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First published online 22 February 2006
doi: 10.1242/dev.02287
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1 Biozentrum der Ludwig-Maximilians-Universität, Grosshadernerstrasse 2,
82152 Planegg-Martinsried, Germany.
2 Centre for Structural and Functional Genomics, Concordia University Montreal,
Quebec H4B 1R6, Canada.
3 Dipartimento di Scienze Cliniche e Biologiche, Università di Torino,
Ospedale S.Luigi, 10043 Orbassano, Torino, Italy.
* Author for correspondence (e-mail: macw{at}zi.biologie.uni-muenchen.de)
Accepted 18 January 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Ameba, Cell cycle, Differentiation, Evolution
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kaiser, 2001), but some gene
families are known to be important in the multicellular development of both of
these groups. Such genes may have initially acquired their developmental
functions in the unicellular ancestors of modern forms. The study of
unicellular differentiation may thus provide insights about the original
functions of such conserved developmental mechanisms.
The Amebozoans are a group of unicellular and facultative multicellular
organisms which separated from the lines leading to animals and plants about
when these split from one another (van der
Peer et al., 2000; Eichinger
et al., 2005). Like most microorganisms, Amebozoans proliferate
indefinitely when provided with an adequate food supply. When they starve,
however, the amebae transform to cysts, dehydrated and encapsulated cells that
can survive for long periods under hostile conditions. If food again becomes
available, the cysts germinate and re-enter the proliferative phase. The best
studied Amebozoan, Dictyostelium discoideum (reviewed by
Kessin, 2001), is unicellular
in the proliferative phase, but shows a primitive form of multicellular
development. Cells aggregate when the nutrition is exhausted and after a short
multicellular migration phase, most of the amebae differentiate as cyst-like
cells called spores. A minority of the cells enter a second differentiation
pathway to form stalk cells; the stalk elevates the spore mass from the
substrate and is thought to facilitate dispersal.
Dictyostelium is probably derived from a purely unicellular
Amebozoan [S. Baldauf, personal communication; see also Alvarez-Curto et al.
(Alvarez-Curto et al., 2005)],
and this implies that its simple multicellularity evolved independently from
that of animals and plants. Dictyostelium nonetheless has orthologs
of important plant and animal differentiation regulators, and these may be in
involved in Dictyostelium development. One example is the homeodomain
gene wariai, a negative regulator of stalk differentiation
(Han and Firtel, 1998);
another is the Dictyostelium ß-catenin ortholog
aardvark, also a repressor of the stalk pathway
(Coates et al., 2002;
Coates, 2003).
The decision for stalk or spore differentiation depends on an interaction
of cell-autonomous properties determined in the unicellular stage with
intracellular signals in the aggregate. If two different
Dictyostelium cultures are mixed just before development, one
frequently observes that the cells of one culture preferentially form stalk,
while the other amebae preferentially make spores; if unmixed, both cultures
would make normally proportioned fruiting bodies. `Pathway preferences' may
result from differences in the composition of the growth medium or in stage of
the growth curve when harvested. Preferences may also be found among the cells
of a single culture, and here they depend on the cell cycle position of
individual amebae at the moment when development begins (reviewed by
MacWilliams et al., 2001).
Preferences are not absolute but relative, so that cells can be placed in a
linear hierarchy from most stalk-preferring to most spore-preferring
(Leach et al., 1973;
Fortunato et al., 2003).
During the multicellular stage, the proportions of spores and stalk cells are
regulated by negative feedback (Rafols et
al., 2001) and theoretical models suggest that pathway preferences
modulate the sensitivity of cells to negative-feedback signals
(Blaschke et al., 1986). Recent
studies suggest that nutritional state and cell cycle position modulate the
sensitivity of cells to the negative feedback regulator DIF, a chlorinated
hydroxyphenone made by cells of spore pathway that promotes stalk
differentiation (Kay and Thompson,
2001).
In a database survey for Dictyostelium genes that might link
pathway preferences with the cell cycle, we came across an ortholog of the
`retinoblastoma-susceptibility gene' Rb, a gene that regulates both
the cell cycle and differentiation in animals and plants (reviewed by
Classon and Harlow, 2002).
