Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720, USA
* Author for correspondence (e-mail: beckendo{at}uclink.berkeley.edu)
Accepted 19 June 2003
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SUMMARY |
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Key words: Embryonic salivary glands, senseless, Lyra, Apoptosis, reaper, hid, Drosophila
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Introduction |
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We are beginning to understand the transcriptional regulation of the three
upstream cell death genes, reaper, hid and grim. Steroid
hormone signaling activates reaper and hid expression during
histolysis of larval salivary glands and midgut
(Jiang et al., 1997).
Drosophila p53 has been shown to activate reaper in response
to irradiation but not in response to developmental cell death in the embryo
(Brodsky et al., 2000
). The Hox
gene Deformed can directly activate reaper expression at the
segmental boundaries in the maxillary segment in the embryo
(Lohmann et al., 2002
). The
only known negative regulator is the Ras-MAPK pathway. It represses
hid and allows the survival of cells in the Drosophila eye
(Kurada and White, 1998
).
Though apoptosis is an essential part of development, there are many
tissues that do not show any cell death during embryogenesis. The lack of cell
death in these tissues can be ascribed to lack of activators of the apoptotic
pathway or the presence of repressors of apoptosis in these tissues. One of
the tissues that does not show any programmed cell death in the embryo is the
embryonic salivary gland (Myat and Andrew,
2000). The salivary glands are derived from 80-100 cells of the
ventral ectoderm in parasegment 2 of the embryo. Previous studies have shown
that the expression of the homeotic gene Sex combs reduced
(Scr) in parasegment 2 is necessary for specification of the salivary
primordium (Panzer et al.,
1992
). Scr is expressed in the entire ectoderm of
parasegment 2, including the cells that will form the salivary placodes.
Embryos mutant for Scr lack salivary glands and overexpression of
Scr in other parasegments of the embryo can lead to ectopic salivary
gland formation (Panzer et al.,
1992
). Ventrally, the two salivary placodes are separated by two
rows of cells that give rise to the salivary ducts. The ventral extent of the
salivary placodes is specified by EGFR signaling that occurs in the cells
closest to the ventral midline of the embryo
(Kuo et al., 1996
). As
germband retraction proceeds, the cells of the salivary placodes invaginate to
form the salivary glands (reviewed by
Bradley et al., 2001
).
One of the Scr-induced transcription factors that is crucial for
salivary gland formation is fork head (fkh). fkh,
which encodes a winged helix transcription factor, is expressed in the
salivary placodes beginning at embryonic stage 10 and continues to be
expressed in the salivary glands throughout embryonic and larval development
(Weigel et al., 1989b).
fkh is necessary for many aspects of salivary morphogenesis,
including the distinction between salivary gland and duct primordia,
invagination of the placodes and survival of salivary placode cells
(Kuo et al., 1996
;
Weigel et al., 1989a
). In
fkh mutant embryos, salivary placodes do not invaginate and undergo
apoptosis as the germband retracts (Myat
and Andrew, 2000
; Weigel et
al., 1989a
).
We examine the role of senseless (sens; Ly -
FlyBase) in salivary gland development. Like fkh, sens, which encodes
a Zn-finger transcription factor is expressed in the salivary glands. The
Zn-finger motifs in SENS show homology to the Zn finger domains of mammalian
GFI-1 protein and to the PAG-3 protein of C. elegans. SENS binds to
the GFI-1 consensus sequence and potentially acts as a transcriptional
repressor (Nolo et al.,
2000).
Previous work has illustrated the role for sens in neuronal
development. It is expressed in the sensory organ precursors in the embryonic
peripheral nervous system, as well as the wing and eye antennal imaginal
discs. sens has been shown to be necessary and sufficient for
neuronal fate specification. Embryos mutant for sens show loss of ES
and Ch neurons in the peripheral nervous system
(Nolo et al., 2000). In the
wing imaginal discs, loss of sens also results in loss of neuronal
fate. In addition, sens is important for specification of R8 cell
fate in the eye ommatidia by preventing rough from being expressed in the R8
precursors (Frankfort et al.,
2001
). Moreover, ectopic expression of sens in the
ectodermal imaginal cells can make these cells take on a neuronal fate
(Nolo et al., 2000
;
Nolo et al., 2001
). Thus,
sens appears to be primarily expressed in cells fated to adopt a
neuronal fate and is necessary for them to maintain their neuronal
identity.
