1 Division of Neuroanatomy (D12), Department of Neuroscience, Osaka University
Graduate School of Medicine, Suita, Osaka 565-0871, Japan
2 Department of Developmental Genetics, National Institute of Genetics, Mishima,
Shizuoka 411-8540, Japan
3 Department of Genetics, The Graduate University for Advanced Studies, Mishima,
Shizuoka 411-8540, Japan
4 Department of Molecular Neurobiology, Institute of Basic Medical Sciences,
University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan
5 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL
35294, USA
6 Department of Physiology, Keio University School of Medicine, 35 Shinanomachi,
Shinjuku, Tokyo 160-8582, Japan
7 Core Research for Evolutional Science and Technology (CREST), Japan Science
and Technology Corporation, Kawaguchi, 332-0012 Japan
* Author for correspondence (e-mail: hidokano{at}sc.itc.keio.ac.jp)
Accepted 10 February 2003
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SUMMARY |
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Key words: Drosophila, Glia, Neuron, repo, tramtrack, pointed, gcm
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INTRODUCTION |
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Glial cells in the Drosophila embryonic nervous system can be
classified into several groups based on their location, morphology and
expression of several marker genes (Ito et
al., 1995). Glial cells in the central nervous system (CNS) are
generated from the mesectoderm and the ventral neuroectoderm. Glial cells of
mesectodermal origin are called midline glia, which unsheathe commissural axon
bundles (Klämbt et al.,
1991
). Other CNS glia arise from glial precursors and neuroglial
precursors in the ventral neuroectoderm, and consist of diverse glial types
such as surface-associated glia located close to the CNS surface,
cortex-associated glia that lie among the neuronal cell bodies in the cortex
and neuropile-associated glia that associate with the axonal structures. Glial
cells in the peripheral nervous system (PNS) are derived from either lateral
neuroblasts in the ventral neuroectoderm or the dorsal epidermal anlage, and
unsheathe afferent and efferent axon bundles.
In Drosophila, all glial cells except the midline glia require the
function of the glial cells missing (gcm) gene for their
cell-fate determination (Hosoya et al.,
1995; Jones et al.,
1995
; Vincent et al.,
1996
). GCM acts as a binary switch between the neuronal and glial
fates; it promotes glial development, while inhibiting neuronal
differentiation (Hosoya et al.,
1995
; Jones et al.,
1995
). GCM induces the expression of three transcription factors,
Reversed Polarity (REPO), Tramtrack p69 (TTK69; an isoform of the ttk
gene product) and PointedP1 (PNTP1; an isoform of the pointed gene
product), in glial cells. Because the loss of function of any of these three
factors is not as severe as the loss of GCM function, each of these factors is
considered to be responsible for only a part of the GCM function. Giesen et
al. (Giesen et al., 1997
)
proposed that glial cell differentiation is achieved by two parallel
processes: the promotion of glial gene expression by PNTP1 and the suppression
of neural properties by TTK69. PNTP1 is an ETS transcription factor that can
activate transcription through ETS binding sites
(O'Neill et al., 1994
;
Albagli et al., 1996
;
Granderath et al., 2000
). It
is expressed in a subset of glial cells, such as longitudinal glia and VUM
glia (Klaes et al., 1994
), as
well as in the ventral ectoderm and tracheal cells
(Mayer and Nüsslein-Volhard,
1988
; Gabay et al.,
1996
). TTK69 is a BTB/POZ domain/zinc-finger type transcription
factor that has been implicated in transcriptional repression during embryonic
segmentation and eye development (Read et
al., 1992
; Brown and Wu,
1993
; Xiong and Montell,
1993
; Li et al.,
1997
). Its expression can be best characterized as non-neuronal;
TTK69 is expressed in all cells except in the neuronal lineage, like the
support cells of the sensory organ and the epidermis
(Harrison and Travers, 1990
;
Brown et al., 1991
;
Read and Manley, 1992
).
Although differential functions for PNTP1 and TTK69 is an attractive idea,
these two factors alone cannot account for the GCM-dependent development of
glial cells, because the PNTP1 and TTK69 double-mutant phenotype is different
from the gcm mutant phenotype
(Giesen et al., 1997). In
addition, the pleiotropy of PNTP1 and TTK69 makes them unlikely candidates for
the glial determinant. Therefore, to characterize the molecular cascades
required for the differentiation of glial cells, we chose to analyze the
repo gene, which is expressed only in GCM-positive glia
(Campbell et al., 1994
;
Xiong et al., 1994
;
Halter et al., 1995
). The
region upstream of the repo transcription start site contains eleven
GCM-binding consensus sequences, and GCM is necessary and sufficient to induce
repo expression in vivo (Hosoya
et al., 1995
; Jones et al.,
1995
; Akiyama et al.,
1996
). Thus, repo is likely a direct target of GCM. REPO
is a paired-like homeodomain protein that specifically binds the ATT sequence
in the CAATTA motif (Halter et al.,
1995
). Although the expression of several glial marker genes is
known to depend on repo activity, how REPO functions as a
transcription factor in glial differentiation is as yet unknown.
