Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA
* Author for correspondence (e-mail: twolff{at}genetics.wustl.edu)
Accepted 27 January 2003
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SUMMARY |
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Key words: Tissue polarity, strabismus, flamingo, Cadherin, prickle, Drosophila eye
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INTRODUCTION |
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We have identified interactions between a subset of tissue polarity genes in the developing Drosophila eye. The compound eye of the fly is a polarized epithelium composed of approximately 800 unit eyes, or ommatidia. Each ommatidium contains 20 cells, including eight photoreceptors (R1-R8). The photosensitive organelles of the photoreceptors, the rhabdomeres, are arranged in a characteristic trapezoid in which photoreceptor R3 defines the `point' of the trapezoid. There are two chiral forms of the trapezoid and they fall on opposite sides of a dorsal-ventral midline known as the equator. In the dorsal hemisphere of the eye, the points of the trapezoids face the dorsal margin while those in the ventral half face the ventral margin of the eye (Fig. 1A).
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A number of tissue polarity genes have been identified, among them
frizzled (fz), prickle (pk),
dishevelled (dsh), diego (dgo),
strabismus (stbm; also known as Van Gogh) and
flamingo (fmi; also known as starry night)
(Zheng et al., 1995;
Gubb et al., 1999
;
Klingensmith et al., 1994
;
Feiguin et al., 2001
;
Wolff and Rubin, 1998
;
Taylor et al., 1998
;
Usui et al., 1999
;
Chae et al., 1999
). We have
focused our efforts on identifying the position of stbm in the tissue
polarity pathway as a means of more precisely defining its role in setting up
polarity in the eye. Flies null for stbm lack an equator because of a
variety of defects in ommatidial orientation and fate misspecification
(Wolff and Rubin, 1998
).
stbm acts cell-autonomously to define R4
(Wolff and Rubin, 1998
), and
it co-localizes with other tissue polarity proteins at the contact between
photoreceptors R3 and R4 (this report)
(Strutt et al., 2002
).
To identify genes that interact with stbm, we carried out a
genetic modifier screen. We identified two tissue polarity genes, fmi
and pk, that dominantly modify the stbm mutant phenotype.
The role of fmi in tissue polarity was identified from its
requirement for polarization of wing hairs
(Usui et al., 1999).
fmi also plays an essential role in the first asymmetric cell
division of the SOP cell lineage in the PNS
(Lu et al., 1999
).
fmi encodes a protein with a seven-pass transmembrane domain, a
unique cytoplasmic tail and nine extracellular cadherin domains
(Usui et al., 1999
;
Chae et al., 1999
). The
extracellular cadherin domains are capable of mediating cell-cell adhesion
while the unique intracellular domain is potentially involved in signal
transduction (Usui et al.,
1999
).
In this paper, we define a role for fmi in directing tissue
polarity in the Drosophila retina. We show that loss-of-function
fmi interacts genetically with both misexpression and
loss-of-function stbm. We have generated an antibody against Stbm and
show that Stbm is apically localized in all cells anterior to, in, and several
rows posterior to, the morphogenetic furrow. Stbm subsequently fades in R8, R2
and R5 and becomes pronounced at the contact between R3 and R4 [also reported
using Sbm-YFP by Strutt et al. (Strutt et
al., 2002)]. We also show that Fmi and Stbm co-localize in early,
but not later, stages of ommatidial development. In addition, we show that
fmi is cell- and ommatidium-autonomously required for ommatidial
polarity [as also reported by others (Yang
et al., 2002
; Das et al.,
2002
; Strutt et al.,
2002
)].
Like fmi, pk also acts globally to influence polarity throughout
the fly (reviewed by Mlodzik,
2000). Pk has several protein interaction domains and binds Dsh
(Tree et al., 2002
). Tree et
al. have suggested this interaction is an essential component of a feedback
loop that asymmetrically localizes Fz and Dsh in wing cells, ultimately
leading to the polarized arrangement of hairs and bristles. Similarly, in the
eye, pk is essential in establishing Fz asymmetry in R3 and R4
(Strutt et al., 2002
). Taylor
et al. have demonstrated a genetic interaction between pk and
stbm in the wing (Taylor et al.,
1998
). Here we show that pk enhances both misexpression
and loss-of-function stbm phenotypes in the eye, and that
localization of Pk is disrupted in stbm mutant ommatidia.
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MATERIALS AND METHODS |
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sev-stbm homozygous flies were crossed to the 250 second and third
chromosome deletion lines that constitute the Bloomington Deficiency Kit. The
degree of eye roughness in F1 transheterozygotes was analyzed under the
dissecting microscope and compared to that of sev-stbm heterozygotes.
A secondary screen, in which adult eyes were fixed and sectioned (as described
by Wolff, 2000) and the
phenotype quantified, was conducted on 27 candidate interactors. Six enhancers
of sev-stbm were confirmed.
