Department of Biology, 071 Gilmer Hall, University of Virginia, Charlottesville, VA 22903, USA
Author for correspondence (e-mail:
condron{at}virginia.edu)
Accepted 3 November 2003
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SUMMARY |
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Key words: Serotonin, Axon guidance, Midline, Eagle, Robo, Transporter
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Introduction |
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Serotonergic neurons in the Drosophila ventral nerve cord (VNC)
are organized in a bilaterally symmetric pattern, with two serotonergic
neurons per hemisegment that extend axons across the midline via the posterior
commissure and branch in the contralateral neuropil
(Valles and White, 1988). The
specification of serotonergic neurons has been well described in the fly
through use of molecular markers (Broadus
and Doe, 1995
; Doe,
1992
; Isshiki et al.,
2001
; Lundell et al.,
1996
; Lundell and Hirsh,
1998
); however, the process of later development remains
relatively uncharacterized. Reuptake of released serotonin by the serotonin
transporter (SerT) is an essential component of serotonergic neuron function
and one of the earliest steps in serotonergic neuron differentiation. A highly
conserved serotonin transporter, SerT, is specifically expressed in
fly serotonergic neurons and is blocked by both fluoxetine (Prozac) and
cocaine (Corey et al., 1994
;
Demchyshyn et al., 1994
).
Expression of SerT precedes the onset of serotonin synthesis in fly (this
study) and grasshopper (Condron,
1999
), as well as synapse formation in mouse
(Bruning et al., 1997
).
In grasshopper, cuts that sever serotonergic neuron contact with the
midline lead to a loss of SerT expression, suggesting that the midline plays a
crucial role in the induction and maintenance of SerT activity
(Condron, 1999). However,
application of Fibroblast Growth Factor 2 (FGF2) to the nerve cord rescues
SerT expression, indicating the presence of a midline-associated FGF-like
signal that induces serotonergic neuron differentiation. In mouse and rat,
specification of serotonergic neurons also requires FGF signaling
(Ye et al., 1998
). Axon
guidance molecules are also likely to play an important role in serotonergic
neuron development, as these neurons must cross the ventral midline and branch
in the contralateral neuropil in order to achieve their final differentiated
state. Additional indications of a relationship between serotonergic neuronal
axon guidance and differentiation come from data showing that a loss of
function in genes of the serotonin biosynthetic pathway impairs axon
pathfinding and alters branching patterns in the periphery
(Budnik et al., 1989
).
We use SerT activity, measured by reuptake of serotonin, as a marker of
serotonergic neuron differentiation. SerT serves as an early and easily
testable step in serotonergic neuron differentiation, because it precedes the
onset of serotonin synthesis. We show that regulation of SerT activity in the
fly embryo, as in the grasshopper, is not only temporally but also physically
related to midline crossing. Additionally, we show that members of the
roundabout (robo) family of axon guidance receptors,
robo2 (lea - FlyBase) and robo3 (robo2/3),
but not robo, are required for serotonergic neuron differentiation.
robo2 and robo3 control axon guidance throughout the CNS by
regulating midline crossing and determining lateral position along specific
longitudinal fascicles (Rajagopalan et
al., 2000a; Rajagopalan et
al., 2000b
; Simpson et al.,
2000a
; Simpson et al.,
2000b
).
Finally, the present study indicates that robo2/3 function in the
same genetic pathway as the zinc-finger transcription factor eagle
(eg), which is required for serotonergic neuron differentiation and
is expressed in all serotonergic neurons of the fly VNC
(Dittrich et al., 1997;
Higashijima et al., 1996
;
Lundell and Hirsh, 1998
). Loss
of eg function causes a loss of both serotonin synthesis and the
biosynthetic enzyme Dopa decarboxylase (Ddc) in a subset of serotonergic
neurons according to the severity of the mutation
(Lundell and Hirsh, 1998
). We
show that a loss of eg also causes a loss of SerT activity. The
pattern of SerT and serotonin loss observed in an eg mutant closely
resembles the phenotype observed in robo2/3 loss-of-function mutants.
A direct relationship between robo2 and eg is suggested by
data showing a loss of Eg expression in robo2/3 mutants and through
genetic rescue experiments.
