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First published online January 10, 2007
doi: 10.1242/10.1242/dev.02754
1 Graduate Program in Neurological Sciences, 1650 Cedar Avenue, Montreal, QC,
H3G 1A4, Canada.
2 Centre for Research in Neuroscience, 1650 Cedar Avenue, Montreal, QC, H3G 1A4,
Canada.
3 Department of Neurology and Neurosurgery, McGill University, 1650 Cedar
Avenue, Montreal, QC, H3G 1A4, Canada.
4 McGill University Health Centre Research Institute, 1650 Cedar Avenue,
Montreal, QC, H3G 1A4, Canada.
* Author for correspondence (e-mail: don.vanmeyel{at}mcgill.ca)
Accepted 22 November 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Drosophila, Glia, Neuron, Fringe, Notch, Delta, Prospero, Axon
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
During nervous system development in Drosophila, glial cells are
important for neuron survival and for the guidance, pruning and ensheathment
of axons; in return, neurons provide trophic support for glia, stimulate glial
proliferation and guide glial migrations
(Booth et al., 2000;
Chotard and Salecker, 2004;
Hidalgo and Griffiths, 2004;
Klambt et al., 2001). Although
progress has been made in understanding cellular aspects of neuron-glial
interactions, the molecular signals that regulate communication between
neurons and glial cells remain largely undetermined. In the ventral nerve cord
(VNC) of Drosophila, several glial classes have been defined based on
their morphology, position, function and additional embryologic and molecular
characteristics (Ito et al.,
1995). The longitudinal glia (LG) lie dorsal to the longitudinal
axon tracts of the VNC (Jacobs et al.,
1989). These axon tracts, called connectives, lie within a dense
neuropil enwrapped by plasma membrane from LG. The close association of LG
with the neuropil provides an excellent system to identify and characterize
neuron-glial signals in vivo.
LG are derived from a glioblast, which gives rise to 10-12 glial progeny
only (Griffiths and Hidalgo,
2004; Jacobs et al.,
1989; Schmidt et al.,
1997). Proliferation within this lineage is controlled in part by
Epidermal growth factor receptor (EGFR) signaling in response to an
axon-derived signal, Vein (Hidalgo et al.,
2001). Mitosis occurs in LG precursors that express the
transcription factor Prospero (Pros) at high levels
(Griffiths and Hidalgo, 2004).
Pros is thought to promote cell division through EGFR-dependent activation of
the MAPK pathway. Of the 10-12 LG at late stages of embryogenesis, only six
continue to express Pros.
Subclasses of glia such as LG are highly specialized in both form and
function, and probably arise from intrinsic genetic programs as well as
extrinsic cues experienced in part through contact with neurons. All glia in
Drosophila, except midline glia, are intrinsically specified by a
regulatory cascade of transcription factors including Glial cells missing
(Gcm), Tramtrack, PointedP1 and Reversed polarity (Repo), which act in concert
to promote glial-specific gene expression
(Jones, 2005). Targets of this
cascade encode the regulator of G-protein signaling Locomotion defects (Loco)
and the transcription factor Retained (Retn)
(Granderath et al., 1999;
Shandala et al., 2002;
Yuasa et al., 2003). However,
Loco and Retn are expressed in only restricted subsets of glia, including LG,
as are a number of other genes, including the Fibroblast growth factor
receptor Heartless (Htl), the transcription factor Distal-less and Pros. The
individual or combined activities of these and other factors are likely to
endow LG with their specialized morphological and functional properties.
Indeed, genetic mutants for retn exhibit defects of LG position and
reduced Loco and Pros expression, while mutants of loco or
htl each have defects in LG membrane morphology
(Granderath et al., 1999;
Shandala et al., 2003;
Shishido et al., 1997).
