1 Department of Molecular and Human Genetics, Baylor College of Medicine, One
Baylor Plaza, Houston, Texas 77030, USA
2 Summer Medical and Research Training (SMART) Program, Baylor College of
Medicine, One Baylor Plaza, Houston, Texas 77030, USA
3 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
* Authors for correspondence (e-mail: crt{at}bcm.tmc.edu and gadi{at}bcm.tmc.edu)
Accepted 22 October 2003
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SUMMARY |
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Key words: Dictyostelium, DIF-1, Pattern formation, Prestalk O cells, dimA, dmtA
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Introduction |
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The developing Dictyostelium slug has a clear anteroposterior
pattern, with the prestalk and prespore cell types arranged into tissues along
this axis. Prestalk cells occupy the anterior quarter and are of two major
types: the prestalk-A (pstA) cells, which are at the very front, and the
prestalk-O (pstO) cells just behind them
(Early et al., 1993;
Jermyn et al., 1989
). The
posterior three-quarters of the slug comprise the prespore zone, and there is
some evidence for the subdivision of that region as well
(Haberstroh and Firtel, 1990
;
Kibler et al., 2003
). The
question of how pattern arises is fundamental to our understanding of
Dictyostelium development.
The chemical nature and cell culture actions of DIF-1 provide a candidate
molecule for the control of Dictyostelium patterning. DIF-1 is a
chlorinated alkyl phenone produced by developing Dictyostelium cells.
DIF-1 can drive amoebae to differentiate as vacuolized stalk cells
(Morris et al., 1987). It also
induces the expression of prestalk markers, represses prespore markers and
prevents cells in culture from differentiating as spores. Consequently, DIF-1
has been considered to be a central regulator of the stalk/spore decision
(Early et al., 1995
;
Early and Williams, 1988
;
Fosnaugh and Loomis, 1991
;
Kay and Jermyn, 1983
).
However, a mutant specifically defective in DIF biosynthesis
(dmtA) has been generated, which develops relatively normally until
the slug stage of development. At that stage, it makes long, thin structures
compared with wild type, which later develop spores and a stalk of sorts
(Thompson and Kay, 2000b). The
only characterized defect in cell type differentiation is a failure to express
a subset of pstO markers (Maeda et al.,
2003
; Thompson and Kay,
2000b
). However, several prestalk markers (including a pstA
marker) and prespore markers are expressed normally. This suggests that DIF-1
is only required for the differentiation of a subset of prestalk cells, the
pstO cells, but not for the differentiation of pstA cells or prespore
cells.
These observations, together with earlier work, suggest that patterning
arises by a mechanism whereby the choice between (at least) the pstO and
prespore fates is driven by a process akin to lateral inhibition
(Clay et al., 1995;
Kay et al., 1999
;
Leach et al., 1973
;
Loomis, 1993
). It is proposed
that as cells enter the mound, they all experience similar concentrations of
DIF-1. Initial intrinsic differences between the cells distinguish between
responding and non-responding populations. Such differences have been noted,
and include cell cycle position and growth history
(Leach et al., 1973
), both of
which bias cell fate choice and affect DIF-1 sensitivity
(Thompson and Kay, 2000a
). As
some of the earliest responses to DIF-1 include the downregulation of DIF
biosynthesis and upregulation of DIF breakdown
(Insall et al., 1992
), two
populations of cells quickly emerge: DIF-1 responding (prestalk) and DIF-1
producing (prespore). Consistent with this idea, prestalk cells ultimately
exhibit the highest levels of DIF-1 breakdown and prespore cells the highest
levels of DIF-1 biosynthesis (Kay et al.,
1993
; Kay and Thompson,
2001
). Finally, once distinct populations of cells arise,
subsequent tissue patterning may occur by sorting out as a result of
differential adhesion and/or cell motility
(Clow et al., 2000
;
Early et al., 1995
;
Matsukuma and Durston, 1979
;
Siu et al., 1983
;
Tasaka and Takeuchi, 1979
;
Traynor et al., 1992
).
