Institut für Neurobiologie, Universität Münster, Badestrasse 9, D-48149 Münster, Germany
Accepted 24 February 2004
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SUMMARY |
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Key words: Schizo, Slit, Endocytosis, Drosophila, Midline crossing, GEF
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Introduction |
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Over recent years our knowledge of the molecular mechanisms underlying this
process in a number of species has greatly advanced
(Grunwald and Klein, 2002;
Yu and Bargmann, 2001
). An
evolutionarily conserved family of proteins called Netrins mediates the
initial attraction of growth cones toward the midline. Netrins are expressed
by the CNS midline cells and are secreted into the extracellular space, where
they presumably form a gradient that guides commissural growth cones toward
the CNS midline (Kennedy et al.,
1994
; Serafini et al.,
1994
). In netrin mutants no attractive signal is
generated and the majority of axons fails to cross the midline in the first
place (Hamelin et al., 1993
;
Harris et al., 1996
;
Hedgecock et al., 1990
;
Mitchell et al., 1996
;
Serafini et al., 1996
). A
similar mutant phenotype is displayed by animals lacking the Netrin receptor
Unc40/Dcc/Fra (Fazeli et al.,
1997
; Keino-Masu et al.,
1996
; Kolodziej et al.,
1996
). As soon as the midline has been crossed, the same Netrin
signal can be interpreted as a repulsive signal by expression of an Unc40/Unc5
heterodimeric receptor (Hong et al.,
1999
; Leonardo et al.,
1997
).
Beside Unc5, which mediates repulsion in response to Netrin, there is a
second repulsive system operating at the CNS midline. In Drosophila,
this repulsive signal is encoded by the gene slit
(Kidd et al., 1999). It
ensures that ipsilateral projecting growth cones never cross the midline and
keeps axons that have crossed the midline once away from the midline. In
homozygous slit mutants, attractive signals predominate and all axons
collapse at the CNS midline (Rothberg et
al., 1988
). Slit is a member of the Leucin Rich Repeat (LRR)-type
family of secreted proteins. It contains four LRR type repeats, six epidermal
growth factor (EGF)-like motifs and a C-terminal cystein-rich region and is
proteolytically cleaved (Brose et al.,
1999
; Brose and
Tessier-Lavigne, 2000
;
Rothberg and Artavanis-Tsakonas,
1992
; Rothberg et al.,
1990
). Like Netrin, Slit is thought to be secreted into the
extracellular space, but direct evidence supporting this notion is lacking.
However, at least the C-terminal portion of the Slit protein is detected as
being fixed to membranes of the secreting midline cells only. The nature of
the Slit receptor expressed by the midline glia is unclear but may involve
heparane sulfate (Hu,
2001
).
The prime function of Slit is to mediate repulsive growth
(Kidd et al., 1999). However,
in a number of cases Slit can act as an attractive guidance cue, too
(Englund et al., 2002
;
Kramer et al., 2001
;
Schimmelpfeng et al., 2001
).
The different functions of Slit are mediated by a conserved family of
transmembrane receptors related to the Roundabout protein
(Battye et al., 1999
;
Brose et al., 1999
;
Kidd et al., 1999
;
Kidd et al., 1998a
;
Li et al., 1999
).
Drosophila possesses three different roundabout genes,
robo, leak (also called robo2) and robo3, which are
expressed in subsets of CNS neurons. The combined action of these
robo genes provides a `Robo-code', which helps to establish the
organization of discrete axon fascicles in the longitudinal connectives
(Rajagopalan et al., 2000a
;
Rajagopalan et al., 2000b
;
Simpson et al., 2000a
;
Simpson et al., 2000b
). Netrin
and Slit signaling do not act independently of each other, and signaling
pathways intersect (Stein and
Tessier-Lavigne, 2001
).