The restriction of gene expression to specific subsets of glia suggests
there may be context-dependent, locally derived regulators that direct aspects
of glial differentiation, including extrinsic molecular signals provided by
axons. The expression of Pros in only six LG, and its absence in the remainder
of the LG with which they share a common lineage, provided an opportunity to
identify and characterize local molecular signals that mediate the
differentiation of glial subtypes. We have found that expression of
fringe (fng) is restricted to a small population of cells in
the VNC that include a subset of LG. Fng is a ß-1,3-N-acetyl-glucosaminyl
(GlcNac) transferase that catalyzes the addition of GlcNac to O-linked fucose
monosaccharides on specific EGF repeats of the extracellular domain of the
Notch (N) receptor (Blair,
2000; Haltiwanger and Stanley,
2002). N is a single-pass transmembrane receptor that has two
known ligands in Drosophila, Serrate (Ser) and Delta (Dl). Fng
modulates signaling through N by reducing the sensitivity of N for Ser and
increasing its sensitivity for Dl (Bruckner
et al., 2000; de Celis and
Bray, 2000; Fleming et al.,
1997; Hicks et al.,
2000; Moloney et al.,
2000; Okajima et al.,
2003; Panin et al.,
1997). Only some of the many developmental events controlled by N
also involve Fng. In Drosophila, Fng is an important determinant of
boundary formation in the wing, eye and leg imaginal discs, and for specifying
polar cell fates during oogenesis (Cho and
Choi, 1998; de Celis et al.,
1998; Dominguez and de Celis,
1998; Grammont and Irvine,
2001; Irvine and Wieschaus,
1994; Klein and Arias,
1998; Rauskolb et al.,
1999; Rauskolb and Irvine,
1999). To date, modulation of N by Fng has not been implicated in
central nervous system (CNS) development in any organism, although Fng
orthologs are expressed in the developing brains of mice and fish
(Ishii et al., 2000;
Moran et al., 1999;
Qiu et al., 2004).
Here we show that Fng is required for subtype-specific Pros expression in LG. Pros expression can be triggered by N ligands derived from neurons but not glia, and this effect can be mimicked by direct activation of the N pathway within glia. N is expressed in LG while Ser and Dl are each restricted to unique subsets of neurons. Our genetic studies in vivo suggest that Fng sensitizes N on LG to axon-derived Dl and that neuron-glial communication through this ligand-receptor pair is required for the proper molecular diversity of glial cell subtypes in the developing nervous system.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
htl-Gal4 (Shishido et al.,
1997), repo-Gal4
(Sepp et al., 2001),
scrt11-6-Gal4 (Boyle et
al., 2006). UAS lines: UAS-fng
(Kim et al., 1995),
UAS-NICD (Go et al.,
1998), UAS-H (Maier
et al., 1999), UAS-Numb
(Yaich et al., 1998),
UAS-Ser (Speicher et al.,
1994), UAS-Dl
(Fleming et al., 1997),
UAS-mCD8::GFP (Lee and Luo,
1999), UAS-Tom (Lai
et al., 2000) and UAS-mycGFP, which has a nuclear
localization signal. Mutant alleles: Df(3L)riXT1 (fngDf),
fngL73, fng13 and fng80
(Correia et al., 2003;
Irvine and Wieschaus, 1994),
fngRF584 (Grammont and
Irvine, 2001), ser+r83k
(Hukriede et al., 1997),
serRx82 (Speicher et
al., 1994), serRev2-11
(Fleming et al., 1990),
locorC56 (Granderath
et al., 1999) here called loco-lacZ, and
DlRev10(Heitzler and
Simpson, 1991).
Immunohistochemistry
Embryos at stages 13-17 were collected at 25°C, dissected to reveal the
VNC, fixed in 4% paraformaldehyde, and stained according to standard
procedures. Antibodies from the Developmental Studies Hybridoma Bank: mouse
anti-prospero (dilution 1:25), mouse anti-BP102 (1:50), mouse C17.9C6 (anti-N,
1:50), rat anti-elav (1:50) and mouse C594.9B (
-Dl, 1:100). Other
antibodies used: rabbit anti-GFP (1:1000, Molecular Probes), rabbit
anti-ß-Gal (1:1000, MP Biomedicals), mouse anti-Gs2 (1:100, Chemicon),
and Cy2-conjugated goat anti-horseradish peroxidase (1:100, Jackson
ImmunoResearch). Rat anti-Ser polyclonal antiserum (1:1000) was a gift from K.
Irvine, and rabbit anti-Dll (1:100) was a gift from S. Carroll.
In situ hybridization
In situ hybridization was performed in whole mount on embryos collected
4-16 hours after egg laying. A digoxigenin-labeled cRNA riboprobe (Roche) was
made from the fng cDNA RE03010. Hybridization was done overnight at
55°C, and visualized using -Dig-AP (1:1000; Roche). For
fluorescence in situ hybridization, anti-Dig-POD (1:100; Roche) and Cy-3
conjugated tyramide reagent were used for probe detection (1:50;
Perkin-Elmer).
