1 Department of Molecular Genetics, Albert Einstein College of Medicine, 1300
Morris Park Avenue, Bronx, NY 10461, USA
2 Howard Hughes Medical Institute, Waksman Institute and Department of Molecular
Biology and Biochemistry, Rutgers, The State University, Piscataway, NJ 08854,
USA
Accepted 19 March 2003
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SUMMARY |
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Key words: Notch, Delta, Fringe, O-fucose, Drosophila eye, Neurogenesis, Lateral inhibition
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INTRODUCTION |
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The receptor protein Notch (N) appears to be the receptor for the lateral
inhibitory signal (Artavanis-Tsakonas et
al., 1999). N is required cell autonomously to suppress neural
fate, and is sufficient to block neural fate specification when activated in
all the cells (Hoppe and Greenspan,
1990
; Heitzler and Simpson,
1991
; Lieber et al.,
1993
; Rebay et al.,
1993
; Struhl et al.,
1993
). These results imply that N activity must remain low in the
cells that take neural fate.
The transmembrane protein Delta (Dl) is a ligand for N and is required to
inhibit neural fate (Kopczynski et al.,
1988; Lehmann et al.,
1981
; Vassin et al.,
1987
). Dl is required cell nonautonomously and is thought to
encode the lateral inhibitory signal
(Fehon et al., 1990
;
Heitzler and Simpson, 1991
).
Such a view predicts that Dl expression should be required in the neural cell
to signal to other cells, and that if Dl in the non-neural cells was able to
activate N in neural precursor cells, neural fate specification would be
prevented by non-neural Dl overexpression. Neither of these predictions has
yet been tested directly.
One way that N activity could be restricted to some cells would be if N was
not expressed in the future neural cells, or if Dl was expressed in the neural
cells only. Such reciprocal expression has been reported for the anchor cell
equivalence group in the nematode C. elegans
(Wilkinson et al., 1994). By
contrast, in Drosophila, N and Dl are expressed homogeneously in both
neural and non-neural cells (Baker,
2000
). One model proposes that N or Dl are modified or associated
with other molecules so that one or both proteins becomes active in only a
subset of the locations where they are expressed. Alternatively, it has been
suggested that homogenous Dl expression reflects spatially uniform mutual
inhibitory signaling, to which non-neural cells make the same contribution as
do neural precursor cells (Muskavitch,
1994
). In support of the idea that non-neural cells also signal,
Dl suppresses neurogenesis in some tissues that lack any neural precursor
cells (Parks and Muskavitch,
1993
). Mutual inhibition would require some other mechanism to
release each neural precursor cell from receiving the homogenous N-activating
signals.
N and Dl function in many developmental processes in addition to neural
fate specification. For example Dl activation of N is important in the
induction of the dorsoventral boundary during wing development
(Doherty et al., 1996;
Irvine, 1999
), in the
induction of proneural development in the morphogenetic furrow of the
developing eye imaginal disc (Baker and Yu,
1997
; Baonza and Freeman,
2001
; Li and Baker,
2001
), in preventing the recruitment of supernumerary
photoreceptor cells to the ommatidia of the developing eye
(Cagan and Ready, 1989
;
Sun and Artavanis-Tsakonas,
1996
), in specifying the difference between the R3 and R4
photoreceptor cells of each ommatidium
(Cooper and Bray, 1999
;
Fanto and Mlodzik, 1999
;
Tomlinson and Struhl, 1999
),
and in specifying the difference between R7 and R1 or R6 photoreceptor cells
of each ommatidium (Cooper and Bray,
2000
; Tomlinson and Struhl,
2001
). In these inductions, ectopic expression of Dl leads to
ectopic activation of N, as predicted if the expression pattern of Dl
determines the spatial pattern of normal induction. One qualification is that
during wing development Dl activity largely depends on modification of N by
the glycosyltransferase Fringe to extend O-fucose glycans
(Bruckner et al., 2000
;
Moloney et al., 2000a
).
