1 Department of Developmental Genetics, National Institute of Genetics, Shizuoka
411-8540, Japan
2 Department of Genetics, Graduate University for Advanced Studies, Mishima,
Shizuoka 411-8540, Japan
Author for correspondence (e-mail:
yhiromi{at}lab.nig.ac.jp
Accepted 22 May 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Eye, Chordotonal organ, Ras signaling
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Drosophila, two regions of the nervous system are known to use
homeogenetic induction: (1) the specification of photoreceptor neurons during
ommatidial assembly in the compound eye
(Tomlinson and Ready, 1987),
and (2) the formation of subepidermal stretch receptor precursors (chordotonal
organ precursors: COPs) in the embryonic peripheral nervous system
(Jarman and Jan, 1995
). In
both processes, cells already specified as photoreceptor neurons or COPs
recruit surrounding cells to assume fates similar to their own. The inducing
signal is a TGF
-like protein Spitz, that acts through EGF receptor to
activate the Ras/MAPK signaling pathway in the induced cells (reviewed by
Zipursky and Rubin, 1994
). The
outcome of the induction is remarkably constant: each ommatidium in the eye
contains precisely eight photoreceptor neurons (R1-R8), and each hemisegment
in the embryo generates exactly eight COPs (C1-C8). In order to achieve such
constancy, the induction process must be exquisitely controlled.
Within the induced cells, the induction is regulated by phosphorylation of
Ets transcription factors Pointed P2 (PNTP2) and YAN, which respectively acts
as positive and negative regulators of neuronal differentiation
(Brunner et al., 1994a;
O'Neill et al., 1994
;
Rebay and Rubin, 1995
). Both
proteins possess an Ets-specific domain called the Pointed domain
(Klämbt, 1993
) that are
phosphorylated by MAPK upon stimulation by the induction signal. This
modification leads to the degradation of YAN and activation of PNTP2 function.
Activation of PNTP2 leads to the production of another isoform produced by the
pointed (pnt) gene, PNTP1, a constitutive transcriptional
activator that is necessary for neuronal development of the induced
photoreceptor cells and COPs (O'Neill et
al., 1994
).
Induction is also regulated by restricting the cells with inducing ability.
Inducing cells are selected from groups of cells, called proneural cluster,
that express a bHLH transcription factor Atonal (reviewed by
Jan and Jan, 1995). In the
larval eye imaginal disc, proneural cluster consists of a moving front of
differentiation called the morphogenetic furrow. From a stripe of cells in the
morphogenetic furrow, evenly spaced R8 photoreceptor neurons are selected
through lateral inhibition. In the embryo, lateral inhibition likewise selects
COPs C1-C5 from Atonal+ proneural clusters. In both tissues, Atonal
expression is linked to inducing ability thorough expression of Rhomboid, a
founding member of the Rhomboid family of intramembrane serine proteases,
which is involved in the proteolytic activation of Spitz EGF (reviewed by
Freeman and Gurdon, 2002
).
Rhomboid+ R8 and C1-C5 secrete Spitz EGF and act as founder cells,
inducing their neighbors to assume photoreceptor neuronal fates and COP fates,
respectively (Jarman et al.,
1993
; Jarman et al.,
1994
; Lage et al.,
1997
; Okabe and Okano,
1997
). R8 then induces the formation of R2 and R5, which in turn
express rhomboid and serve as the secondary source of Spitz signal.
Although in the eye Rhomboid paralog Roughoid plays a major role in induction,
misexpression of Rhomboid causes recruitment of supernumerary photoreceptor
neurons, implying that spatial regulation of Rhomboid is nevertheless
essential for generating the correct ommatidium
(Freeman et al., 1992
;
Wasserman, 2000
). R8/R2/R5 and
C1-C5 constitute EGF signaling centers, inducing R3/R4/R1/R6/R7 and C6-C8,
respectively (Freeman et al.,
1992
; Tomlinson and Ready,
1987
; Lage et al.,
1997
; Okabe and Okano,
1997
). Thus, the transcriptional regulation of rhomboid
constitutes a key element in specifying the cells with inducing ability.
Spitz EGF acts not only as a paracrine inducer, but also has an autocrine
function; inducing cells secreting Spitz themselves receive the Spitz signal
and must respond to it for cell survival
(Tio and Moses, 1997).
Although all cells within the ommatidium require EGF receptor function
(Freeman, 1996
), it is not
known whether or not the Ras signaling pathway downstream of the receptor is
used in the same way in all cells. We found that hyperactivation of
pnt function abrogates the inducing ability of the inducing cells.
Hence, inducing cells must possess a mechanism to escape the inhibitory effect
of pnt. We have identified a novel Ets-related factor EDL (ETS-domain
lacking), containing the Pointed domain but not the ETS domain
(Shilo, 1998
), that may
mediate this mechanism. EDL has also been identified as MAE (modulator of the
activity of ETS), a protein that binds YAN and promotes its phosphorylation by
MAPK (Baker et al., 2001
).
Although such activity suggests a role of EDL/MAE in the induced cells, we
find that EDL/MAE is specifically expressed in cells that act as the induction
center by producing Spitz EGF. Furthermore, we show that EDL/MAE abolishes
transcriptional activation function of PNTP2 through direct binding, rather
than promoting it as suggested by Baker et al.