We show that REPO can act as a transcriptional activator through the CAATTA motif in glial cells, and we define three genes whose expression depends on REPO function. In different types of glial cells, REPO can act alone or cooperate with either TTK69 or PNTP1 to regulate different target genes. Surprisingly, REPO also suppresses neuronal development. We propose that REPO has a cardinal function in glial identity.
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MATERIALS AND METHODS |
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S2 cells cultured in a 60 mm diameter dish were transfected with 500 ng of luciferase reporter, 300 ng of effector vector and 50 ng of Renilla reference reporter (pACT-Rluc) using Lipofectin (GIBCO BRL) or Effectene (QIAGEN). We used the empty vector (pACT) to adjust the total amount of the effector plasmid to 300 ng, except in the AES-luc reporter assay, where the total was 600 ng. Luciferase activity 48 hours after transfection was normalized to values obtained with the Renilla reference reporter. Transfections were carried out at least three times.
Fly stocks
The following mutant alleles were used. repo4e19
(Xiong et al., 1994),
rk264 (repo64)
(Campbell et al., 1994
),
pntD88
(Klämbt, 1993
).
ttk1e11 mutation causes a specific loss of the TTK69
isoform (Xiong and Montell,
1993
; Lai and Li,
1999
) and ttkB330 is a strong hypomorph
(Salzberg et al., 1994
;
Giesen et al., 1997
).
Homozygous mutant embryos were identified using the TM3 [Ubx-lacZ]
balancer or by examining their phenotypes. The enhancer-trap strains
pntrM254 (Klämbt,
1993
), ttk0219
(Lai et al., 1996
) and
locorC56 (Granderath
et al., 1999
) were used to detect the expression of pointed,
ttk and loco, respectively. The Ftz HDS reporter (2X21F)
(Nelson and Laughon, 1993
) and
the M84 strain (Klämbt and Goodman,
1991
) were used as glial markers. Ectopic expression was achieved
using GAL4 enhancer-trap insertions into scabrous
(Kramer et al., 1995
) and
engrailed (a gift from Andrea Brand) loci. The UAS-repo
construct was made by cloning the full-length repo cDNA into the
pUAST vector (Brand and Perrimon,
1993
). The UAS-gcm, UAS-pntp1 and
UAS-ttk69 strains have been described
(Hosoya et al., 1995
;
Klaes et al., 1994
;
Giesen et al., 1997
).
Immunohistochemistry
Anti-REPO antiserum was obtained after immunizing rats with bacterially
expressed REPO (amino acids 25-156)-GST fusion protein. Immunohistochemistry
was performed as described by Patel
(Patel, 1994) with minor
modifications. The following primary antibodies were used: mouse anti-BP102
[obtained from Developmental Studies Hybridoma Bank (DSHB)] at 1:4, mouse
anti-ELAV at 1:4 (obtained from DSHB), anti-REPO rat antiserum at 1:1000, rat
anti-TTK69 at 1:200 (gift from A. Travers) and rabbit
anti-ß-Galactosidase polyclonal antibody (Cappel) at 1:1000. The
following secondary antibodies were used: HRP-conjugated goat anti-mouse,
anti-rat, or anti-rabbit IgG (Jackson ImmunoResearch) at 1:1000, and
FITC-conjugated donkey anti-rat, Cy3-conjugated donkey anti-rabbit (Jackson
ImmunoResearch) at 1:800. Immunofluorescence was visualized with a FLUOVIEW
laser-scanning microscope (Olympus). Embryos were staged according to
Campos-Ortega and Hartenstein
(Campos-Ortega and Hartenstein,
1997
).
In situ hybridization
Whole-mount in situ hybridization was conducted using digoxigenin-labeled
RNA probes, essentially as described in Lehmann and Tautz
(Lehmann and Tautz, 1994).
Full-length loco-cl cDNA was obtained by RT-PCR amplification and was
used as a template.
