et al., 2000;
Wang et al., 1999;
Wilson et al., 2002). In
mammals, developmental control of apoptosis is mediated in many cases by Bcl2
family members, particularly BH3 domain-encoding genes that are regulated in
response to diverse stimuli (Puthalakath
and Strasser, 2002). Our demonstration here of direct regulation
of the BH3 domain-encoding gene egl-1 by a Hox co-factor/Hox protein
complex to initiate programmed cell death in C. elegans suggests the
hypothesis that mammalian BH3 domain-encoding genes may be similarly regulated
by Hox co-factor/Hox complexes as part of normal development of cells and
tissues.
Previously, two pathways have been identified that directly regulate
egl-1 and programmed cell death of specific cells of C.
elegans; both pathways contain genes whose human homologs are oncogenes
(Conradt and Horvitz, 1999;
Metzstein et al., 1996;
Metzstein and Horvitz, 1999;
Thellmann et al., 2003).
Mutations affecting a mammalian homolog of ces-2, hepatic leukemia
factor (HLF), may contribute to oncogenesis by preventing programmed cell
death of malignant cells through an evolutionarily conserved mechanism. HLF is
altered by the rare t(17;19) translocation in children with acute
lymphoblastic leukemia (Inaba et al.,
1992). The E2A-HLF fusion protein generated by the translocation
inhibits programmed cell death of malignant precursor B lymphoblasts
(Inaba et al., 1996). Based
upon the genetic pathway established in C. elegans in which CES-2
acted through the Snail family member CES-1 to regulate programmed cell death,
a human homolog of CES-1, SLUG, was identified and shown to mediate the
actions of the fusion protein in preventing cell death of the malignant cells
(Inukai et al., 1999).
We report here that the third pathway that directly regulates
egl-1 also contains a human oncogene. A human homolog of
ceh-20 is Pbx1, an oncogene initially identified at the
t(1;19) breakpoint in children with acute lymphoblastic leukemia
(Kamps et al., 1990;
Nourse et al., 1990), the most
common form of cancer in children. How mutations of Pbx1 promote
leukemogenesis is not yet known. Some egl-1 homologs act during
normal hematopoiesis to prevent the development of hematopoietic malignancy.
For example, Bid-deficient mice develop a myeloproliferative disease
that progresses to leukemia in many mice
(Zinkel et al., 2003), and
Bad-deficient mice develop diffuse large B cell lymphoma
(Ranger et al., 2003). Hox
genes have many well described functions during normal hematopoiesis and are
frequently affected by chromosomal translocations in hematopoietic cancers
(Grier et al., 2005). How
expression of the BH3 domain-encoding genes is regulated during hematopoietic
development is not yet clear, but direct regulation by Hox co-factor/Hox
complexes of BH3 gene function to promote or prevent programmed cell death is
an intriguing possibility.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/4/641/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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