However, embryonic salivary glands are an exception. Although the cells in
the salivary glands are not neuronal, they do express sens throughout
embryonic development (Nolo et al.,
2000) (this paper). Despite the expression of sens, the
cells of the salivary placodes maintain their ectodermal character and do not
adopt a neuronal fate. This led us to ask two questions: what are the genes
that activate sens expression in the salivary glands, and what role
does sens play in the morphogenesis of the embryonic salivary glands,
a non neuronal tissue?
Our data demonstrate that both the regulation and downstream effectors of sens show significant differences between the PNS and the salivary glands. Although DA:bHLH heterodimers stimulate sens transcription in both tissues, this complex is not needed to start the expression of sens during salivary development. Instead, fkh expression in the salivary placodes initiates sens expression. Then SAGE, a bHLH protein, acts as a novel DA partner to maintain sens expression. Furthermore, we find that sens functions as an anti-apoptotic protein in the salivary glands by preventing the expression of reaper and hid. By blocking these proapoptotic genes, sens allows survival of the salivary gland cells.
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Materials and methods |
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w1118 flies were used as wild-type controls for all the experiments.
Immunocytochemistry
Embryos were collected on molasses/agar plates and dechorionated using 50%
bleach. These embryos were then fixed in a 1:1:2 mixture of PBS, 10%
formaldehyde (Polysciences) and heptane (Sigma) for 30 minutes at room
temperature. Embryos were devitellenized using methanol (Sigma) and stored in
methanol at 4°C prior to immunostaining. Embryos were incubated overnight
at 4°C with one or a combination of the following antibodies: rat
anti-CREB (1:5000) (Andrew et al.,
1997), rabbit anti-FKH (1:3000), rabbit anti-ß-galactosidase
(1:1000, Vector Laboratories) and guinea pig anti-SENSELESS (1:1000)
(Nolo et al., 2000
). The
secondary antibodies used to detect these primary antibodies include
biotinylated goat anti-rabbit IgG (1:200, Vector Laboratories), biotinylated
goat anti-rat IgG (1:200, Vector Laboratories) and biotinylated goat
anti-guinea pig IgG (1:200, Vector Laboratories). These conjugates were then
detected using the Vectastain ABC kit (Vector Laboratories), followed by
incubation with 0.5 mg/ml diaminobenzidine and 0.06% hydrogen peroxide. The
embryos were then cleared with methyl salicylate and photographed using the
Nomarski optics on the Leica DMRB microscope.
For fluorescent staining, embryos were incubated with secondary antibodies conjugated to either Alexa 488 or Alexa 546 (1:500, Molecular Probes) after the primary antibody incubation. The embryos were then cleared in 50% glycerol, followed by 70% glycerol in PBS containing 2% n-propyl gallate (Sigma) and visualized using the Zeiss 510 confocal microscope.
In situ hybridization
Whole-mount in situ hybridization was performed as described by Tautz and
Pfeifle (Tautz and Pfeifle,
1989) with modifications
(Harland, 1991
) using
antisense digoxigenin-labeled probes. The signal was visualized using nitro
blue tetrazolium and BCIP as substrates for alkaline phosphatase. Following
the in situ hybridization, the embryos were immunostained for
ß-galactosidase as described above in the immunocytochemistry protocol.
The embryos were rinsed and cleared in 50% glycerol followed by 70% glycerol
and photographed using Nomarski optics on the Leica DMRB microscope.