Because Fringe is only expressed dorsally, ectopic Dl activates N
predominantly in cells of the dorsal compartment of the developing wing
(Fleming et al., 1997
;
Irvine, 1999
;
Panin et al., 1997
). This
provides at least one precedent for differential activity of modified N
proteins.
We report that mosaic analysis supports the lateral inhibition model of Dl function more than mutual inhibition. This leads us to hypothesize that either N or Dl proteins must be differentially active within the R8 proneural group. We present evidence that R8 cell precursors do not normally respond to Dl, despite expressing N. As one approach to investigating this, we have examined a particular N mutant allele called split, which affects eye and bristle development to a greater degree than other aspects of N function. We discover that the split mutation renders R8 precursors sensitive to Dl, leading to N activity within the R8 cell, and that the consequences of such neural N signaling include defective specification, differentiation and survival both of R8 cells and of other retinal cells that depend on R8 via other signaling pathways. We show that the amino acid substitution responsible for the spl phenotype introduces a site for O-fucosylation into EGF repeat 14 of the N extracellular domain, and that although this glycan is a substrate for the glycosyltransferase Fringe, extension by Fringe is not necessary for N activity. We propose that the spatial pattern of N activity in wild type may be determined by interactions that prevent N activity as much as by interactions that activate N.
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MATERIALS AND METHODS |
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To mutate Thr540 in the 13th EGF repeat of pAc13-15EGFN and pAc13-15EGFspl into I540, the site-directed mutagenesis kit (Clontech) was used. The oligonucleotides used were: p-CCTGAACGATGGAATTTGCCACGACAAGATC (to mutate Thr540 into Ile540) and p-GTGACTGGTGAATACTCAACCAAGTC (to mutate the ScaI site for selecting). Products were verified by sequencing.
Cell culture and transfection
Drosophila melanogaster Schneider cells were kept at 25°C in
Shields and Sang M3 insect medium (Sigma) supplemented with 10%
heat-inactivated fetal bovine serum (FBS) (Sigma) and penicillin (50
U/ml)-streptomycin (50 mg/ml) (Gibco). Cells were transfected using lipofectin
(Lee et al., 1996). The three
EGF repeat proteins were purified from cell media using ProbondTM resin
(Invitrogen).
Labeling EGF fragments of N
After elution of EGF polypeptides from metal chelating beads, the buffer
from 100-400 µl eluant was exchanged with Glyco buffer (50 mM HEPES pH 7.0,
140 mM NaCl, 10 mM MnCl2, 0.2% Tween-20) by concentration in
Centricon filter units, dilution into 400 µl Glyco buffer, and
reconcentration to 20 µl. Labeling reactions were conducted by incubating
this 20 µl EGF polypeptide with 20 µl [14C]UDP-GlcNAc (25
µCi/ml, AP Biotech), 5 µl purified Fringe:His6 (0.1 µg/µl)
(Moloney et al., 2000a) and 5
µl Glyco buffer at 25°C for 2 hours. The reaction mixture was then
boiled in SDS-PAGE sample buffer and run on two parallel gels. One gel was
subject to western blotting, using Mouse anti V5-HRP (Invitrogen) for
detection. The other gel was subject to Fluorography, with Amplify (AP
Biotech) for signal enhancement.
Fly strains
Fly strains are as follows.
md0.5-Lacz [number 181 from Cooper
(Cooper and Bray, 1999)].
Clones were induced by heat shock FLP-mediated recombination of larvae
heterozygous for mutants linked to appropriate FRT chromosomes and FRT
[arm-lacZ] chromosomes (Golic,
1991
; Xu and Rubin,
1993
). Fly stocks were maintained on standard cornmealagar medium
at 25°C. Sections of adult retinas were prepared as described
(Baker et al., 1990
).
Antibodies
Antibody staining was performed as described
(Li and Baker, 2001).
