Centre for Developmental Genetics, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
* Author for correspondence (e-mail: d.strutt{at}sheffield.ac.uk)
Accepted 14 November 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Four-jointed, Drosophila, Planar polarity, Cadherins
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Cloning of fj revealed that it encodes a predicted type II
transmembrane protein, with two putative cleavage sites for signal peptidase
in its transmembrane domain (Villano and
Katz, 1995; Brodsky and
Steller, 1996
). Cleavage at these sites is predicted to result in
release and secretion of the C terminus of Fj. Cleavage of Fj has been
demonstrated in in vitro translation experiments and in larval tissue
(Villano and Katz, 1995
;
Buckles et al., 2001
), and the
C-terminus of Fj is secreted into the supernatant in transfected tissue
culture cells (Buckles et al.,
2001
). As Fj functions non-autonomously, it has been proposed to
act as a secreted signalling molecule, analogous to the cleaved type II
transmembrane protein Hedgehog (Lee et
al., 1992
; Tabata and
Kornberg, 1994
).
The ds and ft genes encode atypical cadherins
(Clark et al., 1995;
Mahoney et al., 1991
), and
have mutant phenotypes similar to fj in both PD patterning and planar
polarity (Adler et al., 1998
;
Casal et al., 2002
;
Yang et al., 2002
;
Rawls et al., 2002
;
Strutt and Strutt, 2002
;
Ma et al., 2003
). Recent
experiments have indicated complex crossregulatory interactions between
fj, ds and ft (Yang et
al., 2002
; Ma et al.,
2003
). Furthermore, Ds and Ft protein localisation is altered on
the boundaries of fj mutant clones
(Strutt and Strutt, 2002
;
Ma et al., 2003
). These
observations suggest that these molecules may act in a common pathway.
Using modified forms of Fj, we now demonstrate that, contrary to previous models, secretion of Fj is not necessary for its functions in planar polarity and PD patterning. Instead Fj shows highest activity when localised to the Golgi, consistent with a role in modulating the activity of other signalling molecules most likely via post-translational modification.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular biology
Constructs were generated by PCR-based methods and amplified regions
verified by sequencing. FjUn is the full-length Fj ORF
(Brodsky and Steller, 1996;
Villano and Katz, 1995
), with
amino acid substitutions V87F and A91M. CD2-Fj is amino acids 1-34 of the CD2
ORF (Williams et al., 1987
)
and amino acids 102-583 of Fj. GNT-Fj is amino acids 1-121 of human GalNAcT3
(Bennett et al., 1996
), a
glycine residue, and amino acids 109-583 of Fj. FjUn-FLAG and
GNT-Fj-FLAG have the FLAG octapeptide at the C terminus. GNT-Fjx1 is amino
acids 1-121 of human GalNAcT3, linker residues GPDL and amino acids 36-437 of
mouse Fjx1 (Ashery-Padan et al.,
1999
). Constructs were inserted into the vectors pUAST
(Brand and Perrimon, 1993
) for
expression in flies and pMK33b for expression in Drosophila S2 cells
under control of the metallothionein promoter.
Genomic rescue constructs were made using P1 clone DS08374, which spans the fj gene. fj-24kb is a 24.4 kb SalI-SpeI fragment, containing 18.3 kb genomic sequences upstream of the fj ORF, and 4.3 kb downstream sequences. fj-14kb is a 14.8 kb KpnI-SpeI fragment, with 8.7 kb sequences upstream of the fj ORF, and 4.3 kb downstream. Both constructs were made in pCasper4. Modified fj isoforms were made by inserting 6 bp (GGCCGC) in the Cavener consensus of the fj gene (GC-GGCCGC-AAAATG), to create a NotI site, and by replacing the N terminus of fj up to the internal BglII site at amino acid 110 with modified Fj sequences. Insertion of this NotI site did not affect the rescue in vivo by an otherwise wild-type fj gene (data not shown).
Antibodies, immunostaining and western blotting
Antibodies were raised in rabbits against the C terminus of Fj, using a
His-tagged bacterially expressed peptide containing amino acids 485-583 and
affinity purified against a GST-tagged peptide for the same region. Rat
antibodies against Fj were raised against a His-tagged peptide containing
amino acids 111-433 and used for immunofluorescence without purification.
