Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
Author for correspondence (e-mail:
kmoses{at}cellbio.emory.edu)
Accepted 23 April 2003
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SUMMARY |
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Key words: MAP kinase, Drosophila, Compound eye
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INTRODUCTION |
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The first cell type specified is the R8 photoreceptor
(Tomlinson and Ready, 1987)
and the central event in the formation of this founding cell is the
progressive restriction of the transcription factor Atonal to individual cells
(Jarman et al., 1994
;
White and Jarman, 2000
).
Atonal expression begins about 10-15 cells anterior to the morphogenetic
furrow, initially in all cell nuclei in a rising gradient
(Fig. 1). Then expression is
lost from some cells and retained in others, to produce the `intermediate
groups' (arrow in Fig. 1E)
(Jarman et al., 1995
). By the
next column, only one cell retains Atonal: the presumptive R8 photoreceptor
cell (Baker et al., 1996
).
|
The formation of a precisely spaced pattern of founder cells in phase 1
involves Notch mediated lateral inhibition
(Cagan and Ready, 1989;
Baker and Zitron, 1995
;
Baker et al., 1996
;
Baker and Yu, 1998
). The role
of the Notch signal is not simple: it initially induces Atonal expression and
only later restricts it (Baker and Zitron,
1995
; Baker et al.,
1996
). Furthermore, this Notch signal is genetically upstream of
Egfr pathway signaling (Chen and Chien,
1999
; White and Jarman,
2000
).
It has been suggested that the Egfr/MAPK signal is required for the
formation of the founding R8 photoreceptor cells in phase 1, either as an
inductive or an inhibitory signal, or both (via positive and/or negative
ligands) (Baker and Rubin,
1989; Baker and Rubin,
1992
; Zak and Shilo,
1992
; Xu and Rubin,
1993
; Freeman,
1996
; Spencer et al.,
1998
). Dominant gain-of-function mutations of Egfr
(EgfrElp) reduce the number of founder cells
(Baker and Rubin, 1989
;
Baker and Rubin, 1992
;
Zak and Shilo, 1992
). Genetic
loss-of-functions tests in mosaic clones were difficult at first, because the
Egfr/Ras signal functions in cell cycle regulation (a MAPK signal is normally
required for cells to pass the G1/S checkpoint) and also later to regulate
apoptosis (Dickson, 1998
;
Freeman, 1998
;
Kurada and White, 1998
;
Assoian and Schwartz, 2001
;
Baker and Yu, 2001
;
Jones and Kazlauskas, 2001
;
Baonza et al., 2002
;
Howe et al., 2002
). To
overcome this operational problem, we removed Egfr function by means
of a temperature-sensitive mutation and found that the R8 cells form in a
nearly normal pattern (Kumar et al.,
1998
). Others then used an alternative approach to derive genetic
loss-of-function mosaic clones for elements of the Egfr pathway (the
Minute technique) and they obtained similar results. Loss-of-function
genetic tests have been carried out for Egfr itself
(Kumar et al., 1998
;
Baonza et al., 2001
;
Yang and Baker, 2001
), the
positive ligands Spitz and Vein (Freeman,
1996
; Tio and Moses,
1997
; Spencer et al.,
1998
), the inhibitory ligand Argos
(Yang and Baker, 2001
), Ras
(Halfar et al., 2001
;
Yang and Baker, 2001
), and Raf
(Yang and Baker, 2001
). In
each case, removing Ras pathway signaling during phase 1 eliminates neither
the initial upregulation of Atonal nor its subsequent downregulation to
(mostly) single founder cells. Indeed, rather than being regulated by the
Egfr/Ras pathway, atonal function is genetically upstream of MAPK
activation (Chen and Chien,
1999
; White and Jarman,
2000
). However, in gain-of-function experiments, there is evidence
that activation of MAPK signaling in the morphogenetic furrow can inhibit
Atonal expression (Kumar et al.,
1998
; Chen and Chien,
1999
). Thus, we conclude that a subset of cells in the furrow can
be specified as R8 founder cells without any direct function of the Egfr/Ras
pathway, and indeed, that pathway activity may be antagonistic to R8 cell
specification at this stage.