Microscopy and imaging
Confocal microscopy was performed using a Yokogawa spinning disk confocal
system (Perkin-Elmer) and an Eclipse TE2000-U microscope (Nikon).
Z-series images were collected using Metamorph software (Molecular
Devices). To better visualize anti-N in LG, the images in
Fig. 3A-F were processed for
3D-deconvolution with Autodeblur software (Autoquant), and 3D-rendering of
stacks was performed using Imaris software (Bitplane AG). Images were compiled
with Adobe Photoshop.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). There are 10-12 LG in each hemisegment
(Griffiths and Hidalgo, 2004),
8-11 of which can be readily counted in Htl-nGFP-expressing embryos
(Fig. 1D). The uncounted LG are
either obscured by other Htl-nGFP cells or occasionally fail to express GFP.
Interestingly, the LG can be further divided into an anterior and posterior
subpopulation. The transcription factor Pros is expressed in the six
anteriormost LG, which are positioned along the connectives, mostly in the
interval between the anterior and posterior commissures
(Fig. 1E,F). With confocal
imaging we found that, in a compressed stack of Z-series images, some
hemisegments appear to show only five Pros-positive LG, because the sixth is
obscured by another. We used in situ hybridization to determine the spatial and temporal expression pattern of fng transcripts in the VNC. Beginning at stage 14-15, fng mRNA was observed in two continuous stripes of cells that lay dorsal to the longitudinal connectives (not shown). This pattern was reminiscent of mature LG, although expression of fng transcript was not observed in the longitudinal glioblast at earlier stages of development, nor in migrating LG precursors. In older embryos, the two rows of fng-positive cells were somewhat discontinuous (Fig. 1G), and expression persisted to stage 17. To confirm whether fng mRNA is expressed in LG, fluorescence in situ hybridization (FISH) was performed (Fig. 1H), and embryos co-labeled with the markers Repo (Fig. 1I) and Pros (not shown). There was clear enrichment of fng labeling surrounding nuclei of the anterior LG. We cannot rule out the possibility that fng transcripts were expressed at very low levels by posterior LG and perhaps a few other cells near the neuropil, consistent with our observations of embryos carrying a lacZ P-element insertion in the fng locus (fngRF584, not shown). Together, the results indicate that fng expression probably begins in all LG after their final division, and becomes progressively enriched in the anterior LG.
Fng is necessary for the correct expression of Pros in LG
Expression of fng in the Pros-expressing LG prompted us to test
whether Pros expression was affected in LG of fng mutant embryos.
Four different fng alleles were studied: fngL73
and fng13 each contain distinct single base-pair mutations
resulting in a premature stop codon, while fng80 and
fngDf are small and large deletions of the fng
locus, respectively (Correia et al.,
2003). In wild-type animals, fng heterozygotes
(Fig. 2A) and fng
hemizygotes (Fig. 2G,J), 5-6 of
the 10-12 LG express Pros in nearly 100% of hemisegments. We tested the
fng alleles in various combinations and found significant losses of
Pros-positive LG (Fig. 2D,H,J),
indicating a recessive defect. Depending on the allelic combination, between
38 and 62% of hemisegments in fng mutants had fewer than five
Pros-positive LG, with an average of 50% (299/597) for all combinations
(Fig. 2J). Of the hemisegments
that exhibited a defect, 53% were reduced to three Pros-positive LG or fewer.
Among the LG that remained Prospositive, the intensity of Pros
immunoreactivity was often reduced in fng mutants relative to wild
type (Fig. 2D,H). In each
hemisegment, Pros is also expressed by a small cluster of neurons
(Doe et al., 1991;
Vaessin et al., 1991). Pros
expression in these neurons was unaffected in fng mutants, indicating
that the effect was specific for LG (Fig.
2D,H).
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The evidence is consistent with the idea that fng is necessary for
maintenance, not initiation, of Pros expression in LG. First, fng
expression begins at stages 14-15 of embryonic development, whereas Pros
expression in LG precursors begins at the two-cell stage of the lineage, at
stage 11-12 (Griffiths and Hidalgo,
2004). Second, although Pros expression was markedly reduced in
fng mutants, low levels could still be observed in five to six cells
in many hemisegments, suggesting Pros expression could be initiated in many
instances, but not maintained.