An alternative way to regulate the repulsive signaling activity is to
modulate the cell surface expression of the Roundabout receptors. In
Drosophila this function is mediated by commissureless.
commissureless mutants are characterized by a complete loss of
commissures resulting from an overactivation of the repulsive signaling
(Georgiou and Tear, 2002;
Keleman et al., 2002
;
Kidd et al., 1998b
;
Tear et al., 1996
).
Commissureless, for which no clear vertebrate homologs have been described
yet, helps to target the Roundabout receptor into the endosome. Thus in
commissureless mutants an excess of Robo is secreted into the cell
membrane, resulting in an exaggerated repulsive signaling
(Keleman et al., 2002
;
Myat et al., 2002
).
Pan-neural expression of Slit leads to a roundabout phenocopy.
Enhanced expression in the midline, however, does not lead to prominent
phenotypes and it had been suggested that efficient post-transcriptional
mechanisms tightly regulate the levels of secreted Slit
(Battye et al., 1999;
Kidd et al., 1999
). Assuming
that such mechanisms are needed to downregulate membrane-bound Slit, either by
increasing endocytosis or decreasing the rate of membrane transport of Slit,
we might expect reduced CNS midline crossing due to an enhanced expression of
the repulsive signal.
A number of extensive phenotypic screens for mutations affecting CNS axon
pattern formation were conducted in Drosophila
(Hummel et al., 1999a;
Seeger et al., 1993
). Beside
mutations in frazzled, slit, roundabout and commissureless
(Seeger et al., 1993
) we have
identified mutations only in two additional genes, weniger and
schizo (siz FlyBase), which showed a prominent loss
of commissural axon tracts (Hummel et al.,
1999a
). netrin schizo double-mutant analyses indicated
that schizo acts in a pathway parallel to netrin directing
commissural growth cones toward the midline or that schizo functions
to repress the repulsive signaling originating from the CNS midline
(Hummel et al., 1999a
).
Here we report a further characterization of the schizo mutant CNS phenotype and demonstrate that schizo acts in the CNS midline glial cells. schizo mutant embryos are characterized by increased Slit activity, whereas overexpression of schizo in the CNS midline cells leads to a mild slit phenocopy. schizo encodes a protein homologous to vertebrate Arf6-GEF proteins, which have been shown to regulate membrane dynamics. Expression of a dominant negative Dynamin protein in the CNS midline cells leads to a block of endocytosis in these cells. Concomitantly a schizo phenocopy develops, supporting the model that the level of Slit expression is regulated by endocytosis.
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Materials and methods |
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Sema2b-myc flies were provided by B. Dickson, Wien;
UAS-netrinB flies were provided by C. Goodman, Berkeley;
UAS-shibireDN flies were obtained from the Bloomington
Stock Center; the loner alleles T1032 and T1015 were provided by E.
Olson.
The P-element EP(3)3375 is inserted in the 78A/B interval about 20 kb
upstream of schizo and complements schizo. The
P-element-induced schizo allele P224 was generated by local
hopping experiments (Tower et al.,
1993).
Histology
Antibody staining was performed as described
(Hummel et al., 1999a). Mab
BP102 was obtained from the Hybridoma bank in Iowa. A polyclonal
ß-galactosidase antibody (Cappel) was used to visualize balancer carrying
embryos and the AA142 expression in midline glia cells. In-situ hybridization
was performed according to Tautz and Pfeifle
(1989
) using a
digoxigenin-labeled RNA probe of the LP01489 cDNA.
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Results |
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|
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schizo interacts with netrin and slit
The most prominent function of schizo is its role in commissure
development. Two major signaling cascades are known to control axonal growth
across the midline. They are initiated by the signaling molecules Netrin and
Slit, which are both secreted by the CNS midline glial cells in the
Drosophila embryo (Battye et al.,
1999; Harris et al.,
1996
; Kidd et al.,
1999
; Mitchell et al.,
1996
).
