Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA
Author for correspondence (e-mail:
steth{at}ifm.liu.se)
Accepted 15 September 2004
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SUMMARY |
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Key words: Drosophila, dachshund, eyes absent, BMP signaling, Combinatorial code, FMRFamide, FMRFa
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Introduction |
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One particularly well-documented example of a network of regulatory genes
controlling organ development is that controlling eye formation in
Drosophila. Genetic analysis of Drosophila eye formation has
identified a conserved core group of transcriptional regulators collectively
known as the retinal determination network (RDN). This network comprises a
hierarchical genetic cascade, wherein twin of eyeless (toy)
activates eyeless (ey)
(Czerny et al., 1999),
ey in turn activates both eyes absent (eya) and
sine oculis (so) (Halder
et al., 1998
; Niimi et al.,
1999
), and eya and so activate dacshund
(dac) expression (Chen et al.,
1997
; Pignoni et al.,
1997
). Extensive reciprocal positive feedback loops between these
genes ensure robust gene expression and potency of the entire network
(Chen et al., 1997
;
Czerny et al., 1999
;
Halder et al., 1995
;
Pignoni et al., 1997
;
Shen and Mardon, 1997
). A
complex of Eya, So and Dac is generally believed to be central to RDN
function, and their coexpression and functional synergism are conserved in
numerous vertebrate tissues (Chen et al.,
1997
; Heanue et al.,
1999
; Ikeda et al.,
2002
; Li et al.,
2003
; Pignoni et al.,
1997
; Xu et al.,
1999
). So and the homologous vertebrate Six family are
transcription factors characterized by a homeodomain and the conserved Six
domain (Kawakami et al.,
2000
). Eya and the vertebrate Eya family are nuclear co-factors
with no known DNA-binding motifs (Bui et
al., 2000
; Ikeda et al.,
2002
; Ohto et al.,
1999
; Silver et al.,
2003
). Recent studies revealed that Eya proteins have an intrinsic
phosphatase activity critical for both their transcriptional activity and
in-vivo function (Li et al.,
2003
; Rayapureddi et al.,
2003
; Tootle et al.,
2003
). Dac and vertebrate Dach1-2 have two conserved Dachshund
domains, one of which may mediate DNA binding directly
(Ikeda et al., 2002
). Binding
studies have shown direct physical interaction between invertebrate and
vertebrate Eya and Six family members
(Heanue et al., 1999
;
Li et al., 2003
;
Pignoni et al., 1997
;
Silver et al., 2003
). The
functional relevance of this interaction has been well demonstrated by mutant
analysis (Li et al., 2003
;
Pignoni et al., 1997
) and by
their strong phenotypic and transcriptional synergy
(Bui et al., 2000
;
Heanue et al., 1999
;
Ikeda et al., 2002
;
Li et al., 2003
;
Pignoni et al., 1997
;
Silver et al., 2003
). Direct
physical interaction between Dac/Dach and Eya has been observed in several
(Chen et al., 1997
;
Heanue et al., 1999
;
Li et al., 2003
), but not all
(Ikeda et al., 2002
;
Silver et al., 2003
),
studies.
In spite of these elaborate hierarchical and reciprocal relationships
between RDN genes in the eye, evidence suggests that their specific function
in photoreceptor neurons may not be identical: eya mutant clones
appear to have a more dramatic effect on the differentiation of photoreceptor
cells than do dac mutant clones
(Mardon et al., 1994;
Pignoni et al., 1997
).
Furthermore, RDN genes have remarkably divergent expression patterns elsewhere
in the Drosophila embryo (Bonini
et al., 1998
; Kammermeier et
al., 2001
; Kumar and Moses,
2001
; Mardon et al.,
1994
). For example, toy, ey and dac are
coexpressed in the developing mushroom bodies of the Drosophila
central nervous system, but eya and so are absent
(Kurusu et al., 2000
;
Martini et al., 2000
;
Noveen et al., 2000
). In
addition, there appears to be no regulatory relationship between toy,
ey or dac in the mushroom bodies
(Kurusu et al., 2000
). Given
the partially overlapping expression patterns of RDN genes in the vertebrate
central nervous system (Caubit et al.,
1999
; Davis et al.,
1999
; Xu et al.,
1997
) it will be important to determine the roles that these genes
play, independently and possibly combinatorially, in neuronal development.
In the Drosophila ventral nerve cord (VNC), a small subset of
neurons expresses the LIM homeodomain gene apterous (ap)
(Lundgren et al., 1995). These
neurons can be subdivided, based upon differential neuropeptide expression and
axon pathfinding (Fig. 1).
ap itself is an important regulator of these diverse properties
(Benveniste et al., 1998
;
Lundgren et al., 1995
) and
thus must be acting combinatorially with other regulators. We previously found
that ap acts with the squeeze (sqz) zinc finger
gene and the BMP pathway to activate expression of the neuropeptide gene
FMRFamide-related (Fmrf) in one subset of ap
neurons, the Tv neurons (Allan et al.,
2003
). Reconstitution of this combinatorial code in other
peptidergic neurons triggered ectopic Fmrf expression in a subset of them.
