1 Department of Molecular Genetics, Albert Einstein College of Medicine, 1300
Morris Park Avenue, Bronx, NY 10461, USA
2 Department of Biology, Department of Biochemistry and Molecular Biology,
Pennsylvania State University, 208 Mueller Laboratory, University Park, PA
16802, USA
Authors for correspondence (e-mail:
zcl1{at}psu.edu
and
baker{at}aecom.yu.edu)
Accepted 28 February 2003
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SUMMARY |
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Key words: Notch, Drosophila eye, Scabrous, Gp150, Endosome
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INTRODUCTION |
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The accompanying paper describes a Notch mutation that specifically
elevates Notch activity in the neural cells. The split mutation
alters glycosylation of the N extracellular domain and leads to inappropriate
N activity within R8 precursor cells in the developing eye
(Li et al., 2003). This
suggests that factors specifically regulating the inactivity of N in neural
cells contribute to the spatial pattern of neurogenesis.
Genetic studies have identified several genes whose mutations interact with
the split allele (Brand and
Campos-Ortega, 1990). One gene has been reported where deletion of
a single allele is sufficient to suppress the spl phenotype. This
gene encodes the secreted protein Scabrous
(Baker et al., 1990
). In
addition in the homozygous absence of sca, the spl mutation
has no detectable effect, i.e. spl mutant and wild-type N behave
indistinguishably. Conversely duplications of sca enhance the
spl phenotype (Rabinow and
Birchler, 1990
). These results indicate that activity of N in
neural cells depends critically on sca. By contrast, none of the
well-known components of N signaling behave as such dose-sensitive genetic
modifiers of spl. Special alleles of Dl were also recovered
as dominant spl suppressors (Brand
and Campos-Ortega, 1990
), consistent with the finding that in
spl the N activity in neural cells is ligand dependent
(Li et al., 2003
).
The molecular role of Scabrous in the Notch pathway is not yet clear
(Baker, 2000;
Justice and Jan, 2002
).
Mutations of sca cause defects in the spacing and number of sensory
mother cells in the epidermis and of R8 precursor cells in the retina, two
founder cell types for adult peripheral nervous system
(Mlodzik et al., 1990
). The
sca mutations act cell nonautonomously. Because N acts cell
autonomously in the specification of these same cell types it was suggested
that sca encoded an extracellular ligand for the receptor protein N
(Baker et al., 1990
). This
hypothesis proved difficult to confirm, however, as sca mutations
affected only a subset of Notch functions, had weaker effects than N null
mutations, and as no direct interaction between the Sca and N proteins was
demonstrated (Baker and Zitron,
1995
; Lee and Baker,
1996
). More recently, other ideas have been proposed: that Sca
acts to scaffold N to the extracellular matrix to downregulate N activity
(Powell et al., 2001
), acts to
preserve epithelial structure within proneural regions and so enhance function
of other N ligands (Renaud and Simpson,
2001
), or acts independently of N to arrest ommatidial rotation
(Chou and Chien, 2002
).
Other findings strongly suggest that Sca and N proteins are closely
associated in vivo. When Sca is overexpressed in the developing wing, N
activity and specification of the wing margin are prevented, even though wing
margin specification is independent of Sca function in the wild type. Sca
protein appears to prevent Dl from activating of N in this ectopic expression
assay. The results strongly suggest that Sca protein targets N signaling,
although not defining the exact role of Sca in normal development
(Lee et al., 2000). In other
experiments, Powell et al. (Powell et al.,
2001
) reported that when ectopically expressed in pupal retina,
Sca protein was preferentially stabilized in cells expressing N and that such
stability depended on EGF repeats 19-26 of the N extracellular domain. Dl and
Ser signal through EGF repeats 10-12 (de
Celis et al., 1993
; Rebay et
al., 1991
). The association with Sca occurred independently of N
signaling activity (Powell et al.,
2001
). Chemical crosslinking of Drosophila embryos
detected Sca protein in a complex with N, consistent with a close association
between the proteins in vivo. Sca protein also appeared to stabilize N protein
on the surface of tissue culture cells
(Powell et al., 2001
). It
remains uncertain whether the interaction is direct or mediated by other
proteins, or where in the cell it occurs.
Another gene required for proper eye and bristle patterning has recently
been described. Mutations at the Gp150 locus cause defects in ommatidial
development and cuticular bristle development that are similar to those seen
in sca homozygotes (Fetchko et
al., 2002). Gp150 protein was originally isolated biochemically as
a phosphoprotein target of the receptor tyrosine phosphatase DPTP10D
(Tian and Zinn, 1994
;
Fashena and Zinn, 1997
).
Recent work shows that Gp150 is located in endosomes and interacts with the
Notch pathway (Fetchko et al.,
2002
).