In order to further understand this patterning process and its control, it
is important to identify the molecular components of the DIF-1 response
pathway and to determine how each component may be influenced by other
signals, such as those determining intrinsic biases. Only a few components of
the DIF-1 signaling pathway have been identified, although several different
mechanisms of signal transduction have been proposed. These include a steroid
hormone type receptor (Insall and Kay,
1990), signaling through intracellular calcium
(Schaap et al., 1996
),
intracellular pH (Gross et al.,
1983
) and the control of nuclear export
(Fukuzawa et al., 2003
).
Studies to identify DIF-responsive transcription factors have also had some
success. A minimal DIF-response element has been described that is both
necessary and sufficient for DIF-induced gene expression in cell culture
(Kawata et al., 1996).
Furthermore, several activities have been identified in cell extracts that
bind to this element in vitro and, ultimately, led to the identification of
the Dictyostelium STAT family of transcription factors
(Fukuzawa et al., 2001
;
Kawata et al., 1997
). Of
these, STATc is tyrosine phosphorylated and translocates to the nucleus of
pstO cells in response to DIF-1, where it represses the activity of a pstA
marker (Fukuzawa et al.,
2001
). However, STATc does not seem to play a role in the
activation of DIF-1 target genes. Expression of the pstO marker
ecmO/lacZ is unaffected in the STATc null mutant, and the mutant
shows little morphological similarity to the DIF-non-producing
dmtA mutant.
We have taken a forward genetic approach to identify mutants in key signaling molecules required to transduce the DIF signal. One such mutant, dimA, shows no response to DIF-1 in all conditions tested and exhibits morphological phenotypes indistinguishable from those of the dmtA mutant. However, key differences lie in the cell autonomous nature of the phenotype and the finding that dimA produces normal levels of DIF-1. As the dimA gene encodes a transcription factor of the bZIP or bRLZ classes, we propose that dimA encodes a key transcriptional regulator required to integrate DIF-1 signaling.
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Materials and methods |
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REMI mutagenesis and DIF-resistant mutant selection
REMI mutagenesis was performed as described
(Kuspa and Loomis, 1992),
except that pools of
5000 mutants were grown in shaken suspension
directly after transformation. Mutant cells were harvested and resuspended at
1x105 cells/ml in stalk salts [10 mM MES (pH 6.2), 1 mM
CaCl2, 2 mM NaCl, 10 mM KCl, 0.5 mg/ml streptomycin sulphate, 30
µg/ml tetracycline]. 3.75x106 cells were plated on tissue
culture dishes at a density of 1.6x104 cells/cm2,
and supplemented with 10 mM 8-Br-cAMP (Sigma) and 100 nM DIF-1. After 48
hours, detergent was added to a final concentration of 0.1% NP40 and 10 mM
EDTA, to eliminate unsporulated cells.
Development and whole-mount lacZ staining
Cells were developed at a density of 6.4x105
cells/cm2 on KK2 (16.1 mM KH2PO4, 3.7 mM
K2HPO4) plates containing 1.5% purified agar (Oxoid)
with or without 100 nM DIF-1. lacZ staining was performed as
described (Dingermann et al.,
1989).
Monolayer assays and lacZ marker quantitation in culture
All stalk and spore cell monolayer assays were performed as described
(Thompson and Kay, 2000a).
Induction of marker gene expression in dissociated cells was performed as
described (Berks and Kay,
1990
), except that 200 µM CaCl2 was added to the
buffer. For induction of lacZ markers in monolayers (I. Sarafimidis,
personal communication), mid-log phase cells were harvested, washed and
resuspended at 1x105 cells/ml in spore medium [20 mM KCl, 20
mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM MES (pH 6.2), 100
µg/ml streptomycin sulphate], containing 2 mM cAMP and 50 µM cerulenin,
with or without 100 nM DIF-1. 50 µl aliquots were added to each well of a
flat-bottomed 96-well tissue culture dish and incubated for 24 hours at
22°C. Cells were lyzed in 50 µl lysis buffer [200 mM HEPES (pH 8.0), 2
mM MgSO4, 4% TritonX-100] containing 2 mM CPRG (Roche).
ß-galactosidase enzyme activity was monitored by measuring the color
change at 575 nm.
