1 Department of Biochemistry and Molecular Biology, M. D. Anderson Cancer
Center, Houston, TX 77030, USA
2 Program in Genes and Development, M. D. Anderson Cancer Center, Houston, TX
77030, USA
3 Program in Developmental Biology, Baylor College of Medicine, Houston, TX
77030, USA
4 Department of Molecular and Human Genetics, Baylor College of Medicine,
Houston, TX 77030, USA
5 Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX
77030, USA
* Author for correspondence (e-mail: ghalder{at}mdanderson.org)
Accepted 19 September 2002
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SUMMARY |
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Key words: Drosophila, Imaginal discs, Cell proliferation, Apoptosis, WW domain-protein
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INTRODUCTION |
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The Drosophila imaginal discs provide an excellent model system to
study how cell proliferation is regulated during organ growth
(Edgar and Lehner, 1996;
Johnston and Gallant, 2002
).
Imaginal discs are epithelial sacs that differentiate into the external
structures of head, thorax and genitalia of the adult fly
(Cohen, 1993
). Each disc
develops from 10-30 precursor cells that proliferate extensively during the
larval stages to give rise to approximately 50,000 cells in case of wing and
eye discs, before differentiating into the corresponding adult structures
during metamorphosis (Bryant,
1978
). The growth of imaginal discs to specific sizes and shapes
is directed by secreted signaling molecules including Decapentaplegic (Dpp), a
TGFß homolog, Wingless (Wg) and Hedgehog (Hh), which act as morphogens to
induce patterning and growth (Day and
Lawrence, 2000
; Lawrence and
Struhl, 1996
; Serrano and
O'Farrell, 1997
). Although these factors may still be expressed,
imaginal disc cells stop proliferating when discs reach their correct size
(Bryant and Levinson, 1985
).
In addition, transplantation experiments revealed that developing discs
transplanted into adult hosts grow until they reach their normal size and
shape but do not grow larger than normal size even though they are not forced
to differentiate (Bryant and Levinson,
1985
; Garcia-Bellido,
1965
). Therefore, mechanisms exist that terminate cell
proliferation when discs have reached their correct size.
The generation of discs with stereotypical sizes and shapes is not the
result of a predetermination of the number of cell divisions of progenitor
cells. This is evident because the size of clones of imaginal cells is
variable and thus not determined
(Postlethwait, 1978), and
cells can compensate for growth defects by extra proliferation. The
flexibility of cell lineages is best illustrated by the observation that
mitotic clones of cells that are induced to grow faster than their neighbors
are significantly larger compared with wild-type clones
(Morata and Ripoll, 1975
).
Notably, after such manipulation of proliferation rates, the final pattern and
size of the adult structures are normal. Moreover, discs can regenerate
missing parts after surgical manipulation
(Bryant, 1978
;
Bryant and Simpson, 1984
) and
when
75% of the progenitor cells of imaginal discs are killed by X-rays,
the remaining cells proliferate and compensate for the loss of cells
(Haynie and Bryant, 1977
).
Hence, cell proliferation is plastic and cells in a developing tissue adjust
their proliferation depending on whether more cells are needed to build a
normal sized structure (Day and Lawrence,
2000
; French et al.,
1976
; Garcia-Bellido and
Garcia-Bellido, 1998
). However, the molecular mechanisms that
direct cells to stop proliferating once the primordium of a structure has
reached the correct size are poorly understood.
In principle, defined organ size can be generated either by regulating the
extent of cell proliferation or by eliminating superfluous cells through
programmed cell death, or both. Only limited amounts of cell death are
observed during imaginal disc growth
(Milan et al., 1997;
Wolff and Ready, 1991
),
indicating that disc size is primarily, albeit not exclusively, controlled at
the level of cell division. Thus, factors must exist that regulate the
decision of imaginal disc cells to re-enter or exit the cell cycle to mediate
growth control.
The Drosophila eye is particularly well suited to identify factors
that regulate cell proliferation. First, the various stages of cell division
and differentiation can be accurately followed in eye imaginal discs. Second,
defects in growth control and differentiation can be easily scored. In the
early growth phase of the eye disc, cell cycles are not synchronized and
proliferating cells are evenly distributed throughout the disc
(Baker, 2001). Later, during
the third larval stage, a wave of differentiation called the morphogenetic
furrow sweeps across the eye disc from posterior to anterior
(Wolff and Ready, 1993
). Cells
anterior to the furrow are developmentally uncommitted and divide
asynchronously, whereas cells within the furrow arrest in the G1 phase of the
cell cycle, synchronize and either start to differentiate into photoreceptor
cells as they leave the furrow or undergo one additional round of cell
division, referred to as the second mitotic wave before differentiating into
the remaining photoreceptor, cone, pigment and bristle cells
(Baker, 2001
;
Dickson and Hafen, 1993
;
Wolff and Ready, 1993
). Thus,
different modes of cell proliferation control can be studied with single cell
resolution.