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Materials and methods |
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Immunohistochemistry
Embryos were collected into Eppendorf tubes, bleached for 1 minute and
washed with salt solution (0.04% NaCl + 0.03% TritonX-100) followed by
Schneider's medium (Sigma). Embryos were then either transferred to a Petri
dish containing Schneider's medium for dissection or to a second Eppendorf
containing Schneider's, where they were dounced gently with a DNA pellet
disrupter. VNCs isolated from dissection or limited douncing were then
transferred to another Petri dish containing Schneider's medium and affixed to
the surface of a glass coverslip. All stage 14 VNCs were dissected manually as
they were too fragile to withstand douncing. Stage 15-17 VNCs were isolated on
the basis of embryo morphology (Campos-Ortega, 1985) when dissected or BP102
staining and serotonergic axon morphology when dounced.
To visualize serotonin transporter (SerT) activity, serotonin (Sigma) was
added to the medium to a final saturating concentration of 10 µM and
incubated for 10 minutes. Media was then removed, replaced with fix (4%
paraformaldehyde/PBS, freshly made) and incubated for 60 minutes at room
temperature. After fixation, coverslips with attached nerve cords could be
transferred out of liquid between washings. VNCs were then processed for
histochemical analysis as described previously
(Condron, 1999). Imaging was
performed on an Olympus BX40 microscope and photographs, DIC and fluorescent,
taken with a Photometrics SenSys camera. All images except those in
Fig. 5 were taken with an
Olympus 40x or 20x lens. ImageIP software was used to capture the
images, and fluorescent images were subjected to one frame deconvolution (95%
removal, 75% gain) using VayTek Hazebuster. Fluorescent channels were stacked
and layouts/labeling were performed in Adobe Photoshop. Fixed samples shown in
Fig. 5 were imaged with a Nikon
Eclipse E800 microscope (100x oil lens, NA=1.3), Hamamatsu ORCA-ER
camera, with a Perkin-Elmer spinning disc confocal unit. One stack of about
100 slices was taken, spaced 0.24 µm, 2x2 binning and with an
exposure time of 500 mseconds. Image reconstructions were performed with
Volocity 2.0. The antibodies used were as follows: 5HT 1:5000 (Immunostar);
ßgal 1:100 (Clontech); BP102 1:1000, ßgal 1:20 (Developmental
Studies Hybridoma Bank, University of Iowa); Mouse anti-Eg 1:40 (gift from
Chris Doe); secondary antibodies 1:2000 (Jackson Labs).
|
![]() |
Results |
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Preliminary screen of midline guidance mutants did not show a disruption of serotonin transporter expression
In order to further investigate the relationship between midline crossing
and serotonergic neuron differentiation, a broad screen of mutants causing
disruptions in midline structures and/or axon guidance was conducted. SerT
activity was analyzed in mutants including commissureless
(comm), robo gain of function and robo2 gain of
function (Fig. 2), as well as
spitz (spi - FlyBase) and single-minded
(sim) (data not shown). Specific overexpression of Robo or Robo2 in
the serotonergic neurons was achieved by driving expression of
UAS-robo (Kidd et al.,
1998a; Kidd et al.,
1998b
) or EP2582 (Rajagopalan
et al., 2000b
; Simpson et al.,
2000a
) under egGal4 (Mz360)
(Dittrich et al., 1997
).
Loss-of-function mutants for robo and slit (sli -
FlyBase; the ligand for robo) were also analyzed.
|
Based on studies in grasshopper, we expected mutants that prevent midline crossing to lack SerT activity. By contrast, the midline mutants analyzed in our initial screen (shown in Fig. 2) displayed normal stage 16 SerT activity. Later examination of other axon guidance mutants, including robo2 and robo3 (see Fig. 4), revealed their role in regulating SerT. However, the negative results obtained from our original screen led us to address the question of midline function differently, by attempting to recapitulate midline cut experiments previously done in the grasshopper. A simple explanation for the lack of serotonergic phenotype in comm and Robo2 gain-of-function mutants, for example, is that midline crossing is not crucial for differentiation in the fly as it is in the grasshopper. Another possibility, however, is that transient functional contact occurs between serotonergic axonal projections and the midline in these mutants that are sufficient to induce normal differentiation.
|
|
|
Possibly, the lack of SerT and serotonin synthesis in robo2/3 loss of function mutants is due to general developmental defects in these nerve cords. To test the state of cellular differentiation in serotonergic neurons lacking SerT activity, robo2 mutant VNCs were double stained for SerT activity and lacZ expressed under the eagle promoter (eg289; Fig. 4E). Cells lacking SerT still stain for lacZ, indicating that loss of robo2 at least does not disrupt the lineage or early specification of serotonergic neurons in the VNC, when the eg promoter is first active.