We have explored the relationship of Sca and Gp150. We find that Gp150 is required for neural Notch activity in the spl mutant, and conclude that the Sca and Gp150 proteins must act in a common pathway, with Gp150 acting downstream in cells that respond to secreted Sca protein. Gp150 is required for all Sca activities yet identified, including those of ectopic expression and association with Notch in vivo. Sca is localized to endosomes along with Gp150. We propose that an endosomal pathway downregulates N activity in neural cells, and that Sca and Gp150 oppose this pathway to permit N activity in a subset of non-neural cells. Accordingly, Sca and Gp150 activate N indirectly, via effects on N downregulation.
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MATERIALS AND METHODS |
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HSsca 41-514 and
HSsca
513-773 transformants were obtained by
transferring the corresponding gene sequences from pUAST plasmids described
previously (Lee et al., 1998
)
into the pCasperHS vector and transforming Drosophila using standard
procedures (Rubin and Spradling,
1982
; Steller and Pirrotta,
1986
).
Drosophilagenetics
Fly stocks were maintained on standard cornmeal-agar medium. Crosses were
performed at 25°C. Genetic mosaics were obtained by heat shock induction
of FLP recombinase as described, using recombinant chromsomes carrying p[FRT]
insertions FRT40, FRT42 or FRT82 as appropriate
(Golic, 1991;
Xu and Rubin, 1993
).
For the ectopic expression of Sca in pupae, white prepupae were collected
and aged 36 hours at 25°C prior to heat shock. Heat shock was at 35°C
for 2 or 3 minutes for HSsca transformants, 36°C for 2 minutes
for the HSsca513-773 transformant, and 34°C for 2
minutes for the HSsca
41-514 transformant.
Histology and immunochemistry
Sections of adult retinas were prepared as described
(Tomlinson and Ready,
1987).
Monoclonal antibodies specific for ß-galactosidase (mAb40-1a) and ELAV
(rat mAb7E8A10) were obtained from the Developmental Studies Hybridoma Bank,
maintained by the University of Iowa, Department of Biological Sciences, Iowa
City IA 52242, USA under contract N01-HD-7-3263 from the NICHD, and used as
described (Li and Baker,
2001). Other antisera were rabbit guinea pig anti-Senseless
(Nolo et al., 2000
), rat
anti-DE-cadherin (Oda et al.,
1994
), mouse and rabbit anti-Scabrous
(Lee et al., 1996
) and guinea
pig anti-Hrs (Lloyd et al.,
2002
), mouse anti-Gp150
(Fetchko et al., 2002
), and
rabbit anti-GFP (Santa Cruz Biotechnology). Secondary antibodies were HRP-,
Cy2- and Cy3-conjugated antisera from Jackson Immunoresearch or FITC- and
Texas Red-conjugated antisera from Vector Laboratories.
Adult wing wholemounts were prepared and (where necessary) pharate adult
wings expanded as described (Couso and
Martinez Arias, 1994).
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RESULTS |
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Comparison of sca, gp150 and sca gp150 mutant eyes revealed only minor differences in internal or external structures, consistent with the model that Sca and Gp150 were required for the same process. In all three genotypes, the spacing of ommatidia and number of photoreceptor cells were abnormal (Fig. 2C-F; data not shown). Adult eyes from the double mutant were also similar to sca (Fig. 2G-J). These findings support the notion that gp150 and sca affect a common process, at least in patterning the development of the Drosophila eye.
Gp150 may have some additional functions to Sca, as gp150
homozygotes show reduced adult viability in comparison with sca
homozygotes, and subtle defects in wing venations that are not seen in
sca null mutations (Fetchko et
al., 2002).
Assessing whether Sca can affect neural patterning independently of Gp150
requires identification of the gp150-null phenotype. The
gp1501 or gp1502
alleles cause eye defects comparable with that of sca nulls, but we
consistently observe the gp1503 or
gp1504 homozygous phenotypes to be slightly
weaker. Neither gp1503 nor
gp1504 encodes detectable protein, and
gp1504 contains an early stop codon
(Fig. 2H,I) (the
gp1503 open reading frame is unchanged although
the protein is not expressed) (Fetchko et
al., 2002). To determine the nature of the
gp1501 and gp1502
mutations, regions of the gp150 gene were PCR amplified. Smaller
products were recovered compared with the controls
(Fig. 3A). Sequence analysis
revealed an identical 1305 bp deletion in both
gp1501 and gp1502
alleles, covering a region of exon 5 and extending into exon 6 resulting in a
stop codon at amino acid position 623 of the gp150-coding region
(Fig. 3B). The identical
changes suggest that gp1501 and
gp1502 may be reisolates of the same mutation.