(Baker et al., 2001
). This
antagonistic action on PNTP2 blocks an autocrine pathway downstream of Spitz
EGF, thereby allowing inducing cells to express their inductive function.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Somatic recombination clones were induced using the FRT technique
(Xu and Rubin, 1993), over
FRT43D, P[w+]47A. Embryos homozygous for
edlL19 were identified using the CyO,
wg-lacZ balancer chromosome. FRT-mediated mitotic recombination clones of
pnt
88
(Klämbt, 1993
) were made
over FRT82B, ub-GFP, P[w+]90D,
RpS32 (also called M(3)w124), and
those for a null allele of rhomboid,
rhomboidP
5
(also called verho-PDelta5)
(Freeman et al., 1992
) were
made over P[w+]70C, RpS174 (also
called M(3)i55), FRT80A.
Ectopic expression of edl was achieved by the GAL4/UAS system
(Brand and Perrimon, 1993),
using the following drivers: elav-GAL4 C155
(Lin and Goodman, 1994
),
sevE-GAL4 K25 (Brunner et
al., 1994a
), CY2 (gift of T. Schüpbach) and
en-GAL4 (gift of A. Brand and N. Perrimon).
UAS-pntP2 (Klaes et al.,
1994
), UAS-phl.gof (also called
D-rafF179)
(Brand and Perrimon, 1994
).
Ras85Dv12.sev (also called
sevE-Ras1Val12)
(Fortini et al., 1992
) has
been described.
Histology
In situ hybridization and antibody staining were performed as described
(O'Neill and Bier, 1994;
Tautz and Pfeifle, 1989
;
Tomlinson and Ready, 1987
)
with minor modifications. Confocal microscopy was done using BioRad MRC 1024
mounted on a Zeiss Axioplan2 microscope. For light microscopy, adult heads
were fixed in 0.1 M cacodylate buffer or phosphate buffer (pH 7.4), 2.5%
glutaraldehyde, 2% paraformaldehyde for 4 hours to overnight. Samples were
postfixed in 1% OsO4 in the same buffer for 3-4 hours, then
dehydrated and embedded in Durcapan (Fluka). Sections (0.5 µm) were cut
using a Reichert microtome, stained with Toluidine Blue and viewed in bright
field microscopy. For scanning microscopy, flies were fixed in 0.1 M
cacodylate buffer (pH 7.4), 2% OsO4 overnight at room temperature.
After dehydration, samples were dried using Peldry II (Ted Pella), coated with
gold palladium in a Denton Desk II sputter coater and photographed in a JEOL
840 SEM.
Molecular analysis
DNA sequences flanking the P-element insertion point in the P17 line were
recovered by plasmid rescue, and were used to initiate a chromosomal walk.
Genomic restriction fragments were used to screen a 4-6 hour embryonic cDNA
library (Novagen). We isolated three classes of cDNAs, represented by N1, N4
and N9, that failed to hybridize with each other. Only N9 showed an expression
pattern similar to the lacZ expression pattern of the P17 enhancer
trap line in the ventral neuroectoderm. Screening of a 4-8 hour embryonic cDNA
library (Brown and Kafatos,
1988) using N9 as a probe resulted in isolation of nine additional
cDNAs, of which clone 115-3A was the longest. These cDNAs define the
edl transcription unit that produces a 1.6 kb transcript. N9, whose
length is 2 kb, possesses an upstream exon not found in other cDNAs. As the
signal produced by a probe unique to N9 was weak, this cDNA appears to
represent a minor transcript of edl. Nucleotide sequences of cDNAs N9
and 115-3A were determined by the chain termination method using Sequenase v.2
(US Biochemical Corporation), and was compared with the genomic sequence
obtained by the Drosophila Genome Project
(FlyBase, 1999
). Within the 37
kb of DNA downstream of the edl gene, there was no potential exon
capable of encoding an ETS domain. The genomic rescue transgene was made by
subcloning a 18 kb XbaI genomic fragment into the pCaSpeR4 vector. To
generate the UAS-edl effector construct, the edl open
reading frame was PCR-amplified from cDNA clone 115-3A using primer
5'-TCAAGAACTCAAACGTTGCG-3' and the T7 primer and subcloned into
the EcoRI site of the pUAST vector
(Brand and Perrimon, 1993
).
Sequences were verified by cycle sequencing according to the instructions of
the manufacturer (ABI). GMR-GAL4 carries the GAL4 coding region in the pGMR
vector (Hay et al., 1994
).
Transfections and CAT assays
Transfections and CAT activity measurements were performed essentially as
described (Pascal and Tjian,
1991; O'Neill et al.,
1994
) with minor modifications. In all transfections, 100 ng of
each expression plasmid were cotransfected along with 2 µg of
E6BCAT and 3 µg of pBluescriptSK(-) (Stratagene) using the
calcium phosphate method except various amount of EDL/MAE expression plasmid
(12.5-200 ng) were used in Fig.
3E. For each plasmid, six (Fig.
3D) or two (Fig.