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RESULTS |
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We constructed a luciferase reporter gene with two REPO-binding sites
(CAATTA-luc) and tested its transcriptional activity in the
Drosophila S2 cell line. Co-transfection with a REPO-expressing
plasmid (pACT-repo) caused a sevenfold increase in luciferase activity
compared with co-transfection with the vector alone (pACT)
(Fig. 1A). REPO lacking most of
its homeodomain, but retaining a putative nuclear localization signal located
from amino acid 1 to 4 of the homeobox (pACT-repobox), was unable to
activate transcription of the reporter gene
(Fig. 1A), despite being
localized to the nucleus (data not shown). Furthermore, transcriptional
activation by REPO was dependent on the presence of the CAATTA motif in the
reporter gene; a single base substitution in this motif resulted in a complete
loss of REPO-dependent transcription (Fig.
1A). We conclude that REPO is a transcriptional activator that can
act through the CAATTA motif.
|
REPO functions as a transcriptional activator in glial cells
To test whether REPO activates transcription through CAATTA sites in vivo,
we examined the expression pattern of the ftz HDS lacZ
reporter gene in the repo mutant background. This reporter gene
(2x21F) carries two copies of a 21 mer containing the CAATTA motif, and is
expressed in all glial cells in the PNS and a subset of CNS glia
(Nelson and Laughon, 1993)
(Fig. 2A,D). The glial
expression of the ftz HDS reporter gene in the PNS overlaps precisely
with REPO-expressing cells. The loss of repo function abolished
lacZ expression in both the PNS and CNS glia
(Fig. 2B,E), even though glial
cells are still present in stage 16 repo mutant embryos
(Halter et al., 1995
).
Expression of the ftz HDS reporter gene in the antenno-maxillary
complex and posterior spiracles, where REPO is not expressed, was unaffected
in repo mutants (data not shown). These results indicate that REPO
acts through the CAATTA site to drive transcription in glial cells.
|
Although ectopic REPO induced the appearance of many non-glial
lacZ-expressing cells in the dorsal epidermis, cells within the CNS
did not respond to ectopic REPO. In fact, even in the wild-type background,
not all REPO-positive glia in the CNS expressed the ftz HDS reporter.
This suggests that the mechanism by which REPO regulates transcription may be
different in the CNS from the one for peripheral glia. One possible scenario
is that the functions of REPO in the CNS requires cooperation with one or more
other factors, and that these interactions preclude REPO from acting through
the CAATTA motif. TTK69 and PNTP1 are good candidates for such co-factors,
because ttk and pointed are both required for the
development of CNS glial cells (Klaes et
al., 1994; Giesen et al.,
1997
). Although repo, ttk and pointed are
expressed in overlapping subsets of CNS glial cells, their expression is
mutually independent; REPO continued to be expressed in the ttk or
pointed mutant background, and lacZ expression levels in
enhancer-trap lines of ttk or pointed were unaffected in
repo mutant embryos (Fig.
3). Moreover, ectopic expression of REPO in the entire
neuroectoderm did not increase the expression of pointed P1 mRNA or
TTK69, nor did ectopic expression of either TTK69 or PNTP1 affect REPO
expression (data not shown). All three genes are most probably regulated
independently, downstream of the glial determinant GCM
(Hosoya et al., 1995
;
Jones et al., 1995
;
Giesen et al., 1997
).
|
|
Expression of loco-c1 depends on both REPO and PNTP1
Although results presented above suggest a synergistic action of REPO and
TTK69 in transcriptional activation, we cannot determine whether REPO and
TTK69 act cooperatively on the same target gene, because the gene responsible
for M84 is not known. We thus studied the regulation of the gene
loco, which encodes a member of the family of Regulator of G-protein
Signaling (Granderath et al.,
1999; Granderath et al.,
2000
). The loco function is required for glial
morphogenesis, and loco-c1, an isoform of loco, is expressed
specifically in REPO-positive glial cells, which also express PNTP1
(Granderath et al., 1999
;
Granderath et al., 2000
). The
expression of a loco reporter gene carrying a glial enhancer element
of loco (Rrk; Fig. 5D)
requires PNTP1 function, as well as an Ets-binding site located within this
glial enhancer element (Granderath et al.,
2000
). Although this loco-reporter gene is expressed
normally in repo mutants
(Granderath et al., 2000
), we
found that in stage 16 repo mutant embryos loco-c1 mRNA was
reduced to undetectable levels (Fig.