First genetic interaction studies of schizo and frazzled
or schizo and netrin function demonstrated a much stronger
commissural phenotype in double mutants than embryos mutant only for
schizo, frazzled or netrin
(Hummel et al., 1999a)
(Fig. 2). The commissural
phenotypes of the double-mutant embryos suggest that schizo is not
acting within the Netrin signaling pathway but may be required for a
Netrin-independent attractive pathway. Alternatively, schizo may be
necessary for suppressing the perception or the generation of a repulsive
signal normally generated by the CNS midline cells.
|
However, since genetic data indicated that schizo and
netrin act independently and netrin RNA expression was found
to be normal in schizo mutants (data not shown), one might speculate
that a reduced repulsive function could compensate for the loss of
schizo, too. The main axonal repulsive signal is encoded by
slit. Slit is an LLR protein secreted by the CNS midline glial cells
(Rothberg et al., 1990). When
schizo function is normally required to downregulate repulsive
signaling, either by affecting the generation of active Slit protein or by
preventing signaling in the commissural growth cones, the mutant
schizo phenotype could be explained by an upregulated Slit signaling.
Thus, one might expect that the schizo commissural phenotype could be
suppressed by a concomitant reduction in the dose of slit function.
We have generated slit-/+;
schizo/ as well as
robo-/+; schizo/
embryos, and in both cases observed a suppression of the schizo CNS
phenotype (Fig. 3D-G). Thus
schizo might be required to negatively regulate Slit signaling.
If schizo is indeed a negative regulator of slit function, we may expect that an increase of schizo gene dose should result in a decrease of active Slit signaling. One might thus be able to enhance the mutant slit phenotype by using a schizo gene duplication. Following mapping of schizo to the base of the left arm of chromosome 3 we utilized a chromosomal translocation of the corresponding part of the third chromosome to the Y chromosome (Tp(3;Y)A81) to generate embryos with three copies of schizo. In an otherwise wild-type background, this triplication of the schizo region did not result in an abnormal CNS phenotype (data not shown). However, when we placed the schizo translocation in a heterozygous slit/+ background, we observed a slit-like phenotype that was never detected in heterozygous slit embryos (Fig. 3H).
schizo encodes an Arf-GEF
To further understand the function of schizo, we mapped the
schizo locus to the chromosomal interval 78A/B using a series of
deficiency chromosomes between the genes poils aux pattes and
knockout (see Materials and methods). To identify the schizo
gene in this chromosomal interval we used P-element-induced schizo
alleles. The chromosomal insertions of the P-elements in l(3)3
(kindly provided by A. Carpenter) and P224 (generated in a local
hopping experiment; see Materials and methods) were determined by inverse PCR
and Southern analyses and suggested that schizo corresponds to CG
32434 (Fig. 4A). The
lethality associated with the P-element-induced l(3)3 schizo mutation
could be reverted by precise excision of the P-element and mutant
l(3)3 embryos displayed a schizo phenotype with reduced
commissures and defective fasciculation in the longitudinal connectives (not
shown). Subsequent sequencing of cDNA clones LP01489, RE44556 and GH10594
isolated by the BDGP showed that the schizo locus encompasses 41 kb
of genomic DNA. At least two different promoters direct the expression of two
isoforms of 1325 amino acids (SchizoP1) and 1313 amino acids (SchizoP2) in
length. Verification of the cloning of schizo was obtained by genetic
rescue experiments (see below). These deduced schizo proteins
correspond to the Iso1 and Iso2 variants of the loner gene, which was
recently identified in a screen for mutations affecting mesoderm development
(Chen et al., 2003). By
contrast to Chen et al. (2003
)
we found that GH10594 is entirely contained within the LP01489 sequence and
found no evidence for a third schizo protein isoform.
|
The molecular identification of schizo allowed us to determine the expression pattern throughout development. schizo expression is already detected in the unfertilized egg, indicating a prominent maternal contribution (Fig. 4). schizo expression stays almost uniform until the end of stage 10. Within the developing nervous system, expression can be noted in the CNS midline cells. In addition, schizo expression can be detected in the epidermis and the visceral mesoderm (Fig. 4C-H).