Measurement of DIF levels
DIF levels were measured by development on agar containing
36Cl, followed by extraction of DIF with
chloroform/methanol, TLC separation and detection on a phosphorimager
(Kay, 1998).
Nucleic acid techniques
For northern blots (Berks and Kay,
1990), RNA integrity and loading were monitored by Methylene Blue
staining of ribsosmal RNAs (large subunit shown in figures).
Electrophoretic mobility shift assays (EMSA)
For dimA expression in E. coli strain BL21, the region
predicted to encode the DNA-binding and dimerization domains (amino acids
545-676) was cloned and expressed as a GST-fusion protein. Coomassie-stained
gels were used to ensure similar amounts of soluble protein from
dimA-expressing and control extracts were assayed. Oligonucleotides
corresponding to sequences proximal to the transcriptional start site of the
ecmO/lacZ reporter gene (Oligo1,
TTTTTATTTTTTTTTTTTTTATTTAAACAGTTACACCCCACAATTTTG; Oligo2,
GATCCAAAATTGTGGGGTGTAACTGTTTAAATAAAAAAAAAAAAAATA) were annealed, labeled, and
EMSA performed as described (Uv et al.,
1994), except that either 0.5 U/ml polydA/dT or 0.5 U/ml polydI/dC
(Roche) was included as a non-specific competitor. For competition assays with
mutant oligonucleotides (mOligo1,
TTTTTATTTTTTTTTTTTTTATTTAAACAGTTAAACACAACAATTTTG;
mOligo2,
GATCCAAAATTGTTGTGTTTAACTGTTTAAATAAAAAAAAAAAAAATA; where
bold letters indicate mutations) were annealed and used.
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Results |
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Second, we examined DIF-1 responsiveness using another monolayer test in
which cells are initially brought to competence to respond to DIF-1 by
treatment with cAMP, before removing the cAMP and incubating in the presence
of DIF-1. Under these conditions, wild-type cells differentiate as stalk cells
but not spores (Fig. 1B).
Again, dimA showed no DIF-1 response
(Fig. 1B), nor did it respond
to the chemically related stalk cell inducers DIF-2 and DIF-3
(Morris et al., 1988) (data
not shown).
Finally, to test whether any of the observed defects in DIF response were
specific to terminal differentiation, we employed two independent tests in
which changes in gene expression were monitored in response to DIF-1. First,
we used a shaken suspension assay (Berks
and Kay, 1990). Cells were developed to the mound stage,
dissociated, and shaken in suspension with cAMP and DIF-1. We observed that
the prestalk markers ecmA and ecmB were induced in wild-type
cells by DIF-1 treatment, and the prespore marker cotB was repressed
(Fig. 1C). Under the same
conditions, dimA cells showed little or no response
to DIF-1 (Fig. 1C). Second, we
used a variation on the monolayer assay in which cells are prevented from
undergoing terminal differentiation because of the continued presence of high
levels of cAMP. Under these conditions, any effects of endogenous DIF-1 are
minimal because its biosynthesis is inhibited by the addition of cerulenin
(Kay, 1998
). The level of the
DIF response was determined by quantification of ß-galactosidase activity
from strains carrying cell-type-specific reporter constructs. Quantification
revealed that the prestalk reporter constructs ecmAO/lacZ and
ecmB/lacZ were efficiently induced by DIF-1 in wild-type cells but
not in dimA cells
(Fig. 1D). Furthermore, the
prespore marker construct cotB/lacZ was strongly repressed by DIF-1
treatment in wild-type cells, but was unaffected in
dimA cells (Fig.
1D). Therefore, in all conditions tested, dimA is
required for cellular responses to DIF-1.
dimA encodes a bZIP or bRLZ transcription factor
We identified the disrupted gene by plasmid rescue and found the insertion
to lie in the second exon of a gene with an 3700 bp ORF
(Fig. 2A). The predicted
protein product of the dimA gene shows strong sequence similarity
over 66 amino acids to the DNA-binding and dimerization domains of bZIP and
bRLZ transcription factors (Fig.