Other primary antibodies used were rabbit anti-ßGAL (Cappel), rabbit
anti-Ci (Alexandre et al.,
1996) and mouse monoclonal anti-Golgi (Calbiochem)
(Stanley et al., 1997
).
Immunofluorescence was carried out using secondary antibodies conjugated to
Cy2, RRX (Jackson), Alexa488 or Alexa568 (Molecular Probes). For western
blotting, HRP-conjugated secondary antibodies (Dako) were used with Dura
(Pierce) chemiluminescent detection.
Tissue culture
Drosophila S2 cells were grown in Schneiders medium (Gibco) with
FCS and transfected with hygromycin-resistant pMK33 vectors by the calcium
phosphate method. Transfected cells were selected by growth in the presence of
200 µg/µl hygromycin for several weeks. Expression was induced by
addition of CuSO4 24 hours prior to harvesting.
Tissue and cell extracts and immunoprecipitations
Third instar larval extracts were prepared by dissecting larval head
complexes (consisting of the imaginal discs and brain plus associated tissue)
into SDS-PAGE sample buffer on ice. The equivalent of 1-2 head complexes were
loaded per lane for western blotting. For immunoprecipitation from S2 cells,
cells were lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA and 1%
Triton X-100 and precipitations carried out in the presence of 1% Triton X-100
using FLAG-M2-Affinity Resin (Sigma).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To study the subcellular localisation of endogenous Fj protein, higher magnification images were taken of expression in the wing pouch (Fig. 1I,J). Fj is principally localised in discrete spots inside the cell, the majority of which colocalise with a Golgi marker. In more basal regions of the cell some large spots are seen that do not colocalise with the Golgi marker (Fig. 1J), and additionally faint staining is also seen in the cytoplasm.
Analysis of modified Fj proteins
Although Fj protein can be cleaved and secreted in vitro and in vivo
(Villano and Katz, 1995;
Buckles et al., 2001
), the
significance of this is unclear. As we observe localisation of Fj protein in
the Golgi (Fig. 1I), this
raised the question of whether cleavage and secretion of Fj is required for
its function. To investigate this, we designed a series of Fj transgenes, in
which Fj localisation and processing is altered
(Fig. 2A).
|
We used two systems to determine if the FjUn, CD2-Fj and GNT-Fj proteins displayed the expected intracellular localisation: either transfection into Drosophila S2 cells, or expression in the large salivary gland cells of transgenic animals using the GAL4-UAS system.
A single major Fj protein band of the expected size, was detected on
western blots of extracts from larvae expressing the Fj constructs expressed
under control of the actin promoter (Fig.
2B). In addition, expressing wild-type Fj produces a minor, lower
molecular weight band, which probably represents the cleaved C terminus.
Prominent higher molecular weight bands are seen in GNT-Fj extracts, which
most likely represent glycosylated forms. In S2 cells, Fj protein is partially
cleaved, and its secreted C terminus can be detected in the supernatant
(Buckles et al., 2001).
Similarly, we were able to detect the C terminus of Fj in the medium of cells
expressing CD2-Fj (data not shown). In addition, expression of FjUn
in S2 cells resulted in a low level of cleavage and secretion, which could be
detected by immunoprecipitation of a tagged form from a large volume of medium
(Fig. 2C), confirming the
SignalP cleavage prediction. Nevertheless, cleavage is impaired relative to
wild-type Fj, as the cleaved C terminus is not detected in FjUn
larval extracts. By contrast, GNT-Fj did not show secretion of the Fj
C-terminus into the medium.
In agreement with the colocalisation of endogenous Fj with a Golgi marker in wing imaginal disc cells (Fig. 1I), immunostaining of ectopically expressed wild-type Fj protein in salivary glands and S2 cells revealed that it overlaps, but does not entirely co-localise with a Golgi marker (Fig. 2D,H). Therefore, as in the wing, some Fj is either in a Golgi compartment not marked by the anti-Golgi antibody used, or is in an adjacent compartment of the secretory pathway. FjUn localises similarly to wild-type in S2 cells (Fig. 2J). GNT-Fj, as expected, is tightly localised to the Golgi (Fig. 2G,K), completely overlapping with the marker used. By contrast, the constitutively-secreted form of Fj (CD2-Fj) shows less colocalisation with the Golgi in both systems (Fig. 2E,I), being seen additionally in many spots within the cell (which may represent secretory vesicles), and in salivary glands being efficiently secreted into the duct (Fig. 2F).
