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First published online 24 November 2005
doi: 10.1242/dev.02168
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1 Department of Cell Biology, Emory University School of Medicine, Atlanta, GA
30322, USA.
2 Pediatric Surgical Research Laboratories, Massachusetts General Hospital,
Harvard Medical School Boston, MA 02114, USA.
3 Department of Pediatric Gastroenterology, Medical College of Wisconsin,
Milwaukee, WI 53226, USA.
Author for correspondence (e-mail:
mosesk{at}hhmi.org)
Accepted 14 October 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, moleskin, Importin 7, Wing, MAP kinase, Cell cycle, Translocation, ERK, Ras
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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25% of human tumors (Hanahan and
Weinberg, 2000
). MAPKs are activated by dual-phosphorylation at a
specific tyrosine and threonine residue in response to many signals that
control cell proliferation, growth, shape, adhesion, fate, death and other
physiological responses (Perrimon,
1994; Schaeffer and Weber,
1999). These signals are often received by trans-membrane RTKs,
such as EGFR, and are transduced by a cascade that includes Ras, Raf (MAPK
kinase kinase) and Mek (MAPK kinase), which dually phosphorylates MAPK
directly to generate phospho-MAPK (pMAPK) (reviewed by
Wassarman et al., 1995). pMAPK
then disassociates from MEK and forms active homodimers
(Cobb and Goldsmith, 2000) that
can phosphorylate targets in the cytoplasm, and/or translocate to the nucleus
to activate nuclear targets, and thus controls gene expression
(Brunet et al., 1999;
Chen et al., 1992;
Gonzalez et al., 1993;
Khokhlatchev et al., 1998;
Lenormand et al., 1993).
Signaling specificity is achieved through several mechanisms, including
restricted ligand or receptor expression, ligand processing, activating and
inactivating ligands, and specific MAPK phosphatases
(Rebay, 2002).
We have reported a regulated cytoplasmic sequestration of pMAPK antigen in
the developing Drosophila compound eye [`MAPK cytoplasmic hold' (see
Kumar et al., 1998)]. We
developed four new reagents to detect MAPK cytoplasmic hold in vivo by a
second independent method to detect nuclear MAPK
(Kumar et al., 2003). All four
use a heat-inducible (hsp70) promoter to drive the expression of
ectopic proteins. The first is a fusion between MAPK and the yeast
transcription factor GAL4 (`MG'). When MG is expressed and translocates to the
nucleus, it can specifically direct the expression of reporter genes. We also
fused the SV40 nuclear localization sequence (NLS) to MG (`NMG'), to drive the
fusion protein into the nuclei of cells as a positive control. The third
construct is the SV40 NLS fused to MAPK alone (no Gal4, or `NM'), to test the
developmental consequences of forcing MAPK into cells nuclei inappropriately
(i.e. breaking cytoplasmic hold). The last reagent is MAPK alone (`M'), as a
control for the simple effects of MAPK overexpression. In the developing eye,
we used these reagents to detect MAPK cytoplasmic hold and show that this hold
is developmentally important in the morphogenetic furrow
(Kumar et al., 2003).
To address the potential role of MAPK cytoplasmic hold in the regulation of
Ras/MAPK signaling in other tissues, we examined the Drosophila wing,
which develops from an epithelial sheet during larval and pupal stages
(Bier, 2000;
de Celis, 2003). In third
larval instar wing imaginal discs, pMAPK antigen is most strongly expressed in
the pro-vein and wing-margin cells (Fig.
1A,B) (see Guichard et al.,
1999; Martin-Blanco et al.,
1999). Loss-of-function mutations in Egfr upstream
functions (Rhomboid, Star, Vein) or downstream effectors [Ras, Raf and the
gene encoding MAPK (rolled, rl)], each result in vein loss and, in
those cases tested, loss of pMAPK antigen in larval wings
(Biggs et al., 1994;
Guichard et al., 1999;
Martin-Blanco et al., 1999).