We and others have observed highly regulated activation of MAPK at the
earliest stage of ommatidial cluster formation (the intermediate groups), by
the use of antibodies specific to diphosphorylated MAPK (dpErk antigen) (see
Fig. 1)
(Gabay et al., 1997;
Kumar et al., 1998
;
Spencer et al., 1998
;
Chen and Chien, 1999
;
Yang and Baker, 2003
). This
regulated activation of MAPK in the furrow is controlled by Egfr
(Dominguez et al., 1998
;
Kumar et al., 1998
;
Chen and Chien, 1999
;
Baonza et al., 2001
;
Halfar et al., 2001
;
Yang and Baker, 2001
). This is
apparently paradoxical: how can the Egfr pathway specifically activate MAPK in
the nascent ommatidial clusters in the morphogenetic furrow, yet also be
dispensable for their foundation? We observed that the activated MAPK (dpErk)
antigen is predominantly cytoplasmic at this stage and thus unable to regulate
nuclear targets, although it may still affect cytoplasmic proteins (see
Fig. 1)
(Kumar et al., 1998
). Thus, we
suggested that while the pathway is activated in a patterned manner, the
nuclear MAPK signal may be blocked at the level of MAPK nuclear translocation
after its phosphorylation, a mechanism that we call `cytoplasmic hold'.
In vertebrates MAPK phosphorylation has been shown to induce a
conformational change and homodimerization
(Zhang et al., 1994;
Wang et al., 1997
;
Cobb and Goldsmith, 2000
).
Although some MAPKs such as mammalian Erk3, are constitutively nuclear
(Cheng et al., 1996
), most
(such as Erk1 and Erk2) are activated in the cytoplasm and then move to the
nucleus (Khokhlatchev et al.,
1998
; Cobb and Goldsmith,
2000
; Robinson et al.,
2002
). In cultured cells, MAPK nuclear translocation follows
phosphorylation within a few minutes (Chen
et al., 1992
; Lenormand et
al., 1993
). Thus, our suggestion that phospho-MAPK may be subject
to developmentally regulated cytoplasmic hold
(Kumar et al., 1998
) was novel
but was based only on the observed subcellular distribution of dpErk antigen,
which might be subject to a number of artifacts.
The experiments presented here address two questions: does MAPK cytoplasmic
hold really occur in the morphogenetic furrow and, if so, what is its
function? We adopted an approach that had been used to demonstrate the nuclear
translocation of Notch (Struhl and Adachi,
1998). We created and expressed proteins with Drosophila
Rolled MAPK fused to an exogenous transcription factor (based on yeast GAL4),
which can activate a reporter gene only if it can reach the nucleus. We report
that the nuclear translocation of MAPK is indeed regulated by a second means
and does not directly follow from phosphorylation during phase 1 (but not in
phase 2). Furthermore, we show that if MAPK cytoplasmic hold is overcome in
phase 1 Atonal expression is reduced. We conclude that MAPK cytoplasmic hold
occurs during phase 1 and is released later to allow Ras pathway signaling to
operate during ommatidial assembly (phase 2). We also conclude that this early
block is necessary for the patterning events that focus Atonal expression to
produce the precise array of R8 photoreceptors.
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MATERIALS AND METHODS |
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Drosophila stocks, mosaic clones and temperature-shift
regimes
Materials were sourced as follows:
UAS:GFP, UAS:lacZ was a gift from J. Fischer
Wild-type Canton-S was a gift from D. Gailey
GMR:Gal4 was a gift from S. L. Zipursky
(Pignoni and Zipursky,
1997)
rl1 (Biggs et al.,
1994), rlSem
(Brunner et al., 1994
) were
gifts from the Bloomington center
Nts is Nl1N-ts1
(Shellenbarger and Mohler,
1975)
ato3, an antigen positive, functional null allele
(Jarman et al., 1995)
Clones were obtained as described previously
(Xu and Rubin, 1993) using FRT
82B, Ub:GFP and eyFLP (Newsome et
al., 2000
)
Nts and hhts shifts were as follows: 1 hour 29°C, 1 hour 37°C, 2 hours 25°C
Egfrtsla shifts were as follows: 22 hours 29°C, 1 hour at 37°C, 2 hours at 29°C
The HS:M and HS:NM time course experiment was carried out as follows: the first larvae were dissected before induction, then kept for 1 hour at 37°C; more larvae were dissected immediately, then kept at 25°C with dissections at 1, 2, 4, 6 and 8 hours.