To gain insights into the mechanisms that regulate cell proliferation during organogenesis, we conducted a genetic screen in Drosophila to identify mutations that affect adult eye size. We describe the identification and phenotypic characterization of a novel gene, shar-pei (shrp), that is required for cells to terminate proliferation once imaginal discs have reached their correct size. Flies with shrp mutant tissues have enlarged structures that contain more cells of normal size. These overgrowths result from an extended period of cell proliferation, accompanied by a decrease in cell death. Based on these observations, we propose that shrp regulates organ growth by promoting both cell cycle exit and apoptosis. As shrp is not required for terminal cell cycle exit, we conclude that control of proliferation arrest during organ growth is separable from, and probably precedes, cell cycle exit during terminal differentiation. The presence of shrp homologs in other species suggests that the mechanisms that control tissue size are evolutionarily conserved.
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MATERIALS AND METHODS |
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Meiotic mapping of shrp with w+ marked P
elements
Flies carrying the shrp1 allele were crossed to flies
carrying one of the five P elements in the 94A-96A region (see fly stocks
above). Transheterozygous virgin females (y w/y w; shrp 1 /
P[w+]) were collected and crossed to males of the
genotype y w eyFLP/Y; st shrp4 ca/TM3
P[w+] Sb. Recombination between
shrp1 and a given P element would produce wild-type
chromosomes that lack shrp1 and the P element. Half of
these chromosomes were recovered over the st shrp4 ca
chromosome and the other half over TM3 P[w+]. The
reciprocal chromosomes carried both shrp1 and the P
element. Recombinant flies of the genotype y w / y w; + / st
shrp4 ca were the only progeny that did not carry a
P[w+] and were identified by their white eyes
among the rest of the red-eyed progeny. The frequency of white-eyed progeny is
thus equal to half the meiotic distance in cM.
Scanning electron microscopy, immunohistochemistry and in-situ
hybridization
Adult flies with heads in which over 90% of cells were mutant, were
processed for SEM by using the hexamethyldisilazane (HMDS) method
(Braet et al., 1997) with
modifications. Flies were fixed for a day in 70% acetone, and washed twice in
100% acetone for 4 hours each. Acetone was then exchanged with HMDS through
two washes in 1:1 acetone:HMDS and two washes in 100% HMDS over 2 days.
Samples were air dried for 1 day prior to sputter coating with 25 nm platinum
alloy. Antibody staining of imaginal discs carried out as described earlier
(Halder et al., 1998
). The
following antibodies were used (dilutions in parenthesis): rat
-Elav
(1:30) (O'Neill et al., 1994
),
rabbit
-Dlg (1:2000) (Cho et al.,
2000
), rabbit
-Drice (1:2000)
(Yoo et al., 2002
), guinea-pig
-Dlg (1:2000) (Woods and Bryant,
1991
), guinea-pig
-Sens (1:1000)
(Nolo et al., 2000
), mouse
-BrdU (1:50, Becton-Dickinson) and mouse
-CycE (1:50)
(Richardson et al., 1995
).
Secondary antibodies were donkey Fab fragments from Jackson Immuno Research.
BrdU incorporation was carried out as described
(de Nooij et al., 1996
) by
incorporating BrdU for 1 hour. For in situ hybridization, Drosophila
cDNA clone RE52745 (ResGen Invitrogen Corp.) was used as template to generate
DIG-labeled RNA probes (Roche), and in situ hybridization was performed as
described (Nolo et al.,
2000
).
FACS analysis and cell counts
To analyze cell cycle and cell size distribution of shrp mutant
and wild-type cell populations, wing imaginal discs containing mutant clones
were dissected from transheterozygous larvae of the genotype y w hsFLP;
FRT82B shrp/FRT82B P(w+) ubi-GFPnls. Clones were
induced 24-48 hours after egg laying (AEL) by administering a heat-shock at
34°C for 30 minutes and discs were dissected 72 hours later. About 80-100
wing discs were dissected in PBS and transferred to 5 ml polystyrene tubes
containing Trypsin-EDTA (Sigma, T-4174): PBS 9:1 v/v and 0.5 µg/ml Hoechst
33342 (Neufeld et al., 1998).
Cells were dissociated for 4 hours by gentle shaking. The dissociated cells
were analyzed on a Becton Dickinson Vantage Fluorescence activated cell sorter
(FACS) and more than 50,000 mutant cells were scored for each sample. Data
were analyzed with the Cell Quest program. For cell counts, wing discs from
y w hsFLP; FRT82B shrp / FRT82B P(w+)
ubi-GFPnls transheterozygous larvae were dissected 48 hours
after clone induction, fixed in PEM (100 mM PIPES, 2 mM MgSO4, 1 mM
EGTA, 4% formaldehyde) on ice for 1 hour, washed briefly in PBT (PBS + 0.3%
Triton X-100) and incubated with Hoechst (0.02 µg/ml) for 25 minutes. The
discs were washed twice in PBT and mounted in Vectashield (Vector Labs). Cell
numbers were determined by counting the nuclei (Hoechst-stained) of cells in
mutant clones (GFP negative) and associated wild-type twin clone
(GFP-positive) on a Zeiss axioplan fluorescence microscope.