To demonstrate the specificity of the fng mutant phenotype for glia, repo-Gal4 was used to drive the expression of a UAS-fng transgene in fng mutants (fng13/fngDf). Pros expression was restored to levels comparable to wild type in the anterior LG (Fig. 2I). The rescue was nearly complete; 89% of hemisegments had five to six Pros-positive anterior LG, and the remaining 11% had four (Fig. 2J). In rescued fng mutants, ectopic Pros-positive cells were rarely observed. However, in a wild-type background, they did appear occasionally (not shown), suggesting that fng is both necessary and sufficient for Pros expression in LG. The sufficiency of Fng for Pros expression will be addressed again below.
The expression of Notch and its ligands at late stages of embryonic VNC development
Previous studies have demonstrated that Fng acts cellautonomously on
nascent N polypeptides within the Golgi
(Bruckner et al., 2000;
Munro and Freeman, 2000).
Therefore, we surmised that Notch should be expressed on LG. Upon
immunostaining embryos at stages 15-17, N was found to be expressed on the
surface of most cell bodies in the VNC and was broadly distributed throughout
the neuropil (Fig. 3A),
consistent with previous studies (Fehon et
al., 1991). Due to this robust expression, and the close
apposition of LG with axons, it was difficult to assess whether N was
expressed in LG. To address this, N expression was examined in embryos that
expressed a membrane-targeted GFP reporter in LG (Repo-Gal4,
UAS-mCD8GFP), and co-stained with antibodies to detect GFP and HRP to
reveal glia and axons, respectively. In optical sections dorsal to the
longitudinal connectives, N co-localized with LG membrane
(Fig. 3B,C). In cross-section,
N was localized primarily to the ventral side of LG (closest to the neuropil),
and to contact sites between adjacent glia
(Fig. 3B',C'). In
addition to LG, N was also found in other glia, and N overlapped with HRP in
the dorsal neuropil (Fig. 3F).
Our results show that N is indeed expressed in LG, where its sensitivity to
ligand could be modulated by Fng.
Dl/Ser ligands are transmembrane proteins that activate N in a contact-dependent manner. If these ligands are required for N activation in LG, one would expect them to be expressed on cells that neighbor the LG or on neurons that project axons that contact LG. Immunochemistry for Dl revealed that at stage 15-17, Dl expression was restricted to a cluster of roughly 20-30 cells with somata that were located at the lateral edge of each hemisegment (Fig. 3G,H), approximately in the middle of the dorsoventral axis. All these cells appeared to be neurons projecting axons in two fascicles, one through each of the two commissures (Fig. 3H). Interestingly, we observed that Dl immunoreactivity was dramatically reduced where these axons met the longitudinal axon tracts and LG (Fig. 3H).
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). We
found that Ser expression was restricted to two or three commissural
interneurons per hemisegment. Each cell extended an axon through either the
anterior or posterior commissure (Fig.
3I), which then projected anteriorly within the longitudinal
connectives. In summary, both Dl and Ser were expressed in restricted populations of neurons in the embryonic VNC but were not observed in LG. By contrast, N was more broadly distributed and exhibited polarized subcellular localization within LG. These findings are consistent with the possibility that expression of Dl or Ser ligands on axons could activate N signaling in LG. Expression of Fng in a subset of glia might confer differential sensitivity to N ligands, and could underlie the acquisition of distinct properties in response to different levels of N signaling.
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;
Schweisguth, 2004). In the
canonical signaling pathway, the N receptor is cleaved upon ligand-binding,
releasing the intracellular domain, NICD. NICD is
transported to the nucleus, where it binds Suppressor of Hairless [Su(H)]. In
the absence of NICD, Su(H) is part of a repressor complex that
includes Hairless (H). NICD replaces H, binds Su(H) and recruits
another co-activator, mastermind, leading to the activation of target gene
transcription. Fng can either increase or decrease activity through the Notch pathway by modulating the sensitivity of the N receptor to ligand. Fng renders N more sensitive to activation by Delta, and less sensitive to activation by Serrate. Therefore, it is possible that the loss of Pros seen in fng mutants is due either to hypo-activity or hyper-activity of the N signaling pathway, depending upon whether the relevant ligand is Dl or Ser, respectively. To test these alternatives, we examined the effects of either inhibiting or activating the Notch pathway specifically in LG.