Conversely, gain-of-function mutations in the pathway lead to increased or
ectopic veins, including a dominant gain-of-function MAPK mutation,
rlSem (Brunner et al.,
1994; Martin-Blanco et al.,
1999). In addition to patterning, wing cells lacking Ras
have growth defects (Prober and Edgar,
2000). Thus, EGFR/Ras/MAPK signaling controls both cell fate (vein
versus intervein) and general cell proliferation at similar times within this
tissue, and the proper regulation of these two independent functions must be
tightly controlled in order for the wing to develop normally.
In this report, we show that pMAPK is held in the cytoplasm of developing
wing vein and margin cells throughout larval and early pupal life, and that
this cytoplasmic hold promotes vein differentiation. We also show that MAPK
translocates to the nucleus of larval wing cells, and this nuclear
translocation promotes the G1/S cell cycle transition. As Egfr is the only
known RTK involved in wing development
(Guichard et al., 1999), our
data suggests a novel pathway bifurcation in Drosophila, where this
single pathway can control both proliferation and differentiation in the
developing wing through control of MAPK subcellular localization.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), mounted
in Vectashield (Vector Labs, H-1000) and imaged by confocal microscopy or by
bright-field microscopy with DAB and Ni/Co intensification. Primary antibodies
were: mouse anti-pMAPK, Sigma (1:625, Sigma M8159)
(Gabay et al., 1997), mouse
anti-HSV-Tag (1:2,000, Novagen, 69171), mouse anti-Cyclin E (1:5, Iowa DBHB),
rabbit anti-ß-galactosidase (1:1,000, Cortex Biochem CA2190), rabbit
activated caspase 3 (1:200, BD Biosciences, 551150)
(Srinivasan et al., 1998),
mouse anti-BrdU (1:50, BD Biosciences, 33281A), rabbit anti-Phospho Histone H3
1:1,000 (Cell Signaling Technologies 9701), rabbit anti-lamin DmO (R836, gift
of Paul Fisher). Secondary antibodies were from Jackson ImmunoResearch: goat
anti-mouse Cy5 (1:500, 115-175-003), goat anti-rabbit TRITC (1:250,
111-025-003), goat anti-mouse HRP (1:40, 115-035-003). DNA was stained using
SYTO24 (1:10,000. Molecular Probes S-7559). Actin was detected with
Rhodamine-phalloidin (1:50, Molecular Probes, A-12380). Adult wings were
dehydrated in ethanol and mounted in DPX (Zeiss). Expression domain areas/cell
numbers were counted using pixel counts in Photoshop.
Drosophila stocks and DNA constructs
The wild-type strain used was Canton-S. MAPK expression construct
transgenics were hs:M, hs:NM, hs:MG and hs:NMG
(Kumar et al., 2003);
hs:rho and hs-rho30a (gifts from E. Bier)
(Guichard et al., 1999);
bsP1292 (a gift from M. Affolter)
(Montagne et al., 1996);
en:GAL4 (a gift from R. Palmer); UAS:msk and
msk5 (Lorenzen et al.,
2001); stg-lacZ
(Lehman et al., 1999);
rl10a and UAS:Argos
(Freeman, 1994);
rlsem (Brunner et al.,
1994); UAS:p35 (FlyBase;
http://flybase.bio.indiana.edu);
UAS:GFP (a gift from J. Fisher); and Su(var)2055
and tkv8 (FlyBase). pBS.Kpn(RBS)MYC-DIM7 contains coding
sequence of Drosophila msk
(Lorenzen et al., 2001) with a
N-terminal MYC epitope tag added (Hsp70-CaSpeR).
Heat shock regimes
hsp70 promoter transient expression and larval analysis
Larvae were incubated at 37°C for 1 hour, then recovered at 25°C
for various times as noted in text.
Pupal analysis
Twenty-three- or 21-hour after puparium formation (APF) pupae were induced
at 37°C for 1 hour and immediately dissected (0H, 23 APF) or recovered at
25°C for 2 hours (2H, 21 APF).
Adult wing analysis
Larvae were aged every 4-8 hours throughout life cycle, induced at 37°C
for 1 hour, then allowed recovery at 25°C until eclosion.