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RESULTS |
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Mutations in shar-pei cause overgrowth of adult
structures
Mutations in shrp were isolated on chromosome arm 3R using
chemical mutagenesis. Complementation tests showed that six mutations
(shrp1-6) that caused a head overgrowth phenotype
fail to complement each other. All mutations showed a very similar phenotype
and caused early larval lethality. Given the nature of the molecular lesions
(see below) it is likely that they are either null alleles or very severe
loss-of-function alleles. All experiments involving cell clones were performed
with at least three independent alleles.
The heads of flies in which over 90% of cells are homozygous mutant for
shrp1 are proportionally larger than other
structures but have a normal overall pattern, including bristles, ocelli and
ommatidia (Fig. 1A-F). All
mutant fly heads have folded head cuticle and eye tissue
(Fig. 1C-F) and over 15% of
flies are severely affected (Fig.
1D). Smaller clones generated by heat-shock induced Flipase
expression do not exhibit this folding phenotype. Folding may therefore be a
secondary consequence of limited space within the pupal case, which does not
allow overgrown tissue to fully expand. In addition to producing structures
that are too big, shrp mutant cells appear to out-compete wild-type
cells. The flies shown in Fig.
1A,B are eyFLP induced genetic mosaics in which clones
are genotypically marked white, heterozygous portions are wild type
and twin clones were eliminated by a cell lethal mutation to increase the
amount of mutant cells in the eye (Newsome
et al., 2000). When white cells proliferate at a normal
rate, heterozygous red cells contribute about 20% of cells in this type of
experiment. In contrast, eyes with white marked
shrp1 mutant clones are predominantly white
(Fig. 1B) and contain fewer
wild-type ommatidia when compared with a control fly shown in
Fig. 1A. Hence, the phenotype
suggests that shrp mutant cells proliferate more rapidly than
wild-type cells (Kirby and Bryant,
1982
; Moberg et al.,
2001
; Simpson and Morata,
1981
).
|
To test whether shrp affects cell proliferation in tissues other
than the head, we induced random clones by heat-shock induced Flipase
expression (Xu and Rubin,
1993). Such mutant clones resulted in overgrowths on thorax,
wings, halteres and legs (Fig.
1G-J, not shown). As observed for the eye and head, these
structures differentiated the correct tissue-specific cell types. We conclude
that shrp is generally required to restrict the size of imaginal
disc-derived adult structures, whereas tissue-specific cell-type specification
and differentiation remain unaffected in shrp mutant cells.
shar-pei mutants produce extra interommatidial cells
To define the developmental basis for the enlarged tissue phenotypes, we
focused on patterning and cell proliferation in the developing eye because the
eye has a precise pattern of cell types and highly regulated cell
proliferation (Baker, 2001;
Kumar and Moses, 2000
;
Wolff and Ready, 1993
). We
first analyzed the pattern of differentiated photoreceptor cells in adult
shrp mutant clones in 1 µm sections
(Fig. 2A). We observed eight
photoreceptors per ommatidium with a normal trapezoidal arrangement,
indicating that this aspect of pattern formation is not affected. However,
spacing between individual photoreceptor clusters was significantly increased
in shrp5 clones when compared with wild-type
areas (Fig. 2A arrowhead). To
test whether the increased space was due to an excess of interommatidial
cells, we stained wild-type and mutant midpupal retinas with an antibody
against Discs-large (Dlg), a protein that localizes to apical junctions and
hence reveals cell outlines (Fig.
2B,C) (Woods and Bryant,
1991
). We found that shrp4 mutant
clones exhibit a dramatic increase in the numbers of interommatidial cells
(Fig. 2B) when compared with
wild type (Fig. 2C). These
extra interommatidial cells differentiated into pigment cells that produced
normal pigmentation when clones were induced in a w+
background (not shown). These data indicate that Shrp regulates cell number
but not differentiation in the retina.