In vivo activity of modified Fj proteins
We have demonstrated that wild-type Fj protein localises in vivo to the
Golgi apparatus, although a subset also appears to be cleaved and secreted. In
addition, we have made modified forms of Fj which are either efficiently
secreted or tightly retained in the Golgi. These constructs were tested for in
vivo function in transgenic flies, by overexpressing using the GAL4-UAS
system.
Expression of wild-type Fj using omb-GAL4 results in a wing
phenotype very similar to loss-of-function phenotypes, as has been shown for
other drivers (Zeidler et al.,
2000; Buckles et al.,
2001
). In wings of both fj mutants and flies
overexpressing Fj, there are proximodistal patterning defects which result in
the anterior and posterior crossveins being closer together (blue line in Fig.
3B and
3D, compare with
3A). In addition, the distal
region of the wing is shorter in omb-GAL4, UAS-fj flies.
Overexpression of FjUn, CD2-Fj and GNTFj all give a similar
phenotype (Fig. 3E,F), despite
their different subcellular localisations. Quantification of the distance
between the two crossveins (the `intervein distance') shows a trend for GNT-Fj
to give the most severe phenotypes, and CD2-Fj the mildest phenotypes
(Fig. 3F). These results
suggest that Fj protein is most active when it is retained in the Golgi,
rather than when it is secreted.
A similar overexpression assay was used to determine whether the modified
forms of Fj were active in planar polarity. If wild-type Fj is expressed at
the poles of the eye using omb-GAL4, then several rows of ommatidia
on the dorsal and ventral edges of the eye are inverted
(Zeidler et al., 1999)
(Fig. 4B). Overexpression of
FjUn, CD2-Fj and GNT-Fj give similar inversions of polarity
(Fig. 4C-E). Thus, all three
modified forms of Fj are active in polarity as well as PD patterning, and,
furthermore, the direction of the polarity phenotypes demonstrate that none of
the three forms are acting as dominant negatives.
|
In addition, we tested if the mouse homologue of Fj, Fjx1
(Ashery-Padan et al., 1999),
could provide Fj activity when overexpressed. However, no overexpression
phenotypes were observed with a variety of drivers, even if the protein was
retained in the Golgi using the GNT retention signal
(Fig. 3F, and not shown).
Rescue of fj mutants by modified forms of Fj
Although Golgi-retained Fj is most active in overexpression assays, the
secreted CD2-Fj still gives significant phenotypes. This could be explained by
the fact that the GAL4-UAS system results in high levels of expression, and
that a large amount of CD2-Fj is always passing through the Golgi/secretory
pathways. Therefore, we developed a more sensitive assay for Fj activity, by
making rescue constructs using genomic DNA from around the fj
locus.
We initially tested a 24 kb rescue construct, which consists of 17 kb of genomic sequence upstream and 4 kb downstream of the fj transcribed region. This transgene appeared to contain most of the regulatory elements required to rescue a fj mutant. One copy of the fj-24kb transgene gave significant rescue of the wing PD patterning defect (Fig. 5A, compare with female wings in Fig. 3A and 3B), whereas there was complete rescue with two copies (Fig. 5B). Rescue of the leg PD patterning defect was also seen with this construct (Fig. 5C-F). Rescue of fj polarity phenotypes are harder to assay, as fj only shows such phenotypes in clones. Nevertheless, we think it likely that fj-24kb contains the enhancers necessary for polarity patterning at least in the eye for two reasons. First, loss-of-function clones of fj-24kb in a fj null background phenocopy fj loss of function clones (Fig. 5G); and second, the mini-white gene present in the rescue construct is expressed in a gradient in the eye, similar to that of fj itself (Fig. 5H).