Flow cytometry and adult wing analysis
hs:MG and hs:NMG larval wing discs (also containing
UAS:GFP) were placed at 37°C for 1 hour, then at 25°C for 3
hours, then incubated for 3 hours in PBTH as described previously
(Neufeld et al., 1998). Discs
were then analyzed on DakoCytomation MoFlo. Data from at least 10,000 cells
each was analyzed using FloJo software v4.5.2 (Tree Star). Adult wings of five
flies from hs:M, hs:NM, hs:rho and hs:rho; hs:msk were
dissected as above and wing hair counts/surface area measurements were
determined as previously described
(Marenda et al., 2003).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Martin-Blanco et al., 1999).
In contrast to a previous report (Guichard
et al., 1999), a more detailed analysis of the subcellular
localization of pMAPK within these cells shows that the pMAPK antigen is
predominately cytoplasmic during this stage (representative examples, L3 vein
in Fig. 1C and margin in
Fig. 1D, arrows show DNA,
arrowheads show pMAPK). pMAPK is also cytoplasmic in veins L4 and L5 (data not
shown). Thus, MAPK cytoplasmic hold occurs in developing larval wing
cells.
If the pMAPK antigen detected in larval wings moved to the nucleus, we
would expect MG-driven GFP reporter expression to mimic pMAPK expression in
the wing at this time. However, MG-driven GFP expression is nearly
complimentary to the cytoplasmic pMAPK antigen expression at this time
(Fig. 1A-B, arrow in
Fig. 1B). To quantify this, we
used phalloidin to visualize individual cells and the coincidence of both
pMAPK staining and MG-driven GFP reporter
(Fig. 1E). In 7140 cells from
five wing pouches, we found that pMAPK and GFP are coincident in 0.02% of
cases. As pMAPK is only expressed in pro-vein and margin cells, our results
suggest that MAPK nuclear translocation occurs predominantly in
non-vein/non-margin territories. To further examine this relationship, we
analyzed the coincidence of MG-driven GFP and the expression of
blistered (bs:lacZ), the Drosophila Serum Response
Factor, which is expressed in the majority of non-vein and non-margin cells
(Fristrom et al., 1994;
Montagne et al., 1996). We
find that in the wing pouch, MG-driven GFP is expressed predominantly in
bs:lacZ domains (Fig.
1F). Analysis of six wing pouches
(Fig. 1G) showed that the
majority (75-95%) of MG-driven GFP nuclei occurs in cells within
bs:lacZ expression domains (as defined by outlined regions in
Fig. 1G, e.g. arrowhead),
although some did not (arrow in Fig.
1G). In total, 61% of the wing pouch area expresses
bs:lacZ. Thus, we conclude that MAPK nuclear translocation in larval
wings occurs predominantly, though not exclusively, in non-vein/non-margin
cells.
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;
Martin-Blanco et al., 1999).
In early pupal development (24-27 hours APF;
Fig. 2A), pMAPK expression is
elevated in pro-vein and wing margin cells, while MG-driven GFP is expressed
predominantly along the wing margin (arrowheads in
Fig. 2A,C,D) and in two rows of
cells that flank the developing veins in the distal pupal wing (arrows in
Fig. 2C,D). At about 28 hours
APF, we (and others) see a loss of pMAPK antigen from vein cells (compare
asterisks in Fig. 2A with 2B)
(Guichard et al., 1999;
Martin-Blanco et al., 1999).
It is possible that pMAPK is first stored in vein cell cytoplasm and becomes
nuclear only at about 28 hours APF. However, we do not observe MG-driven GFP
expression matching the pMAPK expression pattern detected in 24-27 APF pupal
wings at, or after, 28 hours APF (out to 33 hours APF;
Fig. 2A-D). It is possible that
pMAPK is eliminated from the cytoplasm of pupal pro-vein cells by either
dephosphorylation or proteolysis, without nuclear translocation. Again, as in
larval discs, the pMAPK antigen we do detect in pupal wings is predominantly
cytoplasmic (e.g. Fig.
2E,F).