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 IA52242, USA under contract N01-HD-7-3263 from the NICHD. Other antisera
were guinea pig anti-Senseless [number 173 from Nolo et al.
(Nolo et al., 2000
)], rabbit
anti-CM1 [number 202 from Srinivasan et al.
(Srinivasan et al., 1998
)],
rabbit anti-Boss (Kramer et al.,
1991
) and monoclonal anti-Dl
(Parks et al., 1995
).
Secondary antibodies include HRP-, Cy2- or Cy3-conjugated antisera from
Jackson Immunoresearch.
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RESULTS |
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Although cell nonautonomy of Dl function is well established, the focus of
Dl function has never been mapped precisely within proneural groups to
determine whether normal patterning can occur when a single neural precursor
cell is mutant for Dl. It has been reported that single Dl
mutant cells transplanted into wild-type host embryos can take neural fates,
consistent with the mutual inhibition model, but in these experiments the
transplanted cells may not all integrate into proneural regions
(Technau and Campos-Ortega,
1986). In the case of thoracic microchaete bristles a modest bias
against neural specification by cells with lower Dl gene dose, and
increased levels of Dl signal from ectopic microchaete together support a
lateral inhibition model for this class of epidermal sense organ
(Heitzler and Simpson, 1991
;
Heitzler and Simpson,
1993
).
The ideal experiment of removing Dl function from single cells and
determining their fate is difficult to achieve by mitotic recombination
because of perdurance. A single recombinant cell that has lost the Dl
gene may not lose Dl mRNA and protein immediately. A suitable opportunity
arises during Drosophila eye development because of regulation of
both Dl expression and cell cycle progression
(Fig. 1A). Founding R8
photoreceptor neurons are specified during an extended G1 arrest of the cell
cycle (Wolff and Ready, 1993).
Loss of the Dl gene by mitotic recombination would have to occur at
or before the preceding mitosis, anterior to the morphogenetic furrow. Dl
protein levels drop below the threshold of detection before the G1 arrest, and
Dl protein that appears during R8 specification is the product of new
transcription which would be absent from a recombinant cell mutant for
Dl (Parks et al.,
1995
; Baker and Yu,
1998
). Thus, any genetically Dl mutant R8 cell must have
undergone R8 specification in the absence of both the Dl gene and its
products.
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Dl does not activate N in R8 precursor cells
If Dl acts a lateral inhibitor from the R8 cell despite apparently
homogenous expression, perhaps Dl protein expressed in cells neighboring the
R8 precursor is less able to activate N in the R8 precursor than Dl protein in
the R8 precursor is able to activate N in the neighboring cells. We determined
the consequences of ectopic Dl expression to test this model. UAS-Dl
transgenes were expressed posterior to the morphogenetic furrow using the
GMR:Gal4 driver. Ectopic N activation at this stage causes loss of R8 cells
and prevents differentiation of other neural cell types
(Baker et al., 1996) (see
below). Ectopic Dl expression led to abnormal, rough eyes in the adult
(Fig. 2A,B). Labeling imaginal
discs with neural-specific antibodies showed missing photoreceptor cells (data
not shown), but R8-specific markers revealed no change in the number or
distribution of R8 cells (Fig.
2C,D). R8 cells are eliminated when Ser or intracellular effectors
of N signaling are expressed by GMR-Gal4 (not shown). We examined many
different UAS-Dl insertion lines conferring varying levels of Dl function
without observing effects on R8 specification, even though the strongest lines
were associated with ectopic Dl expression levels higher than those of the
endogenous protein. Gal4 lines that drive UAS:Dl expression earlier in eye
development do not eliminate R8 cells either
(Baonza and Freeman, 2001
;
Li and Baker, 2001
) (Y.L. and
N.E.B., unpublished). Because the same UAS-Dl transgenes did activate N during
wing development, leading to formation of ectopic wing margin
(Lee et al., 2000
), did
activate N when expressed anterior to the morphogenetic furrow, leading to
accelerated furrow progression (Li and
Baker, 2001
), and also repressed neurogenesis of photoreceptor
neurons other than R8 cells (data not shown), we conclude that N in R8
precursor cells is particularly insensitive to activation by Dl.