The HS:MG and HS:NMG experiments were carried out as follows: 1 with hour at 37°C then 2 hours at 25°C.
Immunohistochemistry in situ hybridization and SEM
Primary antibodies: rabbit anti-ß-galactosidase (Cortex Biochem),
mouse anti-dpErk (Sigma) (Gabay et al.,
1997), mouse anti-HSV-Tag (Novagen), rabbit anti-Ato
(Jarman et al., 1993
), rabbit
anti-Spalt (a gift from R. Schuh)
(Kuhnlein et al., 1994
), mouse
anti-BarH1 (a gift from K. Saigo)
(Higashijima et al., 1992b
),
guinea pig anti-Sens (a gift from G. Mardon)
(Frankfort et al., 2001
),
mouse anti-Cut (mAb 2B10) (from Developmental Studies Hybridoma Bank), mouse
anti-Pros (mAb MR1A) (from Developmental Studies Hybridoma Bank), rat
anti-Elav (from Developmental Studies Hybridoma Bank) and mouse anti-Glass
(mAb 9B2.1) (from Developmental Studies Hybridoma Bank). Secondary antibodies
were conjugated to FITC or TRITC or Cy5 (Jackson Laboratories). F-actin was
visualized with phalloidin (Molecular Probes). DNA was stained with SYTO24
(Molecular Probes). Immunohistochemistry on imaginal discs was as described
previously (Tomlinson and Ready,
1987
). SEM on adult flies was as described previously
(Tio and Moses, 1997
).
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RESULTS |
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|
Reporter gene activation is not seen in the same cells that show high
level dpErk expression in the furrow
In the morphogenetic furrow, large groups of cells (the `intermediate'
groups) express high levels of dpErk, but this antigen is predominantly
cytoplasmic (Fig. 1)
(Kumar et al., 1998). A simple
hypothesis might be that in the HS:MG experiment described above, the
MG fusion protein requires some time to translocate to the nucleus. This
hypothesis suggests that all the cells that express dpErk cytoplasmically
early would later go on to show reporter gene activation by MG. However, when
we double stained induced HS:MG eye discs for the reporters and
dpErk, we found that the prominent column of dpErk expression intermediate
groups was not followed in a later column by a prominent column of reporter
gene expressing clusters (Fig.
3A,B). We conclude that the majority of the dpErk antigen in the
intermediate groups does not later move to the nucleus and activate gene
expression, rather we suggest that it is probably dephosphorylated within the
next column or two (2-4 hours), in the cytoplasm, without first passing
through the nucleus.
|
In vivo calibration of reporter gene activity
The two reporter genes (UAS:lacZ and UAS:GFP) could
themselves be responsible for the long delay in reporter gene activation seen
by MG induction following the furrow. Indeed, GFP is known to require time to
reveal its expression, because the fluorescent molecule is the product of a
series of enzymatic reactions catalyzed by GFP, which requires time
(Matz et al., 2002). Although
this would seem to be controlled by the much reduced delay seen in the NMG
lines, we calibrated the expression of both reporters in third instar eye
imaginal discs in vivo. We colocalized both ß-galactosidase and GFP
reporters in eye discs with Glass protein itself, with the GAL4 activity
driven directly by GMR or by the MG or NMG fusions
(Fig. 4). As Glass is
responsible for the induction in all three cases, the spatial delay between
Glass antigen and reporter expression in each case is the sum of the delays
imposed by driver expression at the transcriptional level and later and by
reporter gene expression. We make two significant observations: (1) GFP
reporter is expressed in most or all target cells, but only after a delay of
several columns (8-10 hours); (2) ß-galactosidase reporter is expressed
only in a subset of target cells, but its induction is much faster (seen
within one column or 2 hours). Thus, the UAS:lacZ reporter appears to
have a higher threshold of activation than UAS:GFP. These operational
limitations of the two reporter genes must be borne in mind in analyzing the
results reported below.
|
|
|
|
We also tested the hypothesis that Atonal expression might be upstream of
MAPK nuclear translocation. This possibility follows from the observation that
where Atonal is expressed, dpErk antigen is not nuclear
(Fig. 1). To test this, we
derived atonal loss-of-function clones
(Fig. 7E-L) and although the
clones do show the expected developmental defects (including loss of dpErk
antigen, Fig. 9E) (Chen and Chien, 1999), they
do not fill with reporter gene expression, as would be expected if Atonal is
antagonistic to MAPK translocation.