Both mutants replace the C-terminal region (amino acids 620-1051) of the Gp150
protein by a proline-serine-isoleucine peptide. These results are consistent
with the observation that a
90 kDa protein product was detected in
gp1502 mutant tissues
(Fetchko et al., 2002
). It is
possible that gp1502 reflects the null phenotype
for the gene. If gp1502 is dominant-negative and
gp1503 and gp1504
represent the null phenotype, then slightly more severe eye defects in
sca would suggest that some aspect of sca function can occur
in the absence of gp150.
|
|
Gp150 and Sca are required in different cells
To explore how gp150 was required for sca function, we sought to
identify the cells in which gp150 was required using mosaic analysis. Mosaic
analysis using sca mutations showed that the likelihood of normal
ommatidial assembly was reduced unless the R8 cell was genetically wild type
for sca, consistent with a nonautonomous role for sca in
lateral inhibition (Baker et al.,
1990). Previous mosaic analysis with gp150 provided only
limited data for R8 cells (Fetchko et al.,
2002
). Sections were cut through eyes containing
gp1503 homozygous clones, and 90 ommatidia that
were phenotypically normal scored (Table
1). No specific photoreceptor cell type was found to be important
for gp150 function, and ommatidia with R8 cells mutant for
gp150 developed normally with the same probability as ommatidia with
R8 cells wild type for the gp150 locus. Similar results were obtained
from a smaller number of gp1501 and
gp1502 mosaics
(Table 1). The mosaic results
show that gp150 is not required in the same cells as sca, at
least during eye development. They would be consistent with gp150
function in cells that take many fates other than R8, so that no requirement
is detected in any specific ommatidial cell. The data rule out the model that
Gp150 is required for Sca protein synthesis, but are consistent with Gp150
being required for the localization or reception of Sca by other cells.
|
|
Sca deletion proteins were used to investigate further how Sca associates
with N. Sca comprises an N-terminal coiled-coil, previously found to be
sufficient for sca function, and the C-terminal fibrinogen related
domain (FReD) that increases the activity of the protein
(Lee et al., 1998). Flies
transgenic for truncated Sca proteins under control of the heat shock promoter
were prepared. Neither the Sca
41-514 protein encoding the FReD nor the
N-terminal sequences encoded by Sca
513-773 accumulated in N-expressing
cells to the same degree as did full-length Sca
(Fig. 5G,H). There seemed to be
more accumulation with the Sca
41-514 protein, as if the FReD made more
contribution to Sca accumulating in N-expressing cells
(Fig. 5G).
Gp150 and Sca colocalize in late endosomes
Gp150 is located in endosomes where it may interact with endocytosed
extracellular proteins (Fetchko et al.,
2002). We sought to determine whether Sca protein was also found
in endosomes. Although Sca is quantitatively secreted from tissue culture
cells, antibodies detect Sca protein only within cells in epithelial tissues
(Lee et al., 1996
).
Immunoelectron microscopy studies located Sca within large intracellular
vesicles (Baker and Zitron,
1995
). There is evidence that at least some such vesicles contain
endocytosed Sca (Lee et al.,
1996
).
Double labelling using markers for particular parts of the endocytic
pathway were examined by confocal microscopy. One such marker was Rab7, which
associates with the cytoplasmic face of late endosomes
(Entchev et al., 2000). In
tubGal4>rab7-GFP eye discs, most of the Sca protein detected by confocal
microscopy was located in late endosomes surrounded by rab7-GFP
(Fig. 6A). A second marker was
HRS, a protein found in early endosomes and required for maturation of
endosomes into multivesicular bodies
(Komada et al., 1997
;
Lloyd et al., 2002
). HRS and
Sca protein distributions did not overlap in eye disc cells, showing that Sca
is not stably retained in early endosomes
(Fig. 6B). Gp150 protein also
overlaps with rab7-GFP, although Gp150 was found separately from GFP-rab7 in
addition, perhaps in other parts of the endosome pathway
(Fig. 6C). Further double
labelling showed directly that Gp150 is present in the same late endosomes
that were the major location of Sca protein
(Fig. 6D).