Because robo2 gain of function permits maintenance of SerT activity following a midline cut, it seems likely that Robo2, at least, functions autonomously in the serotonergic neurons. However, an alternative possibility suggests that they function indirectly by guiding serotonergic growth cones to a currently unknown differentiation signal in the neuropil. Evidence that this might not be the case comes from data showing that mutations in robo, spitz and sim do not cause defects in serotonergic neuron differentiation, despite a sometimes severe disruption in CNS organization (data not shown).
robo2 and robo3 are required for normal expression of the transcription factor eagle
Normal serotonergic neuron differentiation in the fly requires the orphan
steroid hormone receptor and zinc-finger transcription factor eagle
(eg), which is expressed in the neuroblast that gives rise to the
serotonergic neurons and throughout early stages of serotonergic neuron
differentiation (Dittrich et al.,
1997; Higashijima et al.,
1996
; Lundell and Hirsh,
1998
). Loss of eg function results in a loss of SerT
expression and activity in a percentage of neurons according to the severity
of mutation (see Fig. 7,
compare the eg hypomorphic allele, egMZ360, to
the eg null allele, eg18b). Serotonin synthesis
is also disrupted in eg mutants
(Lundell and Hirsh, 1998
).
The pattern of SerT loss observed in eg mutants is almost
identical to that observed in robo2/3 mutants. To examine the
relationship between robo2/3 and eg further, robo2
and robo3 mutant nerve cords were stained with an anti-Eg monoclonal
antibody. Wild-type serotonergic neurons express Eg until stage 17
(Fig. 5A)
(Dittrich et al., 1997)
although message is lost by stage 14
(Higashijima et al., 1996
). In
an eg hypomorph (egMz360), all Eg staining is
absent, and a loss of SerT is observed in
30% of VNC hemisegments
(Fig. 5B, see
Fig. 7). A loss of Eg
expression is also observed in robo2
(Fig. 5C) and robo3
mutants (data not shown). Eg expression is lost in 100% of those cells lacking
SerT activity but remains in those cells positive for SerT
(Fig. 5C; n=110 and
112 hemisegments for robo2 and robo3 mutants, respectively).
These data suggest that Robo2/3 function in the same genetic pathway as Eg,
and that the loss of SerT seen in robo2/3 mutants is due to a loss of
Eg, as Eg is required for SerT expression.
Finally, as both eg hypomorphs and robo2 loss-of-function mutants show a loss of SerT activity in only a percentage of neurons (see Fig. 7), a robo2 loss of function/eg hypomorph double mutant line was analyzed for any increases in phenotype severity. There is not an additive effect of robo2 loss of function and eg hypomorph in the double mutant (see Fig. 7). These data provide further evidence that Robo2 functionally cooperates with Eg, because a partial loss of eg function does not exacerbate the loss of SerT in a robo2 mutant.
Intriguingly, we observe an effect of Robo2 expression on the mediolateral position of the serotonergic cell bodies. This is most markedly seen in the robo2 loss-of-function mutant hemisegment (Fig. 5C), where the cell bodies are positioned close to the midline, and in the Robo2 gain-of-function hemisegment (Fig. 5D), where the cell bodies are shifted laterally. A similar positional shift is observed in the Unc5 gain-of-function (Fig. 3D) and comm mutants (Fig. 2B). Therefore, the midline axon guidance signaling pathways may also play a role in cell body positioning.
Robo2 expression rescues an eagle hypomorphic phenotype and Eagle expression rescues a robo2 loss of function phenotype
To investigate the interaction between Robo2 and Eg, combinations of
mutations at both loci were examined. Comparison of SerT expression in VNCs
with varying levels of Robo2 and Eg reveals a dose-sensitive interaction
between the two genes (Fig.
6B-E). Overexpression of one copy of EP2582 (Robo2) with one copy
of the egGal4 driver prevents the midline crossing of serotonergic
neurons but does not disrupt SerT expression
(Fig. 6C). If one copy of
EP2582 (Robo2 gain of function) is expressed with two egGal4 drivers,
a loss of SerT results (Fig.