|
We see no major effect on Atonal or Elav expression at t=0 for the control wild-type or HS:M (Fig. 8A,B), or any later time point (not shown). However, we do see a clear change in the HS:NM larvae: Atonal expression is reduced ahead of the furrow where it normally ramps up, in the furrow itself, where gaps appear in Atonal expression between the intermediate groups (arrow in Fig. 8C) and after the furrow where Atonal is reduced or undetectable in the single R8 founder cells. In addition, the normal actin driven tight constriction of cells apical surfaces in the furrow (Fig. 8E) is much reduced (Fig. 8F). Furthermore, although Elav normally first appears in pairs of cells (the future R2 and R5 photoreceptors, Fig. 8G) in HS:NM larvae at t=0, Elav is ectopically expressed (at a low level) in earlier columns, in the R8 cells (which have lost their Atonal expression). At t=1 hour in HS:NM larvae the ectopic Elav expression is lost and Atonal expression rebounds, even beyond the levels and extent normally seen (Fig. 8D). At t=2 hours and later in HS:NM, the expression patterns of both Atonal and Elav recover and are indistinguishable from the controls (data not shown). In all cases, the pattern of dpErk antigen is indistinguishable from wild-type (data not shown).
|
Thus, we observe clear consequences in the developing furrow of adding a
strong NLS to MAPK: Atonal expression is lost and R8 cells begin to
differentiate precociously as neurons (they express Elav early). This is
consistent with a crucial function for the cytoplasmic hold of activated MAPK
in the morphogenetic furrow of the developing fly eye and others data showing
genetic interactions between atonal and Ras pathway signaling
(Dokucu et al., 1996;
Chanut et al., 2002
). This
pair of experiments is controlled for other variables (promoter strength and
so on) that may have a bearing on expression levels. We cannot claim that
cytoplasmic hold can block pathway signaling at any expression level, and thus
the observations of ourselves and others that pathway gain-of-function can
block Atonal expression may be due to different levels of expression in those
conditions (Kumar et al.,
1998
; Chen and Chien,
1999
).
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DISCUSSION |
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Consistent with our suggestion that the pathway signal is blocked in the
intermediate groups (phase 1), we see reporter gene expression only later, in
the future R8 cells in the last two columns of Atonal expression and later in
cells as they are recruited into the assembling ommatidia (phase 2). This is
consistent with normal Egfr pathway activity in the downregulation of Atonal
at the end of phase 1 and then again at later stages (phase 2), when
successive Ras pathway signals recruit each cell type that follows the
founding R8 cell (which is specified by other means). Although we cannot
detect MAPK nuclear translocation early in the furrow (in the intermediate
groups) with either of our reagents (the transcription factor fusion or the
epitope tag) we cannot formally exclude the possibility that there is some
lower level of nuclear MAPK at these stages that is below the limits of our
two detection systems. Similarly, we cannot exclude the possibility that there
are cytoplasmic functions for phosphorylated MAPK at these stages. However,
our results are consistent with two Egfr pathway functions in the developing
R8 cells at this time (as Atonal expression ends): for the maintained
expression of differentiation markers (Boss and Elav) and for later cell
survival (Kumar et al.,
1998).
Furthermore, through the addition of a constitutive NLS, we have driven MAPK into the nuclei of cells in phase 1, thus overcoming MAPK cytoplasmic hold. This results in a rapid downregulation of Atonal and the precocious neural differentiation of the R8 photoreceptors (Fig. 10). Taken together with our observation of the first nuclear translocation of MAPK as Atonal is downregulated in normal development (above), we suggest that the Egfr/Ras pathway may normally contribute to the end of phase 1 by ending Atonal expression.
|
What is the developmental purpose of this block of MAPK signaling in the
furrow? Anterior to the furrow, MAPK cytoplasmic hold cannot function, or it
would prevent the MAPK signaling required for the G1/S transition and thus
halt cell proliferation. Perhaps this is one reason why all cells in phase 1
exit the cell cycle (Ready et al.,
1976; Wolff and Ready,
1991
; Thomas et al.,
1994
). However, new data suggest that the Egfr pathway does
function in the furrow to maintain G1 arrest (visualized as increased cyclin B
expression) (Yang and Baker,
2003
). This could be mediated through some low level of nuclear
MAPK at this stage or possibly through cytoplasmic targets for MAPK signaling.