To inhibit the N pathway, htl-Gal4 was used to express two
different N antagonists: the Hairless (H) transcriptional repressor
(Morel et al., 2001) and Numb,
which promotes endocytosis of N and limits exposure to ligand
(Berdnik et al., 2002;
Jafar-Nejad et al., 2002).
Expression of UAS-H in LG resulted in a near complete loss of Pros
expression (Fig. 4A).
Seventy-eight percent of hemisegments failed to express Pros at all, and none
expressed the normal number of five to six
(Fig. 4J). The overall number
or position of LG was unaltered (Fig.
4B). This result suggests that Pros expression in LG is regulated,
either directly or indirectly, through canonical Notch signaling involving
transcription activation. Expression of UAS-Numb was also a potent
inhibitor of Pros expression (Fig.
4C,J) (Griffiths and Hidalgo,
2004).
To determine whether the effect of N inhibition was specific for Pros
expression, we sought markers other than Pros that distinguish the anterior LG
from posterior LG. Glutamine synthetase 2 (Gs2) has been shown previously to
be expressed in LG (Freeman et al.,
2003). With in situ hybridization, we found Gs2 transcript limited
to the anterior six LG in each hemisegment in stage 16-17 embryos (not shown).
Immunochemistry with an anti-Gs2 antibody confirmed this specific pattern of
expression (Fig. 4D,E).
Glutamine synthetase is expressed in glia and converts the neurotransmitter
glutamate into glutamine, which is then transported back into neurons. The
limited expression of Gs2 in the Pros-positive anterior LG indicates that they
are indeed functionally distinct from the Prosnegative posterior LG.
Inhibition of N signaling by misexpression of Numb with htl-Gal4
caused no changes in Gs2 expression (Fig.
4F), consistent with the idea that Pros expression is specifically
regulated by N signaling, that Gs2 is regulated independently, and that
inhibition of N in this context does not simply convert anterior LG to
posterior LG.
Is N activity sufficient to induce Pros expression in LG that do not
normally express it? To test this, htl-Gal4 was used to express a
constitutively active form of N (UAS-NICD).
NICD was indeed sufficient to drive Pros in most of the posterior
LG, and sometimes all of them, without altering their number or their general
positioning (Fig. 4G,H),
confirming the results of others
(Griffiths and Hidalgo, 2004).
We confirmed that the ectopic Pros-positive cells were indeed LG, as they
coexpressed Distal-less, a transcription factor expressed exclusively in LG
and only one additional cell (not shown). Together with the results of N
inhibition, these data indicate that activation of Notch signaling promotes
the maintenance of Pros expression in the anterior LG. As the fng
mutant phenotype resembled that caused by N inhibition, it is likely that Fng
increases N activity in the anterior LG. As fng transcripts were
enriched in the anterior LG, but not in posterior LG, we reasoned that perhaps
Pros is not expressed in posterior LG because Fng is not there to heighten the
sensitivity of these cells to ligand. Consistent with this idea, expression of
UAS-fng with htl-Gal4 was sufficient to promote
ectopic expression in posterior LG (Fig.
4J, Fig. 5A).
Pros expression is upregulated in glia upon panneuronal misexpression of Dl or Ser
What factors in addition to Fng might limit N-dependent expression of Pros
to the anterior LG? If the restricted expression of Dl on subsets of axons
were to limit N activation, we predicted that misexpression of Dl in all axons
would lead to excessive N activation and ectopic Pros expression. To test
this, a UAS-Dl transgene was misexpressed using
elavC155-Gal4, which drives expression in all
postmitotic neurons but is not expressed earlier in neuronal lineages and is
not expressed in glia (Lin and Goodman,
1994). Misexpression of Dl did indeed lead to ectopic expression
of Pros (Fig. 5B).
Interestingly, co-labeling for Distal-less revealed that the effect was
specific for the posterior LG. In posterior LG, one to three extra
Pros-positive cells were evident in all hemisegments examined
(Fig. 4J,
Fig. 5B,C).
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-HRP
(Fig. 5B) and -BP102
immunostaining (not shown).