5B,C), whereas such embryos exhibit robust expression of a
pnt-lacZ reporter gene in glial cells
(Fig. 3E). It is thus likely
that proper expression of loco depends on both pointed and
repo function.
|
As loco expression requires both repo and
pointed, we studied the effects of co-expressing REPO and PNTP1. The
loco genomic fragment in AEE-luc reporter was extended 0.7
kb in the 5' direction, to include the Ets-binding site through which
PNTP1 acts (AES-luc, Fig.
5D). In S2 cells transfected with a plasmid that directs REPO
expression, a fivefold increase in this reporter gene expression was achieved,
compared with transfection with the vector alone
(Fig. 5F). Although the
expression of PNTP1 had little effect on the reporter gene, co-expression of
PNTP1 with REPO resulted in a significantly higher level of reporter
expression than the expression of REPO alone
(Fig. 5F). Taken together with
the previous work (Granderath et al.,
2000), these results show that both PNTP1 and REPO acts through
their respective binding sites in the loco promoter to activate
transcription.
Synergistic effect of REPO and PNTP1 was also observed in vivo. While ectopic expression of either PNTP1 or REPO in the neuroectoderm caused only a minor increase in the number of cells that expressed a loco enhancer-trap strain (rC56), co-expression of REPO and PNTP1 in the neuroectoderm caused a dramatic increase in the response (Fig. 5I-K). When misexpression was directed using the engrailed-GAL4 driver, co-expression of REPO and PNTP1 caused a fivefold increase in the number of rC56-expressing cells compared with the expression of REPO or PNTP1 alone (Fig. 5L). Such a synergistic effect was not observed between REPO and TTK69, or between PNTP1 and TTK69 (data not shown). We conclude that the glial expression of loco is regulated by the cooperation of REPO and PNTP1.
REPO also suppresses neuronal characteristics
In addition to promoting glial differentiation, the glial determinant GCM
also inhibits neuronal differentiation
(Hosoya et al., 1995;
Jones et al., 1995
). This
function is probably mediated by glial transcription factors that operate
downstream of GCM. Because the results presented above established that REPO
cooperates with TTK69 and PNTP1 to direct the expression of glial-specific
genes in the CNS, we tested whether these proteins also function to inhibit
neuronal differentiation.
Ectopic expression of GCM throughout the neuroectoderm has a profound
effect on neuronal differentiation; the number of cells that express the
neuron-specific marker ELAV is reduced to 5-15% of that in normal embryos
(Hosoya et al., 1995). In
stage 13 embryos, ectopic expression of REPO or TTK69 alone caused,
respectively, little or a modest reduction in the number of ELAV-positive
cells (Fig. 6A,C,D). When these
two proteins were co-expressed, however, neuronal differentiation was severely
blocked, surpassing the inhibition achieved by the ectopic expression of GCM
(Fig. 6B,E). Thus, REPO
cooperates with TTK69 not only to promote glial development, but also to
inhibit neuronal differentiation.
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DISCUSSION |
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Despite the glia-specific expression of the ftz HDS reporter gene,
the CAATTA motif is not a specific target site of REPO. FTZ and EN bind the
CAATTA motif (Desplan et al.,
1988; Schier and Gehring,
1992
), which also resembles the consensus binding sequence for
ANTP and UBX (Müller et al.,
1988
; Ekker et al.,
1992
). Why do other homeodomain proteins fail to drive the
ftz HDS reporter gene in vivo? Recent results show that homeodomain
proteins require co-factors to activate the transcription of their target
genes. Co-factors, such as EXD and FTZF1, are also DNA-binding proteins that
require specific binding sites in the target gene
(Chan et al., 1994
;
van Dijk and Murre, 1994
;
Guichet et al., 1997
;
Yu et al., 1997
). Homeodomain
proteins other than REPO may be incapable of activating the ftz HDS
reporter gene because it does not have binding sites for their co-factors.
The behavior of the ftz HDS reporter gene suggests that the
requirement for co-factors may also apply to REPO. Although the ectopic
expression of REPO induced the ectopic expression of the ftz HDS
reporter gene in the periphery, it did not affect the expression pattern in
the CNS. Thus, REPO cannot be the single factor responsible for the activation
of the ftz HDS reporter gene. Indeed, the expression pattern of the
ftz HDS reporter gene is altered by changing nucleotides outside the
CAATTA motif (Nelson and Laughon,
1993), indicating that ftz HDS contains binding sites for
factors other than REPO. The simplest interpretation is that such factors are
present in the periphery, but not in the CNS.