However, because only a fraction of peptidergic neurons are `responsive',
additional factors probably contribute to Fmrf expression. Here, we find that
dac and eya are expressed in ap neurons and play
critical roles in Fmrf regulation and ap-axon pathfinding. In
dac and eya mutants, ap neurons are generated in
normal positions and numbers, thus allowing us to address the specific role
that each gene plays during neuronal differentiation with single cell
resolution. In the VNC, Dac expression is restricted to a subset of
interneurons and peptidergic neurons, with no expression observed in
motoneurons or glia. Eya shows an early phase of expression in subsets of VNC
cells, but rapidly becomes restricted to a subset of ap neurons.
Expression and mutant analyses show that both Dac and Eya are present in the
Fmrf-expressing Tv cells and that both are essential for proper Fmrf
expression. However, mutant and misexpression analyses indicate that Dac and
Eya have very different functions within ap neurons. dac has
a weak effect on Fmrf expression but, when misexpressed together with
ap, it potently triggers ectopic Fmrf expression in many peptidergic
neurons and motoneurons. This ectopic Fmrf expression is dependent upon BMP
signaling, indicating that dac acts as a potent member of an
ap/sqz/BMP/dac combinatorial code that activates
Fmrf expression in postmitotic neurons. By contrast to the weak effect of
dac mutation, Fmrf expression is almost entirely lost in eya
mutants. However, eya does not act combinatorially with ap
and BMP signaling to trigger ectopic Fmrf expression. Instead, eya
appears to play a dual role in Tv neurons, controlling both axon pathfinding
and BMP signaling. Thus, our data show that despite being coexpressed in a
single identified neuron, dac and eya perform entirely
different functions with a common phenotypic outcome: the activation of Fmrf
expression.
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Materials and methods |
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Immunohistochemistry
Antibodies used were: -c-Myc mAb 9E10 (1:30), concentrated
-ß-gal mAb 40-1a (1:20),
-Dac mAb dac2-3 (1:25),
-Eya mAb 10H6 (1:250) (all from Developmental Studies Hybridoma Bank);
rabbit
-proFmrf (1:2000) (Chin et
al., 1990
), rabbit
-ß-gal (1:5000, ICN-Cappel), rabbit
-pMad (1:2000) (Tanimoto et al.,
2000
), rabbit
-Glutactin (1:300)
(Olson et al., 1990
), rabbit
-GFP (1:500, Molecular Probes). Immunolabeling was carried out as
previously described (Allan et al.,
2003
).
Analysis of enhancer trap lines
Expression analysis of the 577 second-chromosome lethal lines identified by
the BDGP project (Spradling et al.,
1999) was carried out using X-gal and anti-ß-gal staining. Of
these lines, several showed restricted patterns of expression in the VNC. One
of them was a lacZ insertion in dac, referred to as
dacP.
Confocal imaging and data acquisition
A Zeiss LSM 510 confocal microscope was used to collect data for all
images; confocal stacks were merged using LSM 510 software. Where
immunolabeling was compared for levels of expression, wild-type and mutant
tissue was stained and analyzed on the same slide. Images were 2 µm
thick (Fig. 2B,C;
Fig. 3E-L;
Fig. 4I-L,R,S; Fig. 5A-L;
Fig. 6G; insets in
Fig. 6) or 5 µm thick
(Fig. 3B-C'';
Fig. 4E-H',O-Q;
Fig. 6H). Images of the entire
VNC (Fig. 2A;
Fig. 3D;
Fig. 4A-D,M,N;
Fig. 6A-F,I-L) consisted of 1.2
µm-thick steps through the entire VNC (30-40 µm), which were merged to
obtain the final image. The intensity index used to quantify Fmrf expression
levels in dac mutants and rescues
(Fig. 4T) was obtained as
previously described (Hewes et al.,
2003
). Statistical analysis was performed using Microsoft Excel.
Where appropriate, images were false colored to help color-blind readers.
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Results |
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Peptidergic neurons can be subdivided into two groups
Previous studies had identified several genes acting to specify Tv cell
identity. ap and the Krüppel-type zinc finger gene
squeeze (sqz) act together to make the Tv cell competent to
express Fmrf (Allan et al.,
2003). However, Fmrf expression is not triggered until a
target-derived retrograde signal, mediated by the BMP ligand Glass bottom boat
(Gbb) and the type-II BMP receptor Wishful thinking (Wit), activates the BMP
pathway within the Tv cell (Allan et al.,
2003
; Marques et al.,
2003
). Additionally, the bHLH gene dimmed
(dimm), which specifies generic aspects of peptidergic cellular
identity, is also required for wild-type levels of Fmrf expression
(Hewes et al., 2003
).