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The spl mutant eyes are smaller, have reduced numbers of
ommatidia, and frequently lack ommatidial cells
(Cagan and Ready, 1989). The
spl mutation was reported not to affect specification of bristle
precursor cells but altered their differentiation, causing both missing and
duplicated external bristle shafts (Lees
and Waddington, 1942
).
Previously, mosaic analysis determined that in the eye the spl
phenotype depended on the genotype of R8 cells. Mosaic ommatidia with
spl mutant R8 cells developed normally only 40% of the time
(Baker et al., 1990). As N is
normally inactive in R8 precursor cells, the mosaic analysis indicates
inappropriate N activity in R8 cells. The spl phenotype was
investigated further to determine the nature of the N activity.
As described previously, the smaller eyes of spl mutants are
associated both with fewer ommatidia and with ommatidia containing less than
the normal complement of differentiated cells
(Cagan and Ready, 1989;
Campos-Ortega and Knust,
1990
). When molecular markers for R8 specification are examined,
fewer R8 cells were seen, with greater separation than in wild type
(Baker et al., 1990
;
Nagel and Preiss, 1999
)
(Fig. 3A,B). In addition, we
noticed that the expression level of R8 genes varied within individual cells.
One example is the nuclear protein Senseless, which is required for proper R8
differentiation (Frankfort et al.,
2001
). Whereas in wild type each R8 cell expresses a uniform level
of the Senseless protein appropriate for its developmental age, in the
spl mutant Senseless expression levels varied between normal and much
lower levels, as though some cells were adopting R8 fate less successfully
than others (Fig. 3A,B). In
addition, the proneural groups from which R8 cells emerge frequently contained
fewer cells and lower levels of Senseless than in wild type
(Fig. 3A,B).
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If cell death was the primary effect of spl, then preventing cell
death would rescue the spl phenotype. We found, however, that many R8 cells
were missing in spl; GMRp35 eye discs, similar to
spl (Fig. 3D,E). Other
photoreceptor cells were also missing from many of the ommatidia. In a few
cases, we observed ommatidia where the R8 cells were absent but other
photoreceptor cell types present (Fig.
3D,E). As all other R cells depend on R8 for recruitment
(Jarman et al., 1994), this
should not be observed unless some R8 precursor cells stop differentiating
after recruiting other R cells. Taken together, these results indicate that
the spl mutation causes the failure to specify and maintain R8 cells
and other photoreceptor cells. In addition, a proportion of R8 cells, other
photoreceptor cells and unspecified cells undergo apoptosis.
Elevating N signaling in R8 cells mimics the spl
phenotype
If N signaling in the R8 cells is the basis of the spl phenotype,
ectopic activation of N signaling in the wild-type R8 cells should mimic
spl. The UAS/Gal4 target gene expression system was used to elevate N
signaling only in R8 cells. The Gal4 driver G109-68 was used to express the N
intracellular domain specifically in R8 cells
(White and Jarman, 2000). R8
cells were missing or expressed lower levels of the R8 marker Boss
(Fig. 4A,B). Despite the R8
specific expression, other photoreceptor cells were also absent, cell death
was elevated and the adult eyes were small and rough
(Fig. 4A,B; data not shown).
The phenotype was similar to that of the spl mutation, but stronger
(Fig. 4C). Similar defects were
obtained with a range of lower penetrances when G109-68 was used to drive R8
expression of N intracellular domain from a weaker UAS insertion line
(Fig. 4D), R8 expression of
full-length N (Fig. 4E) or R8
expression of the N target gene E(spl)-m
(Fig. 4F). These eye discs
closely resembled those from spl mutants
(Fig. 4C). These results show
that N activity in R8 cells reduces the neural differentiation and survival of
other ommatidial cells as a secondary consequence of abnormal R8 development.