3E) transfections were performed in parallel and the resulting
data were averaged. Expression plasmid pPacEdl was generated by amplifying the
open reading frame of edl from the N9 cDNA clone using primers
5'-ACGGAAGCCATATGCAAGTGGAATC-3' and
5'-GAATCCTCGAGATATGTACAAC-3', and subcloning into pPacUbx+Nde
after digestion by NdeI and XhoI. Other expression plasmids
are described in O'Neill et al. (O'Neill
et al., 1994
), and were generous gifts from I. Rebay. The pPacEdl
clone used in Fig. 3D has a
single base mutation causing a conservative amino acid change (K to R) at
position 159. Identical results were obtained in small scale experiments using
a pPacEdl clone without this mutation.
|
Electrophoretic mobility shift experiments
Proteins Myc-tagged at the N terminus were made by in vitro
transcription/translation using TNT T7 Quick kit (Promega) and templates
generated by PCR. Primers for the 5' end contained a T7 promoter
sequence for in vitro transcription. Electophoretic mobility shift experiments
were carried out using DIG Gel Shift Kit (Roche) with minor modifications.
Oligonucleotides containing EBS sequences with or without a mutation (EBS* or
EBS) (Albagli et al., 1996)
were self-annealed and 3'-end labeled with DIG-11-ddUTP using terminal
transferase. Each 12 µl reaction contained 0.5 µl of protein solutions
(or the sequential dilutions for myc-Edl), 0.6 ng labeled EBS probe and 0.5
µl anti-Myc monoclonal antibody (9E10). Maximum amount of unlabeled probes
used for a competition assay was 12.5 ng per reaction. Solutions were mixed on
ice and left for 20-25 minutes at room temperature. The order of mixing did
not cause any significant changes in the results. Samples were then resolved
on a 0.25x TBE, 2.5% glycerol, 5.25% polyacrylamid gel
preelectrophoresed at 4°C for 1 hour at 80 V. The gel was electroblotted
onto GeneScreen Plus membrane (NEN), crosslinked by UV and subjected to a
chemiluminescent detection using CDP-Star (Roche) as a substrate. Signals were
recorded by Lumi-Imager (Boehringer-Mannheim).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Analysis of edl mutants revealed that in both the eye and
chordotonal organ, the loss of edl reduced the efficiency of
Spitz-mediated induction. In retinal sections of
edlJV/Df(2R)P34 and
edlJV/L19 animals, about 3% of ommatidia showed
loss of photoreceptor cells, of the R1-R6 and R7 photoreceptor subtype
(Fig. 2A,L). The R8 cell, which
most strongly expresses edl expression within the ommatidium, was
always present, even in ommatidia where other photoreceptor cells were missing
(Fig. 2A, inset). A similar
phenotype was seen in edlL19 mutant clones, which
entirely lack edl function. This phenotype was almost completely
rescued by an edl+ transgene
(Fig. 2L). The requirement of
edl was more pronounced when the level of the inducing signal was
compromised. Star is a dosage-sensitive component of Spitz-mediated
induction in the eye, and is required for the transport of Spitz EGF to the
Golgi apparatus (Heberlein and Rubin,
1991; Kolodkin et al.,
1994
; Lee et al.,
2001
). In Star-/+ animals, 30% of ommatidia
show a reduction in the number of photoreceptor neurons, with the average
number of R1-R7 cells reduced per ommatidium of 0.39
(Fig. 2B,D,M). When
edlJV/L19 mutation
was placed in the Star-/+ background, 65% of ommatidia
lacked at least one neuron, with 1.71 photoreceptor cells missing per
ommatidium on average (Fig.
2C,E,M). Similarly, the edl mutation enhanced the
reduction in the number of photoreceptor neurons in a hypomorphic allele of
spitz (Fig. 2F,G,M).
These synergistic effects of edl and Star/spitz
suggest that edl participates in the induction of R1-R7 by Spitz
EGF.
|
Spitz EGF acts through the EGF receptor, resulting in the activation of the
Ras/MAPK signaling leading to the phosphorylation of Ets proteins YAN and
PNTP2. Baker et al. (Baker et al.,
2001) have reported that EDL/MAE promotes the MAPK-mediated
phosphorylation of the repressor protein YAN, thus leading to its
inactivation, and is also required for the transcriptional activation by
PNTP2. Although the reduction in Spitz-mediated induction observed in
edl mutants appears consistent with the role of EDL/MAE in promoting
MAPK signaling, two lines of evidences argues against such model of EDL/MAE
action. First, the expression of edl in chordotonal organs is
detectable only in COPs C1-C5, which form using the proneural activity of
atonal and do not require EGF receptor function for their
specification (Okabe and Okano,
1997
). It is also unlikely that edl acts solely by
regulating YAN activity, because loss of edl function had an effect
in the absence of YAN; while a null allele of yan
(aop1)has increased number of scolopidia in Lch5,
introduction of edl mutation caused a clear reduction in the number
of scolopidia formed (Fig.
2J,K,O,P). EDL/MAE thus has targets other than repressor protein
YAN. These results indicate that the mechanism by which EDL/MAE participates
in Spitz-mediated induction is different from the one proposed by Baker et al.
(Baker et al., 2001
).
EDL/MAE inhibits transcriptional activation by PNTP2
As our genetic analysis indicated that EDL/MAE has targets other than YAN,
we investigated the effect of EDL/MAE on PNTP2 activity. To test whether
EDL/MAE directly binds PNTP2, we performed pull-down assays on bacterially
produced GST-fusion proteins. EDL/MAE bound to the N-terminal region of the
PNTP2 protein (PNTP2 1-276), which contains the Pointed domain and is specific
to the PNTP2 isoform, consistent with the result obtained by Baker et al.