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DISCUSSION |
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We suggest that neural cells in the spl mutant mimic a subset of non-neural cells that approach neural fate in wild-type development, and that Sca and Gp150 chiefly contribute to N signaling in such cells. We propose that during lateral inhibition to select neural precursor cells, activation of N signaling is only one part of the story. Inactivation of N signaling in cells taking the neural fate is also required. We suggest that neural cells in which N is inactive have passed through a transient stage in which a low level of incipient N signaling is a normal occurrence prior to neural determination (Fig. 7). In our model, Sca and Gp150 normally function to sustain N activity in potential neural cells (or to block or delay N inactivation in potential neural cells). Accordingly, Sca and Gp150 increase N signaling by the same mechanism both in wild-type cells on the verge of neural specification and in spl mutant cells struggling to maintain N inactivity. This model predicts that absence of Sca or Gp150 could lead to N inactivity in too many cells and specification of extra neural precursor cells. This is consistent with the sca and gp150 mutant phenotypes. Our model is consistent with the presence of Sca and Gp150 in endosomes, as it posits that they regulate inactive N molecules, not the process of N activation that occurs at the cell surface.
|
The current data focuses attention on possible roles of endosomes in N
signaling. Both Sca and Gp150 proteins are found predominantly in endosomes,
where Gp150 is required for Sca location or stability, and for Sca function.
This suggests that Sca and Gp150 promote N function, or prevent N
inactivation, through an effect on endosomes. Gp150 is thought to be
transported to late endosomes directly from the Golgi
(Fetchko et al., 2002). Sca is
thought to reach late endosomes after uptake from outside the cell, because in
cultured cells all the Sca is secreted
(Lee et al., 1996
). Several
studies indicate that Sca can be taken up into other cells in vivo
(Chou and Chien, 2002
;
Lee et al., 1996
). Notably,
the subcellular distribution of Sca proteins shows little dependence on
dynamin function, suggesting a dynamin-independent mode of uptake
(Chou and Chien, 2002
) (Y.L.,
unpublished).
The pathway of N activation in which ligands trigger proteolytic cleavages
to release the intracellular domain is thought to occur at the cell surface,
and none of these reactions is thought to involve endosomes
(Chung and Struhl, 2001;
Lopez-Schier and St Johnston,
2002
). N activation by trans-endocytosis of the N extracellular
domain has been proposed, but this involves endosomes in the signal sending
cell, which is not where mosaic analysis finds Gp150 to be required
(Parks et al., 2000
).
Endocytosis has been proposed both to downregulate N activity and to promote N
activity by removing inactive and inhibitory forms of both N and its ligands
from the cell surface (Berdnik et al.,
2002
; Seugnet et al.,
1997
). Although our data are probably consistent with previous
models for Sca function in increasing the sensitivity or range of N signaling
(Baker and Zitron, 1995
;
Renaud and Simpson, 2001
),
both the idea that sca and gp150 are most important in cells
where N signaling would otherwise be downregulated, and the location of their
products away from the cell surface supports the view that these proteins
specifically affect a downregulatory mechanism, rather than acting directly on
N activation. As the ectopic N activity in the spl mutant depends on
Dl (Li et al., 2003
),
we infer that sca and gp150 promote ligand-dependent N
activation.
Several new models can be proposed. One model is that either before or
after Dl binding, endocytosis reduces the amount of surface N available for
activation. Sca and Gp150 might antagonize such endocytosis, or permit
endocytosed N to be activated, either by permitting -secretase to act
on endocytosed intermediates or by their return to the cell surface. A second
model incorporates the observation that in addition to activating N signaling
on neighboring cells, N ligands can `cis-inactivate' N signaling in the same
cell (Doherty et al., 1996
;
Doherty et al., 1997
;
Jacobsen et al., 1998
;
Klein et al., 1997
;
Micchelli et al., 1997
).
Protection of neural cells from N activation by Dl might reflect an increased
cis-inactivation in neural cells. In this model, Sca and Gp150 would
antagonize cis-inactivation, e.g. by removing Dl or N from cis-inactivatory
interactions at the cell surface or in endosomes. Interestingly, Dl is also
present in Gp150-positive vesicles. Elevated intracellular Dl levels have been
observed in gp150 mutants, suggesting that intracellular Dl may
antagonize N signaling (Fetchko et al.,
2002
).
One problem for these models is that changes in the cell surface levels of
N or Dl have not been detected during the selection of neural cells. It
remains possible that there are changes in subsets of the detectable N or Dl
proteins that are somehow particularly important for signaling. It is
interesting to note that endocytosis is also implicated in N regulation within
neural stem cell lineages. Asymmetric divisions during sensory organ lineages
deliver Numb protein to particular daughter cells, where Numb then inhibits N
signaling through binding to N and to -adaptin, an adaptor for
endocytosis via clathrin-coated pits. Although presumed to promote N
endocytosis, numb and
-adaptin result in no detectable reduction in N
protein levels despite blocking N activity
(Berdnik et al., 2002
). In
nematodes, endocytosis has been proposed to permit downregulation of the
N-homolog lin-12 by Ras (Shaye
and Greenwald, 2002
). Perhaps endosomes provide an environment
where N signaling components are neither degraded nor removed permanently from
the cell surface, but rerouted or modified to change their signaling
properties.
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ACKNOWLEDGMENTS |
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Footnotes |
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