schizo functions in the CNS midline to control commissural crossing
The commissural schizo phenotype does not allow the deduction of
cell type in which Schizo normally acts. To test the cell-type requirement we
employed the GAL4 system and established UAS-schizoP1 and
UAS-schizoP2 transgenic flies. Expression of the different
schizo proteins was directed in the CNS midline cells of mutant
schizo embryos using the sim-GAL4 or sli-GAL4
driver strains (Scholz et al.,
1997). In both cases expression could rescue the schizo
mutant CNS phenotype indicating that Schizo acts in the midline glial cells,
which express both Slit and Netrin (Fig.
5).
|
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Discussion |
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Two major signaling molecules guide commissural growth cones
Over the last years a number of elegant genetic and biochemical studies
have revealed the key components guiding commissural growth cones toward and
across the CNS midline (Dickson,
2002). In all complex metazoan organisms two conserved major
signaling molecules are involved. Netrin proteins are secreted by the CNS
midline cells, from where they presumably form a gradient that helps to orient
the growth of commissural axons toward the CNS midline
(Tessier-Lavigne and Goodman,
1996
). In addition, the CNS midline cells express a repulsive
signal that is encoded by the gene slit
(Brose and Tessier-Lavigne,
2000
; Wong et al.,
2002
). Like Netrin, Slit is supposed to be secreted; however,
antibodies directed against the C-terminal part always detect Slit on the
membrane of the midline glial cells. Possibly the N-terminal portion of Slit
may be released from the CNS midline to form a gradient similar to the one
proposed for Netrin (Brose et al.,
1999
; Brose and
Tessier-Lavigne, 2000
;
Schimmelpfeng et al., 2001
).
Given the phenotypic analyses, such a gradient appears probable
(Murray and Whitington, 1999
;
Rajagopalan et al., 2000a
;
Rajagopalan et al., 2000b
;
Simpson et al., 2000a
;
Simpson et al., 2000b
). Both
signaling molecules act on conserved receptor systems, the activity of which
is tightly regulated in a coordinated fashion
(Stein and Tessier-Lavigne,
2001
). Furthermore, both systems share downstream components,
indicating that Netrin and Slit function is closely interwoven
(Yu et al., 2002
).
Regulation of slit expression
The expression of guidance cues is often dynamic and has to be
developmentally regulated, either on a transcriptional or a
post-transcriptional level. Recent data have identified transcriptional
factors regulating the expression of Slit in the CNS midline cells. The
initial formation of these cells requires the function of the PAS bHLH
transcription factor single-minded (sim), which is also
involved in the regulation of slit expression
(Wharton and Crews, 1993;
Wharton et al., 1994
). In
addition, the slit promoter also contains binding sites for the SOX
HMG domain protein Fish-hook (Fsh) and the POU domain protein Drifter (Dfr)
(Ma et al., 2000
). All three
genes have been shown to act in concert to regulate Slit expression
(Ma et al., 2000
).
An independent level of regulation of slit expression appears to
be mediated by the transcription factor Lola, which affects the expression
level of slit (Crowner et al.,
2002). Similarly, the Slit receptors, which are encoded by the
Robo gene family are subject to intense transcriptional control
(Crowner et al., 2002
;
Zlatic et al., 2003
).
Recently the relevance of post-transcriptional regulation of Roundabout by
Commissureless was shown (Keleman et al.,
2002; Myat et al.,
2002
). Commissureless was shown to act as a cytoplasmic sorting
receptor for Roundabout, diverting it from the synthetic to the late endosomal
compartment (Keleman et al.,
2002
). In the absence of Commissureless, all Roundabout is
transported to the cell surface, where it binds Slit to induce repulsive
signaling. Thus the commissureless mutant phenotype, which is
characterized by a loss of axon commissure, can be explained by an increased
repulsive signaling originating from the CNS midline.