As a control, we tested the effects of misexpression of Dl in LG rather
than neurons. By contrast to neuron-derived Dl, which increased Pros
expression, glial-derived Dl suppressed Pros expression and mimicked N
inhibition (Fig. 5D). This
result could be a consequence of cis-inhibition of N signaling in LG by
coexpression of Dl. Cis-inhibition of N is a phenomenon that has been observed
previously, but the mechanism by which it operates remains poorly understood
(de Celis and Bray, 2000;
Jacobsen et al., 1998;
Ladi et al., 2005;
Sakamoto et al., 2002).
These results confirm that Notch ligands derived from neurons, but not glia, are capable of driving ectopic Pros expression specifically in posterior LG. We next asked whether Dl or Ser, like Fng, were required for the maintenance of endogenous Pros expression in the anterior LG. As Fng is known to sensitize N to the ligand Dl, and as Fng function in the anterior LG correlates with N activation and not N inhibition, we hypothesized that Dl, and not Ser, was the relevant ligand. We first tested trans-heterozygotes for fng and Dl and found no reduction of Pros expression (not shown). Homozygous Dl mutants could not be studied because of the earlier roles Dl and N have in neurogenesis, whereby certain cells of the developing neurectoderm adopt neural fate while inhibiting others from doing the same. Instead, we tested whether the loss of Pros expression observed in fng mutants could be further reduced by simultaneous reduction of Dl. Indeed, fng mutants heterozygous for Dlrev10 had dramatically lower levels of Pros than did fng mutants alone (Fig. 2J, Fig. 5E), with 93% of hemisegments failing to express the correct number of five to six Pros-positive LG, and 82% with three or fewer. To test whether Ser was required, Pros expression was examined in Ser mutants (Serrev2-11/SerRx82), which was feasible because Ser is not required for neurogenesis. Pros expression was unaffected in Ser mutants (Fig. 2J, Fig. 5F), suggesting either that Ser has no function in Pros expression or that it is redundant with Dl. However, given that Fng renders cells less sensitive to N activation by Ser and more sensitive to Dl, we believe that Ser is unlikely to play a role and that Dl is the N ligand important for the maintenance of Pros in anterior LG.
N signaling in LG through neuron-glia interactions
The data suggested that Dl-bearing axons could activate N signaling in LG.
In addition, Dl immunoreactivity was dramatically reduced at the position
where these axons intersect with the longitudinal axon tracts and LG. It is
possible that this reduction in Dl immunoreactivity resulted from increased
endocytosis of Dl in the vicinity of the neuropil. Endocytosis of Dl into
ligand-bearing cells is a crucial step in reception of the N signal by
N-expressing cells (Le Borgne et al.,
2005a). Dl endocytosis is promoted by the activity of the E3
ubiquitin ligases Neuralized and Mind bomb
(Lai et al., 2001;
Lai et al., 2005;
Le Borgne et al., 2005b;
Pavlopoulos et al., 2001;
Pitsouli and Delidakis, 2005;
Wang and Struhl, 2005).
Neuralized is inhibited by interactions with members of the Brd class family
of proteins (Bardin and Schweisguth,
2006; De Renzis et al.,
2006). Brd proteins block Neuralized from interactions with Dl and
prevent Dl endocytosis, thereby inhibiting N signaling in a
non-cell-autonomous manner. To examine whether N signaling in LG occurred
through neuron-glia interactions, we used the Brd protein Twin of m4 (Tom) to
block Dl endocytosis in neurons, and examined the effects on Pros expression
in LG. As the population of Dl-expressing neurons remains poorly defined, and
there are no Gal4 lines specific for these cells, we used two alternatives.
The first, elavC155-Gal4, expresses in all postmitotic
neurons, whereas the second, scrt11-6-Gal4,
expresses at moderate levels in most, perhaps all, neuroblasts, ganglion
mother cells and postmitotic neurons. Misexpression of Tom with
elavC155-Gal4 had a mild effect on Pros, with 13% of
hemisegments showing fewer than five to six Prospositive cells (not shown).