Pan-neuronal misexpression of ap and sqz can trigger ectopic
Fmrf expression, but only in a subset of peptidergic neurons: the Va and dMP2
neurons (previously described as Vap neurons)
(Allan et al., 2003
). All these
cells have active BMP signaling, as detected by immunoreactivity to the
phosphorylated receptor-Smad protein Mothers against dpp (pMad; Mad -
FlyBase). From these studies, we proposed a simple model wherein an
ap/sqz/BMP combinatorial code would be sufficient to activate Fmrf in
all peptidergic neurons (Allan et al.,
2003
).
To test this hypothesis, we examined immunoreactivity to pMad in the
majority of peptidergic neurons, using the c929-GAL4 line
(Hewes et al., 2003)
(Fig. 2A-C). Certain
peptidergic cells, such as the corazonin cells
(Fig. 2B), showed no evidence
of BMP activity. However, in addition to the Tv, Va and dMP2 peptidergic
neurons (Allan et al., 2003
;
Miguel-Aliaga and Thor, 2004
),
we found that a number of peptidergic cells stained for pMad, but were
refractory to ap/sqz misexpression. These include a lateral
cluster of peptidergic cells in abdominal segments, here referred to as Plc
(peptidergic lateral cluster; Fig.
2C). This indicates that pMad-positive peptidergic cells in the
Drosophila VNC can be subdivided into two subclasses: those that
respond to ap/sqz by triggering Fmrf expression, and those
that are refractory. Thus, other factors besides ap, sqz, dimm and
the BMP pathway are probably necessary for proper Fmrf expression
(Fig. 2D).
Dachshund and Eyes Absent are expressed in `responsive' peptidergic neurons
To understand why only a subset of peptidergic cells trigger Fmrf in
response to the ap/sqz/BMP code, we attempted to identify
additional genes expressed in subsets of peptidergic cells, including the Tv
cells. To this end, we analyzed the expression of a number of enhancer trap
lines (see Materials and methods). We found that P-element transposon
insertions (lacZ or GAL4) in the dac gene revealed
dac expression in a large population of interneurons, with no
evidence of expression in either glia (repoGAL4) or
motoneurons (pMad; Fig.
3A-C''). Importantly, however, we observed dac
expression in a lateral group of cells in the three thoracic segments
(Fig. 3A). Using antibodies to
Dac, and the Fmrf-lacZ and apGAL4 reporter lines,
we found that Dac was expressed in all four ap-cluster cells at stage
15 (not shown). However, from stage 16 onward, Dac expression was restricted
to three of the four cells in the ap-cluster
(Fig. 3E). In order to identify
which ap-cluster cells expressed Dac, we co-labeled for
c929-GAL4 (restricted to the peptidergic Tv, Tvb of the
ap-cluster and dAp cells) (Hewes
et al., 2003) and Fmrf-lacZ (to distinguish the Tv cell)
(Fig. 3G). We found that Dac
was absent from the Tvb and dAp cells (c929-GAL4-positive,
Fmrf-lacZ-negative, Fig.
3G), and thus was selectively expressed in the Tv, Tva and Tvc
cells. Dac expression was initiated postmitotically in ap-neurons,
but it was rapidly activated by stage 15 as ap-neurons emerged (not
shown). We found that Dac expression, as visualized by Dac,
dacP (a lacZ insertion in dac) or
dacGAL4, was initiated postmitotically in the majority of
neurons, a notion that is substantiated by the onset of expression in
ap-neurons, and by the expression of Dac in the pCC interneuron but
not in its sibling, the aCC motoneuron
(Fig. 3C, arrow).
Next, we examined whether pMad and Dac expression coincided in peptidergic cells, utilizing the c929-GAL4 reporter to identify VNC peptidergic neurons. pMad/Dac coexpression was restricted to a small subset of peptidergic neurons: the Va and dMP2 cells (Fig. 3C'',I-J), as well as a posterior cluster (Pc) of peptidergic neurons (not shown), all of which exit the VNC. In contrast, neither Dac nor pMad were expressed in several other peptidergic neurons, such as the Crz neurons (Fig. 3K) or the Tvb or dAp cells (Fig. 3G). In the clusters of lateral abdominal peptidergic cells (Fig. 2A,D; Fig. 3L), Dac and pMad expression was mutually exclusive; the pMad+ Plc cells did not express Dac (Fig. 2C, arrow; Fig. 3L, arrowhead) while Dac was expressed in two neighboring pMad-negative peptidergic cells, herein referred to as the ventral intermediate (Vi) neurons (Fig. 3L, arrow).
dac encodes a transcriptional co-factor that plays key roles
during Drosophila imaginal disc development
(Mardon et al., 1994). In the
developing eye, dac function within the retinal determination gene
network is intimately linked to that of the homeobox gene sine oculis
(so) and the transcriptional co-factor eyes absent
(eya) (Hsiao et al.,
2001
). We analyzed the expression of solacZ
(so7) and eya (anti-Eya). As previously
described, there is an early phase of both solacZ and Eya
expression in subsets of VNC cells between stages 13 and 15
(Kumar and Moses, 2001
) (not
shown). ap-neurons could first be discriminated at stage 15.