TUNEL staining
After fixation, embryos were immunostained with rabbit anti-FKH and rabbit
anti-ß-Galactosidase, followed by a fluorescent detection using goat
anti-rabbit IgG-Alexa 488 (Molecular Probes, 1:500). The immunostained embryos
were then processed for TUNEL staining as described below. Embryos were
treated with 4 µg/ml of proteinase K for 5 minutes at room temperature and
then postfixed with 1:1 mixture of 10% formaldehyde and PBS. TUNEL staining
was performed using the Intergen Apoptag Kit. Briefly, the embryos were
incubated with equilibration buffer for 1 hour, followed by an overnight
incubation with terminal deoxynucleotidyl transferase (TdT) at 37°C. To
detect TUNEL staining, the embryos were incubated with rhodamine-conjugated
anti-digoxigenin antibody at 4°C overnight. The embryos were then cleared
with 50% glycerol, followed by 2% n-propyl gallate in 70% glycerol and imaged
using the Zeiss Confocal microscope.
RNA interference
The primers used to PCR amplify sage from genomic DNA were:
5'-ATGACGGATCAACTGCTGAGCTCCA-3',
5'-CGCTCCCCAATATCGTTGCCA-3'. The genomic sage fragment
(845 bp) was then cloned into pBluescript and used to make dsRNA for RNA
interference using the protocol described by Kennerdell and Carthew
(Kennerdell and Carthew,
1998). The dsRNA as well as injection buffer was injected into pre
cellularization w1118 embryos. The embryos were
aged overnight at 18°C. They were then fixed with 1:1:2 mixture of PBS,
10% formaldehyde and heptane for 30 minutes and manually devitellenized. The
devitellenized embryos were then immunostained as described above.
GST pulldown
The sage cDNA was obtained using an embryonic cDNA library as a
template for PCR. The primers were as shown above but with different
restriction sites (SalI and NcoI) flanking the primers to
facilitate cloning into pGEX. The PCR fragment was about 800 bp and was
ligated into the pGEM Teasy vector (Promega) and then inserted into a
SalI-NcoI cut pGEX-2TKN vector (modified version of pGEX
vector from Amersham Biosciences) and transformed in BL-21 cells. SAGE protein
is expected to be around 30 kDa and GST-SAGE was found to be around 60 kDa. We
also made a truncated version of SAGE that lacks the C-terminal bHLH domain
(SAGE ) that was around 50 kDa. The GST-conjugated proteins were bound
to GST-agarose beads overnight. The beads were rinsed and stored at
4°C.
The Promega WGA-in vitro transcription and translation kit was used to make
DA protein from pBS-da
(Cronmiller and Cummings,
1993). The in vitro translated DA protein, labeled using
35S-methionine, migrated in an SDS gel at about 80 kDa, which
agrees with the previously documented molecular weight of DA
(Cronmiller and Cummings,
1993
).
For the GST-pulldown assay, DA was incubated with beads coupled with
GST-alone, GST-SAGE or GST-SAGE overnight at 4°C. The beads were
washed and eluted with 20 mM glutathione. The eluates were electrophoresed on
a denaturing gel and visualized by autoradiography.
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Results |
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sens expression in the salivary glands is dependent on bHLH
proteins
Because the salivary glands are the only non neural tissue in the embryo to
express sens, we were curious to see how different the regulation of
sens transcription is in this tissue. In the PNS, DA forms
heterodimeric complexes with proneural bHLH proteins. These complexes are
necessary for both the initiation and maintenance of sens expression
in the sensory organ precursors (Nolo et
al., 2000). The proneural genes achaete, scute, lethal of
scute, asense and atonal are mainly expressed in the proneural
clusters and are absent from the salivary placodes
(Brand et al., 1993
;
Cabrera et al., 1987
;
Jarman et al., 1993
;
Romani et al., 1987
;
Vaessin et al., 1994
). By
contrast, da expression is ubiquitous in the early embryo and is
upregulated in the salivary glands of older embryos
(Cronmiller and Cummings,
1993
) (V.C. and S.K.B., unpublished), suggesting that da
might be involved in regulating the expression of sens in the
salivary placodes. If so, da mutants would have a salivary phenotype
similar to sens mutants. In confirmation of this hypothesis, salivary
glands in da mutants were smaller than in wild-type embryos
(Fig. 2A,B). In situ
hybridization showed that the levels of sens mRNA (and protein, data
not shown) were dramatically reduced in the salivary glands of da
mutants (Fig. 2E,F), suggesting
that DA regulates sens in the both the PNS and salivary gland.