MAPK cytoplasmic hold and vein fate
Taken together, our data suggest that at the stages we observed, pMAPK
antigen is predominantly cytoplasmic, and most often detected in cells in
which MG-driven GFP is not. We therefore decided to test the hypothesis that
the different subcellular localization of MAPK may direct different cellular
outcomes in the developing wing. We have shown that we can express high levels
of nuclear-directed MAPK (hs:NM) in the developing eye
(Kumar et al., 2003). In order
to examine the effect of increased MAPK nuclear translocation in the wing, we
transiently expressed both normal (hs:M) and nuclear-directed MAPK
(hs:NM) for 1 hour at various times during development. Such
expression of hs:M has no affect on the pattern of veins in the adult
wing (Fig. 3A). As this only
increases total MAPK, and NOT activated (or pMAPK), this suggests that MAPK
levels are not limiting in the wing at this time. Importantly, similar ectopic
expression of hs:NM also has no effect on vein patterning
(Fig. 3B), consistent with the
hypothesis that nuclear MAPK does not induce vein cell fate. Similar results
were obtained when both hs:M and hs:NM are raised
continuously at elevated temperatures (29°C, data not shown).
Ectopic expression of hs:NM (but not hs:M) does produce
blisters in 1-5% of adult wings when transiently expressed between late L3 and
mid pupation (96-168 hours after egg deposition, data not shown). Further
analysis of these adult wings showed that while ectopic hs:NM did not
increase the overall size of the adult wing when compared with hs:M
controls (Fig. 3E), it did
significantly increase the number of cells within the wing when compared with
hs:M (Fig. 3F) [as
determined by wing hair counts in a defined area of the wing (cells per unit
area, or cell density)], as each cell secretes one hair
(Meyer et al., 2000). Thus,
although increasing nuclear MAPK does not induce vein hypertrophy (as observed
by induction of vein-promoting factors such as rhomboid, see below),
it does increase cell division. We suggest that increased cell division,
constrained within an unchanged surface area, can occasionally warp the
epithelium, thus forming a blister.
Though expression of hs:NM causes increased cell division, it
remains possible that increasing nuclear MAPK through hs:NM merely
phenocopies the effect of increasing the amplitude/level of Ras signaling, as
opposed to specifically increasing nuclear directed signaling downstream of
receptor activation. To rule this out, we expressed the upstream EGFR
activator rhomboid (rho)
(Guichard et al., 1999) to
increase the amplitude of pathway signaling. Overexpression of rho
(hs:rho) increases pMAPK antigen in larval and pupal wings
(Guichard et al., 1999). This
increased pMAPK leads to vein hypertrophy (arrow in
Fig. 3C)
(Guichard et al., 1999), but
not increased wing area or cell division
(Fig. 3E,F). Thus, excess
nuclear MAPK (induced by hs:NM) has a different phenotype (division)
than excess cytoplasmic pMAPK induced by hs:rho (differentiation). We
suggest a simple hypothesis to explain this difference: cytoplasmic pMAPK
induces vein differentiation, while nuclear MAPK induces proliferation.
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; Lorenzen et al.,
2001). Ectopic Msk expression has been previously shown to bind to
pMAPK, and to rescue loss of nuclear pMAPK in the developing
Drosophila embryo (Baker et al.,
2002; Lorenzen et al.,
2001). We also show that ectopic expression of Msk in larval wings
is sufficient to induce increased MAPK nuclear translocation (see below).
We examined the effect on pMAPK expression in pupal wings that transiently
overexpress both Msk and Rho. In these wings, ectopic Rhomboid increases and
alters pMAPK expression (compare asterisk in
Fig. 3G with 3H), in a
previously reported pattern (Guichard et
al., 1999). This ectopic pMAPK persists in pupal wings for at
least 2 hours after induction by hs:rho
(Fig. 3I). Ectopic Msk combined
with ectopic Rhomboid (hs:rho; hs:msk) suppresses the
Rhomboid-induced increased pMAPK, both immediately after induction (compare
asterisks in Fig. 3H with 3J)
and 2 hours after induction (compare asterisks in
Fig. 3I with 3K), suggesting
that Msk is limiting in the developing wing. As Msk is a pMAPK nuclear import
co-factor, we suggest that the addition of ectopic Msk increases the rate of
nuclear translocation of pMAPK, after which nuclear phosphatase (or protease)
activity eliminates the antigen. Consistent with our model, the extra veins in
the adult wing induced by ectopic Rhomboid expression are strongly suppressed
by the addition of ectopic Msk (Fig.