Expression of solacZ was not observed in an
ap-cluster at any stage (not shown). As the lineage generating
ap-neurons is unknown, we could not determine whether
solacZ was expressed in the ap-neuron precursors.
By contrast, Eya expression was observed within a subset of
ap-neurons, the four ap-cluster cells and the dAp cells,
even as they first emerged (Fig.
3D,F,H). Remarkably, by stage 16, the expression of Eya within the
VNC was entirely restricted to these ap-neurons
(Fig. 3D).
Dac and Eya were expressed in partially overlapping subsets of ap-neurons. The Tv, Tva and Tvc cells expressed both Dac and Eya. However, in the Tvb and dAp cells, Eya was expressed without Dac. With respect to the ability of ap/sqz/BMP to trigger Fmrf expression ectopically in the VNC peptidergic compartment, we found that all `responsive' peptidergic cells (the dAp, Va, dMP2 cells) expressed either Dac or Eya, whereas `non-responsive' peptidergic cells (such as the Plc and Crz cells) did not (Fig. 3M). pMad staining, indicative of active BMP signaling, also contributes to the definition of the responsive/non-responsive peptidergic compartments. pMad was evident in the responsive Va and dMP2 cells, which expressed Dac and responded to ap/sqz alone. pMad was absent from the responsive dAp cells, which expressed Eya and responded to ap/sqz only when co-misexpressed with BMP signaling. In the non-responsive population, certain cells (such as the Plc cells) had pMad but did not express Dac or Eya, while others (such as the Crz cells) had neither Dac/Eya nor pMad. The expression of these markers within the VNC peptidergic compartment is summarized in Fig. 3M.
Dachshund and Eyes Absent are important for FMRFamide-related expression but play different roles in ap-neurons
To test whether dac and eya play any roles in the
specification of Fmrf-Tv neurons, we analyzed mutants for each gene. In
dac mutants (Fig. 5C)
we found that ap-cluster cells were generated and that Tv neurons
showed normal innervation of the DNH and pMad staining
(Fig. 4F,J). However, there was
a small but numerically significant loss of Fmrf expression (97% in wild type
compared with 94% in dac mutants; P<0.05)
(Fig. 4A,B). Moreover,
quantification of their Fmrf expression levels revealed that Fmrf expression
was consistently weaker in dac mutants compared with that of wild
type (P<0.0001) (Fig.
4T). Upon rescue of dac mutants, by re-introduction of
UAS-dac from apGAL4, we observed a clear
upregulation of Fmrf expression above wild-type levels (P<0.0001
compared with control) (Fig.
4T). This supports a cell-autonomous role for dac in
controlling high-level expression of Fmrf in Tv neurons.
By contrast, in eya mutants
(Fig. 5J), Fmrf expression was
severely reduced, with only 32% of Tv cells expressing Fmrf compared with 97%
in wild type (P<0.0001) (Fig.
4A,C). We had routinely used apGAL4 as a
marker for ap-neurons and, although apGAL4 is a
strong ap allele, we had not seen evidence of genetic interactions
between ap and either sqz, dac or BMP signaling
(Allan et al., 2003) (not
shown). However, upon comparing Fmrf expression in eya mutants in the
presence or absence of apGAL4, we found that this
ap allele enhanced the eya phenotype; Fmrf was expressed in
only 6% of Tv neurons in an eya null, ap heterozygous
background, compared with 32% for an eya null, ap wild-type
background. This genetic interaction did not result from regulation of
ap by eya, or vice versa, because ap expression was
normal in eya mutant ap-cluster cells, and vice versa
(Fig. 4I,K;
Fig. 5G,H,J). eya
mutants also displayed a severe pathfinding phenotype with a nearly complete
failure of DNH innnervation: 19% DNH innervation in eya mutants
compared with 100% in controls (Fig.
4E',G'). As predicted, this failure to reach the DNH
in eya mutants resulted in the nearly complete loss of pMad in the
ap-cluster (26% pMad staining of Tv cells compared with 99% in
controls; P<0.0001) (Fig.