However, unlike the PNS, salivary gland sens expression initiates in
the absence of da (Fig.
2C,D).
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In the sensory organ precursors of the PNS, sens is necessary to
maintain the expression of the proneural genes
(Nolo et al., 2000).
Similarly, we find that sage RNA is decreased in sens
mutants (Fig. 3B,F), suggesting
a positive feedback loop between sens and sage. However,
expression of da appears to be unaffected in sens mutants
(data not shown).
fkh is necessary for initiation of sens expression
in the salivary glands
Although da and sage are necessary for maintaining
sens expression, initiation of sens in the salivary placodes
did not depend on either of these genes. As sens expression in the
salivary placodes initiates at stage 11, later than primary Scr
target genes, we thought sens might be indirectly activated by
Scr through one of these primary targets. As expected, we found that
sens expression is absent in Scr mutant embryos.
sens expression was unchanged in embryos mutant for several
Scr-regulated early transcription factors such as huckebein
(Fig. 4C), trachealess
and eyegone (data not shown). However, fkh mutant embryos
show a complete absence of sens expression in the salivary placodes
and never express sens at the later stages. The expression of
sens in the PNS is unaffected in these mutants
(Fig. 4B). da and
sage RNAs were unchanged at stages 10 and 11 in fkh mutants,
indicating that the lack of sens is not due to the effects on
sage or da expression. There was a slight reduction in
sage RNA at stage 12 (data not shown), which may be due to the
positive feedback loop between sens and sage in the salivary
placodes. Thus, sens expression in the salivary placodes is initiated
by fkh and is maintained at high levels throughout embryogenesis by
da and sage.
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sens is necessary for the survival of cells in the embryonic
salivary glands
The apoptosis observed in sens mutants may be a result of other
problems with the salivary glands or may be due to the anti-apoptotic role of
sens in the salivary glands. These two possibilities can be
differentiated by examining the salivary glands in sens mutant
embryos where the cell death pathway has been blocked. If the cell death in
the sens mutant salivary glands is a secondary effect, blocking cell
death would not lead to rescue of sens phenotype, whereas if
sens functions in the cell death pathway, then the rescue of cell
death in sens mutants should result in normal morphogenesis of
salivary glands. One of the methods to rescue embryonic cell death is by
expressing an anti-apoptotic protein P35 in the embryo using the UASGAL4
system. P35 is a baculovirus protein that is homologous to the IAPs and has
been shown to rescue apoptotic cells in wide variety of organisms including
Drosophila embryos and other Drosophila tissues
(Hay et al., 1994;
Rabizadeh et al., 1993
;
Sugimoto et al., 1994
;
Xue and Horvitz, 1995
). When
we expressed P35 ubiquitously in sens mutant embryos using
arm-GAL4, the salivary glands were normal in size and went through
normal morphogenesis (Fig. 6C,
see Fig. 8). The rescued
salivary glands also showed normal expression of late salivary gland markers
such as slalom, indicating that they were functioning normally (data
not shown). However, most of the cells in these glands continued to show high
expression of reaper at stage 13 and later
(Fig. 6F,G). Based on the
reaper expression in these rescued glands, it appears that the cells
on the medial side of the salivary glands did not express reaper and
were probably the cells that survive to form the small salivary glands in
sens mutant embryos.
|
|
sens represses the transcription of reaper in the
salivary glands
Previous studies have demonstrated that the 11 kb region upstream from the
reaper transcription start site is responsive to apoptotic signals
(Brodsky et al., 2000). We used
flies carrying 11 kb of reaper promoter fused to ß-galactosidase
(Rpr-11-lacZ) to test whether sens represses the reporter
activity. In wild type embryos, Rpr-11-lacZ drives very low
expression in the dorsal posterior part of the salivary placode and some
expression in the invaginated portion of the salivary gland at stage 12
(Fig. 9A',A''). There is very low expression observed in later embryos
(Fig. 9C',C''). In
sens mutant embryos, the expression of the Rpr-11-LacZ
fragment in the salivary placodes is dramatically increased and expression
remains elevated throughout embryogenesis
(Fig. 9B',B'',
D',D''). These results indicate that sens
directly or indirectly represses the expression of reaper in the
normal salivary glands.