Rb was discovered as a tumor suppressor that is inactivated, directly
or indirectly, in most cancers (Sherr,
1996). In the cell cycle, the protein pRb blocks the G1/S
transition through its interaction with transcription factors of the E2F
family. E2Fs target many genes required in the S phase, and pRb both
sequesters `activator E2Fs' and is recruited to the promoter by `inhibitor
E2Fs' where it participates in inhibitory chromatin remodeling complexes
(reviewed by Trimarchi and Lees,
2002). pRb is neutralized at the end of G1 when it is
phosphorylated by cyclin-dependent kinases, which leads it to dissociate from
E2Fs of both classes.
The role of Rb in cell differentiation is perhaps best illustrated
by the phenotype of the Rb-null mouse; this is superficially normal
in early development, but dies before birth with massive defects in many
differentiated tissues, including muscle, blood and nerves
(Zacksenhaus et al., 1996).
During normal development, Rb is strongly expressed in these tissues
(Szekely et al., 1992;
Jiang et al., 1997) and pRb
interacts with tissue-specific transcription factors to promote both cell
cycle withdrawal and the expression of terminal genes
(Gu et al., 1993;
Novitch et al., 1999;
Thomas et al., 2001;
Chen et al., 1996;
Cole et al., 2003;
Iavarone et al., 2004;
Toma et al., 2000;
Batsché et al., 2005).
Rb may also have additional roles in mammalian differentiation. There
are intriguing reports of Rb acting as a `selector gene', favoring
white over brown adipose differentiation
(Hansen et al., 2004) or
specifically inhibiting neurendocrine cell fate in the lung
(Wikenheiser-Brokamp, 2004);
the Rb-related gene p130 favors neuronal rather than glial
differentiation (Jori et al.,
2001). In a particularly fascinating case, pRb may referee the
interaction of activin and nodal signaling by competing with goosecoid for
binding to the transcription factor PU.1
(Konishi et al., 1999).
Rb-family genes also affect differentiation in Drosophila
(Du and Dyson, 1999),
Caenorhabditis (Lu and Horwitz,
1998; Myers and Greenwald,
2005) and Arabidopsis
(Ebel et al., 2004).
The widespread role Rb-family genes in multicellular development encouraged us to investigate the role of the Dictyostelium retinoblastoma ortholog rblA in the development of this Amebozoan. A priori, any of a variety of functions were conceivable, ranging from cell-cycle control, regulation of cell cycle exit and selector gene function. Our results suggest that the major role of rblA may be to modulate pathway preference. rblA also affects the Dictyostelium growth-development transition, but its action here is unusual: whereas Rb in animals and plants promotes cell cycle exit and differentiation, rblA inhibits the onset of development. We interpret this as an adaptation specific to social amebae, and suggest that in ancestral Amebozoans, the role of Rb in development was similar to that in higher organisms.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and derivatives were used in all experiments. For
disruption of rblA, exponentially growing cells were electroporated
with 10-20 µg of transforming DNA in 0.1 cm cuvettes
(Pang et al., 1999) and
selected in HL5 containing 10 µg/ml blasticidin. For the reporter- and
overexpression constructs, we used the transformation method previously
described (Wetterauer et al.,
1996) and modified (Deichsel
et al., 1999). Cell cycle synchronization, BrdU staining and
ß-galactosidase measurements were performed as previously described
(MacWilliams et al., 2001).
Measurement of the DIF response was as described
(Thompson and Kay, 2000).
Vector construction
The rblA disruption construct contains the first 903 bp of the
rblA-coding region, followed by a blasticidin-resistance cassette
(Adachi, 1994) and bp 1699-2949 of rblA. The reporter
rblA::
pgal contains 1126 bp of genomic sequence upstream of
the rblA ATG plus the first 21 rblA codons, with upstream
XbaI and downstream BglII sites, fused to the reporter
i-pgal (Gaudet et al.,
2001). The overexpression vector A15::rblA contains the
actin 15 promotor followed by eight actin codons, a
BglII/BamHI fusion, 10 codons specifying a myc
epitope, a BglII site, followed by the entire rblA-coding
region and 
1500 bp of downstream genomic sequence, culminating in an
actin 8 terminator. The cassette was assembled in V18tn5 turbo
(Deichsel et al., 1999).