4I,K). To analyze axon pathfinding in eya mutants without
altering ap gene dosage, we used the ap enhancer construct
apC-
-lacZ (Lundgren et al.,
1995
) instead of apGAL4. Unfortunately, unlike
the membrane-targeted UAS-myc-EGFPF, the
-lacZ reporter did not reproducibly reveal the Tv axon terminals
in the DNH. Thus, we could not address DNH innervation in eya mutants
without using apGAL4. However, since ap is not
important for Tv pathfinding (Allan et al.,
2003
; Benveniste et al.,
1998
), it is unlikely that the severe Tv-axon pathfinding observed
in eya was exacerbated by the removal of one copy of ap. We
did find a remarkably strong ectopic midline crossing of dAp axons in
eya mutants using
-lacZ, most evident in abdominal
segments: 96% of segments showed at least one dAp axon crossing the midline in
eya mutants, compared with 0% in controls (n=24 segments;
Fig. 4O-Q). This demonstrates
that eya is critical for axon pathfinding even in the presence of
wild-type ap. The ventral pair of ap-neurons (vAp) did not
express eya and did not show any apparent defects in pathfinding
(Fig. 4O-Q).
In the embryo, Eya is expressed in certain regions of the lateral mesoderm
and in dorsal, anterior structures, and has been shown to be important for
embryonic head morphogenesis (Bonini et
al., 1998). In spite of these other roles for eya during
embryogenesis, we found that reintroducing UAS-eya from
apGAL4 in eya mutants rescued DNH innervation to
96% (Fig. 4G',H'),
rescued pMad staining of the Tv cell to 95%
(Fig. 4K,L), and rescued Fmrf
to 85% (Fig. 4C,D; all
P<0.0001, compared with eya mutants). These data support
a cell-autonomous role for eya in controlling Tv-axon pathfinding and
Fmrf expression.
In summary, dac and eya act cell-autonomously to regulate
crucial, yet different, aspects of Tv cell differentiation. dac is
important for high-level Fmrf expression but does not affect pathfinding.
eya regulates axon pathfinding of a subset of ap-neurons,
including the Tv and dAp cells. We also observed a genetic interaction between
eya and ap with respect to Fmrf expression. Given that
ap regulates Fmrf gene expression directly by binding to its
enhancer (Benveniste et al.,
1998), the genetic interaction observed between eya and
ap suggests a direct regulation of Fmrf gene expression by
eya.
In addition to pathfinding, Eyes Absent controls BMP signaling
These eya mutant results did not discriminate between an effect
for eya directly on Fmrf, or indirectly on Fmrf via its control of
Tv-axon pathfinding to the DNH. Fmrf expression in the Tv neurons is crucially
dependent on a target-derived BMP signal mediated by the BMP ligand Gbb, which
is accessed by Tv axons at the DNH (Allan
et al., 2003; Marques et al.,
2003
). Fmrf expression is lost when Tv-axon pathfinding is
disrupted by UAS-robo misexpression
(apGAL4/UAS-robo), forcing Tv axons to avoid the midline
and DNH. However, Fmrf expression can be efficiently restored in these
misguided Tv neurons by providing the Gbb ligand cell-autonomously
(apGAL4/UAS-robo, UAS-gbb)
(Allan et al., 2003
). The
severe pathfinding defects observed in eya mutants raised the
possibility that loss of Fmrf solely reflected a loss of DNH innervation and
access to Gbb. Is the loss of Fmrf in eya mutants secondary to these
axon-pathfinding defects, or does eya regulate other aspects of Tv
cell differentiation?
To resolve this issue, we tested whether Fmrf expression could be restored
in eya mutants by providing gbb cell-autonomously. Even
though UAS-gbb rescues gbb mutants and misguided Tv neurons
(Allan et al., 2003),
UAS-gbb failed to rescue Fmrf expression in eya mutants
(Fig. 4N). Surprisingly, we
also noted only a partial rescue of pMad staining in Tv neurons; 46% pMad in
gbb-rescued eya mutants compared with 26% in eya
mutants and 98% pMad in gbb-rescued gbb mutants
(P<0.0001) (Fig.
4S) (Allan et al.,
2003
). This suggested that two aspects of the competence to
respond to BMP signaling were affected in eya mutants. First, the
inability of gbb to rescue pMad activation reflects the functional
absence of a component of the BMP signaling pathway upstream of pMad in
eya mutants. This component may be the BMP type-II receptor Wit,
which mediates BMP retrograde signaling in Tv neurons. Unfortunately, the Wit
antibody is not sufficiently sensitive to test this hypothesis directly.
Second, the complete failure to rescue of Fmrf expression with gbb,
in spite of its partial rescue of pMad, suggested that a downstream component
of the BMP signaling pathway that leads to Fmrf expression was additionally
affected in eya mutants. Our observations in eya mutants,
that remaining pMad-positive Tv neurons were frequently Fmrf-negative, is
consistent with this hypothesis (26% were positive for pMad staining, whereas
only 6% expressed Fmrf; P<0.0001). To test this idea directly, we
bypassed the Wit receptor by driving activated BMP type I receptors from
apGAL4 in an eya mutant background. In spite of a
full rescue of pMad in Tv cells (100% compared with 26% in eya
mutants, P<0.0001) (Fig.