A fj-14kb transgene (7 kb genomic sequences upstream and 4 kb downstream of the fj gene) also gave partial rescue of the fj wing PD patterning defect (Fig. 6C, compare with males in Fig. 6A,B). The degree of rescue was similar for several independent insertions, and was not significantly improved by increasing the copy number (Fig. 6D). No rescue of the leg PD patterning defect was seen (data not shown). The pattern of gene expression driven by this 14 kb region of genomic DNA was tested by inserting a lacZ gene in place of the 5' end of fj (lacZ(fj)-14kb). As expected from the rescue phenotypes, expression was observed in the wing pouch of the third instar imaginal disc (data not shown).
|
Interestingly, most of the transgenic lines carrying a single copy of the GNT-fj-14kb transgene had a dominant PD patterning defect in the wing and leg (Fig. 6F, and not shown). In flies with two copies of the transgene, the wing phenotype was as strong as that of fj null mutants (Fig. 6G). No dominant phenotypes were observed with multiple insertions of fj-14kb, again suggesting that GNT-Fj protein has greater activity. Therefore, these results confirm those seen with the overexpression assay, supporting the conclusion that constitutive secretion of Fj reduces its activity, while Golgi-retained Fj is hyperactive.
However, it should be noted that there are two possible explanations for the higher activity of GNT-fj-14kb. The first is that the Golgi-retained protein is itself biochemically more active. The second is that the Golgi-retained protein in some way causes endogenous Fj to be more active, perhaps by promoting its secretion. Two experiments rule out this second possibility. Loss-of-function clones of GNT-fj-14kb in the eye give a non-autonomous polarity phenotype, even in a fj mutant background (Fig, 6H). Furthermore, overexpression of GNTFj using the omb-GAL4 driver in a fj mutant background, still results in strong inversions of ommatidial polarity on the polar boundary of the eye (Fig. 6I). Therefore these results indicate that the activity of GNT-fj-14kb is not dependent on endogenous Fj and furthermore that it does not act non-autonomously by promoting secretion of endogenous Fj.
Interaction of fj with ft and ds
Our results show that Fj does not need to be secreted to function, but that
it nevertheless acts non-autonomously. Thus, Fj must be acting intracellularly
to modify the activity of one or more other proteins that then signal outside
the cell. As ft and ds have similar mutant phenotypes to
fj in PD patterning and planar polarity, they are good candidates for
being targets of Fj activity.
First, we looked for genetic interactions between fj and weak
allele combinations of ft and ds. Double mutants of
fj and ft had a wing PD patterning phenotype that was
stronger than either of the single mutants
(Fig. 6B,
Fig. 7A,B). The PD patterning
defect of ds fj double mutants was harder to assess, as there was no
posterior crossvein; but there was no obvious reduction in the length of the
total wing compared with either single mutant (Figs
6B,
7C,D). However, we did observe
reproducible defects in wing trichome polarity in ds fj double
mutants. Defects in the polarity of wing trichomes are normally only observed
in fj mutant clones, but not in fj homozygous animals
(Zeidler et al., 2000)
(Fig. 7E). Similarly, the weak
ds combination
ds1/dsUA071 does not
show significant polarity defects (Fig.
7F). However, in ds1
fjd1/dsUA071
fjd1 double mutants, reproducible hair polarity
swirls occur in one region of the wing, between veins 2 and 3, above the
anterior crossvein (compare Fig.
7E-G). In addition,
fjd1/fjd1 or
ds1/dsUA071 mutants
give only rare planar polarity defects in the eye, in which less than 1% of
ommatidia are inverted (Zeidler et al.,
2000
; Strutt and Strutt,
2002
) (Fig. 7H,I),
while in the double mutant 16% of ommatidia are inverted
(Fig. 7J). These synergistic
interactions are consistent with the hypothesis that fj acts in a
common pathway with ds and ft.
Dominant genetic interactions were also observed between fj and ft/ds. Removing one copy of either ft or ds enhances the wing PD patterning phenotype of fjd1/fjd1 mutants (Fig. 7K), and enhances the dominant overexpression phenotype caused by the GNT-fj-14kb transgene (Fig. 7L). Conversely, removing one copy of fj enhances the weak ds1/dsUA071 eye polarity phenotype from 0.3% to 8% ±2 inverted ommatidia (data not shown).