Measurement of nuclear DNA content
Measurements were performed essentially as described previously
(Weijer et al., 1984).
Axenically grown cells or spores harvested from bacterial plates were
collected by centrifugation, resuspended in 70% ethanol and maintained at
4°C until needed. For staining, cells or spores were collected once again
by centrifugation and suspended in 1 µg/ml DAPI. In most experiments, cells
and spores were mixed and viewed in a chamber made of two coverslips separated
by spacers. Images were captured using a Zeiss 63x/1.25/oil objective, a
Till Photonics Polychrome IV illuminator, a Sensicam 370-KL cooled CCD camera
and Tillvision software. A short through-focus film was obtained from each
field and the best frame selected for each cell or spore. Polygon ROIs were
drawn about the nucleus and the cell body as a whole, and average and
integrated pixel values measured; this allowed correction of nuclear
fluorescence for cytosolic background. Controls were performed to confirm that
the DAPI concentration was sufficient to saturate the fluorescence of both
spore and cell nuclei, and that fading during image acquisition was
negligible. To compare wild-type and rblA-null spores, each was mixed
with wild-type vegetative cells, and the spore measurements were normalized to
the mean nuclear fluorescence of the cells. In the comparison of wild-type and
rblA-overexpressing cells, wild-type spores were similarly used as an
internal standard.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The alignment of the predicted protein with sequences from different
organisms revealed two regions of high similarity. One, near the N terminus
(Fig. 1A,B), encompasses the
pRb interaction domains with the transcription factor SP1 and the pRb kinase
cdk4/cyclin D (Connell-Crowley et al.,
1997). Here, the sequence is 20% identical and 35% similar to
human pRb. The remaining region (23% identical/38% similar) encompasses the
pfam RbA and RbB domains (Marchler-Bauer
et al., 2003) (Fig.
1A,C). Eighteen out of the 27 amino acids important for generation
of the `pocket motif' are conserved, as are 10 of the 12 residues that
interact with the LXCXE motif (Umen and
Goodenough, 2001; Lee et al.,
1998). In its overall size, rblA resembles the related
pocket proteins p107 and p130 more closely than pRb
(Fig. 1D), and the resemblance
to the latter is also greater in pairwise blastP matches of the N-terminal
domains (expected values of 4e-07 and 6e-05 versus 9e-1). The match is better
to pRb in the spacing of the RbA and RbB boxes, and the closer resemblance in
this region is also detected by the blastP algorithm (3e-18 versus 2e-10 and
2e-11). rblA lacks two conserved features of p107 and p130, a cdk
inhibitor domain and a bipartite B box
(Classon and Dyson, 2001) and
in this aspect is also closer to pRb.
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To determine whether rblA expression is cell-type specific or
common to all developing cells, we constructed a reporter,
rblA::pgal, in which genomic sequences upstream from
rblA drive a short-lived ß-galactosidase reporter. Using a
sensitive chemiluminescent assay, we could detect ß-galactosidase
activity in vegetative cells; this increased about 200x during
development (Fig. 2B). When
allowance is made for differences in developmental timing and the
developmental asynchrony that is common in transformants, the pattern is
consistent with that seen in the rblA northern blot. As the construct
contains all but 300 bp of the intergenic region separating rblA from
the divergently transcribed upstream neighbor, talA, it seemed
possible that its behavior could be influenced by talA promoter
elements. The temporal regulation of talA is very different from that
of rblA, however; it is expressed at moderate levels in vegetative
cells, peaks at 6-9 hours of development and subsequently decreases (A.