4K,R), Fmrf expression was only poorly rescued to 36%, compared
with 6% in eya mutants (P<0.0001)
(Fig. 4M). This contrasts with
the ability of these activated type I receptors to rescue gbb and
wit mutants fully (Allan et al.,
2003
), and indicates that eya controls a component of the
pathway downstream of pMad that is essential for activating Fmrf expression.
This component may be Eya itself or some other unknown regulatory factor that
directly controls Fmrf expression.
In summary, eya plays multiple roles in the Tv neuron. eya is necessary for Tv innervation of the DNH, as well as normal pathfinding of dAp neurons along the ap-fascicle. In addition, eya is required in Tv neurons for the activation of pMad in response to gbb, as well as for the activation of Fmrf expression following pMad nuclear accumulation.
Dachshund, but not Eyes Absent, is in part regulated by other genes specifying FMRFamide-related cell fate
We next addressed whether the genes controlling Fmrf expression regulate
one another. As shown above, there was no effect on apGAL4
reporter activity or ap cell numbers in either dac or
eya mutants (Fig.
5A,C,D). Additionally, dac did not regulate Eya
(Fig. 5I). However, we did note
a partial loss of Dac expression in one ap-cluster cell in
eya mutants (Fig. 5D).
This cell was probably the Tva or Tvc cell, because Dac was never lost in the
Tv cell, identified as the cell with highest apGAL4
activity (Fig. 5D; note pMad
staining in cell of highest apGAL4 activity in
Fig. 4I,J,L). We found no
evidence that the late (stage-17) activation of the BMP pathway was important
for the maintenance of either Dac or Eya expression
(Fig. 5E,K). In sqz
mutants, Eya expression was evident within every ap-cluster cell
(Fig. 5L), including the extra
ap cells that we typically observed in sqz mutants
(Allan et al., 2003) (not
shown). However, we did observe a partial loss of Dac in sqz mutants;
it was typically lost from one ap-cluster cell
(Fig. 5F). In independent
studies, we have found that sqz regulates the identity and number of
ap-cluster cells through an interaction with the Notch pathway,
resulting in the generation of additional Tvb cells within each
ap-cluster in sqz mutants (D.W.A. and S.T., unpublished).
Dac is not normally expressed in the Tvb cell, so we propose that the loss of
Dac in one extra cell per ap-cluster in sqz mutants is due
to the generation of an extra Tvb cell, rather than the result of a direct
effect of sqz on Dac expression. Given these early effects of
sqz function on ap-cluster cell identity via the Notch
pathway, we did not examine sqz expression in either Dac or Eya
mutants, which are expressed exclusively postmitotically and were not found to
modulate the number of ap cells generated.
Finally, we observed that in ap mutants, Dac expression was often maintained in the Tvb neuron (56%), indicating that ap normally contributes to the repression of dac in Tvb neurons. Because ap does not normally prevent Dac expression in the other neurons of the ap cluster, additional factors must make the ap-mediated repression of Dac context-dependent.
Dachshund, but not Eyes Absent, acts in a combinatorial code to trigger ectopic FMRFamide-related expression
The expression patterns of Dac and Eya, together with their roles in Fmrf
regulation, suggested that they are the missing factors in pMad-positive,
peptidergic cells that are non-responsive to the ap/sqz/BMP
combinatorial code. To test this notion we addressed the sufficiency of
dac and eya to activate Fmrf expression ectopically, either
alone, in combination with one another, or together with the previously
identified Fmrf regulators. This was tested in peptidergic cells
(c929-GAL4), in ap-neurons (apGAL4) and
in all postmitotic neurons (elavGAL4).
First, we examined the effects of UAS-eya misexpression. UAS-eya failed to trigger ectopic Fmrf expression when driven from any GAL4 driver, in spite of its ability to rescue eya mutants and its robust expression in our misexpression conditions (verified by anti-Eya). This held true whether eya was misexpressed alone or in combination with either dac, ap or sqz, using any of the three GAL4-drivers (n=8 VNCs; not shown). eya mutant analysis indicated that eya was necessary for competence of the Tv neuron to respond to the Gbb ligand. To address whether eya is sufficient to confer Gbb-responsiveness on other neurons, we misexpressed UAS-eya in combination with UAS-gbb and either dac or ap [elavGAL4/UAS-gbb; UAS-eya (UAS-dac or UAS-ap)]. However, we did not observe any ectopic pMad staining or any ectopic Fmrf expression (n=6 VNCs; not shown). Thus, although eya is critical for wild-type Fmrf expression and Gbb responsiveness in Tv cells, it is neither sufficient to activate Fmrf nor sufficient to promote pMad phosphorylation in response to Gbb outside ap-neurons.