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The extra interommatidial cells could be due to excess cell proliferation,
increased spacing of photoreceptor clusters during patterning, lack of
apoptosis or a combination thereof. In wild-type flies, interommatidial cells
are initially produced in excess but the extra cells are later eliminated by
apoptosis during pupal development in a process that requires cell-cell
signaling (Rusconi et al.,
2000). This system generates a very precise retinal lattice. To
determine whether shrp mutant cells initiate the apoptotic program,
we stained shrp mosaic pupal retinas with an antibody that detects
the activated form of Drice, a caspase that triggers the apoptotic program and
specifically marks cells undergoing apoptosis
(Yoo et al., 2002
). We
detected many Drice-positive cells in wild-type retinal tissue, but none were
found in shrp3 mutant territories
(Fig. 2D,E). Importantly, all
Drice-positive cells were wild type. This suggests that the apoptotic pathway
is blocked in shrp mutant cells and that this block occurs upstream
of Drice activation. We conclude that shrp mutant cells do not
receive or are resistant to signals that induce apoptosis.
To test directly whether lack of apoptosis is sufficient to produce the
shrp mutant phenotype, we compared the phenotype of shrp
mutant retinas with that of wild-type retinas in which apoptosis was blocked
by expressing the apoptosis inhibitor p35
(Hay et al., 1994). Ectopic
expression of p35 eliminates most, if not all, normally occurring cell death
in the retina (Hay et al.,
1994
) and results in extra interommatidial cells
(Fig. 2F). However, the number
of additional cells is significantly less than that observed in
shrp4 mutant clones.
(Fig. 2B). Therefore, while
lack of apoptosis allows additional cells to survive, it is not sufficient to
explain the amount of extra interommatidial cells generated in shrp
mutants.
To investigate whether the extra interommatidial cells are due to abnormal
ommatidial spacing during patterning, we stained developing mosaic eye
imaginal discs for the neuronal marker Elav
(Robinow and White, 1988) and
the R8 marker Senseless (Sens) (Frankfort
et al., 2001
; Nolo et al.,
2000
). Elav is expressed in all differentiating photoreceptor
cells and outlines differentiating photoreceptor clusters, while Sens is a
marker for early pattern formation and ommatidial spacing, as well as R8
photoreceptors. Mutant ommatidial clusters have normal numbers of
differentiating photoreceptor cells per ommatidium and are initially spaced
correctly (Fig. 2G-I). However,
at later stages in more posterior clones, spacing between photoreceptor
clusters is increased (Fig.
2J-L). Thus, early retinal pattern formation is normal in
shrp mutants.
shar-pei cell-autonomously restricts cell proliferation of
uncommitted cells
To test directly whether shrp affects cell proliferation, we
monitored the distribution of cell cycle progression in mutant third larval
eye discs by bromodeoxyuridine (BrdU) incorporation
(Fig. 3). In wild-type discs,
BrdU-incorporating cells are randomly distributed in front of the
morphogenetic furrow (Fig. 3A).
In the furrow, cells synchronously arrest in G1 and do not incorporate BrdU
(Fig. 3A, arrow). Posterior to
the furrow, cells go through a synchronous S phase in the second mitotic wave,
revealed as a band of cells incorporating BrdU
(Fig. 3A, arrowhead). Few
BrdU-positive cells are found posterior to the second mitotic wave
(Fig. 3A).
shrp1 mutant cells also synchronize their cell
cycles in the furrow and progress normally through the second mitotic wave
(Fig. 3B). However, in contrast
to wild-type cells, shrp1 mutant cells still
display BrdU incorporation after the second mitotic wave
(Fig. 3B, asterisk). The extra
DNA synthesis is followed by cell division, as revealed by ectopic expression
of phosphorylated histone H3 (PH3), which marks chromosomes during mitosis
(not shown). This phenotype of shrp is cell autonomous, because only
mutant cells undergo extra rounds of cell proliferation
(Fig. 3C,D), and all
territories of mutant cells show the excess interommatidial cell phenotype in
pupal retinas, whereas non-mutant tissue appears wild type. Extra cell
proliferation continues into the pupal stage but ceases by 24 hours after
pupariation (not shown). Double labeling with BrdU and antibodies against Elav
to detect differentiating photoreceptor cells revealed that only Elav-negative
cells incorporated BrdU (Fig.
3E,F). Therefore, shrp is required to arrest cell
proliferation in developmentally uncommitted cells after the second mitotic
wave, but is not required for cell cycle arrest of differentiating
photoreceptor cells. The ectopic proliferation produces extra interommatidial
cells, which together with the lack of apoptosis, are sufficient to explain
the overgrowth phenotypes observed in pupal and adult retinas.