However, although cyclin B expression is elevated posterior to the furrow in
all cells other than R8 in Egfr pathway loss-of-function mutant clones, the
leading edge of cyclin B expression does not advance (it is not expressed
earlier) (Yang and Baker,
2003
). Thus, it may be that the role of Egfr pathway signals in
maintaining G1 arrest is later than the end of Atonal expression (i.e. in
phase 2, not in phase 1).
We suggest that the founder cells have a special developmental function to
fulfill in phase 1: they must act as organizing centers for lateral inhibition
to produce the spaced pattern of R8 cells. If the founder cells did not
inhibit their neighbors most or all cells in phase 1 might rapidly
differentiate as photoreceptors, resulting in disorder. This type of disorder
is observed when the Egfr/MAPK pathway is ectopically activated ahead of the
furrow, when photoreceptor differentiation becomes independent of Atonal and
R8 fate (Baonza et al., 2001).
Our model may also explain the loss of ommatidia seen in
EgfrElp gain-of-function mutants
(Baker and Rubin, 1989
;
Baker and Rubin, 1992
;
Zak and Shilo, 1992
;
Lesokhin et al., 1999
). Excess
Ras/MAPK pathway signals may reduce Atonal expression and thus the number of
R8 founder cells. Our results lead us to predict that G1 cell-cycle arrest may
be found in other cases in which a subset of progenitor cells is selected by
lateral inhibition through active Notch pathway signaling and repression of
Ras/MAPK signaling. In summary, our data are consistent with a model in which
Egfr/MAPK signaling functions in ommatidial assembly but not directly in
founder cell specification. We propose that MAPK cytoplasmic hold is
restricted to the morphogenetic furrow, and does not happen anterior to the
furrow (the proliferative phase) or posterior (during ommatidial assembly, or
phase 2). It appears to be coincident with the regulated G1 arrest seen in the
furrow.
It is interesting to note that our observed pattern of MAPKGal4/VP16 is very different from our (and others) observation of the pattern of MAPK phosphorylation (dpErk antigen); indeed, they are almost exclusive. We and many others observe that the predominant expression of dpErk in the developing eye is in the intermediate groups in the furrow, and yet we (and, we believe, the data of others) show little detectable signal function there. Furthermore, we and others have shown in many ways that MAPK signaling is absolutely required for ommatidial assembly posterior to the furrow, yet no one can detect much dpErk at that stage. Perhaps MAPK cytoplasmic hold can explain this paradox as well: where MAPK is anchored in the cytoplasm (in the furrow) it can be phosphorylated by MEK but is protected from abundant phosphatase activity that waits in the nucleus. Thus, the pathway is blocked, there is no negative feedback and the antigen builds up to high (and easily detected) levels for several hours. Later (during ommatidial assembly) it is possible be that there is no cytoplasmic hold, so MAPK passes rapidly to the nucleus after its phosphorylation by MEK, where the signal is passed, negative feedback is triggered and the antigen is cleared by phosphatase. Thus, in vivo the dpErk stain may actually be a stain not for pathway activity per se, but predominantly for MAPK cytoplasmic hold!
Our findings indicate that MAPK nuclear translocation is regulated in vivo by some mechanism in addition to, and regulated separately from, its phosphorylation state. We do not propose that MAPK phosphorylation is not required for MAPK nuclear translocation, only that phosphorylation is not always sufficient. What might the mechanism for MAPK cytoplasmic hold be? The simplest hypothesis is that some anchoring factor sequesters activated MAPK in the cytoplasm until a second developmental signal permits its release. This could provide for a point of signal transduction pathway integration. However, we suggest an alternative model: activated MAPK cannot translocate to the nucleus in the intermediate groups not because it is held fast by some negative anchoring factor, but because it lacks some specific positive factor, such as an import factor.
In summary, the results presented here demonstrate that there is the dual regulation of MAPK signal transduction, both through its phosphorylation by MEK and independently through the control of nuclear translocation. Such dual regulation may be important in many developmental events through which a subset of founder cells must first be specified. As such events involve lateral inhibition and cell contact, this mechanism may not be observable in tissue culture systems.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Present address: Department of Biochemistry and Biophysics, University of
California, San Francisco, CA 94143, USA
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