We then investigated whether ds acts upstream or downstream of fj, by testing whether the effects of overexpressing Fj were still retained in a strong ds mutant. Overexpressing GNT-Fj using omb-GAL4 in the eye causes ommatidia on the ventral edge of the eye to be inverted (Fig. 4E), whereas ommatidia in a ds mutant eye are randomised in dorsal-ventral polarity (Fig. 7M). Interestingly, overexpressing GNT-Fj in a ds background results in a ds-like phenotype (Fig. 7N), showing that in this context ds is epistatic to fj. In addition, the PD patterning defect caused by overexpressing Fj in the wing (Fig. 3E) is less severe in a ds mutant background (data not shown), again suggesting that Ds activity is necessary for Fj function. Overall, these results support the conclusion that Fj acts via Ds in both planar polarity and PD patterning.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thus, our results show that rather than acting in an analogous manner to
the cleaved type II transmembrane protein Hedgehog, a better model for Fj
function may be the type II transmembrane protein Fringe (Fng). Fng is
Golgi-localised and acts as a glycosyltransferase enzyme to
post-translationally modify the receptor Notch (N)
(Brückner et al., 2000;
Munro and Freeman, 2000
). This
renders N more sensitive to its ligand Delta, and less sensitive to the ligand
Serrate. In the case of Fj, there are no molecular homologies that give any
clues as to a possible enzymatic activity. Consequently, we cannot be certain
of the precise location of its function. However, as our results show that if
Fj is tethered in the Golgi, it has higher than normal activity, the Golgi
seems most likely to be its preferred site of action.
An important question is whether Fj cleavage has any functional significance. As forced retention of Fj in the Golgi causes hyperactivity, it is possible that cleavage and secretion could be a mechanism to downregulate Fj activity during normal development. However, further experiments will be required to determine if the cleavage step is temporally or spatially regulated.
The mouse homologue of Fj (Fjx1) has also been proposed to act as a
secreted molecule, on the basis of a hydrophobic stretch at the N terminus
that might represent a signal peptide and the presence of predicted signal
peptidase cleavage sites (Ashery-Padan et
al., 1999). However, we note that the hydophobic region is not at
the extreme N terminus and is sufficiently long to be predicted to be a
transmembrane domain by the Kyte and Doolittle
(Kyte and Doolittle, 1982
) and
TMHMM (Sonnhammer et al.,
1998
) algorithms. This structure suggests that Fjx1 may also be a
type II transmembrane protein. Consistent with this, in tissue culture
experiments, we fail to observe secretion of the C-terminal region of Fjx1
into the medium (data not shown). However, our failure to detect any activity
of Fjx1 when overexpressed in flies suggests that there may be a divergence of
function between the fly and vertebrate proteins.
In Drosophila, the atypical cadherins Ft and Ds are good
candidates for being the ultimate targets of fj activity. They are
required for both planar polarity and PD patterning, and have similar mutant
phenotypes to fj. In addition, we now show that fj interacts
genetically with ds and ft in both planar polarity and PD
patterning. Interestingly, ds fj double mutants were previously
reported to have surprisingly strong phenotypes, which were qualitatively
different to those of the single mutants, including duplications or
transformations of limb structures
(Waddington, 1943). However,
we do not see such phenotypes in any of our double mutant combinations,
suggesting that the duplications/transformations may be specific to the
combination of chromosomes used in Waddington's experiment. Our results
instead show that mutations in fj enhance the phenotypes of both
ft and ds hypomorphic mutations, suggesting that these genes
act in a common pathway.
Epistasis experiments further demonstrate that ds is required to
mediate fj function, and therefore ds acts downstream of
fj; this is in agreement with previous data based on clonal analysis
of ds and fj (Yang et
al., 2002). Interestingly, recent experiments have also revealed a
role for fj in regulating the intracellular distribution of Ds and Ft
(Strutt and Strutt, 2002
;
Ma et al., 2003
). In wild-type
tissue, Ds and Ft colocalise at apicolateral membranes, and their localisation
is mutually dependent. Inside fj mutant clones, Ds and Ft
localisation is largely unaltered. However, in the row of mutant cells
immediately adjacent to wild-type tissue, Ft and Ds preferentially accumulate
on the boundary between
fj+/fj- cells. In
addition, cells inside the fj clones appear to be `rounded-up',
suggesting that they prefer to adhere to each other rather than to non-mutant
cells (Strutt and Strutt,
2002
). Thus, it is thought that fj modulates the activity
and intermolecular binding properties of Ft and Ds
(Strutt and Strutt, 2002
;
Ma et al., 2003
).