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shar-pei regulates Cyclin E levels
Cyclin E is limiting for S-phase initiation and progression during imaginal
disc development and several tumor suppressor genes negatively regulate its
activity or levels (de Nooij et al.,
1996; Duman-Scheel et al.,
2002
; Lane et al.,
1996
; Moberg et al.,
2001
; Neufeld et al.,
1998
; Richardson et al.,
1995
). Cyclin E levels are upregulated in
shrp1 mutant cells in the second mitotic wave and
posterior to it (Fig. 4A-C
arrows). Elevated levels were also observed just anterior to the second
mitotic wave, although this effect was not as pronounced. The effect on Cyclin
E is cell autonomous and observed in most or all mutant cells, even though
only a fraction of them are actively progressing through S phase
(Fig. 3C,D). Thus, the effect
of Shrp on cell proliferation arrest may be mediated by regulating the levels
of Cyclin E.
|
shar-pei mutant cells show accelerated cell proliferation
during disc development
Our data show that although shrp mutant cells are able to exit the
cell cycle during cell differentiation, they are delayed in arresting cell
proliferation at the end of eye imaginal disc growth. To determine whether
shrp has a function in uncommitted cells anterior to the
morphogenetic furrow, we wanted to measure whether mutant eye discs were
already larger than wild-type before ommatidial clusters are specified.
Because initial spacing of photoreceptor clusters is normal in shrp
mutant eye discs (Fig. 2G-I),
the final number of ommatidia provides a measure of the number of cells
present in mutant eye discs before R8 cells are specified in the morphogenetic
furrow. We thus determined and compared the numbers of ommatidia in wild-type
and mutant retinas (n=18 each) by counting clusters of photoreceptor
cells revealed by Elav-Gal4 driven GFP expression
(Lee and Luo, 1999). Mutant
retinas contained an average of 913 ommatidial clusters (s.d.=40), whereas
wild-type retinas had an average of 776 (s.d.=45) photoreceptor clusters
(Fig. 5). The two groups are
significantly different by t-test (P<0.001). We conclude
that shrp mutant eye discs are already larger than normal at the time
when the positions of ommatidia are specified in the morphogenetic furrow.
Shrp thus functions in uncommitted cells anterior to the morphogenetic
furrow.
|
To test whether shrp affects the rate of cell proliferation during
the growth phase of imaginal discs, we compared cell numbers in mutant clones
and their associated twin clones in third instar wing discs
(Fig. 6A). To reduce
variability in the proliferation rate of wild-type twin clones, we used
isogenized FRT 82B ubi-GFP chromosomes to generate mitotic clones.
Cell numbers in shrp3 mutant clones were almost always
larger than their twin clones, and the difference in cell numbers was
significant when assessed using a t-test (P<0.001). The
same experiment with a second allele, shrp4, showed
similar differences (not shown). By contrast, cell numbers in clones of the
isogenized wild-type chromosome on which the shrp mutations were
induced during the mutagenesis screen were similar and not significantly
different from the corresponding ubi-GFP/ubi-GFP twin clones
(Fig. 6B). Based on these cell
counts and assuming exponential proliferation, the cell division rate of
shrp mutant cells is 1.10 times faster than that of wild-type cells.
Our data thus indicate that shrp mutant cells proliferated more. This
phenotype is also manifest in mosaic adult eyes, where
shrp1 mutant cells out-compete wild-type cells
(Fig. 1A,B). Determination of
the distribution of cell cycle phases in third instar wing discs using FACS
analysis (Neufeld et al.,
1998) showed that the population of shrp4
mutant cells has the same distribution of cell cycle phases as the wild-type
cells (Fig. 6C). Thus,
shrp mutant cells do not accelerate a particular step in the cell
cycle. Rather, mutant cells show an even acceleration of cell cycle
progression.
|
Manipulating the activity of cell growth regulators such as components of
the insulin receptor signaling pathway results in larger organs because of
more and larger cells (Johnston and
Gallant, 2002; Potter and Xu,
2001
; Prober and Edgar,
2001
; Stocker and Hafen,
2000
; Tapon et al.,
2001
; Weinkove and Leevers,
2000
). To determine whether shrp also affects cell size,
we stained mosaic wing discs with antibodies against Dlg to detect apical cell
outlines. Cells in shrp3 mutant clones showed normal cell
sizes, as judged by cell outlines (Fig.
6E-G) and had normal height as judged by the thickness of the wing
disc epithelium in the mutant clones (not shown). In addition, rhabdomeres of
mutant photoreceptor cells were of normal diameter
(Fig. 2A), and shrp
mutant cone and pigment cells are of normal size at the pupal stage
(Fig. 2B,C). Furthermore,
forward light scatter (FSC) data, a measurement of cell size collected by FACS
analysis confirmed that mutant cells have normal size
(Fig. 6D). Therefore, Shrp does
not regulate cell size. Rather, extra proliferation of shrp mutant
cells is induced by stimulation of cell growth and cell cycle progression,
resulting in balanced growth and extra cells that are of normal size.
shar-pei encodes a conserved WW-domain containing
protein
To identify the shrp gene, we first mapped the lethality
associated with the mutations through deficiency mapping. We found that the
shrp mutations failed to complement the deficiencies
Df(3R)hh (93F;094D) and Df(3R)M95A (94D;095A), placing
shrp within the 94D interval. Male recombination mapping further
mapped shrp to a 150 kb interval between klingon
(Butler et al., 1997) and
hedgehog (Lee et al.,
1992
). Meiotic recombination mapping with several P elements as
dominant markers in the region
(http://flypush.imgen.bcm.tmc.edu/pscreen)
(Fig. 7A), further placed
shrp within a 90 kb interval (Fig.