6D) because the egGal4 driver
(egMz360) is a hypomorphic allele of eg
constructed from a P-element insertion into the eg locus
(Dittrich et al., 1997;
Lundell and Hirsh, 1998
). A
loss of SerT appears only in the egMz360 homozygous
condition (Fig. 6B). However,
when two copies of EP2582 (Robo2 gain of function++) are expressed with two
egGal4 drivers (homozygous egMz360), the
eg hypomorphic phenotype is rescued. This high dose of Robo2 rescues
both SerT (Fig. 6E) and Eg
(Fig. 5D) expression in an
eg hypomorph. The function of Robo2 in midline axon guidance is
therefore separable from its function in regulating serotonergic cell fate, as
a low level of Robo2 overexpression prevents midline crossing but only a high
dose rescues SerT in eg hypomorphs. Initial consideration of this
rescue may seem to contradict previous results showing that robo2
regulates Eg expression (Fig.
5C). However, the rescue of an eg hypomorph by Robo2 gain
of function (Fig. 5D,
Fig. 6E) seems to be due to
some unique property of the hypomorphic allele, as not only SerT but also Eg
expression is restored (further addressed in Discussion). These results can be
seen quantitatively in Fig.
7.
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![]() |
Discussion |
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Induction of serotonin transporter expression and midline crossing
By visualizing serotonergic axonal projections with tau-lacZ, we
determined that SerT expression begins at the end of stage 15, just after
growth cones complete midline crossing and reach the contralateral side. This
temporal correlation between midline crossing and SerT induction suggests that
the midline is important for serotonergic neuron differentiation in the fly,
as it is in the grasshopper (Condron,
1999). Further evidence for the importance of the midline comes
from our data showing that in wild-type cords, axons physically separated from
the midline fail to express SerT. These results recapitulate similar
experiments in the grasshopper. Additionally, when the repulsive axon guidance
receptor Unc5 is expressed in serotonergic neurons, a partial loss of SerT
expression is observed. Although these results suggest a role for the midline
in serotonergic neuron differentiation, it remains unclear whether this role
is temporally restricted as it is in the grasshopper, and, additionally, what
factors act as the presumptive midline signal. FGF signaling in the
grasshopper is crucial for SerT induction
(Condron, 1999
), and plays a
role in the differentiation of vertebrate serotonergic neurons
(Ye et al., 1998
). In the fly,
experiments indicate that FGF signaling also appears to be important for SerT
regulation (J.A.C., M. Levin, E.M.U. and B.G.C., unpublished).
One problem with interpreting the role for the midline is the lack of an
abnormal serotonergic phenotype in mutants for the master regulatory gene
sim, where midline cells fail to properly differentiate
(Nambu et al., 1990;
Nambu et al., 1991
). It is
difficult to speculate about what factors may allow normal differentiation in
the absence of normal midline cells, as there are many changes in gene
regulation throughout sim mutants
(Xiao et al., 1996
). Although
our results suggest a role for the midline in serotonergic neuron
differentiation, it is likely to be more complicated than a simple switch
acting to induce differentiation.
robo2 and robo3 play a role in serotonergic neuron differentiation
Our data show that a loss of robo2 or robo3 causes a loss
of SerT expression, suggesting that Robo2/3 function positively to
regulate serotonergic neuron differentiation. A positive role for Robo2 is
further supported by our results showing that overexpression of Robo2 prevents
a loss of SerT in neurons physically separated from the midline. Possibly,
Robo2 functions downstream of the midline signal required for SerT induction
and thus allows differentiation to proceed in the absence of such a signal. An
alternative hypothesis suggests that Robo2/3 function indirectly to induce
SerT, by guiding serotonergic axons to an unknown signal in the contralateral
neuropil. Such indirect signaling occurs in the developing vertebrate CNS
where trophic support is required by commissural axons at the floorplate, an
intermediate axonal target (Wang and
Tessier-Lavigne, 1999). Although we cannot rule out the
possibility that Robo2/3 act indirectly to regulate serotonergic neuron
differentiation at this time, several lines of evidence suggest a more direct
role. Our data shows that overexpression of Robo2 not only spares SerT loss
following a midline cut but also rescues an eg hypomorph, and
furthermore, that an Eg gain of function rescues a robo2 loss of
function; these results strongly suggests that Robo2 functions autonomously in
the serotonergic neurons. Additionally, SerT loss is not seen in other
guidance mutants that disrupt midline crossing or cause general
disorganization of the CNS. However, it is difficult to clearly resolve the
presence of Robo2/3 protein specifically in the serotonergic neurons because
of the broad distribution of neuronal processes and the fact that serotonergic
neuron branching does not correspond simply to any Fas2 pathway where axons
are known to express Robo2/3.