Mueller-Taubenberger, personal communication). We conclude that the reporter
reflects rblA promoter activity. Using X-gal as a substrate, reporter
expression was not detectable during proliferation, but readily visible in
multicellular stages (Fig. 2C)
in positions where cells of the spore pathway are characteristically found
(Fig. 3). In differentiation,
rblA is essentially specific for the spore pathway.
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1.2 kb truncated message fragment (not shown). If
this is translated, the product would be a fragment of the N-terminal domain,
which in pRb inhibits substrate binding
(Goodrich, 2003), and is not
predicted to lead to a partial-activity phenotype. Nonetheless, there is a
formal possibility that some features of the null phenotype result from this
message.
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) and
the spore-specific reporter spiA-gal
(Richardson and Loomis, 1992),
as well as the prestalk-specific construct ecmA-gal
(Early et al., 1993). The
staining patterns were all normal (not shown); rblA is not necessary
in either the stalk or spore differentiation pathways.
Proliferating Dictyostelium cells have a long G2 phase, while G1
is not normally detectable (Weijer et al.,
1984). A recent study (Chen et
al., 2004) suggests that spores arrest in G1, and we wondered
rblA expression in the spore pathway might be responsible. In this
case, one would expect the G1/S block to be abolished in rblA-null
cells, so that rblA-null spores would have twice as much DNA as the
wild type. We compared the nuclear DNA contents of rblA-null and
wild-type spores using the nuclei of wild-type cells as an internal standard.
The relative fluorescence values (mean±s.d.) were 0.98±0.04 for
wild-type spores, 1.07±0.04 for rblA-null spores and 1 for the
standard. The data thus do not support the idea that rblA brings
about G1 arrest during spore differentiation. It appears, moreover, that,
under our conditions, wild-type spores have the same nuclear DNA content as
proliferating cells (Fig. 4A),
i.e. they are predominantly/exclusively G2. As we used a different
Dictyostelium strain from Chen and co-workers (Ax2 versus Ax4),
estimated DNA content using a different dye (DAPI versus propidium iodide) and
employed a different measurement principle (nuclear fluorescence via imaging,
versus whole-cell fluorescence via flow cytometry), it is not immediately
apparent why our results differ from theirs.
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Pathway preference is normally determined by the composition of the growth
medium, the stage of the growth curve and the cell cycle phase, and we wished
to determine whether these factors influence rblA expression. In
cells carrying the rblA::pgal gal reporter, which had
been synchronized by cold release, reporter expression (measured by
chemoluminescence) was maximal 3 hours before S phase, defined as the peak
nuclear of BrdU incorporation (Fig.
6). Given a doubling time of 10-12 hours, a 30-minute M phase and
a negligible G1 (Weijer et al.,
1984), rblA is maximally expressed in the late G2 phase,
when cells have a strong prespore-differentiation preference
(Thompson and Kay, 2000;
MacWilliams et al., 2001). We
then diluted stationary-phase cells into normal or glucose-free media. The
reporter expression increased sharply after dilution in both media and
decreased again as the cells approached stationary phase
(Fig. 7); the maximal activity
was about threefold higher in glucose-containing than in glucose-free media.
rblA expression is thus highest in cells growing in normal medium,
intermediate during growth without glucose, and lowest in stationary phase
cells; this is exactly the order of spore-formation preferences found in
classical studies (Leach et al.,
1973).
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Pathway preferences are thought to act by modulating the sensitivity of
cells to a negative-feedback regulator of stalk-spore proportioning
(Blaschke et al., 1986) and
recent work suggests that both cell cycle position and composition of the
growth medium modulate the sensitivity of cells to the negative-feedback
regulator DIF (Kay and Thompson,
2001). We accordingly wished to determine whether
rblA-null cells show altered DIF responsiveness. Wild-type and
rblA-null cells were transformed with the stalk-cell specific
reporter ecmB-gal (Early et al.,
1993), and the DIF response was measured in monolayers
(Thompson and Kay, 2000) using
ß-galactosidase activity as a measure of stalk induction
(Fig. 9). rblA-null
cells appeared to be about threefold more sensitive than wild type; the
difference was significant at P<0.05.