An interesting point to note is that both ds and ft show
planar polarity phenotypes as homozygotes
(Adler et al., 1998;
Yang et al., 2002
;
Rawls et al., 2002
), whereas
fj only shows polarity phenotypes on the boundaries of mutant clones
(Zeidler et al., 1999
;
Zeidler et al., 2000
). The
fj phenotypes have been explained by models in which fj acts
redundantly to regulate the production of a gradient, the direction of which
determines polarity. Thus, in homozygotes the direction of the gradient is
unchanged, and animals show no major defects; but at clone boundaries there is
a discontinuity in the direction of the gradient, leading to inversions of
polarity. We can now extend this model to suppose that Fj may modulate Ds/Ft
activity, but that it does not act as a simple on-off switch; rather Ds/Ft
retain some activity even when Fj is not present.
In the absence of a known enzymatic function for Fj, the mechanism by which it might modulate Ft and Ds activity remains uncertain. But we speculate that as Fj acts intracellularly, it is possible that it promotes or mediates the post-translational modification of Ds and/or Ft proteins, and that these molecules mediate the non-autonomous signalling functions of Fj. However, the large size of the Ft and Ds gene products (5147 and 3380 amino acids, respectively) renders the analysis of their post-translational modification highly challenging.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adler, P., Charlton, J. and Liu, J. (1998).
Mutations in the cadherin superfamily member gene dachsous cause a
tissue polarity phenotype by altering frizzled signaling.
Development 125,959
-968.
Alexandre, C., Jacinto, A. and Ingham, P. W. (1996). Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the Cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins. Genes Dev. 10,2003 -2013.[Abstract]
Ashery-Padan, R., Alvarez-Bolado, G., Klamt, B., Gessler, M. and Gruss, P. (1999). Fjx1, the murine homologue of the Drosophila four-jointed gene, codes for a putative secreted protein expressed in restricted domains of the developing and adult brain. Mech. Dev. 80,213 -217.[CrossRef][Medline]
Bennett, E. P., Hassan, H. and Clausen, H.
(1996). cDNA cloning and expression of a novel human
UDP-N-acetyl-alpha-D-galactosamine. Polypeptide
N-acetylgalactosaminyltransferase, GalNAc-t3. J. Biol.
Chem. 271,17006
-17012.
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Brodsky, M. H. and Steller, H. (1996). Positional information along the dorsal-ventral axis of the Drosophila eye: graded expression of the four-jointed gene. Dev. Biol. 173,428 -446.[CrossRef][Medline]
Brückner, K., Perez, L., Clausen, H. and Cohen, S. (2000). Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406,411 -415.[CrossRef][Medline]
Buckles, G. R., Rauskolb, C., Villano, J. L. and Katz, F. N. (2001). four-jointed interacts with dachs, abelson and enabled and feeds back onto the Notch pathway to affect growth and segmentation in the Drosophila leg. Development 128,3533 -3542.[Medline]
Casal, J., Struhl, G. and Lawrence, P. (2002). Developmental compartments and planar polarity in Drosophila. Curr. Biol. 12,1189 .[CrossRef][Medline]
Clark, H. F., Brentrup, D., Schneitz, K., Bieber, A., Goodman, C. and Noll, M. (1995). Dachsous encodes a member of the cadherin superfamily that controls imaginal disc morphogenesis in Drosophila. Genes Dev. 9,1530 -1542.[Abstract]
Ito, K., Awano, W., Suzuki, K., Hiromi, Y. and Yamamoto, D.
(1997). The Drosophila mushroom body is a quadruple
structure of clonal units each of which contains a virtually identical set of
neurones and glial cells. Development
124,761
-771.