Robo2 and Robo3 appear to act as overall regulators of differentiation
rather than specific regulators of SerT, as robo2/3 mutants not only
lose SerT expression (mRNA and reuptake activity) but also have defects in
serotonin synthesis later in development. Thus, the role of Robo2/3 in
serotonergic neuron differentiation parallels that of other genes, including
eg and the LIM-homeodomain transcription factor islet, that
cause both a loss of SerT as well as serotonin synthesis when disrupted
(J.A.C. and B.G.C., unpublished) (Lundell
and Hirsh, 1998; Thor and
Thomas, 1997
). Our data further indicate that robo2/3 are
not required in the formation of the serotonergic neurons from their
progenitor neuroblast 7-3. All serotonergic neurons in a robo2/3
mutant express eg-lacZ, even those with a loss of SerT expression
(Fig. 4E). This may at first
appear to contradict our result showing a loss of Eg at stage 16 in these
mutants, as eg expression must have occurred in order to produce
lacZ. We hypothesize that lacZ staining in stage 16
robo2 mutants is likely to be due to a lengthy persistence of
lacZ rather than continued expression of eg, as eg
mRNA is not detectable by in situ hybridization after stage 14
(Higashijima et al., 1996
).
Most probably, eg-lacZ expression in robo2/3
mutants occurred in the progenitors of serotonergic neurons when other
factors, such as engrailed (en), are known to control
eg expression (Dittrich et al.,
1997
; Matsuzaki and Saigo,
1996
). Even in eg mutants, all serotonergic neurons
continue to express eg-lacZ, despite a disruption in SerT,
serotonin synthesis and expression of Ddc
(Lundell and Hirsh, 1998
).
Thus, a robo2/3 mutant, like an eg mutant, does not affect
the early specification of serotonergic neurons, including early eg
expression, but instead affects later maturation.
Interestingly, we observe an effect of robo2/3, but not
robo, on serotonergic neuron differentiation. Disparities between
Robo and Robo2/3 function have been previously observed in the lateral
positioning of axons where only Robo2/3 appear to play a role
(Rajagopalan et al., 2000b;
Simpson et al., 2000a
), and in
dendritic guidance, synapse formation and midline crossing, where all three
Robo receptors have separable functions
(Godenschwege et al., 2002
;
Rajagopalan et al., 2000a
;
Simpson et al., 2000b
).
Furthermore, Robo2 and Robo3 show greater homology to each other than to Robo
(Rajagopalan et al., 2000b
;
Simpson et al., 2000b
).
Robo2/3 have cytoplasmic domains that diverge from Robo, and lack two motifs
considered important for Robo signaling
(Bashaw et al., 2000
;
Rajagopalan et al., 2000b
;
Simpson et al., 2000b
).
Possibly, Robo2 and Robo3 regulate a Robo-independent signaling cascade that
is critical for serotonergic neuron differentiation. Additionally, a loss of
slit, the ligand for all three Robo receptors, does not perturb SerT
expression, indicating that either another ligand exists or the function of
Robo2/3 in serotonergic neuron differentiation is ligand independent. In
C. elegans, some activities of the Robo homolog SAX-3 are thought to
be Slit independent (Hao et al.,
2001
).
The transmembrane protein Comm has been shown to negatively regulate the
levels of all three Robo receptors (Kidd
et al., 1998b; Rajagopalan et
al., 2000a
; Tear et al.,
1996
). After midline crossing, Comm expression decreases and Robo
levels increase in order to prevent inappropriate midline crossing. In
serotonergic neuron differentiation, Comm may play a role in regulating
Robo2/3, such that levels of both Robo2/3 increase following midline crossing
and thereby permit differentiation to proceed. To test this possibility, we
expressed Comm using egGal4 to specifically induce a loss of Robo2/3
in the serotonergic neurons. In our experiments, expression of Comm caused a
loss of SerT activity in only a few cells and with low penetrance (data not
shown). We believe that this is due to expression of Comm at levels
insufficient for total loss of Robo2/3. Alternatively, other regulators of
Robo2/3 may exist. However, neither a loss of Comm nor overexpression of
Robo2/3 results in precocious serotonergic neuron differentiation, indicating
a requirement for other signals.