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Rb-deficient vertebrate myoblasts can sometimes be induced to differentiate
by growth factor withdrawal, but the myofibrils are unstable and the nuclei
re-enter S-phase if replaced in normal growth medium
(Gu et al., 1993). Wild-type
Dictyostelium cells that have begun development will resume
vegetative growth if nutrients are re-supplied, but only after a period of
several hours (Soll and Wadell,
1975). We wondered if this delay might reflect the stabilization
of cell cycle withdrawal by rblA. We therefore allowed wild-type and
rblA-null cells to develop on filters for 10 hours, after which they
were washed free and resuspended in growth medium. Both strains resumed growth
after 5 hours (see Fig. S2 in the supplementary material), at which point BrdU
labeling also resumed (not shown). No difference between the strains was
apparent.
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; Huang et al.,
1988; Goodrich et al.,
1991), we reasoned that overexpressing rblA might create
a stable G1 phase, and that this would provide strong evidence that
rblA is functionally a retinoblastoma-family gene. We constructed a
vector (A15::rblA) in which rblA-coding sequences
are under the control of the Dictyostelium actin15 promoter, which is
widely used as a strong and unspecific promoter in Dictyostelium
studies. Transformed clones grew exceptionally slowly, though the cells
appeared healthy under phase-contrast optics. Clones were stained in situ with
DAPI and the nuclear DNA contents measured, using wild-type spores as an
internal standard. The results suggested that the cells were predominately G1,
though the distribution also included some G2 cells
(Fig. 4B). Thus, rblA
can produce a long-lasting G1 phase in proliferating Dictyostelium
cells when expressed at non-physiological levels. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Very little has been published on potential rblA-interacting
molecules in Dictyostelium, but a survey of genomic data
(www.dictybase.org)
suggests some conservation of proteins whose animal orthologs participate in
Rb-containing complexes. There is a fairly clear E2F homolog (DDB0216397) and
a related gene (DDB0220101) with closer affinities to the E2F-binding partner
DP. Two particularly notable genes are RbbD (DDB0232003) and
lin9 (DDB0232078). The human orthologs are RbAp48 and LIN9; the
former is found in several complexes involved in pRb-mediated repression
(Lai et al., 2001;
Nicolas et al., 2001;
Vaute et al., 2002) while the
human ortholog LIN9 interacts with pRb in osteoblast differentiation
(Gagrica et al., 2004). The
Caenorhabditis orthologs lin-53 and lin-9 interact
with Rb in the synMuvB group to regulated vulva differentiation
(Lu and Horwitz, 1998;
Beitel et al., 2000). The
Drosophila orthologs CAF1p55 and Mip130 are found with pRb in the
dREAM complex, which localizes to transcriptionally silent chromatin and
represses differentiation- and sex-specific genes
(Korenjak et al., 2004;
Taylor-Harding et al., 2004).
Orthologs of RbbD and lin9 have also been described in
plants (Ach et al., 1997;
Bhatt et al., 2004). A fourth
potential pRb-interactor in Dictyostelium is RbbB
(DDB0220639), the vertebrate ortholog of which, Rbbp2, recruits HDACs to
promoters repressed by Rb/E2F (Gray et
al., 2005). Dictyostelium has a histone methyltransferase
(Chubb et al., 2006), two
putative histone deacetylases (DDB0189724; DDB0190980) and genes for four
chromatin-remodeling ATPases of the SNF2 group, including one apparent SWI/SNF
homolog (DDB022695) the vertebrate relatives of which mediate
retinoblastoma-repression of cyclin A and the polo-like kinase
(Siddiqui et al., 2003;
Gunawardena et al., 2004) and
the plant homologs of which are extensively involved in the control of
development (Sarnowski et al.,
2005; Zhou et al.,
2003).