3D). In addition, hs:rho; hs:msk wings are significantly
larger than either hs:rho alone
(Fig. 3E), or even
hs:M or hs:NM wings (Fig.
3E). Although inducing ectopic nuclear MAPK with hs:NM
shows increased cell division (as evidenced by increased cell density) with no
increase in organ size (suggesting that there is no coordinate increase in
cell growth in these wings, and that the extra cells may be smaller than wild
type cells), promoting MAPK nuclear translocation with hs:rho;
hs:msk increases wing area without an increase in cell density
(Fig. 3F). This suggests that
there is a coordinate increase in both cell division and cell growth in these
wings, and that there are now more cells of a normal size present. Thus, in
both cases (hs:NM and hs:rho; hs:msk) cell division is
increased. This is consistent with nuclear translocation of MAPK promoting
cell division. As the Ras pathway is also known to control cell growth, our
data may suggest that the signal(s) for inducing growth versus cell division
by the Ras pathway split upstream of the nuclear translocation of MAPK.
Importantly, increasing MAPK nuclear translocation with hs:rho;
hs:msk did not broadly inhibit differentiation (inter-vein cells
differentiated normally, as did wing bristles for each cell) but, rather,
specifically inhibited vein cell formation, suggesting that cytoplasmic pMAPK
is specific to vein cell fate (see Discussion).
Taken together, these data suggest that the elevated cytoplasmic pMAPK induced by ectopic Rhomboid directs more cells to vein fate, and that the reduction in pMAPK when we co-overexpress Msk reverses this and induces cells to divide. We suggest that the simplest interpretation is that cytoplasmic, not nuclear, pMAPK directs wing cells to vein fate, while nuclear MAPK directs cells towards proliferation.
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This posterior ectopic expression of Msk eliminates pMAPK antigen within pro-vein and margin cells in the posterior domain of the wing pouch (compare anterior arrows and posterior arrowheads in Fig. 4D,E). Surprisingly, posterior Msk expression disrupts the anteroposterior compartment boundary, as determined by GFP marking (arrow in Fig. 4F). High levels of cell death can disrupt development, and cause cells to cease to respect compartment boundaries. As we see high levels of cell death in Msk overexpression wings (see below), we suggest this may explain this disruption. However, even when we block cell death with p35, we still observe loss of pMAPK in the posterior wing pouch (see below), suggesting that cell death alone is not the cause of lost pMAPK in this genotype.
Taken together, these experiments suggest that ectopic Msk can increase MAPK nuclear translocation and overcome cytoplasmic hold, and that some nuclear enzyme then rapidly eliminates the pMAPK antigen (most likely a phosphatase).
MAPK nuclear translocation promotes cell proliferation
In cultured CCL39 cells, MAPK cytoplasmic tethering inhibits the ability of
cells to enter S phase (Brunet et al.,
1999), suggesting that MAPK nuclear translocation is important for
cell cycle entry. In the developing Drosophila larval wing, elevated
Ras signaling similarly promotes G1/S progression
(Prober and Edgar, 2000), and
MAPK loss-of-function mutations suppress this progression
(Karim and Rubin, 1998). Taken
together, these data suggest that the G1/S transition in the developing larval
wing may require MAPK nuclear translocation. As cell proliferation in the wing
is better understood in larval rather than pupal stages, we focused our
analyses at this stage.
In larval wing discs, margin cells are non-proliferative [the zone of
non-proliferating cells (ZNC)]
(Milán et al., 1996;
O'Brochta and Bryant, 1985),
and markers of S-phase (BrdU) and M-phase (phospho-histone H3 antigen, pH3)
are reduced in this territory
(Milán et al., 1996).
Similarly, MG-driven GFP is also reduced in margin territories, indicating
that it too may be a marker for proliferation. However, MG-driven GFP is not
in the same cells as either BrdU or pH3 (brackets in 5A,B). In the developing
eye, MG-driven GFP follows the transcription of MG with a delay of 4-6 hours
(Kumar et al., 2003). Thus,
our observed non-coincidence of GFP with either BrdU or pH3 in the developing
wing may simply be due to this time lag.