7B). Based on the annotation of the Drosophila genome
(Adams et al., 2000
), we
amplified and sequenced six predicted and conserved open reading frames in
that region (Fig. 7B) and found
mutations in CG13831 in all shrp alleles
(Fig. 7C,D). The full-length
shrp cDNA encodes a protein of 607 amino acids with two WW domains
(Fig. 7C,D). WW domains are
protein-protein interaction domains that bind to short proline-rich motifs,
functionally resembling SH3 domains
(Macias et al., 2002
).
|
Database searches revealed that parts of Shrp are conserved in humans, mice
(WW45) (Valverde, 2000), and
C. elegans (T10H10.3) (Fig.
7C,D). In addition to the highly conserved WW domains, these
proteins share a C-terminal domain that is specific for Shrp and not found in
other proteins (Fig. 7C,D).
Phylogenic analysis by neighbor joining of 202 WW domains
(http://www.Bork.EMBL-Heidelberg.DE/Modules/ww/)
revealed that the two WW domains of Drosophila Shrp and vertebrate
WW45 are more closely related to each other than to other WW domains.
Furthermore, only single copies of this gene were detected in all four
species. We conclude that Shrp, WW45 and T10H10.3 are orthologous.
The alleles shrp1-5 have point mutations that result in STOP codons, which truncate the proteins N-terminal to the WW domains (Fig. 7C,D). The sixth allele (shrp6) has a 20 bp deletion that results in a frameshift between the WW domains and the Shrp-specific domain, resulting in the addition of 76 unique residues thereby effectively removing the Shrp specific domain. All six alleles are purely recessive and homozygous lethal at the first/second instar stage, and show the same lethal phase when heterozygous over Df(3R)hh. In addition, all alleles show similar phenotypes in mitotic clones in discs and adults. Thus, they all appear to be null alleles for shrp function. Because the frameshift allele shrp6 is recessive and behaves as a null allele, the position of its lesion suggests that the conserved C-terminal domain is essential for Shrp function. In situ hybridization of imaginal discs revealed that shrp is ubiquitously expressed in the eye, wing and leg discs (Fig. 7E,F). This is consistent with our findings that shrp is required in all imaginal disc-derived tissues for proper cell proliferation arrest.
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DISCUSSION |
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Pattern formation appears to progress normally in shrp mutant clones. In the eye, clones show the normal complement and morphology of photoreceptor and cone cells. Clones on head, thorax, halteres and wings show normal patterning of bristles and other tissue-specific structures such as ocelli, wing margin bristles, haltere specific sense organs and tissue-specific cell type differentiation. In addition, mutant cell clones in wing discs respect both the AP and DV compartment boundaries and clone borders within compartments are jagged, indicating that cell affinities are not affected by the loss of shrp function. In summary, Shrp specifically regulates cell number by promoting cell proliferation arrest and apoptosis, but is not required for pattern formation or cell type differentiation.
Regulation of cell proliferation arrest during organ growth versus
terminal differentiation
Shrp function is specifically required for the timely proliferation arrest
of developmentally uncommitted cells, but not for terminal cell cycle exit
during cell differentiation. In shrp mutant eye discs, cell cycle
arrest in differentiating photoreceptor cells still occurs normally, and
ectopic BrdU incorporation is confined to developmentally uncommitted cells.
Consistent with this observation, we did not observe duplicated photoreceptors
or cone cells in the eye or duplicated bristles on the head thorax or along
the wing margin. Thus, shrp is not required for cells to exit the
cell cycle during terminal differentiation. Rather, shrp is required
for proliferation arrest before cells are induced to differentiate into
specific cell types. Therefore, more precursor cells are generated in
shrp mutants, which then differentiate normally but produce adult
organs that are too big. For example, shrp mutant eyes have more
ommatidia, indicating that mutant eye discs contain more cells than normal
when ommatidial cell clusters are specified. The observation that
shrp mutant clones produce overgrowths on disc-derived structures,
including eyes, heads, wings, halteres, legs and thoraxes suggests that
shrp function is ubiquitously required. The ubiquitous expression
pattern of shrp in imaginal discs supports this conclusion. In the
eye, we also observe an increase in the number of pigment cells per
ommatidium, while the other cell types are present in normal numbers per
ommatidium. This is because cell types are specified sequentially during eye
development. First, some cells are specified as photoreceptors, while the
other cells remain uncommitted. These uncommitted cells ectopically
proliferate in shrp mutants and produce more interommatidial cells.