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Discussion |
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As sens is a fkh target and because both sens
and fkh embryos show extensive salivary apoptosis, we thought that
apoptosis in fkh mutants might be caused by lack of sens.
Because rescuing cell death in fkh mutants does not rescue normal
morphogenesis (Myat and Andrew,
2000), our model was that sens normally protects salivary
cells from cell death, and other fkh target genes direct the cell
movements and shape changes needed to form the salivary gland. However, the
apoptosis of the salivary placodes in fkh mutants could not be
rescued by ubiquitous expression of sens. There are two explanations
for this result. The first possibility is that we did not overexpress
sens at high enough levels to overcome cell death. However, we do not
believe that to be the case because we used the same
arm-GAL4:UAS-sens combination to rescue the sens
phenotype. Furthermore, arm-Gal4:UAS-P35 rescues cell death in
sens mutants. Thus, we favor the second possibility, that loss of
fkh leads to multiple proapoptotic changes, only one of which is the
failure to activate sens.
da and sage maintain sens expression in the
salivary glands
Although FKH can initiate expression of sens in the salivary
placodes, we show that both DA and SAGE are required for high level
sens expression at later stages. DA is also known to control the
expression of sens in the PNS. There, it partners with the proteins
of the ACHAETE-SCUTE Complex or with ATONAL to regulate sens
expression (Nolo et al.,
2000). For sens regulation in the salivary primordium, we
have identified a new DA partner, SAGE, which belongs to the bHLH proteins of
the Mesp family (Moore et al.,
2000
; Peyrefitte et al., 2000). Our results are the first to
demonstrate the ability of Mesp family members to heterodimerize with DA. We
have shown using RNAi that absence of sage leads to a decrease in the
size of the glands and a reduction in levels of SENS. In turn, SENS appears to
positively regulate the levels of sage mRNA in the salivary glands.
The existence of this positive feedback loop leads to the question of which
protein, SAGE or SENS, is the true antagonist of apoptosis in the salivary
glands. The presence of sage mRNA in sens mutants sheds some
light on this issue. In sens mutants, high levels of
Rpr-11-lacZ are induced at stage 12, in the salivary placodes. At
this stage, sens mutant embryos still express sage and
da mRNA in the placodes at normal levels
(Fig. 3E). Reduction in
sage mRNA is not observed until stages 13-14, by which time the
salivary glands of sens mutants are already reduced in size
(Fig. 3F). These results
indicate that sens, not sage, is necessary to maintain the
survival of the salivary gland cells.
A summary of these regulatory interactions and a comparison with the
regulation in the peripheral nervous system is provided in
Fig. 10. A similar circuit
controls the regulation of expression of Gfi1, the vertebrate ortholog of
sens, in the inner ear cells of mice. The bHLH protein Math1 (Atoh1 -
Mouse Genome Informatics), a homolog of atonal, is necessary to
maintain Gfi1 mRNA, but not for its initiation in the inner ear cells. It
would be interesting to examine if fkh family members are involved in
this case to initiate the Gfi1 expression. However, the feedback regulation of
sens onto sage or proneural genes (this paper)
(Nolo et al., 2000) is not
observed between Gfi1 and Math1 (Wallis et
al., 2003
).
|
The anti-apoptotic role of sens, though tissue specific, appears
to be conserved through evolution. Previous studies have shown that Gfi1, the
vertebrate ortholog of sens can prevent apoptosis by repressing Bax
(Grimes et al., 1996b).