(Baker et al., 2001)
(Fig. 3A,B). Neither the region
common to the PNTP1 and PNTP2 isoforms (PNTP1 223-623), nor the PNTP1-specific
region (PNTP1 1-223) captured EDL/MAE (Fig.
3B). PNTP2 bound EDL/MAE much more efficiently than did YAN
(Fig. 3B). MAPK, which is
encoded by the rolled (rl) gene
(Brunner et al., 1994b
), and
D-jun (Jra FlyBase), which has been shown to act synergistically with
PNT in transcription assays (Treier et
al., 1995
), showed only background levels of binding
(Fig. 3B).
To address the functional significance of the binding of EDL/MAE to PNTP2,
we tested the effect of EDL/MAE on the DNA binding activity of PNTP2. Mobility
shift experiments revealed that PNTP2 can bind DNA in the presence of EDL/MAE,
making a ternary complex with EDL/MAE and DNA
(Fig. 3C). Such a ternary
complex was not detected with PNTP1 protein, consistent with the finding that
EDL/MAE does not bind PNTP1 (Fig.
3A,B). We then asked whether EDL/MAE affects transcriptional
activation function of PNTP2 in a culture cell transfection assay
(O'Neill et al., 1994). When
PNTP2 was expressed in Drosophila Schneider cells, it activated
transcription of a reporter gene harboring Ets-binding sites
(Fig. 3D). Transcriptional
activation by PNTP2 was enhanced by co-transfection with a plasmid encoding an
activated form of Ras. However, in both cases, co-expression of EDL/MAE
completely suppressed the transcriptional activation by PNTP2
(Fig. 3D). This suppression was
dose dependent, and was specific to PNTP2, as EDL/MAE did not have such an
inhibitory effect on PNTP1 (Fig.
3D,E). We conclude that EDL/MAE can bind PNTP2 and inactivate its
function as a transcription activator. This activity is opposite to the one
proposed by Baker et al. (Baker et al.,
2001
), who reported that EDL/MAE potentiates transcriptional
activation by PNTP2 in monkey Cos-7 cells.
EDL/MAE antagonizes pnt function in vivo
To further study the role of EDL/MAE activity in vivo, we examined the
effect of ectopic expression of edl. In the developing eye,
pnt is required for the neuronal differentiation of photoreceptor
cells, whereas yan is a negative regulator
(O'Neill et al., 1994;
Rebay and Rubin, 1995
). If
EDL/MAE inactivates PNTP2 activity, we would expect that misexpression of
EDL/MAE in presumptive neurons would inhibit their neuronal differentiation.
However, if EDL/MAE promoted MAPK-mediated phosphorylation of PNTP2 and YAN,
misexpression of EDL/MAE should produce extra neurons. When edl was
expressed in all neurons, the size of the compound eye was severely reduced
(Fig. 4A,B). Hardly any
photoreceptor neurons were present, and most of the retina was occupied by
pigment cells (Fig. 4C). In the
eye imaginal disc, a massive reduction of differentiating neurons was seen
from the earliest stages of ommatidial assembly
(Fig. 4D,E). The effect on
neuronal specification was cell-type specific; we found little or no
expression of markers for the recruited cells R1-R7, but R8 specification was
largely unaffected (Fig. 4F,G, and data not shown). This phenotype was indistinguishable from the
pnt mutant phenotype (Fig.
4H,I), consistent with the idea that ectopic EDL/MAE blocks
pnt function. However, misexpression of edl in non-neuronal
cone cells did not transform these cells towards a neuronal fate, as seen
following the activation of Ras signaling
(Fig. 4J-M). Furthermore, the
suppressive effect of EDL/MAE on neuronal differentiation could be seen even
in the presence of activated Ras; when edl was co-expressed with
activated Ras, edl completely suppressed the ectopic neurons produced
by Ras activation (Fig. 4J-Q).
Thus, EDL/MAE can inhibit neuronal differentiation either downstream or
parallel to Ras activation, consistent with EDL/MAE having an inhibitory
effect on the transcriptional activation by PNTP2.
|
Overexpression of pnt interferes with induction
The inhibitory effect of EDL/MAE on transcriptional activation by PNTP2
suggests that EDL/MAE normally functions by suppressing PNTP2 function. As
edl expression is most prominent at inducing cells in both
chordotonal organ and the eye, EDL may exert its effect on PNTP2 function in
inducing cells that produce Spiz. Inducing cells not only secrete Spitz EGF,
but they also receive Spiz and thereby activate the downstream Ras/MAPK
signaling pathway, which could result in PNT activation. Although the
activation of PNTP2 is an obligatory step of neuronal specification of induced
cells, the consequence of PNT activation in inducing cells has not been
studied. EDL expression in inducing cells raises a possibility that these
cells need to lower PNTP2 activity to ensure normal development. We thus
examined the effects of hyperactivation of pnt on COP formation and
photoreceptor recruitment.