Successively, the remaining cell types are recruited in normal numbers, but at
the end, too many cells are leftover which differentiate into excessive
numbers of pigment cells, the last cell type to differentiate.
The requirements of Shrp are distinct from those of genes required for cell
cycle exit during terminal differentiation. Dacapo (dap), a
Drosophila homolog of the Kip family of cyclin-dependent kinase
inhibitors, is induced when cells exit the cell cycle prior to terminal
differentiation (de Nooij et al.,
1996; Lane et al.,
1996
). In dap mutant embryos, cells go through one extra
round of cell division just prior to terminal differentiation. In the
developing eye, dap expression is induced in differentiating
photoreceptor cells, but not in developmentally uncommitted cells
(de Nooij et al., 1996
;
Lane et al., 1996
). Adult
dap mutant eye clones do not show gross abnormalities or extra cells,
but rare escapers show duplications of bristles on notum and wing margin
(Lane et al., 1996
).
Downregulation of positive cell cycle regulators such as Cyclin E
(Crack et al., 2002
;
de Nooij et al., 1996
;
Du and Dyson, 1999
;
Knoblich et al., 1994
;
Richardson et al., 1995
;
Richardson et al., 1993
) and
other negative regulators of cell proliferation such as Rbf
(de Nooij et al., 1996
;
Du and Dyson, 1999
;
Du et al., 1996a
) may act
redundantly with Dap to promote cell cycle arrest. Nonetheless, while Dap is
upregulated specifically in cells that withdraw from the cell cycle prior to
terminal cell differentiation, Shrp is required in developmentally uncommitted
cells during the growth phase of organs before terminal differentiation. The
requirements for Dap and Shrp functions are thus spatially and temporally
distinct.
We propose that the arrest of cell proliferation during imaginal disc
development is controlled by several genetically separable mechanisms. First,
cells stop proliferating when imaginal discs have reached their correct sizes
(Bryant and Levinson, 1985;
Garcia-Bellido, 1965
). This
process requires Shrp function. Second, cells permanently exit the cell cycle
during terminal differentiation. Because terminal cell cycle exit is part of
cell differentiation, it is directly regulated by patterning mechanisms that
determine the identity and position of each individual cell. This regulation
is governed by tissue-and cell-type specific enhancers of cell cycle
regulators such as dacapo, cyclin E and string, which all
have complex cis- regulatory regions
(Duman-Scheel et al., 2002
;
Jones et al., 2000
;
Lehman et al., 1999
;
Liu et al., 2002
;
Meyer et al., 2002
).
Similarly, patterned regulation of cell cycle progression may occur before
terminal differentiation, as is observed in the second mitotic wave in the eye
and in the zone of non-proliferation along the presumptive wing margin in the
wing disc (O'Brochta and Bryant,
1985
). None of these processes are affected in shrp
mutants. Thus, the direct control of cell cycle progression by patterning
mechanisms acts epistatically to the control of cell proliferation observed
during organ growth and can impose cell cycle arrest on cells that otherwise
may continue to proliferate. Therefore, shrp mutations do not
deregulate cell proliferation of terminally arrested cells, and cells
differentiate normally. In summary, Shrp identifies a molecular mechanism that
is required to arrest cell proliferation during organ growth and that appears
to be distinct from the ones used to arrest cells during terminal cell
differentiation.
The function of shar-pei during proliferation arrest
What are the downstream effectors of Shrp that are deregulated in
shrp mutants and induce cell proliferation? shrp mutant
clones behind the second mitotic wave in eye discs show elevated levels of
Cyclin E. Notably, Cyclin E was elevated in all developmentally uncommitted
cells of the clones, apparently irrespective of the phase of the cell cycle.
The effect on Cyclin E levels may thus be direct and not just a reflection of
the ectopic cell proliferation observed in mutant clones. Precise regulation
of Cyclin E expression and activity is crucial as ectopic expression of Cyclin
E induces entry into S phase and limited cell proliferation in imaginal discs
and embryos (Knoblich et al.,
1994; Neufeld et al.,
1998
; Richardson et al.,
1995
). Several other negative regulators of cell proliferation
directly regulate the levels of Cyclin E activity. Dap directly inhibits
Cyclin E/Cdk2 complexes (de Nooij et al.,
1996
; Lane et al.,
1996
), and Archipelago is required for degradation of Cyclin E
(Moberg et al., 2001
). The
regulation of Cyclin E is thus likely to be an important downstream effect of
Shrp function.
Ectopic expression of Cyclin E alone, however, is not sufficient to
generate the number of extra cells observed in shrp mutant tissues
(Neufeld et al., 1998;
Richardson et al., 1995
).