Furthermore, Gfi1 knockout mice show increased apoptosis in the inner
ear neurons (Wallis et al.,
2003
). The C. elegans homolog, PAG-3 mutants
also shows increased apoptosis but it is not clear if the apoptosis is a
consequence of improper cell fate specification
(Cameron et al., 2002
).
Gfi1 has been shown to be a transcriptional repressor
(Zweidler-Mckay et al., 1996).
SENS lacks the SNAG repressor domain that is present in Gfi1
(Grimes et al., 1996a
;
Nolo et al., 2000
). Therefore,
sens could be either a repressor or an activator in
Drosophila. Previous studies have shown that sens represses
the expression of rough in the eye imaginal discs and of
E(spl) in the PNS (Frankfort et
al., 2001
; Nolo et al.,
2000
; Nolo et al.,
2001
). We have shown that sens can repress
Rpr-11-lacZ in normal salivary glands, perhaps acting directly as a
repressor or indirectly by inducing the expression of another repressor. By
contrast, sens is necessary for maintaining the expression of
proneural genes in the PNS and sage in the salivary primordium. There
are potential SENS-binding sites upstream or downstream of hid, sage
and the proneural genes, as well as in the 11 kb promoter of reaper,
suggesting that sens could be directly regulating all these genes.
Further studies will be needed to understand whether sens acts as
both a transcriptional repressor or activator and whether it requires specific
co-factors for these distinct functions.
Why is there a need for sens in the embryonic salivary
glands?
The need for sens in the developing salivary gland specifically to
prevent cell death raises the question of why these cells need special
protection. We suggest two possibilities, one related to the initial
specification of the salivary placodes and the other related to cell cycle and
cytoskeletal rearrangements required for proper morphogenesis.
It is possible that the salivary gland cells are at risk of death because
of similarities between these cells and cells in other segments that are fated
to die. The involvement of homeotic genes might provide a common theme between
the salivary placode cells and other apoptotic cells. Deformed
induces apoptosis at the boundary of the mandibular and maxillary lobes by
activating reaper (Lohmann et
al., 2002). Similarly Abdominal B (Abd-B) can
activate reaper at the boundaries of abdominal segments A6/A7 and
A7/A8 (Lohmann et al., 2002
).
It is therefore possible that Scr, which is needed to specify the
salivary primordium, can bind and activate reaper in the labial
segment. In support of this hypothesis, low levels of reaper are
expressed in the dorsal posterior part of the salivary placode. Removal of the
sens repression would then reveal the presence of a strong activator
of reaper transcription. Interestingly, though Deformed and
Abd-B are expressed throughout their respective segments, apoptosis
is limited to the boundaries, indicating the presence of activators at the
boundaries or repressors in the rest of the segment. In parasegment 2,
sens might be an analogous repressor, antagonizing Scr
induction of reaper and hid in the salivary primordium.
Alternatively, this predisposition to apoptosis might be due to intrinsic
aspects of salivary gland morphogenesis that occur after the cells are
specified. The salivary placode is unique in that its cells exit the mitotic
cell cycle early, at cycle 15 rather than at cycle 16 as the rest of the
epidermis does. Shortly thereafter, as the cells are invaginating into the
embryo, they are the first cells to enter the endoreplication cycle.
Furthermore, they are the only cells that appear to enter endoreplication from
G2 instead of G1 (Smith and Orr-Weaver,
1991). We imagine that these unusual changes in cell cycle or the
simultaneous occurrence of cell cycle changes and cytoskeletal rearrangements
in the invaginating salivary placodes might sensitize checkpoints that have
the potential to cause apoptosis. Consistent with this idea, the small piece
of the reaper promoter that contains the p53 response element is
active in wild type salivary glands
(Brodsky et al., 2000
),
suggesting that p53 may be induced in the salivary primordium and push these
cells to the brink of cell death. If so, sens would be necessary to
counter p53 and prevent strong induction of reaper throughout the
salivary placodes.
In either scenario, the induction of reaper and hid would result in apoptosis of the salivary primordium. Therefore, the presence of sens to repress proapoptotic genes is crucial for the survival of the salivary glands during embryogenesis.
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ACKNOWLEDGMENTS |
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