Using additional target genes of REPO, we provided further evidence that
the transcriptional regulation by REPO involves co-factors. Although the
enhancer-trap line M84 and the loco gene were both dependent on REPO
function, and could be expressed precociously and ectopically upon
mis-expression of REPO, much stronger responses were obtained when REPO was
co-expressed with TTK69 or PNTP. Endogenous expression of M84 and the
loco gene occurs in cells that co-express REPO and TTK69 or PNTP1,
respectively. TTK69 and PNTP1 are thus good candidates for REPO co-factors.
Together with an earlier study (Granderath
et al., 2000) our results show that REPO and PNTP1 cooperate on
loco expression through their binding sites in the loco
promoter. Likewise the synergism between REPO and TTK69 may also occur on the
promoter of their target genes.
Our conclusion that the expression of M84 and loco are achieved by
a cooperation of REPO and TTK69/PNTP1 does not rule out the possibility that
these genes are also direct targets of GCM. In fact, reporter genes driven by
loco enhancer elements are expressed normally in stage 14
repo mutant embryos, indicating that other factor(s) activate their
transcription at the onset of gliogenesis
(Campbell et al., 1994;
Granderath et al., 2000
).
Because the loco enhancer element contains GCM-binding sites, GCM can
directly regulate loco
(Granderath et al., 2000
).
However, as the expression of GCM in glia is transient, transcription
initiated by GCM must be sustained by other factors. REPO and PNTP1 are the
best candidates for factors that maintain loco expression throughout
glial development and functioning.
The synergistic effect of REPO and TTK69 on M84 marker expression suggests
a positive role of TTK69 on glial differentiation. As the major function of
TTK69 has been thought to be the inhibition of neuronal differentiation
through transcriptional repression (Brown
et al., 1991; Read et al.,
1992
; Xiong and Montell,
1993
; Giesen et al.,
1997
), the positive action of TTK69 on glial gene expression could
be an indirect effect through repressing transcription of a repressor for M84
expression. However, TTK69 can activate transcription in yeast cells
(Yu et al., 1999
), suggesting
that TTK69 may also promote transcription, depending on the cellular context.
Recent studies also implicate a role of TTK69 in cell proliferation, through
controlling the expression of regulators of the cell cycle
(Badenhorst, 2001
;
Baonza et al., 2002
).
Badenhorst (Badenhorst, 2001
)
has shown that overexpression of TTK69 results in the inhibition of glial
development, accompanied by the repression of the S-phase cyclin and glial
proliferation. As we observe an increase in the number of cells that express
M84 glial marker upon co-expression of TTK69 and REPO, our result cannot be
accounted for by the ability of TTK69 to inhibit glial cell cycle. Whereas
ectopic expression of TTK69 reduces the expression of the endogenous
repo gene (Badenhorst,
2001
), our misexpression paradigm provides exogenous REPO through
the GAL4/UAS control. Thus the existence of REPO might modify the activity of
TTK69, so that it plays a positive role on glial development.
Repression of neuronal differentiation during glial development
Glial fate determination involves not only the promotion of glial
differentiation but also the suppression of neuronal properties. Because
ectopic GCM can induce neurogenesis in certain contexts
(Akiyama-Oda et al., 1998;
Van de Bor et al., 2002
), it
is unlikely that GCM directly represses neuronal differentiation. TTK69 has
been proposed to inhibit neuronal differentiation, mainly because of its
loss-of-function phenotype in the sensory organ
(Giesen et al., 1997
). Here,
we have shown that the co-expression of REPO and TTK69 has a potent
neuron-suppressing activity, and further demonstrated that the repo
mutant permits neuronal differentiation even when GCM is overexpressed. This
strongly suggests that REPO functions not only to activate the transcription
of glial genes, but also to prevent the neuronal differentiation of
presumptive glial cells (Fig.
8).
If glia and neuron represent two mutually exclusive cell states that must
be chosen between early in development, it is somewhat strange that
suppression of neuronal development should be carried out by proteins that are
expressed throughout glial differentiation. The existence of continuous
suppression of neuronal properties in glia suggests that cells within the
nervous system may retain the potential to become neurons or glia throughout
their cellular history. This idea is supported by the observation that GCM is
able to transform post-mitotic neurons into glia
(Jones et al., 1995).
Conversely, in the vertebrate nervous system, glial cells (astrocytes and
oligodendrocyte-precursor) can respond to environmental signals and function
as neural stem cells, generating neurons
(Doetsch et al., 1999
;
Kondo and Raff, 2000
). The
role of REPO and TTK69 may be to suppress the ability of glia to respond to
cues that would cause them to change into neurons or neural precursors.
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ACKNOWLEDGMENTS |
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