Artificial acceleration of the cell cycle by ectopic expression of specific
cell cycle regulators such as E2F accelerates cell division, but does not
stimulate cell growth rates, and cells divide when they are smaller
(Neufeld et al., 1998
). This
results in an increase in cell number and a concomitant decrease in cell size,
yet does not affect the overall tissue size. Thus, cell cycle progression is
not sufficient to drive cell and organ growth. Conversely, stimulating cell
growth alone produces larger organs, but also affects cell size
(Johnston and Gallant, 2002
;
Potter and Xu, 2001
;
Prober and Edgar, 2001
;
Stocker and Hafen, 2000
;
Tapon et al., 2001
;
Weinkove and Leevers, 2000
).
For example, artificially stimulating the activities of Ras, Myc or insulin
receptor signaling produces more and bigger cells and thus bigger but
otherwise normal flies. Thus, cell proliferation during organ growth requires
coordinate stimulation of cell cycle progression and cell growth to produce
normal sized cells. Because shrp mutant cells maintain normal size,
Shrp appears to be required to regulate cell growth and cell cycle progression
coordinately. Thus, Shrp probably regulates other targets driving cell cycle
and cell growth in addition to Cyclin E.
Several other mutations have been described that fail to arrest imaginal
disc growth and were thus classified as tumor suppressor genes
(Gateff, 1994;
Turenchalk et al., 1999
;
Watson et al., 1994
). These
include discs-large (dlg)
(Woods and Bryant, 1991
),
lethal giant larva (lgl)
(Gateff, 1978
) and
scribble (scrib) (Bilder
and Perrimon, 2000
), encoding proteins which form an architectural
complex localized to septate junctions. Mutations in these genes disrupt
septate junctions and apical-basal polarity of epithelial cells and result in
neoplastic overgrowth of imaginal discs
(Bilder et al., 2000
;
De Lorenzo et al., 1999
;
Johnston and Gallant, 2002
).
Mutations in a second group of Drosophila tumor suppressor genes
cause hyperplastic overgrowth of imaginal discs that retain their single
layered epithelial structure. These include warts/lats, which encode
a kinase that regulates the activity of Cdc2
(Justice et al., 1995
;
Tao et al., 1999
;
Xu et al., 1995
);
fat, a large Cadherin (Mahoney et
al., 1991
); hyperplastic discs, a E3 ubiquitin ligase
(Mansfield et al., 1994
); and
discs overgrown, a Drosophila homolog of Casein kinase
I
/
(Zilian et al.,
1999
). The imaginal disc overgrowth in mutants of both groups
occurs during an extended larval period, and embryonic requirements for these
genes appear to be provided by maternal contributions
(Bilder et al., 2000
;
Bryant et al., 1988
;
Mansfield et al., 1994
). By
contrast, homozygous shrp mutant animals die as first/second instar
larvae, which do not show gross morphological defects. Thus, zygotic
expression of shrp is required for early larval viability, whereas
that of other tumor suppressor genes is not. Cells homozygous mutant for
neoplastic or hyperplastic tumor suppressor genes generally differentiate
abnormally and show defects in cell morphology and/or pattern formation
(Agrawal et al., 1995
;
Bilder and Perrimon, 2000
;
Bryant et al., 1988
;
Justice et al., 1995
;
Martin et al., 1977
;
Woods and Bryant, 1991
;
Xu et al., 1995
;
Zilian et al., 1999
). These
phenotypes are thus different from those of shrp mutant cell clones,
which overproliferate but differentiate with normal cell morphology and
patterning. In addition to these differences, clones of cells homozygous
mutant for shrp proliferate more rapidly and have reduced apoptosis,
while cells mutant for most other tumor suppressor genes have reduced
viability and a decreased rate of cell proliferation
(Bryant, 1987
;
Mansfield et al., 1994
;
Woods and Bryant, 1989
). Only
fat and warts/lats mutant cell clones proliferate faster
(Garoia et al., 2000
) (M. K.
S., unpublished), similar to shrp mutant cells. However, the
phenotypes of shrp, fat and warts/lats differ, as
fat and warts/lats affect the morphology and pattern of
adult tissues in addition to cell number. Therefore, shrp affects
cell number more specifically than these other mutants, and future work will
have to establish whether and how Shrp interacts with other tumor suppressor
gene products to control tissue size.
In summary, our studies provide evidence that Shrp functions in the decision of imaginal disc cells to terminate proliferation and to exit the cell cycle once the correct disc size is attained. The determination of the effectors of Shrp action should reveal mechanisms by which cell growth and cell cycle progression are coordinately regulated during organ growth and how cells arrest proliferation once organs have reached their correct size. The presence of Shrp homologs in mouse and human suggest the existence of a conserved organ size control mechanism in mammals. The characterization of Shrp function should therefore provide valuable insights into the mechanisms that underlie tissue size regulation and cause disproportionate growth and tumorigenesis when defective.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
While this manuscript was under review, Tapon et al.
(Tapon et al., 2002) also
reported the characterization of this gene.
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