When the PNTP2 isoform and an activated form of Raf (MAPKKK) was expressed in the posterior compartment of each segment in the embryo, the number of scolopidia in Lch5 was reduced from the normal number of five to three or four in 71% of hemisegments (n=60) (Fig. 5G,H). This phenotype is similar to the edl mutant phenotype, supporting the idea that the role of edl is to repress PNT activity in inducing cells. We also hyperactivated pnt in all cells posterior to the morphogenetic furrow in the eye imaginal disc. Most ommatidial clusters in the early stages of ommatidial assembly contained fewer than the normal number of neurons, an effect that is opposite to the known role of pnt in promoting neuronal identity in induced cells (Fig. 5A,B). Cells that failed to initiate neural differentiation were of specific cell types; the specification of R3/R4/R1/R6 was severely disrupted, whereas the R8 cell was still present (Fig. 5C-F). Although a general disruptive effect on differentiation cannot be completely ruled out, these results suggest that hyperactivation of PNT may reduce the ability of inducing cells to recruit additional photoreceptors and COPs.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
These results contrasts with that of Baker et al.
(Baker et al., 2001) who showed
that EDL/MAE promotes transcriptional activation by PNTP2 protein in monkey
Cos-7 cells. Our EDL/MAE misexpression experiments in vivo support the idea
that EDL/MAE antagonizes, rather than promotes, PNTP2 activity. The effects of
EDL/MAE misexpression cannot be explained by the promotion of phosphorylation
of YAN, because phosphorylation causes the inactivation of YAN
(O'Neill et al., 1994
), and
loss of yan produces effects that are opposite to what we have
observed by EDL/MAE misexpression (Lai et al., 1992;
Tei et al., 1992
;
Okabe and Okano, 1997
). The
opposite effects of EDL/MAE on PNTP2-mediated transcription may be due to the
difference in the cell lines employed in the transfection assays. It is also
possible that EDL/MAE activity is used differently in diverse tissues; for
example, the effect seen on the ventral denticle belts in the embryonic
cuticle (Baker et al., 2001
)
may be due to the promotion of YAN inactivation within the ventral
neuroectoderm, allowing PNTP1 to function in the specification of medial fates
(Kim and Crews, 1993
).
Regulation of homeogenetic induction by EDL/MAE
Within the developmental contexts examined in this study, edl
expression appear to be confined to cells with the ability to induce other
cells using Spitz EGF (Fig.
1D,E). This suggests that EDL may have a role in regulating
induction by Spitz. Secreted Spitz acts not only on the induced cells, but is
also received by the inducing cells themselves. Although the molecular events
leading to the activation of PNT within the induced cells is well established,
whether the same regulatory cascade operates within the inducing cells had not
been studied. We found that hyperactivation of PNT in inducing cells has a
deleterious effect on induction; in the embryo, COP C3 loses expression of
rhomboid, a factor that is essential for the production of Spitz EGF.
Although inducing cells are positioned so that they receive highest levels of
Spitz EGF that they produce, they may possess a mechanism to prevent
hyperactivation of PNT. The phenotypes of the edl loss-of-function
mutants and the effect of PNT hyperactivation are similar in both ommatidial
and chordotonal organ development (Figs
2,
5). EDL/MAE is thus likely to
be a part of the machinery that antagonizes PNTP2 to prevent the negative
effect of PNT on induction in the inducing cells
(Fig. 6).
|
This raises the question when and where PNT uses the activity to curb
induction. During both ommatidial assembly and the development of the
chordotonal system, PNT promotes neuronal development in the induced cells. We
suggest that PNT may also suppress inducing ability in such cells. This would
create a negative feedback loop so that cell, once induced, does not itself
acquire inducing ability. Although such mechanism would be effective in
preventing uncontrolled spread of homeogenetic induction, the need for such
regulatory system arises only if induced cells also have the opportunity to
acquire inducing ability. This is indeed the case for R2/R5; these cells form
via induction by R8, and then express rhomboid and become a secondary
source of Spitz EGF. Other cells, such as R3/R4 could also potentially become
inducers, because they have probably resided within the proneural cluster
prior to the onset of induction and experienced Atonal expression, which
promotes rhomboid expression
(Lage et al., 1997) M.O.,
unpublished). Repressive effect of PNT on rhomboid would thus be a
mechanism to safeguard against the potential activation of rhomboid
by Atonal within the proneural cluster. PNT may cause this repression via
activating expression of a repressor or by acting as a repressor itself.
The inhibition of rhomboid expression is not the only way that PNT
negatively regulates induction. In the eye, a rhomboid paralog
roughoid plays a critical role in generating mature Spitz EGF
(Urban et al., 2001). It is
possible that roughoid may also be regulated by PNT to control
induction. Furthermore, upon activation of Ras signaling induced cells produce
negative regulators of the Ras pathway, such as Sprouty, Argos and Kekkon,
generating negative feedback loops (Casci
et al., 1999
; Ghiglione et
al., 1999
; Golembo et al.,
1996
; Kramer et al.,
1999
; Schweitzer et al., 1995a). Because Argos is a secreted
antagonist of Spitz EGF, its production by inducing cells could be detrimental
for induction. The inhibition of PNT function by EDL/MAE may also serve to
reduce Argos production in the inducing cells, allowing efficient
induction.