They bolster the conclusion from mosaic analysis, that all aspects of the
spl mutant phenotype depend on N activity in R8 cells, and suggest
that such activity is mediated by N intracellular domain and E(spl) expression
in the same way as canonical N signaling. We were unable to detect expression
of N target genes from the E(spl)-C in R8 cells from the spl mutant
using antibodies (data not shown). Low level or transient expression might be
effective, however, as it is also difficult to detect E(spl) and Ato or Sens
proteins in the same cells during N signaling in wild type
(Baker et al., 1996
;
Dokucu et al., 1996
).
Interestingly, prolonged expression and stability of the E(spl) m8 protein
enhances the spl phenotype in the E(spl)D mutant
(Tietze et al., 1992)
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Anterior to the morphogenetic furrow, N signaling enhances proneural gene
function to promote neurogenesis. By activating N, Dl relieves the baseline
repression function of Su (H) protein and reduces levels of two proteins,
hairy and extramacrochaete, that reduce proneural gene function
(Baonza and Freeman, 2001;
Li and Baker, 2001
). Loss of
proneural enhancement is associated with reduced levels of the proneural
protein atonal and with gaps in the proneural intermediate groups
(Baker and Yu, 1997
). Atonal
and Senseless expression are reduced in spl mutant eye discs, perhaps
indicating an effect of spl on proneural enhancement
(Nagel and Preiss, 1999
)
(Fig. 3C). Alternatively,
spl might affect Atonal expression nonautonomously, through signals
such as Hh, Dpp or Sca that diffuse anteriorly from differentiating cells to
regulate Atonal expression (Curtiss and
Mlodzik, 2000
; Greenwood and
Struhl, 1999
; Lee et al.,
1996
). These signals may be affected in spl mutants where
the cells that produce them cells differentiate abnormally and die.
Cell autonomous and nonautonomous features of the spl phenotype were distinguished to identify direct and indirect effects of the spl mutation. Unlike spl homozygotes, we found that atonal levels were normal in intermediate groups in homozygous spl clones, although the number of R8 cell precursors was reduced posterior to the furrow (Fig. 6A). In addition, levels of Senseless, a target genes whose expression reflects levels of Ato function were also normal in intermediate groups. As seen with Atonal, fewer than normal R8 precursor cells expressed Senseless posterior to the morphogenetic furrow (Fig. 6B). These results show that spl autonomously affects the specification and differentiation of R8 cells, but has no autonomous effect on proneural intermediate groups. We find no evidence that spl affects N activity during proneural enhancement and attribute the non-autonomous effect on intermediate groups seen in eye discs wholly mutant for spl to defective induction of Ato by posterior-to-anterior signals.
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After R4 specification, N promotes R7 specification within an R7
equivalence group that also produces R1 and R6 cells
(Cooper and Bray, 2000;
Tomlinson and Struhl, 2001
).
If Spl elevates N activity during R7 specification, R1 and R6 cells will be
transformed into R7 cell fate. In this case multiple R7 cells should be found
in spl mutant ommatidia. In sections through the adult retinas of
spl mutants, cells that had the morphology of ectopic R7 cells were
seen in 19/141 ommatidia examined (Fig.
6E) (Cagan et al.,
1992
). If these cells were R1/6 cells transformed by elevated N
activity, we would expect cell autonomous transformation of R1 or R6 by
spl in genetic mosaics. Out of 233 ommatidia mosaic for
spl/spl and spl/+ cells, one showed a spl
mutant cell in the R6 position that had R7-like morphology. By contrast, 205
of the mosaic ommatidia were constructed completely normally and contained 98
R1 cells and 94 R6 cells that were genetically spl/spl.