Misexpression of UAS-dac alone in peptidergic cells using c929-GAL4 triggered little or no ectopic Fmrf expression (Fig. 6A). By contrast, UAS-dac/UAS-ap co-misexpression within peptidergic cells triggered ectopic Fmrf expression, even within the pMad-positive `non-responsive' peptidergic cells, such as the peptidergic lateral cluster (Plc) cells (Fig. 6B). We found that this ectopic Fmrf expression was dependent upon BMP signaling, because UAS-dac/UAS-ap co-misexpression in a wit mutant background failed to trigger ectopic Fmrf (Fig. 6C). Thus, dac and ap co-expression is sufficient to trigger Fmrf expression within pMad+ peptidergic cells. We did not observe ectopic Fmrf activation within the pMad-negative population of peptidergic cells, such as the Crz or dAp cells. However, co-misexpression of UAS-dac/UAS-ap together with ectopic BMP signaling using UAS-tkvA, UAS-saxA triggered ectopic expression of Fmrf in these normally pMad-negative peptidergic cells: the Crz, Tvb and dAp cells (Fig. 6D). Thus, dac can act with ap and BMP signaling to trigger ectopic Fmrf expression in the majority of VNC peptidergic neurons.
Given its potency to trigger Fmrf in peptidergic neurons, we wished to
assess the sufficiency of this `code' to drive Fmrf expression beyond the
peptidergic cell population. Pan-neuronal misexpression of UAS-dac,
using elavGAL4, triggered ectopic Fmrf expression that was
limited to Pc peptidergic cells (Fig.
6E). By contrast, pan-neuronal co-misexpression of both
UAS-ap and UAS-dac triggered extensive ectopic Fmrf
expression (Fig. 6F). Most, if
not all, of the neurons that ectopically expressed Fmrf were pMad-positive
(Fig. 6G). Thus,
ap/dac co-misexpression is capable of inducing Fmrf expression in
motoneurons. Using HB9-GAL4, which is expressed in the majority of
motoneurons (Broiher and Skeath, 2002), we found that Fmrf expression could
indeed be triggered in defined motoneurons, such as the RP1 and RP4 cells
(Fig. 6H). We were unable to
test the potency of dac/ap/BMP in all neurons, due to
lethality when activating the BMP pathway ectopically throughout the VNC
(Allan et al., 2003).
We next tested the sufficiency of UAS-dac to activate Fmrf within ap-neurons. As expected, UAS-dac alone had no effect in ap-neurons (Fig. 6I). As apGAL4 is an allele of ap, we co-misexpressed UAS-dac and UAS-ap to test whether a higher level of ap expression might work, but again saw no effect (not shown). As the only pMad+ ap-neuron is the Tv cell, we activated the BMP pathway ectopically together with UAS-dac alone, or together with UAS-ap. This led to ectopic expression of Fmrf in the majority of ap-neurons, including the four ap-cluster cells (Fig. 6J,K). This strong effect of ectopic dac/BMP within ap-neurons allowed us to address whether eya is crucial for this ectopic Fmrf expression in all ap neurons, as it is for wild-type Fmrf expression. We misexpressed the same four transgenes in an eya mutant background and found that removing eya from ap-neurons led to loss of both ectopic and endogenous Fmrf expression (Fig. 6L). Since both Dac and pMad expression were clearly observed ectopically in all ap-neurons, failure to trigger Fmrf in this case was not due to a failure to drive the transgenes at sufficient levels (not shown). Fmrf expression was also absent from Tv neurons, indicating that the eya mutant phenotype cannot be rescued by the addition of other Fmrf regulators. Given these results, we analyzed Eya expression when UAS-dac and UAS-ap were misexpressed pan-neuronally from elavGAL4. In spite of the extensive ectopic Fmrf expression, Eya expression itself was unaltered from wild type (not shown).
In summary, although eya was critical for endogenous Fmrf expression, it was not sufficient to activate Fmrf ectopically in any tested scenario, whether alone or combinatorially. By contrast, dac was a potent activator of Fmrf expression, particularly in combination with ap in many postmitotic neurons, including motoneurons. dac/ap-mediated ectopic expression was entirely dependent upon BMP signaling (in all neurons) and also upon eya in the neurons that normally express Eya.
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Discussion |
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|
Apterous, Eyes Absent and axon pathfinding
Our findings illustrate the fact that regulators acting within a
postmitotic neuron can act together in a combinatorial fashion to specify one
aspect of neuronal identity (Fmrf expression, in this case). However, some of
these regulators can simultaneously function in combinatorial sub-codes to
control other aspects of neuronal identity; the additional roles of
ap and eya in Tv axon pathfinding may be one such example.