Here we have shown that schizo acts in a rather similar way to commissureless; however, rather than affecting the Roundabout receptor, schizo appears to act on the expression of the Slit ligand. First we found that the triplication of the schizo gene interfered with slit function and that reduction of slit expression in schizo mutant embryos rescued the schizo mutant phenotype. Finally, expression of a schizo transgene in the Slit-expressing CNS midline cells (1) was able to rescue the schizo mutant phenotype and (2) could induce a slit phenocopy when expressed in wild-type embryos. The deduced nature of the Schizo protein suggests that it affects Slit expression by post-transcriptional mechanisms.
Schizo, an Arf6-GEF, regulates ligand presentation
Guanine-nucleotide exchange factors (GEFs) help to convert the inactive
GDP-bound form of small GTPases into a GTP-bound active form. Schizo is a new
Sec7 domain containing GEF, which shows 40% homology to human
Arf-GEP100. Arf-GEP100 localizes to endosomal membranes
(Someya et al., 2001) and
promotes GDP/GTP exchange on ARF6. The small GTPase ARF6 is a plasma
membrane-localized protein and functions in the regulation of membrane
ruffling, cell motility, aspects of endocytosis and exocytosis, membrane
recycling, reorganization of the cortical actin cytoskeleton and activation of
phospholipase D (Kondo et al.,
2000
; Radhakrishna et al.,
1999
; Randazzo et al.,
2000
; Turner and Brown,
2001
). In Drosophila Arf6 is remarkably well conserved,
being more than 96% identical to the human counterpart (not shown).
One aspect that might hint at how Schizo regulates Slit expression is the
role of ARF6 in endocytosis and exocytosis. The function of ARF6 in
endocytosis is twofold. It either regulates clathrin-mediated endocytosis at
the apical surface of polarized epithelial cells
(Altschuler et al., 1999;
Palacios et al., 2001
) or it
is able to regulate non-clathrin-mediated endocytosis and the recycling
pathway in non-polarized cells (Brown et
al., 2001
). ARF6 has also been postulated to play a role in
Ca2+-activated dense core vesicle (DCV) exocytosis by regulating
phosphatidylinositol(4,5) biphosphate (PIP2)
(Aikawa and Martin, 2003
).
Overexpression of a UAS-ARF6 construct in midline glia cells does not result
in a schizo-like phenotype (data not shown), whereas expression of a
dominant negative form of Arf6 results in a phenocopy of several phenotypes
associated with the schizo mutant
(Fig. 6)
(Chen et al., 2003
). This
suggests that Arf6 might also be involved in the regulation of Slit
expression.
Coordinated expression of Slit and Netrin
In-vivo Slit and Netrin are both expressed by the same CNS midline cells
and their expression needs to be in an intricate balance. The importance of
this balance and not the individual expression levels is highlighted by the
fact that we were able to rescue the schizo mutant phenotype by both
increased Netrin expression or reduced Slit expression. Within the midline
glia, however, Schizo appears to primarily affect Slit expression either by
inducing its endocytosis and subsequent degradation or by blocking exocytosis
and thus release of Slit.
The latter case would suggest that Slit and Netrin are brought to the
membrane of the midline glial cells in distinct vesicle populations, whereas
the former case would require a specific membrane receptor for the Slit
protein expressed by the CNS midline glia. Given the fact that the secreted
Slit protein is found at very high levels at the midline glial cell membrane,
this appears probable. Moreover, expression of a dominant negative Shibire
protein in the midline glia leads to a schizo phenocopy.
shibire encodes the Drosophila dynamin and is required for
endocytosis and a block of shibire function leads to a block of
endocytosis (Moline et al.,
1999), which might result in higher levels of Slit expression.
Thus, regulation of membrane dynamics appears crucial in controlling the
function of the signaling molecule Slit.
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ACKNOWLEDGMENTS |
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Footnotes |
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