These results indicate that R7-like morphology of cells in spl
mutants does not result from cell autonomous effects on R1 or R6. An
alternative possibility is that extra R7-like cells result from indirect
effects of spl mutations on receptor tyrosine kinase signaling. In
addition to N, R7 specification also requires activation of Sevenless and EGFR
by ligands expressed from R8 and other cells. Ectopic activation of these
receptors can transform R3, R4 and non-neuronal cone cells into R7
(Freeman, 1996
;
Tio et al., 1994
;
Zipursky and Rubin, 1994
). In
any case, our results provide no evidence of elevated N activity in
spl mutant R1/6 cells.
In summary, we see no evidence for elevated N activity in three examples of inductive N function during eye development, consistent with the notion that the spl mutation is relatively specific for the inactive N protein in R8 precursor cells.
spl leads to an extra O-fucosylation site
The spl mutation is caused by a missense mutation affecting EGF
repeat 14 of the extracellular domain, replacing Ile578 with a Thr. As others
have noted, Thr is the consensus amino acid present at this position in 16 of
the 36 EGF repeats from N (Hartley et al.,
1987; Kelley et al.,
1987
). Another four EGF repeats have Ser at the corresponding
position. It has therefore been unclear why the Ile578Thr substitution should
mutate N function. One possibility is that Thr578 introduces a glycosylation
site, and recently O-fucosylation has been identified as a novel modification
of EGF repeat proteins including N
(Moloney et al., 2000b
).
The O-fucose modification is specifically found on epidermal
growth factor-like repeats (Harris and
Spellman, 1993). A consensus sequence for O-fucosylation
derived from comparison of blood clotting proteins is
C2XXGGS/TC3, where C2 and C3 are
the second and third conserved cysteine in the EGF repeat, and X represents
any amino acid (Wang and Spellman,
1998
). Site-directed mutagenesis has shown that Gly at the -1 and
-2 positions are not essential for fucosylation
(Wang and Spellman, 1998
). The
corresponding EGF repeat 14 sequence is C2RNRGIC3 from
wild type, and C2RNRGTC3 from spl, raising the
possibility of O-fucosylation of EGF repeat 14 on the split mutant
protein.
In order to test whether spl introduced an additional fucosylation site into N, sequences corresponding to parts of the N extracellular domain were expressed and purified from Drosophila Schneider line 2 cells. A region including the 13th, 14th and 15th EGF repeats flanked by V5 epitope and His6 tags was secreted from SL2 cells using the BiP signal peptide. EGF repeat 13 has a potential O-fucosylation site at Thr540. Thr540 was substituted with Ile in some constructs so that Thr578 would be the only possible site for O-fucosylation in the spl-derived EGF13-15 protein (Fig. 7A).
|
The spl mutant phenotype does not depend on extension of
O-fucose by Fringe
Several models can be proposed to account for the change in N activity in
the spl mutant. One possibility is that EGF repeat 14 has a normal
role in preventing N activation by Dl in R8 precursor cells from wild type. In
this case, mutating EGF repeat 14 would interfere with the normal blocking
function, allowing R8 cells to respond to Dl in the spl mutant. It is
possible that O-fucosylation might contribute to inactivating EGF
repeat 14, although it is also possible other mutations not altering
glycosylation would have the same effect. Alternatively,
O-fucosylation of EGF repeat14 might introduce a novel functional
site on N that promotes N activity in R8 precursor cells. As extension of the
O-fucose chain by Fringe increases N sensitivity to Dl during wing
development, it is plausible that fringe might participate in the
spl mutant eye also. In this case the spl mutant phenotype
is expected to depend on fng.
The role of fringe was investigated by inducing clones of cells
mutant for fringe in eye discs from wild type and from spl
mutants. As reported previously, cells lacking fringe are defective
in dorsoventral patterning, but R8 specification occurs almost normally, as
evidenced by the Senseless pattern (Fig.