Due to the inherent delay of the GAL4-UAS system, perhaps
elavC155-Gal4 activated the expression of
UAS-Tom too late to strongly inhibit N activation upon axon-glial
contact. By contrast, misexpression of UAS-Tom with
scrt11-6-Gal4 caused 55% of hemisegments to
exhibit fewer than five to six Prospositive cells
(Fig. 4J,
Fig. 5G). There was increased
expression of Dl on neuron cell bodies and axons (not shown), consistent with
inhibition of Dl endocytosis. Immunoreactivity was still reduced on distal
portions of axons, perhaps reflecting incomplete inhibition of Dl endocytosis.
This may account for milder effects caused by Tom misexpression in neurons
than those caused by direct blockage of N signaling in LG using H or Numb
(Fig. 4A,C,J). Nevertheless,
our finding that inhibition of Dl endocytosis in neurons reduced Pros
expression in LG provides strong evidence in vivo for Dl-N signaling through
neuron-glial interactions.
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During differentiation of neuronal lineages, N acts to distinguish
asymmetric and unique fates of sibling cells. As
scrt11-6-Gal4 was used to express Tom in neuronal
lineages, it had the potential to inhibit Dl endocytosis, influence N
signaling and cause defects of sibling cell fate determination. If axon
guidance defects arose as a consequence of altered cell fates, they could
interrupt neuron-glial interactions and thereby indirectly influence Dl-N
signaling. Although the cell bodies and axon projections of the Dl neurons
appeared normal (not shown), as did axon patterning in general
(Fig. 5G), we looked for
additional evidence that N-dependent cell fate decisions were unaffected. We
tested whether overexpression of Tom with
scrt11-6-Gal4 caused defects in cell fate by
studying the expression of Even skipped (Eve). The cell fates of the RP2
motoneuron and its sibling (RP2sib), in addition to other neurons that are
also distinguishable by anti-Eve immunochemistry, are dependent on N signaling
(Doe et al., 1988;
Frasch et al., 1988;
O'Connor-Giles and Skeath,
2003), and can be used to indicate whether N-dependent cell fate
decisions in neuronal lineages are intact. For example, when the strongly
expressing scabrous-Gal4 was used to misexpress Tom in
neural lineages, losses of Eve expression in RP2 neurons were observed (not
shown). By contrast, overexpression of Tom with
scrt11-6-Gal4 caused no changes in Eve expression: RP2 and
other Eve-positive neurons such as the U neurons were all present in normal
numbers and positions (Fig.
5H). Together, the data suggest that the timely inhibition of Dl
endocytosis in neurons can specifically block N signaling and Pros expression
in LG.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
The role of Pros in mature LG is poorly understood, but it has been proposed
to retain mitotic potential in these cells for use in repair or remodeling of
the nervous system in subsequent larval or adult stages
(Griffiths and Hidalgo, 2004;
Hidalgo and Griffiths, 2004).
It will be important to determine the consequences of lost Pros expression
from mature anterior LG, and whether additional features and functions of the
anterior LG are controlled by N signaling from axons.
The importance of glycosylation for N function has been demonstrated in
vivo. The addition of O-linked fucose to EGF repeats in the N extracellular
domain is essential for all N activities and is mediated by
O-fucosyltransferase-1 (O-fut1) (Okajima
and Irvine, 2002; Sasamura et
al., 2003; Shi and Stanley,
2003). By contrast, Fng is selectively used in specific
developmental contexts, and has been best studied in the formation of borders
among cells in developing imaginal tissues
(Cho and Choi, 1998;
de Celis et al., 1998;
Dominguez and de Celis, 1998;
Irvine and Wieschaus, 1994;
Klein and Arias, 1998;
Panin et al., 1997;
Rauskolb et al., 1999;
Rauskolb and Irvine, 1999).
Fng catalyzes the addition of GlcNac to O-linked fucose, to which galactose is
then added. The resulting trisaccharide is the minimal O-fucose glycan to
support Fng modulation of Notch signaling
(Haltiwanger and Stanley,
2002). Fng activity reduces the sensitivity of N for the ligand
Ser but increases its sensitivity for Dl. By contrast with imaginal discs, in
which modulation of N sensitivity to both ligands appears to be important,
loss of Fng in LG resulted in reduced N activation only, consistent with
reduced response to Dl. Expression of Pros in LG can be triggered by Dl
derived from neurons but not glia, and this effect can be mimicked by direct
activation of the N pathway within glia. Our genetic experiments implicate
neuron-derived Dl as the relevant N ligand for Pros expression in anterior LG,
consistent with the ability of Fng to sensitize N to signaling by Dl. Enriched
Fng expression in the anterior LG probably renders them differentially
sensitive to sustained N signaling from Dl-expressing axons.