To analyze the cell cycle more precisely, we used FACS to determine the cell-cycle phase of those cells expressing MG-driven GFP, following a 1-hour induction and 6 hour recovery time (Fig. 5E). We sorted for GFP and then compared the DNA content profiles of the two cell populations (GFP– control cells with little or no MAPK nuclear translocation versus GFP+ cells where MAPK nuclear translocation has occurred). The GFP+ cell population has a slightly elevated fraction in G2 and M phase, mostly at the expense of the pool in G1 (Fig. 5E). Although these results are consistent with a function of MAPK nuclear translocation in triggering proliferation, it remains possible that MG-driven GFP is a consequence, not a cause of proliferation. To test this, we increased MAPK nuclear translocation using NMG, while simultaneously driving GFP reporter expression (hs:NMG, UAS:GFP). We induced NMG for 1 hour, followed by 6 hours recovery, and see a dramatic reduction in the fraction of GFP+ cells in G1, while greatly raising the fraction in S and G2/M (Fig. 5F), suggesting that nuclear translocation of MAPK is sufficient to induce proliferation. When we allowed these larvae to recover for 24 hours, the fraction of GFP+ cells in G2/M rose, at the expense of the pool in G1 and S (Fig. 5G). This suggests that MAPK nuclear translocation is sufficient to induce S-phase transition in wing cells, and after the initial nuclear MAPK-induced transition to S-phase, cells then progress normally through the division cycle (at least as far as G2).
However, it could be that upon induction of NMG, a block in G2/M occurs, and this allows cells to build up in S phase. To rule this out, we expressed both hs:MG and hs:NMG, and analyzed pH3 staining. We do not observe fewer pH3-positive nuclei in hs:NMG discs versus hs:MG controls (Fig. 5C,D), as would be expected if a block in G2/M existed in hs:NMG discs. Indeed, we generally see more pH3-positive nuclei in hs:NMG wing pouches when compared with hs:MG controls, along with increased pH3 staining in the ZNC (compare nuclei in brackets of Fig. 5C and 5D). These data are consistent with ectopic nuclear MAPK inducing cell proliferation, even in populations of cells that are normally non-proliferative.
We also used continuous posterior-compartment driven Msk expression (en::msk) as a second test to determine the role of MAPK nuclear translocation in wing cell proliferation. Again, the fraction of GFP+, S-phase cells is increased (27% versus 16% for the control, anterior compartment GFP– cells), as is the fraction in G2/M (37% versus 32%), at the expense of cells in G1 (Fig. 5H). As Msk is continuously available in this experiment, we interpret this as a summation of the transient 6 and 24 hour effects seen with NMG. Consistent with this, in en::msk discs we also see elevated posterior compartment expression of the S-phase limiting factor Cyclin E (compare Fig. 5I with 5M), the M-phase limiting factor String (stg:lacZ, compare Fig. 5J to 5N) and the S-phase marker BrdU (compare Fig. 5K to 5O). Taken together, these data suggest that MAPK nuclear translocation does indeed normally promote S-phase transition in developing wing cells.
Elevated proliferation in the posterior compartment might be expected to
produce adult wings with enlarged posteriors (the `J.Lo wing'). However,
prolonged and elevated expression of Msk induces caspase-dependent cell death
(Fig. 5P) and the resulting
adult wings are severely disrupted, with nearly normal anterior compartments
and severely reduced posteriors (the `Twiggy wing'). These wings display loss
of posterior tissue, including distal regions of veins L4 and L5, and fused
posterior and anterior crossveins (en::msk in
Fig. 6A)
(Baker et al., 2002).
Msk prevents vein fate
Reduction of EGFR pathway function via loss of one copy of the gene
encoding MAPK (rl10A,
Fig. 6F)
(Biggs et al., 1994) strongly
suppresses the Msk-induced Twiggy wing, consistent with the Msk overexpression
phenotype being dependant on MAPK (see Discussion). If Msk is limiting in the
wing (as we suggest above), then msk gene dose should affect vein
formation. msk gain-of-function should suppress vein formation, while
msk loss-of-function should enhance vein formation.