8A) (Cho and Choi,
1998; Papayannopoulos et al.,
1998
; Dominguez and de Celis, 1999). We do see occasional
aberrations in R8 spacing pattern, however. In the spl mutant, many
R8 precursors are absent or show reduced Senseless expression levels. These
features of the spl mutant phenotype were slightly enhanced in
spl mutant cells that were also mutant for fringe. In a
sample of fringe clones in spl mutants, 72% of R8 cells
showed reduced or absent Senseless expression, compared with 66% in control
clones (Fig. 8B). There was no
significant difference between clones in the dorsal or ventral parts of the
eye. It is difficult to determine whether the enhancement is significant, as
we did occasionally see subtle R8 spacing defects in fng clones in a
background wild type for spl. In any case these data show that
presence of the O-fucosylation site on EGF repeat 14 of N is
sufficient to affect R8 development, without further extension of any
carbohydrate chains by Fringe.
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DISCUSSION |
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Altered sensitivity of neural cells to Dl
Inactivity of N in R8 precursor cells is not a passive event defined by
absence of ligands, because even ubiquitous Dl overexpression fails to
activate N in R8 precursor cells. By contrast, a recessive mutation, the
split allele of N, now permits N to be activated by Dl in R8
precursor cells but has little or no effect on N signaling in many other
contexts. The Dl protein in non-R8 cells is in an active form, because it can
activate R8-cell N in the spl mutant.
The spl mutant affects development of many retinal cell types. There is an R8 cell deficit, many other retinal cells are missing, cell death is elevated and additional cells may take R7 fate. The initiation and maintenance of atonal expression is deficient even before R8 specification begins. Mosaic analysis demonstrates that all these defects depend on the genotype of R8 cells only. Therefore N is activated in spl mutant R8 cells. Other cells must be affected indirectly as a consequence of the abnormal R8 cells. In confirmation of this, activation of the N signal transduction pathway solely in R8 cells recapitulates the spl phenotype, including the effects on other cell types.
The notion that many cells might be affected indirectly in spl
mutants is consistent with the role of R8 cells in founding each ommatidium.
R8 cells initiate the cascade of EGF receptor-mediated inductions that recruit
most of the retinal cell types, and are required for the survival of
unspecified cells (Jarman et al.,
1994; Jarman et al.,
1995
). The effectiveness with which R8 cells carry out these roles
depends on the level of atonal expression in the R8 precursors
(White and Jarman, 2000
).
Reduced atonal expression in the ato2 mutant, which is
defective in ato autoregulation, reduces recruitment of other cell fates
because EGF receptor is activated in fewer surrounding cells. Elevating atonal
expression by targeted expression in R8 using the G109-68 driver leads to
activation of EGF receptor in more cells than normal and recruitment of excess
outer photoreceptor cells (White and
Jarman, 2000
). Thus, losses of many other cells are an expected
consequence of the reduced atonal expression that we demonstrate in
spl mutant R8 cells.
In addition to producing ligands for the EGF receptor, R8 and other
photoreceptor cells also secrete Hh, the primary signal moving the
morphogenetic furrow across the eye disc
(Ma et al., 1993). Altering
atonal levels in R8 has further phenotypic effects through altered Hh
signaling (White and Jarman,
2000
). We propose that defective Hh signaling is the likely
explanation of non-autonomous effects of spl on the initiation of
atonal expression in the morphogenetic furrow.
The spl mutation also affects differentiation of sensory bristles
in the epidermis (Lees and Waddington,
1942). As in R8 cells in the eye, sensory organ precursor cells
are specified by lateral inhibition but not inhibited by ectopic Dl expression
(Hartenstein and Posakony,
1990
; Heitzler and Simpson,
1991
) (Y.L. and N.E.B., unpublished). N signaling is important in
cell fate specification within the lineage of cells descended from sensory
organ precursors (Hartenstein and
Posakony, 1990
; Zeng et al.,
1998
). It is plausible that aberrant N signaling might be
responsible for bristle defects in spl mutants, although we have not
examined this directly.