Although induction in Drosophila eye and the chordotonal organ
discussed here is `homeogenetic' in the sense that both the inducing cell and
the induced cell are of the same cell type (photoreceptor neurons or COPs),
they differ in genetic and molecular properties. Although neuronal
specification of founder cells R8 and C1-C5 requires atonal function
but not pnt, induced cells R1-R7 and C6-C8 depend on PNT activation
and need atonal only indirectly. In addition, the induction itself
generates a dichotomy between cells with inducing ability and those without,
because induced cells acquire a different character (lack of inducing ability)
from the inducing cell. Inducing cells, however, are prevented from expressing
these characteristics through the repression of PNT function by EDL/MAE. Other
instances of homeogenetic induction may also possess such properties, in order
to generate cellular diversity, rather than equivalence. During the
development of muscle progenitors in Drosophila, the size of the
inductive field is defined by a group of cells similar to the proneural
cluster; a small number of founder muscles are selected based on the activity
of the bHLH transcription factor lethal of scute
(Carmena et al., 1995) and
EGF-mediated induction (Buff et al.,
1998
). Because edl is also expressed in a subset of
muscle progenitors (data not shown), it may act in founder muscle selection in
a similar way as it does in the eye and chordotonal organs.
Previous phylogenetic analysis revealed that the Ets protein family
originated early during metazoan evolution and most of the functional
diversity was already established prior to the separation of protostomes and
deuterostomes (Degnan et al.,
1993; Laudet et al.,
1993
; Laudet et al.,
1999
; Lautenberger et al.,
1992
; Price and Lai,
1999
). Although it is likely that such an ancestral Ets protein
already contained a Pointed domain, the Pointed domain of EDL/MAE could not be
classified as similar to any of the previously known Ets protein subclasses
(Fig. 1G). This suggests that
EDL/MAE-like protein may have already existed before the divergence of Ets
proteins. It is tempting to speculate that EDL/MAE or EDL/MAE-like proteins
may regulate inductive processes in other developmental processes in
Drosophila and vertebrates.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: MRC Centre for Developmental Neurobiology, King's College
London, London, UK
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albagli, O., Klaes, A., Ferreira, E., Leprince, D. and Klambt, C. (1996). Function of ets genes is conserved between vertebrates and Drosophila. Mech. Dev. 59, 29-40.[CrossRef][Medline]
Baker, D. A., Mille-Baker, B., Wainwright, S. M., Ish-Horowicz, D. and Dibb, N. J. (2001). Mae mediates MAP kinase phosphorylation of Ets transcription factors in Drosophila. Nature 17,330 -334.
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Brand, A. H. and Perrimon, N. (1994). Raf acts downstream of the EGF receptor to determine dorsoventral polarity during Drosophila oogenesis. Genes Dev. 8, 629-639.[Abstract]
Brown, N. H. and Kafatos, F. C. (1988). Functional cDNA libraries from Drosophila embryos. J. Mol. Biol. 203,425 -437.[Medline]
Brunner, D., Ducker, K., Oellers, N., Hafen, E., Scholz, H. and Klämbt, C. (1994a). The ETS domain protein pointed-P2 is a target of MAP kinase in the sevenless signal transduction pathway. Nature 370,386 -389.[CrossRef][Medline]
Brunner, D., Oellers, N., Szabad, J., Biggs, W. H., 3rd, Zipursky, S. L. and Hafen, E. (1994b). A gain-of-function mutation in Drosophila MAP kinase activates multiple receptor tyrosine kinase signaling pathways. Cell 76,875 -888.[Medline]
Buff, E., Carmena, A., Gisselbrecht, S., Jimenez, F. and
Michelson, A. M. (1998). Signalling by the Drosophila
epidermal growth factor receptor is required for the specification and
diversification of embryonic muscle progenitors.
Development 125,2075
-2086.
Carmena, A., Bate, M. and Jimenez, F. (1995). Lethal of scute, a proneural gene, participates in the specification of muscle progenitors during Drosophila embryogenesis. Genes Dev. 9,2373 -2383.[Abstract]
Casci, T., Vinos, J. and Freeman, M. (1999). Sprouty, an intracellular inhibitor of Ras signaling. Cell 96,655 -665.[Medline]
Degnan, B. M., Degnan, S. M., Naganuma, T. and Morse, D. E. (1993). The ets multigene family is conserved throughout the Metazoa. Nucleic Acids Res. 21,3479 -3484.[Abstract]
Dittmer, J. and Nordheim, A. (1998). Ets transcription factors and human disease. Biochim. Biophys. Acta 1377,F1 -11.[CrossRef][Medline]
Edlund, T. and Jessell, T. M. (1999). Progression from extrinsic to intrinsic signaling in cell fate specification: a view from the nervous system. Cell 96,211 -224.[Medline]
FlyBase (1999). The FlyBase database of the
Drosophila Genome Projects and community literature. The FlyBase Consortium.
Nucleic Acids Res. 27,85
-88.
Fortini, M. E., Simon, M. A. and Rubin, G. M. (1992). Signalling by the sevenless protein tyrosine kinase is mimicked by Ras1 activation. Nature 355,559 -561.[CrossRef][Medline]
Freeman, M. (1996). Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye. Cell 87,651 -660.[Medline]
Freeman, M. and Gurdon, J. B. (2002). Regulatory principles of developmental ignaling. Annu. Rev. Cell Dev. Biol. 18,515 -539.[CrossRef][Medline]
Freeman, M., Kimmel, B. E. and Rubin, G. M.
(1992). Identifying targets of the rough homeobox gene
of Drosophila: evidence that rhomboid functions in eye development.
Development 116,335
-346.