In abdominal hemisegments, Ap is expressed in the two vAp and the single dAp
neurons. Normally, the axons of these neurons join a common ipsilateral
longitudinal fascicle running the length of the VNC. Previous studies have
revealed that ap is important for proper ap-axon
fasciculation as well as for their avoidance of the midline
(Lundgren et al., 1995). Eya
is not expressed in vAp neurons, and our results indicated that it
specifically controls dAp pathfinding. The eya mutant phenotype only
partially phenocopies the ap phenotype, since eya affects
midline crossing but not fasciculation; once dAp neurons have aberrantly
crossed the midline they join the contralateral ap-fascicle. Neither
the ap nor the eya mutant phenotypes are due to any apparent
crossregulation between these two genes. Surprisingly, our findings indicated
that different genetic mechanisms underlie the indistinguishable,
ap-dependent axon pathfinding of dAp and vAp neurons; dAp axons
crucially depend upon eya to avoid crossing the midline, whereas vAp
axons neither express eya nor depend upon it.
An instructive and additive code for Fmrf expression
Together with previous findings (Allan
et al., 2003; Benveniste et
al., 1998
; Hewes et al.,
2003
; Marques et al.,
2003
) our results indicate that Fmrf expression is
triggered by the combinatorial action of ap, sqz, dimm, dac, eya and
BMP signaling. However, with the exception of BMP signaling,
none of these factors are absolutely necessary for endogenous Fmrf
expression - in all mutants, expression of Fmrf is not lost from all Tv
neurons. Similarly, although misexpression of a partial code can lead to
ectopic Fmrf expression, its expression levels are consistently
weaker than those seen in Tv neurons. Thus, it appears that a partial code is
sufficient for some level of Fmrf expression: the ectopic expression of
Fmrf in BMP-positive RP neurons - cells that do not express sqz,
eya or dimm - in response to dac and ap is one
such example. However, the complete code
(ap/sqz/dimm/dac/eya/BMP)
appears to be necessary for wild-type (high) levels of expression, as seen in
the Tv neurons. It is possible that the simultaneous misexpression of all
these factors would lead to robust ectopic Fmrf expression in all
neurons. Due to obvious technical limitations, we have not been able to test
this idea.
Eyes Absent: a pivotal integrator of multiple signal transduction networks?
Multiple signal transduction inputs/outputs appear to revolve around Eya.
First, phosphorylation of Eya by the Ras/MAPK pathway has been found to
regulate Eya activity and synergy with So
(Hsiao et al., 2001;
Silver et al., 2003
). Second,
the transcriptional activity of Eya itself depends upon an intrinsic tyrosine
phosphatase activity (Li et al.,
2003
) that is also required for ectopic eye induction in
Drosophila (Rayapureddi et al.,
2003
; Tootle et al.,
2003
). The target(s) of Eya phosphatase activity are currently
unknown. Third, we find that Eya regulates the BMP pathway in Tv neurons and
pMad cannot be reactivated in eya mutants even by cell-autonomous
introduction of the BMP ligand Gbb. A probable explanation for this result is
that eya regulates the expression or activity of the BMP type
receptors Wit, Tkv or Sax. When the BMP pathway is dominantly activated by the
use of activated type I receptors, nuclear pMad is restored. However, this
still does not reactivate Fmrf expression, indicating that Eya additionally
plays important roles downstream of pMad activation. One interpretation of
these findings is that Eya acts directly on the Fmrf gene. However,
it is also tempting to speculate that Eya may act to modulate pMad activity
directly. There are several reasons for this proposal. It is known that
several other kinase pathways, such as MAPK, can phosphorylate Smad proteins
on residues other than those phosphorylated by TGFß/BMP type I receptors
(Derynck and Zhang, 2003
). The
in-vivo roles of such modifications are unclear, but in-vitro evidence points
to both repression and activation of Smad activity
(Brown et al., 1999
;
Engel et al., 1999
;
Kretzschmar et al., 1999
).
Nevertheless, given its nuclear localization and phosphatase activity, it is
possible that Eya acts to de-phosphorylate inhibitory residues in pMad. A
regulatory circuitry between MAPK (and other kinases), Eya and the
TGFß/BMP pathway is an intriguing possibility. Moreover, recent studies
reveal that vertebrate orthologs of Dac can physically interact with the Smad
complex, thereby affecting TGF-ß signaling
(Kida et al., 2004
;
Wu et al., 2003
). Together
with these previous findings, our results point to a model wherein Eya and Dac
play central roles in integrating input from, and controlling the activity of,
multiple signal transduction networks. Determination of the precise mechanisms
by which Eya and Dac orchestrate these events should enhance our understanding
of how both intrinsic and extrinsic signals intersect to affect cellular
differentiation.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Department of Neurology, Enders 211, The Children's
Hospital, 320 Longwood Avenue, Boston, MA 02115, USA
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