To examine Msk gain-of-function, we analyzed overexpression of the negative
ligand Argos in the posterior compartment of the wing, which leads to vein
loss 100% in vein L4, and 90% in vein L5 (n=50, asterisks in
Fig. 6B)
(Freeman et al., 1992). When
Msk and Argos are co-expressed, Msk enhances the vein loss phenotype of Argos
to 100% in L4 and 100% in L5 (n=50, compare arrow in
Fig. 6G and 6B). Similarly,
overexpression of the nuclear ETS domain transcription factor Pointed P2
(PntP2, a positive MAPK effector) (Brunner
et al., 1994; O'Neill et al.,
1994) induces vein loss in 0% in L4 and 89% in L5 (n=54,
asterisks in Fig. 6C)
(Wessells et al., 1999),
consistent with our suggestion that MAPK nuclear function antagonizes vein
fate. Co-expression of Msk and PntP2 further enhances this vein loss to 97% in
L4 and 100% in L5 (n=34, Fig.
6H).
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). Similarly,
trans-heterozygous rlSem/msk5 wings also show
enhancement of the rolled extra vein phenotype (compare arrows in
Fig. 6E with 6J). Furthermore,
Msk gain of function suppresses the extra veins caused by both
hs-rho30a and UAS:rlSem expression
(see Fig. S1E-G in the supplementary material). Though these effects may reflect additive genetic phenotypes as opposed to true genetic interactions, when taken together, our gain-of-function and loss-of-function data suggest that Msk normally functions to restrict vein formation. We suggest this is because gain of msk function leads to increased nuclear MAPK (vein loss), while loss of msk leads to increased cytoplasmic MAPK (extra veins). These data are consistent with our suggestion above: that vein formation through MAPK occurs through a cytoplasmic, rather than a nuclear target.
Msk vein loss is not due to cell death
As posterior overexpression of Msk leads to extensive cell death, and
ultimately the Twiggy wing, vein loss could be due to tissue loss in the
posterior compartment. To block any cell death associated with Msk
overexpression, we co-expressed Msk with the baculovirus pan-caspase inhibitor
p35. Posterior ectopic expression of p35 has no effect on the expression of
pMAPK in the larval wing (compare Fig. 7A
with 7C). Surprisingly, p35 expression leads to vein loss in the
adult wing (asterisk in Fig.
7G). Importantly, posterior co-expression of Msk with p35 leads to
a similar loss of pMAPK expression in larval wings as that observed with
ectopic expression of Msk alone (compare
Fig. 7D with 7B and 7A),
suggesting that cell death is not the cause of lost pMAPK expression in larval
wings expressing Msk in this tissue. Furthermore, the adult vein loss observed
with p35 expression is significantly enhanced by co-expression with Msk
(compare Fig. 7H with 7F and
7G), resulting in severely distorted wings with enlarged posterior
compartments compared with the control anterior compartments (the `J.Lo
wing'). We suggest that co-expression of p35 with Msk blocks Msk-induced cell
death, thus increasing proliferation through the nuclear translocation of MAPK
in the posterior compartment, but also decreasing tissue loss associated with
cell death, resulting in an enlarged posterior wing.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kumar et al., 1998). Here, we
report the existence and contribution of MAPK cytoplasmic hold in the
developing Drosophila wing. We observe a difference in cytoplasmic
versus nuclear function of MAPK, and suggest that in the developing wing, MAPK
subcellular localization controls the difference between vein specification
(cytoplasmic MAPK) and proliferation (nuclear MAPK).
|
Overexpression of either CycE or Stg leads to increased proliferation in
Drosophila wings (Neufeld et al.,
1998); however, there is little to no effect on vein formation,
with no vein loss in either case (see Fig. S1A,B in the supplementary
material). Similarly, inhibiting cell proliferation by over-expressing either
the cyclin-dependant kinase inhibitor dacapo, or the S-phase inhibitor p21 had
no significant effect on vein formation (see Fig. S1C,D in the supplementary
material). This is consistent with a direct effect for MAPK cytoplasmic hold
on vein differentiation.
There are a number of known cytoplasmic targets of MAPK, including p90RSK,
cPLA2 and Myosin light chain kinase
(Ebisuya et al., 2005).