robo2 functions with the transcription factor eagle to regulate serotonergic neuron differentiation
In both a robo2 and a robo3 loss-of-function mutant,
expression of the zinc-finger transcription factor eg is lost in the
same cells that lose SerT expression. Additionally, overexpression of Robo2
rescues the loss of SerT observed in an eg hypomorph in a
dose-sensitive manner. Finally, Eg gain of function rescues the SerT loss seen
in robo2 loss-of-function mutants. These results indicate that
Robo2/3 function in the same genetic pathway as Eg to control serotonergic
neuron differentiation. Although our results suggest that Robo2/3 regulate Eg
in stage 16 embryos, other genes such as en and hunchback
(hb) also have an established role in regulating Eg during
serotonergic neuron differentiation
(Dittrich et al., 1997;
Matsuzaki and Saigo, 1996
;
Novotny et al., 2002
). At
present, it remains unclear if Robo2/3 cooperate with these genes to regulate
Eg expression.
Additionally, in both robo2 and robo3 loss-of-function
mutants only a percentage of neurons lose SerT expression (and serotonin
synthesis), indicating the presence of a redundant mechanism for serotonergic
neuron differentiation. The pattern of SerT and serotonin loss in
robo2/3 mutants appears random and differs between nerve cords. At
this point, it remains unclear why differentiation is affected in only some
cells and not others, or what factors allow remaining cells to maintain normal
SerT expression. One possibility is that cells must maintain a threshold level
of Eg expression to differentiate properly. This is supported by differences
in the degree of SerT loss according to the severity of the mutation in
eg, as a hypomorphic allele displays a loss of SerT in 30% of
hemisegments while a null allele displays closer to 80% loss of SerT. Many
studies have also suggested that a combinatorial code of transcription factors
act to specify serotonergic properties
(Dittrich et al., 1997
;
Thor and Thomas, 1997
). First,
loss-of-function mutations in several genes required for differentiation,
including eg, en and hb show an incomplete loss of SerT
phenotype (Lundell et al.,
1996
; Lundell and Hirsh,
1998
; Novotny et al.,
2002
). Second, if Eg is inappropriately expressed throughout the
nervous system, only a few ectopic serotonin positive cells appear. These
ectopic serotonergic cells always express the transcription factor
hkb (Dittrich et al.,
1997
). Robo2 and Robo3 may also function redundantly. Further
studies should indicate the relationship of Robo2/3 to other genes
involved in serotonergic neuron differentiation, and the mechanism by which
Robo2/3 regulate Eg expression.
One question that readily follows from our observations is how does Robo2 influence Eg expression? Robo2 and Robo3 are cell-surface axon guidance receptors, while Eg is a transcription factor. It is likely that other factors interact with both Robo2/3 and Eg to mediate their roles in serotonergic neuron differentiation. Although their relationship remains obscure, data indicate that Robo2 may regulate Eg post-transcriptionally. In a series of real-time RT-PCR experiments, no difference in eg mRNA levels was detected when EP2582 (UAS-robo2) was expressed using egGal4, scabrousGal4 or elavGal4 (data not shown), suggesting that Robo2 is insufficient to induce ectopic Eg expression. However, when Robo2 is overexpressed, a rescue of Eg protein expression is seen in egMz360 hypomorphs (Fig. 6E). Through the same series of PCR experiments, we discovered that the egMz360 allele produces mRNA, although no Eg staining is observed. This suggests that the Gal4 insertion responsible for the egMz360 allele affects Eg protein expression, which in turn causes a disruption in SerT expression. Thus, expression of Robo2 appears to somehow rescue Eg protein expression in an egMz360 hypomorph sufficiently to rescue SerT activity. At this point, the mechanism of such a post-transcriptional rescue is unclear. Identifying the genetic and intracellular links between Robo2, Robo3 and Eg with more molecular approaches such as RNAi studies will probably reveal how Robo2/3 regulate not only Eg but eventually serotonergic neuron differentiation as well.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Office of Science Policy and Planning, NINDS, NIH,
Bethesda, MD 20892, USA
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