2B), which are required for the activation and repression of gene
expression in response to a wide variety of signals
(Hurst, 1995
;
Jakoby et al., 2002
).
|
dimA and dmtA show similar developmental phenotypes
The experiments with cells in culture show that dimA is required
for each response to DIF-1 that we have investigated. To determine the
function of dimA in normal development, we investigated the
developmental phenotype of the dimA mutant,
comparing it as appropriate with the dmtA mutant,
which is specifically defective in DIF-1 synthesis.
dimA cells grow normally in axenic medium, but when starved on buffered agar exhibit clear morphological defects. Aggregation takes place with relatively normal timing, although there is a tendency for the streams to break up (data not shown). However, clear defects are observed at the finger and slug stages of development. The dimA mutant fingers are extremely long and thin, resulting in the formation of similarly defective migratory slugs (Fig. 3D). Furthermore, after a period of migration, dimA slugs tend to break apart (Fig. 3E). Finally, at the time of fruiting body formation, rather than the stalk lifting the sorus from the agar, as in the wild-type, stalks and spores lie on the surface of the agar, resulting in plates with an untidy appearance (Fig. 3F). Time-lapse microscopy reveals that this is due to both the collapse of the comparatively fine stalks and repeated attempts at culmination (data not shown). The overall morphology of the mutant is strikingly similar to that of the `DIF-less' dmtA mutant (Fig. 3G-I), supporting the notion that dimA, like dmtA, functions in the DIF signal transduction pathway.
|
First, we found that the timing of the initiation of development appears normal, as indicated by the repression of cprD transcripts. Both the wild type and the mutant express cprD during growth, and downregulate its transcripts during the first 6 hours of development (Fig. 4A). Second, we tested the expression of cell-type-specific products, namely the expression of the prestalk markers ecmA and ecmB (which can be induced by DIF-1) and prespore marker cotB (which can be repressed by DIF-1). Expression of these markers in the mutant was unaffected both in terms of timing and levels of expression (Fig. 4A). Therefore, like the dmtA mutant, the DIF non-responsive mutant dimA expresses prestalk and prespore markers.
|
Finally, we tested whether the dimA gene is expressed in a
cell-type-specific manner. mRNA was extracted from separated prestalk and
prespore cells at the slug stage of development
(Ratner and Borth, 1983), and
dimA transcripts were detected by northern blot. We found
dimA mRNA in both prestalk and prespore cells, with the highest
levels of expression in prespore cells
(Fig. 4B). The developmental
timing and broad expression of dimA is therefore consistent with a
role in DIF-1 signaling.
dimA exhibits defects in pstO cell differentiation
Although prestalk and prespore transcripts were detected on northern blots,
this gives no information about spatial patterns of gene expression. As the
dmtA mutant shows non-cell autonomous defects in
pstO differentiation, whereas pstA differentiation appears normal
(Thompson and Kay, 2000b), we
tested whether dimA exhibits similar defects. First, as the
ecmAO/lacZ marker is expressed in both pstA and pstO cells in
wild-type slugs, a shortening of its zone of expression would be expected in
the mutant as a result of expression in pstA cells but not in pstO cells.
Indeed, similar results were described in dmtA mutant slugs
(Thompson and Kay, 2000b
).
Consistent with this, we found the ecmAO/lacZ staining region in
dimA mutant slugs to be approximately 50% shorter
than that of wild-type control transformants
(Fig. 5A-D).
|
dimA produces DIF-1 and dmtA responds to DIF-1
All the above results support a model in which dimA is required to
transduce the DIF-1 signal. However, an alternate explanation for the
similarity between the phenotypes of the dimA and
dmtA mutants is that the DIF response is required
for DIF production, or vice versa. We therefore sought to determine whether
dimA produces DIF-1 and whether the
dmtA mutant responds to DIF-1. First, we measured
DIF-1 production in dimA cells by developing the
mutant on agar containing 36Cl, before extraction
of organic compounds and TLC separation. The results, in
Fig. 6A, clearly illustrate
that dimA produces DIF-1 and its breakdown product
DIF-3. Although the levels are slightly lower in the mutant than in the wild
type, this is likely to be due to the slight developmental delay exhibited by
dimA, especially at later stages of development.