Fujita, S. C., Zipursky, S. L., Benzer, S., Ferrus, A. and Shotwell, S. L. (1982). Monoclonal antibodies against the Drosophila nervous system. Proc. Natl. Acad. Sci. USA 79,7929 -7933.[Abstract]
Ghiglione, C., Carraway, K. L., 3rd, Amundadottir, L. T., Boswell, R. E., Perrimon, N. and Duffy, J. B. (1999). The transmembrane molecule kekkon 1 acts in a feedback loop to negatively regulate the activity of the Drosophila EGF receptor during oogenesis. Cell 96,847 -856.[Medline]
Golembo, M., Schweitzer, R., Freeman, M. and Shilo, B. Z.
(1996). Argos transcription is induced by the Drosophila
EGF receptor pathway to form an inhibitory feedback loop.
Development 122,223
-230.
Graves, B. J. and Petersen, J. M. (1998). Specificity within the ets family of transcription factors. Adv. Cancer Res. 75,1 -55.[Medline]
Guan, K. L. and Dixon, J. E. (1991). Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192,262 -267.[Medline]
Hay, B. A., Wolff, T. and Rubin, G. M. (1994).
Expression of baculovirus P35 prevents cell death in Drosophila.
Development 120,2121
-2129.
Heberlein, U. and Rubin, G. M. (1991). Star is required in a subset of photoreceptor cells in the developing Drosophila retina and displays dosage sensitive interactions with rough. Dev. Biol. 144,353 -361.[Medline]
Jan, Y. N. and Jan, L. Y. (1995). Maggot's hair and bug's eye: role of cell interactions and intrinsic factors in cell fate specification. Neuron 14, 1-5.[Medline]
Jarman, A. P. and Jan, Y. N. (1995). Multiple roles for proneural genes in Drosophila neurogenesis. In Neural Cell Specification: Molecular Mechanisms and Neurotherapeutic Implications (ed. B. H. J. Juurlink, P. H. Krone, W. M. Kulyk, V. M. K. Verge and J. R. Doucette), pp. 97-104. New York: Plenum Press.
Jarman, A. P., Grau, Y., Jan, L. Y. and Jan, Y. N. (1993). Atonal is a proneural gene that directs chordotonal organ formation in the Drosophila peripheral nervous system. Cell 73,1307 -1321.[Medline]
Jarman, A. P., Grell, E. H., Ackerman, L., Jan, L. Y. and Jan, Y. N. (1994). Atonal is the proneural gene for Drosophila photoreceptors. Nature 369,398 -400.[CrossRef][Medline]
Kim, S. H. and Crews, S. T. (1993). Influence
of Drosophila ventral epidermal development by the CNS midline cells and spitz
class genes. Development
118,893
-901.
Klaes, A., Menne, T., Stollewerk, A., Scholz, H. and Klämbt, C. (1994). The Ets transcription factors encoded by the Drosophila gene pointed direct glial cell differentiation in the embryonic CNS. Cell 78,149 -160.[Medline]
Klämbt, C. (1993). The Drosophila gene
pointed encodes two ETS-like proteins which are involved in the development of
the midline glial cells. Development
117,163
-176.
Kolodkin, A. L., Pickup, A. T., Lin, D. M., Goodman, C. S. and
Banerjee, U. (1994). Characterization of Star and
its interactions with sevenless and EGF receptor during
photoreceptor cell development in Drosophila.
Development 120,1731
-1745.
Kramer, S., Okabe, M., Hacohen, N., Krasnow, M. A. and Hiromi,
Y. (1999). Sprouty: a common antagonist of FGF and EGF
signaling pathways in Drosophila. Development
126,2515
-2525.
Lage, P., Jan, Y. N. and Jarman, A. P. (1997). Requirement for EGF receptor signalling in neural recruitment during formation of Drosophila chordotonal sense organ clusters. Curr. Biol. 7,166 -175.[Medline]
Lai, Z. C. and Rubin, G. M. (1992). Negative control of photoreceptor development in Drosophila by the product of the yan gene, an ETS domain protein. Cell 70,609 -620.[Medline]
Laudet, V., Niel, C., Duterque-Coquillaud, M., Leprince, D. and Stehelin, D. (1993). Evolution of the ets gene family. Biochem. Biophys. Res. Commun. 190, 8-14.[CrossRef][Medline]
Laudet, V., Hanni, C., Stehelin, D. and Duterque-Coquillaud, M. (1999). Molecular phylogeny of the ETS gene family. Oncogene 18,1351 -1359.[CrossRef][Medline]
Lautenberger, J. A., Burdett, L. A., Gunnell, M. A., Qi, S., Watson, D. K., O'Brien, S. J. and Papas, T. S. (1992). Genomic dispersal of the ets gene family during metazoan evolution. Oncogene 7,1713 -1719.[Medline]
Lee, J. R., Urban, S., Garvey, C. F. and Freeman, M. (2001). Regulated intracellular ligand transport and proteolysis control EGF signal activation in Drosophila. Cell 107,161 -171.[CrossRef][Medline]
Lin, D. M. and Goodman, C. S. (1994). Ectopic and increased expression of Fasciclin II alters motoneuron growth cone guidance. Neuron 13,507 -523.[Medline]
Mangold, O. and Spemann, H. (1927). Über Induktion von Medullarplatte durch Medullarplatte im jüngeren Keim, ein Beispiel homöogenetischer oder assimilatorischer Induktion. W. Roux's Arch. Entw. Org. 111,341 -422.