Dictyostelium has a `minimal set' of cell cycle genes, inviting
comparison with Ostreococcus (Robbens et
al., 2005). There are three cyclins of the cell-cycle group,
cycA (DDB0231774), cycB (DDB0185035) and cycD
(DDB0231773), and one cyclin-dependent kinase, cdk1 (DDB0185028), in
the superfamily defined by animal Cdks 1/2 and plant Cdks A/B. There is no
cdk4/6 homolog, and no E-type cyclin, as in plants.
rblA in Dictyostelium development
Dictyostelium expresses rblA primarily in cells of the
spore differentiation pathway. During growth, when the promoter is very weakly
expressed, its activity is correlated with the `spore differentiation
preference' of cells: it is highest in the late cell cycle
(Fig. 5), in the exponential
phase of the growth curve and in cells grown in rich medium
(Fig. 6). During development,
the promoter activity increases 200-fold and can be demonstrated by X-gal
staining in the periphery of tight aggregates (where prespore gene expression
begins), in the prespore zones of slugs and in the nascent spores
(Fig. 3). The message is
detectable in northern blots only during the period of cell-type-specific gene
expression.
Although the expression pattern suggests a function in spore differentiation, rblA-null mutants make spores which are grossly indistinguishable from those of the wild type, and withstand dessication and freezing; rblA is thus dispensable for spore formation. Experiments with cell-type-specific reporters demonstrate, furthermore, that rblA-null slugs have normal or near-normal ratios of prestalk and prespore cell types. Although Dictyostelium spores have been reported to show G1 arrest, spores from rblA-null strains do not differ in nuclear DNA content from the wild type.
As rblA expression is correlated with spore differentiation
preference, we finally considered the possibility that rblA might be
involved in this elusive phenomenon. We found in fact that rblA-null
cells are strongly enriched in the prestalk zones of chimeric slugs
(Fig. 4), while they are
depleted in the prespore zones and spores. When rblA-null cells are
given a free choice of the stalk and spore pathways, they show a strong
preference for stalk differentiation. Such preferences are well known in
Dictyostelium, and have been explained as differences in sensitivity
to the stalk-cell inducer DIF (Thompson
and Kay, 2000). We therefore tested the DIF responsiveness of
rblA-null cells and found them significantly more sensitive than the
wild type (Fig. 7). Our data
suggest that the factors controlling pathway preference in
Dictyostelium influence rblA promoter activity, and that
rblA in turn controls the responsiveness of cells to DIF.
rblA in the Dictyostelium cell cycle and at the growth-development transition
rblA has only relatively subtle effects during cell growth and at
the growth-development transition. In contrast to mammalian cells, where a
triple knockout of all three Rb-family genes reduces the cell cycle length by
about 35% (Sage et al., 2000),
deletion of the single Rb ortholog of Dictyostelium has no
significant effect on the generation time. In this respect,
Dictyostelium resembles the unicellular alga Chlamydomonas,
in which cells deficient in mat3 show unchanged mass doubling rate
(Umen and Goodenough, 2001).
This difference presumably reflects a fundamental difference in multicellular
and unicellular life style; in microbes, the cells proliferate as fast as
nutrition (or in the case of algae, light) allows, while in multicellular
organisms, proliferation is regulated by growth factor signaling and is
usually slower than metabolic resources alone would sustain. Removing
Rb family genes in mammalian cells thus eliminates a regulatory level
that is not functional in unicellular organisms. In Dictyostelium, we
nonetheless found that rblA can block the cell cycle in the presence
of nutrition if expressed at nonphysiological levels; raw material is thus
present from which multicellular-type controls could be fashioned.
Proliferating rblA-null cells are significantly smaller than
wild-type cells (Fig. 8).
Similar phenomena are seen in triple-Rb family-knockout mammalian
cells and Chlamydomonas; in all three cases, the volume reduction is
50%. The mechanism underlying this effect is unknown.
Given the dramatic increase in rblA expression in development, we
were surprised to find that RblA-null cells initiate development
earlier and complete development more rapidly than the wild type
(Fig. 9). The
growth-development transition in Dictyostelium is a complex process
in which the yakA kinase plays a central role
(Souza et al., 1998;
Souza et al., 1999). Both the
rblA-null and the yakA overexpressor cease growth
prematurely, so that one could speculate that rblA null suppresses
yakA. The relationship between these two signals is not
straightforward, however, as yakA also affects cell size and, here,
rblA-null resembles the yakA-null.