The final divisions of the six LG precursors that give rise to 12 LG are
thought to be symmetric, with low levels of Pros first distributed evenly
between sibling cells after division. However, Pros is maintained and in fact
upregulated in the anterior LG, and downregulated in sibling LG that migrate
posteriorly (Griffiths and Hidalgo,
2004). We observed that fng transcripts first appear to
be expressed in all LG, then become enriched in the anterior LG and reduced in
the posterior LG. We speculate that refinement of fng expression may
involve a positive feedback mechanism to consolidate and enhance N signaling
in the anterior LG, as we have preliminary evidence to suggest that N
signaling can positively influence fng expression in the LG (G.B.T.,
D.J.vM. and Jennie Yang, unpublished).
Like Pros, Gs2 is specifically expressed in the anterior LG but not posterior LG, indicating that these are functionally distinct glial subtypes with respect to their ability to recycle the neurotransmitter glutamate. The specificity of N signaling for Pros but not Gs2 indicates that N signaling is unlikely to influence cell fate decisions in the LG lineage and that Fng is unlikely to be the primary determinant of anterior versus posterior LG identity. Rather, Fng probably serves to consolidate this distinction through sustained N signaling.
NICD was a potent activator of Pros expression in the posterior LG. This has led us to consider what factors limit Pros expression to the anterior LG in wild-type animals, as posterior LG are indeed capable of expressing Pros in response to constitutive N activity. First, based on our analysis of fng mutants and Fng misexpression, we propose that Fng is a major determinant. Our finding that misexpression of Fng causes ectopic Pros in posterior LG supports the argument that Dl-expressing axons do not contact the anterior LG only. It is likely that they make contact with at least some of the posterior LG. Therefore, in wild-type animals, in which Fng is reduced on posterior LG, contact from the subset of Dl axons is alone not sufficient to drive Pros expression. Second, misexpression of Dl in all postmitotic neurons led to ectopic expression of Pros in posterior LG, indicating that the restricted expression of Dl on a subset of neurons also limits N activation. Third, N appears to be expressed in most or all LG, though we have also found that overexpression of full-length N caused ectopic expression of Pros (not shown). From these data we propose a threshold model for N activation in LG that invokes a combination of factors, including Fng-regulated N sensitivity, exposure of N to ligand, N expression levels, and perhaps others. Increasing any of these factors can provide sufficient signaling for ectopic Pros induction in posterior LG. In wild-type embryos, these factors are also likely to combine with one another in the anterior LG to achieve supra-threshold N signaling and sustained Pros expression during normal development.
Signaling through N is important for glial cell development in
Drosophila, although it is context-dependent. Both an embryonic
sensory lineage and the subperineurial CNS glial lineage utilize N activation
to promote Gcm expression and glial fate
(Udolph et al., 2001;
Umesono et al., 2002). By
contrast, in the sensory organ of adult flies, antagonism of N leads to Gcm
expression in the glial precursor cell
(Van De Bor and Giangrande,
2001). In vertebrates, signaling through Notch receptors promotes
the differentiation of peripheral glia
(Morrison et al., 2000),
astrocytes (Ge et al., 2002;
Grandbarbe et al., 2003;
Tanigaki et al., 2001),
Müller glia (Bernardos et al.,
2005; Furukawa et al.,
2000), Bergmann glia (Eiraku
et al., 2005; Lutolf et al.,
2002; Weller et al.,
2006), radial glia (Dang et
al., 2006; Gaiano et al.,
2000; Yoon et al.,
2004), oligodendrocyte precursors
(Grandbarbe et al., 2003;
Park and Appel, 2003) and
mature oligodendrocytes (Hu et al.,
2003). An Fng ortholog, lunatic fringe, is expressed in the
developing mouse brain in a pattern consistent with glial progenitors
(Ishii et al., 2000). It will
be interesting to determine whether Fng-related proteins in vertebrates have a
role in glial cell differentiation, and whether they too can modulate N
sensitivity and the context of N signaling between neurons and glia.
|
ACKNOWLEDGMENTS |
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REFERENCES |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
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