However, it is important to consider that some cytoplasmic target proteins for
MAPK may first be phosphorylated in the cytoplasm and then translocate to the
nucleus, or be inhibited from doing so, such as SV40 T-antigen and
Xenopus nucleoplasmin (Johnson et
al., 2004). In fact, Hasson et al. have recently reported that the
co-repressor Groucho is directly phosphorylated by MAPK, and this
phosphorylation weakens its repressor activity, leading to extra veins
(Hasson et al., 2005).
Groucho, though it functions as a nuclear transcription factor, may be
phosphorylated in the cytoplasm in pro-vein cells, where it can then
translocate to the nucleus to affect changes in Notch transcription, leading
to vein formation.
Mechanism of cytoplasmic hold: pMAPK anchoring vs import sequestration
Recent reports suggest that MAPK cytoplasmic hold may perform similar
functions in mammals (Ebisuya et al.,
2005). In vertebrate cells, expression of the death effector
PEA-15 can sequester pMAPK in the cytoplasm
(Formstecher et al., 2001).
After treatment with Retinoic acid, embryonic stem and carcinoma cells stop
proliferating, restrict the nuclear entry of pMAPK and differentiate into
primitive endoderm (Smith et al.,
2004). In the mouse embryo, pMAPK is detected in the cytoplasm
rather than the nuclei of cells receiving FGF signals
(Corson et al., 2003). A family
of proteins called SEFs antagonize MAPK signaling
(Fürthauer et al., 2002).
More recently, SEF has been found to act directly to hold pMAPK in the
cytoplasm, suggesting a mechanism for FGF pathway attenuation through MAPK
cytoplasmic hold (Torii et al.,
2004; Tsang and Dawid,
2004). We have been unable to identify any homolog of PEA-15 or
SEF outside the chordates by conventional bioinformatic techniques. However, a
fly protein with a function that is very similar to SEF would fit the MAPK
cytoplasmic hold phenomena we observe in the eye and wing.
While anchoring of pMAPK has been shown to restrict MAPK nuclear entry in cell culture, it remains possible that pMAPK nuclear import could be prevented by removing a required nuclear import co-factor. Thus, by cytoplasmic sequestration of Msk (for example), pMAPK would be unable to translocate into the nucleus, and pMAPK cytoplasmic hold would be achieved.
Regardless of the mechanism, MAPK cytoplasmic hold may be a conserved mechanism necessary for the differentiation of certain developing tissues in many taxa, and proper control of MAPK subcellular localization may act as a developmental signal to determine the proliferative state of a cell.
Mammalian importin 7 is reported to import several proteins into the
nucleus, including histone H1, core histones, HIV-1 reverse transcription
complexes and the glucocorticoid receptor
(Baake et al., 2001;
Fassati et al., 2003;
Freedman and Yamamoto, 2004;
Jäkel et al., 1999).
However, our data suggest that MAPK is a crucial target for the phenotypes we
observe in wings overexpressing Msk: (1) a null mutation in
Drosophila MAPK strongly suppresses the en::msk adult wing
phenotype; (2) increased nuclear MAPK is observed after overexpression of Msk
in larval wings; (3) loss-of-function mutations in Drosophila Histone
H1 (Su(var)205) have no effect on the en::msk phenotype
(data not shown); (4) loss-of-function mutations in members of other vein
promoting pathways (thick veins, tkv8) have no effect on
the en::msk adult wing (data not shown).
In the developing compound eye, breaking MAPK cytoplasmic hold in cells
within the morphogenetic furrow results in reduced expression of Atonal, which
is required for the initiation of differentiation in the developing eye
(Kumar et al., 2003). Taken
together with our new data from the developing wing, we suggest that MAPK
cytoplasmic hold may be generally required for the cell cycle arrest necessary
for the initiation of differentiation, thus defining a novel bifurcation in
the Ras pathway to control different cellular outcomes. Finally, the
regulation of MAPK cytoplasmic hold may help to distinguish the MAPK signals
for cell fate from those for cell proliferation.
|
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/133/1/43/DC1
* Present address: Howard Hughes Medical Institute, Janelia Farm Research
Campus, Ashburn, VA 20147, USA 
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