Mlodzik, M. and Hiromi, Y. (1992). Enhancer trap method in Drosophila: its application to neurobiology. Methods Neurosci. 9,397 -414.
Morimoto, A. M., Jordan, K. C., Tietze, K., Britton, J. S.,
O'Neill, E. M. and Ruohola-Baker, H. (1996) Pointed, an ETS
domain transcription factor, negatively regulates the EGF receptor pathway in
Drosophila oogenesis. Development
122,3745
-3754.
Okabe, M. and Okano, H. (1997). Two-step
induction of chordotonal organ precursors in Drosophila embryogenesis.
Development 124,1045
-1053.
O'Neill, E. M., Rebay, I., Tjian, R. and Rubin, G. M. (1994). The activities of two Ets-related transcription factors required for Drosophila eye development are modulated by the Ras/MAPK pathway. Cell 78,137 -147.[Medline]
O'Neill, J. W. and Bier, E. (1994). Double-label in situ hybridization using biotin and digoxigenin-tagged RNA probes. Biotechniques 17, 870, 874-875.[Medline]
Pascal, E. and Tjian, R. (1991). Different activation domains of Sp1 govern formation of multimers and mediate transcriptional synergism. Genes Dev. 5,1646 -1656.[Abstract]
Peverali, F. A., Isaksson, A., Papavassiliou, A. A., Plastina, P., Staszewski, L. M., Mlodzik, M. and Bohmann, D. (1996). Phosphorylation of Drosophila Jun by the MAP kinase rolled regulates photoreceptor differentiation. EMBO J. 15,3943 -3950.[Abstract]
Price, M. D. and Lai, Z. C. (1999). The yan gene is highly conserved in Drosophila and its expression suggests a complex role throughout development. Dev. Genes Evolution 209,207 -217.[CrossRef]
Rebay, I. and Rubin, G. M. (1995). Yan functions as a general inhibitor of differentiation and is negatively regulated by activation of the Ras1/MAPK pathway. Cell 81,857 -866.[Medline]
Scholz, H., Deatrick, J., Klaes, A. and Klämbt, C.
(1993) Genetic dissection of pointed, a Drosophila gene encoding
two ETS-related proteins. Genetics
135,455
-468.
Schweitzer, R., Howes, R., Smith, R., Shilo, B. Z. and Freeman, M. (1995). Inhibition of Drosophila EGF receptor activation by the secreted protein Argos. Nature 376,699 -702.[CrossRef][Medline]
Shilo, B. Z. (1998). Flies over Crete:
Drosophila molecular biology. Kolymbari, Crete, July 12-18, 1998.
EMBO J. 17,6769
-6771.
Tang, P., Steck, P. A. and Yung, W. K. (1997). The autocrine loop of TGF-alpha/EGFR and brain tumors. J. Neurooncol. 35,303 -314.[Medline]
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85.[Medline]
Tei, H., Nihonmatsu, I., Yokokura, T., Ueda, R., Sano, Y., Okuda, T., Sato, K., Hirata, K., Fujita, S. C. and Yamamoto, D. (1992). pokkuri, a Drosophila gene encoding an E-26-specific (Ets) domain protein, prevents overproduction of the R7 photoreceptor. Proc. Natl. Acad. Sci. USA 89,6856 -6860.[Abstract]
Tio, M. and Moses, K. (1997). The Drosophila
TGF alpha homolog Spitz acts in photoreceptor recruitment in the developing
retina. Development 124,343
-351.
Tomlinson, A. and Ready, D. F. (1987). Neuronal differentiation in the Drosophila ommatidium. Dev. Biol. 120,366 -376.
Tower, J., Karpen, G. H., Craig, N. and Spradling, A. C.
(1993). Preferential transposition of Drosophila P elements to
nearby chromosomal sites. Genetics
133,347
-359.
Treier, M., Bohmann, D. and Mlodzik, M. (1995). JUN cooperates with the ETS domain protein pointed to induce photoreceptor R7 fate in the Drosophila eye. Cell 83,753 -760.[Medline]
Urban, S., Lee, J. R. and Freeman, M. (2001). Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases. Cell 107,173 -182.[CrossRef][Medline]
Wasserman, J. D., Urban, S. and Freeman, M.
(2000). A family of rhomboid-like genes: Drosophila
rhomboid-1 and roughoid/rhomboid-3 cooperate to
activate EGF receptor signaling. Genes Dev.
14,1651
-1663.
Wasylyk, B., Hagman, J. and Gutierrez-Hartmann, A. (1998) Ets transcription factors: nuclear effectors of the Ras-MAP-kinase signaling pathway. Trends Biochem. Sci. 23,213 -216.[CrossRef][Medline]
Xu, T. and Rubin, G. M. (1993). Analysis of
genetic mosaics in developing and adult Drosophila tissues.
Development 117,1223
-1237.
Zipursky, S. L. and Rubin, G. M. (1994). Determination of neuronal cell fate: lessons from the R7 neuron of Drosophila. Annu. Rev. Neurosci. 17,373 -397.[CrossRef][Medline]
Zwick, E., Bange, J. and Ullrich, A. (2002). Receptor tyrosine kinases as targets for anticancer drugs. Trends Mol. Med. 8,17 -23.[CrossRef][Medline]