EGF repeat modification in wild-type and mutant N
The substitution of Thr for Ile578 in the spl mutation has been
known for some time (Hartley et al.,
1987; Kelley et al.,
1987
). Here, we show that the spl mutation introduces a
site for O-fucosylation into EGF repeat 14 of the N extracellular domain. This
site is fucosylated in SL2 cells and provides a substrate for the further
action of Fringe.
Comparisons of O-fucosylation sites on clotting factors identified
a consensus sequence, C2XXGGS/TC3
(Wang and Spellman, 1998).
Similar sequences are found in eleven EGF repeats of N, although little is
known about which EGF repeats are actually modified in vivo
(Moloney et al., 2000b
).
However, site-directed mutagenesis of Factor IX and other proteins indicated
that Gly residues at the -1 and -2 positions of the consensus were not
essential for fucosylation (Panin et al.,
2002
; Wang and Spellman,
1998
). This raises the possibility that some of the other EGF
repeats that contain C2XXXXS/TC3 sequences might be
fucosylated. Indeed EGF repeat 25, which contains
C2QNGAS/TC3, is fucosylated by Drosophila SL2
cells and a substrate for Fringe (Panin et
al., 2002
). We report here that SL2 cells fucosylate the sequence
C2RNRGTC3 in the spl mutant EGF repeat 14 and
the sequence C2LNDGTC3 in wild-type EGF repeat 13. In
light of these results, it seems possible that many of the 22 N EGF repeats
that contain C2XXXXS/TC3 sequences might be fucosylated.
These include the sequence C2QNEGSC3 in EGF repeat 12,
required for Dl to bind and activate N
(Rebay et al., 1991
;
de Celis et al., 1993
). It is
important to note that the efficiency of O-fucosylation at all these
sites is unknown, as well as the efficiency with which O-fucose is
extended by Fringe, so that it is possible that even within the same cell
individual N molecules may carry different combinations of O-fucose
and of extended O-fucose glycans.
During eye development, fng mutants have little direct effect on
R8 specification. In addition, fng was not required for the
spl mutant phenotype. This means that N function during R8
specification is little affected by any extension of O-fucose chains
that occurs, unlike N function during wing development. It is possible that
O-fucose monosaccharides affect N function during eye development,
with or without modification to polysaccharide forms. Consistent with this
interpretation, O-fucosylation has been found to be important for
many aspects of N function, including others not dependent on Fringe
(Okajima and Irvine,
2002).
Taken together, our studies suggest that introduction of an O-fucosylation site into EGF repeat 14 confers sensitivity to Dl on N expressed in R8 precursors, but has little effect on N activity in many other cells. One interpretation is that additional O-fucosylation of N increases sensitivity to ligand, so that N activation occurs in R8 precursors. Our finding that in the wild type R8 cells are insensitive to Dl also suggest another possibility: that EGF repeat 14 has a normal function inhibiting signaling, and that this function is disrupted by O-fucosylation. These two models cannot be distinguished definitively on the basis of current data. The model that EGF repeat 14 has a normal function blocking N signaling in R8 cells is supported by the recessive genetics of the spl mutation, however, because in heterozygous cells that contain wild-type and O-fucosylated EGF repeat 14, the wild-type protein continues to maintain N inactivity in R8 cells. As EGF repeat 12, which is essential for many aspects of N signaling, contains a potential O-fucosylation site, one very simplistic hypothesis is that whereas O-fucosylated EGF repeats promote N activity, during lateral inhibition EGF repeats lacking this modification inhibit N activity. We suggest that during lateral inhibition of neural cells the spatial pattern of N activity is determined by insensitivity of presumptive neural cells to N ligands, and that such insensitivity is regulated by modifications or interactions of EGF repeats on the N extracellular domain.
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ACKNOWLEDGMENTS |
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