Dictyostelium, retinoblastoma and evolution
The retinoblastoma-susceptibility gene Rb was discovered as a
vertebrate tumor suppressor, the protein product of which, pRb, inhibits or
represses many genes required at the G1/S transition or in S phase. Vertebrate
Rb is upregulated during the differentiation of multiple tissues; it
is required for normal differentiation in many of these and, in several, pRb
is known to synergize with tissue-specific transcription factors. These
interactions often have a strong positive-feedback or feed-forward character.
They are usually specific to the hypophosphorylated form of pRb, which occurs
only in G1, and in addition to promoting the expression of terminal
differentiation genes, the interacting factors often induce either pRb itself
or other inhibitors of the G1/S transition, such as p27, which potentate the
interaction by increasing the fraction of pRb in the hypophosphorylated state.
In addition, the interacting partners may induce further transcription factors
with which pRb also interacts (Novitch et
al., 1999). In such systems, the effect of pRb upregulation is to
make cell cycle exit irreversible (Thomas
et al., 2004). Reinoblastoma family genes are also upregulated in
the development of Drosophila
(Keller et al., 2005) and
maize (Huntley et al.,
1998).
Consistent with this pattern, rblA in Dictyostelium is
dramatically upregulated during the formation of spores. As in other
organisms, this may well be under positive-feedback control, as spores are
formed by cells that have higher initial rblA expression. In contrast
to these conserved features, however, the relationship between rblA
and cell cycle exit is not typical. Retinoblastoma-deficient mutants show
supernumerary cell cycle activity in differentiating tissues in vertebrates
(Lee et al., 1992),
Drosophila (Du and Dyson,
1999), Caenorhabditis
(Lu and Horwitz, 1998) and
plants (Ebel et al., 2004). In
Dictyostelium, however, rblA-null cells do not linger in the
proliferative phase but enter development prematurely. Moreover, the signal
that initiates in Dictyostelium, starvation, does not induce
rblA but represses it.
These anomalous features can plausibly be regarded as adaptations to the
way of life of the social ameba. Dictyostelium is almost certainly
derived from ancestors that were solitary in both proliferative and
developmental stages. Solitary Amebozoans generally encyst when starved, and
these cysts may resemble Dictyostelium spores in function
(Mazur et al., 1995) and in
aspects of morphology (Chavez-Munguia et
al., 2005). It thus seems likely that encystment in the non-social
ancestors of Dictyostelium involved an Rb-family gene. In solitary
amebae, starvation induces 100% of the population to encyst; and the role of
Rb in this process may have differed little from the one familiar in
multicellular organisms today. In particular, starvation may have initiated
Rb expression, and Rb expression may have activated mutually
reinforcing processes of cell cycle withdrawal and differentiation.
In social amebae, there are two differentiated cell types, and a mechanism
is needed to allocate two these pathways. Positive-feedback systems are
predestined to work as switches, and a proportioning system could have been
constructed simply by coupling the Rb-driven encystement programs of the
individual amebae via a diffusible negative regulator
(Gierer and Meinhardt, 1972;
Lewis et al., 1977). Here, the
details can have great selective consequences, as stalk cells die while spores
remain in the gene pool. If starvation induces Rb in such a system,
then the proportioning mechanism will preferentially shunt starving cells to
the spore pathway, while amebae that, for whatever reason, have done
particularly well in the competition for nutritional resources will be
diverted to the `altruistic' stalk fate. Here, it seems likely that fitness
could be improved by reversing the relationship between starvation and
Rb expression. Starvation must of course continue to control cell
cycle exit, but alternatives to Rb could be found for this role.
This scenario thus suggests that in the evolution of the social amebae, the link of Rb to cell cycle exit was lost while the relationship with differentiation was retained. This is consistent with the idea that the connections between retinoblastoma-family genes and specific differentiation pathways are particularly stable in evolution. Control of differentiation may thus be an ancient function of retinoblastoma signaling.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/7/1287/DC1
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REFERENCES |
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