Second, we tested the response of the dmtA mutant
to DIF-1 in 8-Br-cAMP monolayers. The dmtA mutant
shows a response indistinguishable from that of wild-type cells, either when
measured in terms of stalk cell induction
(Fig. 6B) or spore cell
repression (Fig. 6C).
|
dimA exhibits cell autonomous defects
Although there are great similarities between the developmental phenotypes
of the dimA and dmtA
mutants, if dimA is defective in DIF-1 responses,
then any defects would be predicted to be cell autonomous. We therefore tested
this hypothesis.
First, we compared the effects of exogenously added DIF-1 on the
development of the dmtA and
dimA mutants. Consistent with previous reports
(Thompson and Kay, 2000b), the
developmental defects of the dmtA mutant are
effectively rescued by development on DIF-1 agar
(Thompson and Kay, 2000b
)
(Fig. 7A;g-i). However, despite
morphological similarity with dmtA, the
dimA mutant is not rescued by development on agar
containing 100 nM DIF-1. Most notably, dimA slugs
remain long, thin and broken (Fig.
7A;d,e), whereas dmtA slugs are of
wild-type appearance (compare with Fig.
7A;g,h; and see Fig.
3). In addition, the culmination defects are rescued in the
dmtA mutant but not in
dimA (Fig.
7A;c,f,i). Furthermore, unlike wild-type cells, which show a
developmental delay on DIF agar, dimA cells appear
largely unaffected. Therefore, the morphological defects of the
dimA mutant are unaffected by exogenously added
DIF-1. This is consistent with the idea that dimA is required to
transduce the DIF-1 signal rather than to produce it.
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Discussion |
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How is dimA activity regulated?
DIF-1 induces prestalk markers and represses prespore markers both in vivo
and in cell culture (Berks and Kay,
1990; Thompson and Kay,
2000b
; Williams et al.,
1987
). However, there are no reports of common elements required
to mediate the transcriptional effects of DIF in prestalk and prespore
promoters. It was therefore unknown whether both target gene activation and
repression were mediated by the same transcription factors. The results
described in this paper suggest that dimA is a common factor in both
pathways, as it is required for both the activation and repression of
DIF-responsive gene expression. This raises the question of how the activity
of DimA might be controlled in order to function as both an activator and
repressor. One possibility arises from the sequence similarity of DimA to
bZIP/bRLZ transcription factors. bZIP/bRLZ proteins bind DNA as obligate
dimers. Their ability to form heterodimers and the choice of partners is
important in the regulation of their activity
(Lee, 1992
). As DimA
represents the first functionally characterized bZIP/bRLZ transcription factor
in Dictyostelium, it is unknown whether it is also able to form
heterodimers. However, searches of the public databases reveal that a number
of related proteins are likely to be encoded by the Dictyostelium
genome (C.R.L.T. and G.S., unpublished).
bZIP/bRLZ transcription factors have been described in a wide variety of
organisms (Chinenov and Kerppola,
2001; Hurst, 1995
;
Jakoby et al., 2002
). The
largest number of putative bZIP/bRLZ proteins has been identified in plants
(Jakoby et al., 2002
);
however, to date, the signaling pathways regulating most bZIP transcription
factors in plants are largely uncharacterized. Similarly, little is known
about the genes required for DIF signaling beyond dmtA (signal
production) and dimA (transcription factor). However, a number of
factors that influence DIF signaling, or correlate with cell fate choice, have
already been described. These include intracellular calcium levels,
intracellular pH, growth history and cell cycle position
(Azhar et al., 2001
;
Gomer and Firtel, 1987
;
Gross et al., 1983
;
Leach et al., 1973
;
Schaap et al., 1996
;
Thompson and Kay, 2000a
;
Weijer et al., 1984
). It will
therefore be of interest to determine whether these factors affect
dimA directly, both to further our understanding of DIF signaling and
to shed light on the regulation of bZIP/bRLZ activity in other organisms. Our
selection strategy provides a means to identify other genes required for DIF
signal transduction and should enable us to identify such factors.
Does DIF play a role in prespore cells?
DIF-1 has been widely viewed as a prestalk inducer, but most DIF-1
biosynthesis takes place in prespore cells
(Kay and Thompson, 2001). This
scheme requires that prespore cells become somewhat DIF-1 insensitive.