Kyte, J. and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157,105 -132.[Medline]
Lecuit, T., Brook, W. J., Ng, M., Calleja, M., Sun, H. and Cohen, S. M. (1996). Two distinct mechanisms for long-range patterning by Decapentaplegic in the Drosophila wing. Nature 381,387 -393.[CrossRef][Medline]
Lee, J. J., von Kessler, D. P., Parks, S. and Beachy, P. A. (1992). Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog. Cell 71,33 -50.[Medline]
Ma, D., Yang, C. H., McNeill, H., Simon, M. A. and Axelrod, J. D. (2003). Fidelity in planar cell polarity signalling. Nature 421,543 -547.[CrossRef][Medline]
Mahoney, P. A., Weber, U., Onofrechuk, P., Biessmann, H., Bryant, P. J. and Goodman, C. S. (1991). The fat tumor suppressor gene in Drosophila encodes a novel member of the cadherin gene superfamily. Cell 67,853 -868.[Medline]
Munro, S. and Freeman, M. (2000). The Notch signalling regulator Fringe acts in the Golgi apparatus and requires the glycosyltransferase signature motif DxD. Curr. Biol. 10,813 -820.[CrossRef][Medline]
Nielsen, H., Engelbrecht, J., Brunak, S. and von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Prot. Eng. 10,1 -6.[Abstract]
Rawls, A. S., Guinto, J. B. and Wolff, T. (2002). The cadherins Fat and Dachsous regulate dorsal/ventral signaling in the Drosophila eye. Curr. Biol. 12,1021 -1026.[CrossRef][Medline]
Sonnhammer, E. L. L., von Heijne, G. and Krogh, A. (1998). A hidden Markov model for predicting transmembrane helices in protein sequences. In Sixth International Conference on Intelligent Systems for Molecular Biology (ed. J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff and C. Sensen), pp.175 -182. Menlo Park, CA: AAAI Press.
Stanley, H., Botas, J. and Malhotra, V. (1997).
The mechanism of Golgi segregation during mitosis is cell type-specific.
Proc. Natl. Acad. Sci. USA
94,14467
-14470.
Strutt, H. and Strutt, D. (2002). Nonautonomous planar polarity patterning in Drosophila: dishevelled-independent functions of frizzled. Dev. Cell 3,851 -863.[Medline]
Tabata, T. and Kornberg, T. B. (1994). Hedgehog is a signaling protein with a key role in patterning Drosophila imaginal discs. Cell 76,89 -102.[Medline]
Tokunaga, C. and Gerhart, J. C. (1976). The effect of growth and joint formation on bristle pattern in D. melanogaster. J. Exp. Zool. 198, 79-96.[Medline]
Villano, J. L. and Katz, F. N. (1995).
four-jointed is required for intermediate growth in the
proximal-distal axis in Drosophila.
Development 121,2767
-2777.
Vincent, J. P., Girdham, C. H. and O'Farrell, P. H. (1994). A cell-autonomous, ubiquitous marker for the analysis of Drosophila genetic mosaics. Dev. Biol. 164,328 -331.[CrossRef][Medline]
von Heijne, G. (1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14,4683 -4690.[Abstract]
Waddington, C. H. (1943). The development of some `leg genes' in Drosophila. J. Genet. 45, 29-43.
Williams, A. F., Barclay, A. N., Clark, S. J., Paterson, D. J. and Willis, A. C. (1987). Similarities in sequences and cellular expression between rat CD2 and CD4 antigens. J. Exp. Med. 165,368 -380.[Abstract]
Wolff, T. and Ready, D. F. (1993). Pattern formation in the Drosophila retina. In The Development of Drosophila melanogaster (ed. M. Bate and A. Martinez-Arias), pp. 1277-1326. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Xu, T. and Rubin, G. M. (1993). Analysis of
genetic mosaics in developing and adult Drosophila tissues.
Development 117,1223
-1237.
Yang, C.-h., Axelrod, J. D. and Simon, M. A. (2002). Regulation of Frizzled by Fat-like cadherins during planar polarity signalling in the Drosophila compound eye. Cell 108,675 -688.[Medline]
Zeidler, M. P., Perrimon, N. and Strutt, D. I. (1999). The four-jointed gene is required in the Drosophila eye for ommatidial polarity specification. Curr. Biol. 9,1363 -1372.[CrossRef][Medline]
Zeidler, M. P., Perrimon, N. and Strutt, D. I. (2000). Multiple rôles for four-jointed in planar polarity and limb patterning. Dev. Biol. 228,181 -196.[CrossRef][Medline]