However, prespore cells do not lose their ability to respond to DIF-1
altogether. For example, dissociated prespore cells downregulate prespore
markers when treated with DIF-1 (Berks and
Kay, 1990
). Furthermore, low levels of DIF-1 have been reported to
stimulate the expression of prespore markers in cell culture
(Oohata, 1995
). The results
presented here provide evidence that dimA is the link between the
prespore and prestalk responses to DIF-1. First, dimA is required for
the repression of the prespore gene cotB, in addition to being
required for the activation of prestalk markers. Second, dimA
transcripts are expressed in both prestalk and prespore cells at the slug
stage of development, and prespore cells express the highest levels of
dimA. Finally, the dimA mutant exhibits cell autonomous
defects in both prestalk and anterior prespore differentiation. Taken
together, these results suggest that dimA is present in and required
for normal prespore cell differentiation.
If dimA is indeed dedicated to the regulation of DIF-1 signaling, as might be inferred from the phenotypic similarities between the dimA and dmtA mutants, then our results suggest a novel role for DIF signaling in prespore cell differentiation.
DIF-1 signaling and pstO cell function
Studies of the patterns of marker gene expression in the
dmtA mutant suggest that DIF-1 signaling is
required for the normal differentiation of pstO cells, but not for the
differentiation of pstA cells (Thompson
and Kay, 2000b). As we found the classical markers of these cell
types to be poorly expressed in the AX4 parental strain of the dimA
mutant, we were unable to test this directly (C.R.L.T. and G.S., unpublished).
Nevertheless, the patterns of expression of more robustly expressed prestalk
and prespore markers reveal the only detectable cell-type defects in
dimA to be consistent with defects in pstO cell
differentiation, whereas pstA differentiation is unaffected. These
observations strengthen the idea that DIF-1 is only required for the
differentiation of pstO cells.
Little is known about the role of pstO cells during normal development. However, these studies further highlight the possibility that the defects in pstO cell differentiation in the dmtA and dimA mutants can explain the major morphological defects visible at the slug stage of development. For example, as mutant slugs tend to break apart, it might be proposed that pstO cells play a role in maintaining slug integrity. In order to understand the role of pstO cells during normal development, it will be important to identify the compliment of genes expressed specifically in this cell type. As it seems likely that a number of these genes will be directly regulated by dimA, this mutant provides another valuable tool for the study of pstO differentiation and function.
DNA binding and dimA target genes
Although bacterially expressed DimA protein binds a fragment from the
ecmO promoter, it is unclear whether this binding is functionally
relevant in the context of the minimal region required for pstO gene
expression, as in subsequent mutational studies we were unable to pinpoint the
exact residues bound (see Materials and methods; C.R.L.T and G.S.,
unpublished). However, we do not believe the binding to be non-specific, as it
can be detected in the presence of excess non-specific DNA. It is more likely
to reflect that at present we do not know (1) whether the ecmO
promoter is a direct DimA target gene, (2) whether DimA normally binds DNA as
a homodimer, or, consequently, (3) the optimal DimA-binding site. Therefore,
in order to understand DimA DNA binding and its regulation, it will first be
important to identify true DimA target genes, and to define the DimA- or
DIF-response elements in these.
dimA and pattern formation
It has been proposed that pstO cells initially differentiate scattered
amongst prespore cells in response to DIF-1
(Early et al., 1995;
Thompson and Kay, 2000b
), and
then subsequently sort out as a result of differential adhesion or chemotaxis
(Clow et al., 2000
;
Early et al., 1995
;
Matsukuma and Durston, 1979
;
Siu et al., 1983
;
Tasaka and Takeuchi, 1979
;
Traynor et al., 1992
). The
identification and study of the dimA mutant,
together with studies on the dmtA mutant, provide
important tools to dissect the regulation of this developmental mechanism in
Dictyostelium. For example, an understanding of how the various
inputs might generate stochastic differences in dimA activation could
explain why a subset of cells adopt the pstO rather than the prespore cell
fate. As this developmental mechanism is likely to be used in other organisms,
these studies will provide insights into conserved features of its mechanism
and regulation.
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ACKNOWLEDGMENTS |
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