Phenotypic analyses
Adult eyes were fixed, embedded and sectioned according to standard
protocol (Wolff, 2000). The
number of ommatidia and eyes scored is as follows:
Df(2R)E3363/sev-stbm14-1: 383 ommatidia from 5 eyes;
Df(2R)Jp4/sev-stbm14-1: 480 ommatidia from 3 eyes; Roote
276/sev-stbm14-1: 533 ommatidia from 3 eyes; Roote
2-42/sev-stbm14-1: 557 ommatidia from 3 eyes;
Df(2L)s1402/sev-stbm14-1: 608 ommatidia from 3 eyes;
Df(2R)pk78k/sev-stbm14-1: 497 ommatidia from 3 eyes;
fmifrz3/sev-stbm14-1: 1135 ommatidia from 14
eyes; fmi192/sev-stbm14-1: 877 ommatidia from
15 eyes; fmiE59/sev-stbm14-1: 872 ommatidia
from 12 eyes; fmifrz3/fmifrz3: 1788 ommatidia
from 17 eyes;
stbm153,fmifrz3/stbm153,fmifrz3:
1024 ommatidia from 15 eyes;
stbm153,+/stbm153,fmifrz3:
1123 ommatidia from 16 eyes;
+,fmifrz3/stbm153,fmifrz3: 976
ommatidia from 10 eyes; EGUF-fmi192: 1612 ommatidia from
18 eyes; fmi192/fmi192 mosaic clones: 792
ommatidia from 23 clones in 23 eyes;
pkpk1/sev-stbm14-1: 1030 ommatidia from 11
eyes; and pkpk1, stbm153/pkpk1,
stbm153: 729 ommatidia from 7 eyes.
Antibody generation
The anti-Stbm polyclonal antibody was raised against the N-terminal 143
amino acids of the protein. A 429 base pair PCR product was generated and
subcloned in frame into the EcoRI/XhoI site of pGEX-4T-1
(Pharmacia). The fusion protein was purified on glutathioneagarose beads and
used to immunize rabbits. Immunization and subsequent production were carried
out by Pocono Rabbit Farm and Laboratory, Inc.
Immunohistology
Third instar larval eye discs were dissected and processed as described
previously (Wolff, 2000).
Tissue was incubated in primary antibody overnight at 4°C at
concentrations of 1:10 for anti-Fmi [mouse monoclonal; generous gift from T.
Uemura (Usui et al., 1999
)],
1:600 for anti-Pk [rabbit polyclonal; generous gift from D. Tree and J.
Axelrod (Tree et al., 2002
)],
1:500 for anti-Stbm (rabbit polyclonal, see above) and 1:10 for anti-Armadillo
(Arm) antibody (mouse monclonal, Developmental Studies Hybridoma Bank).
Secondary antibodies conjugated to Alexafluor fluorescent dyes were used
(Molecular Probes).
Fmi localization was studied in fzKD4A null larval escapers. For immunostaining of Fmi in shi2ts animals, third instar larvae were heat shocked at 31°C for 1 hour, eye discs removed and fixed immediately and immunostained as described above. Recovery experiments were conducted by allowing the larvae to recover at room temperature for 1, 2 or 6 hours following heat shock.
pkeq has not been characterized at the molecular level, however it fails to complement known pkpk-sple alleles. This allele was chosen over pkpk1 to characterize Stbm localization because it produces an obvious eye phenotype, unlike the pkpk1 allele.
Fluorescent images were collected using a Leica TCS SP2 confocal microscope.
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RESULTS |
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In an F1 genetic modifier screen, 250 deletion lines from the Bloomington deficiency kit, which uncover approximately 70-75% of the second and third chromosomes (Berkeley Drosophila Genome Project), were crossed to sev-stbm and the progeny scored for dominant modification of the sev-stbm phenotype. Six deficiency lines were identified as dominant enhancers of the sev-stbm phenotype (Table 1); no suppressors were identified. We have identified the interacting gene in two of these six deletions (Bloomington deficiencies Df(2R)E3363 and Roote 276).
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Das et al. (Das et al.,
2002) reported that fmiE59 mutant clones have
a cell death phenotype in which 20% of fmi ommatidia lack
photoreceptors. This is in contrast to observations that neither
fmiE45/fmiE59 transheterozygotes
(Strutt et al., 2002
) nor
ommatidia in fmi192 clones (reported here) exhibit a
photoreceptor death phenotype. While the basis of this difference is not
known, it is interesting that stbm, fmi double homozygotes are
missing photoreceptors (Table
3), perhaps suggesting a previously unrecognized role for these
loci in photoreceptor specification or survival. [A small percentage of
ommatidia are missing photoreceptors in EGUF-fmi eyes, however this
is an artifact of the EGUF system
(Rawls et al., 2002
).]
fmi acts autonomously within ommatidia to establish
polarity
Gene products that act at a distance generally exert their influence in a
non-autonomous fashion, while gene products that exert their effects
intracellularly act autonomously. fmi has been shown to act
autonomously in the wing (Chae et al.,
1999). Since tissue-specific differences have been observed in the
autonomy of some gene products (for example, fz), we analyzed the
polarity of ommatidia in and near fmi mutant clones to confirm that
fmi also acts autonomously in the eye. This analysis demonstrated
that the presence or absence of fmi does not affect neighboring
ommatidia, suggesting that fmi acts autonomously within ommatidia. In
other words, genetically mutant and mosaic ommatidia have no effect on
wild-type ommatidia outside the clone, nor does wild-type tissue rescue
ommatidia that are mutant or mosaic for fmi
(Fig. 3A,B). Similar findings
reported by Das et al. support the autonomous requirement for Fmi in
developing ommatidia (Das et al.,
2002
).
To determine if fmi acts in one specific photoreceptor to
establish the orientation of an ommatidium, we carried out an analysis of
mosaic ommatidia, ommatidia that contain a mixture of wild-type and mutant
photoreceptors. We scored these ommatidia to determine the requirement for Fmi
in each photoreceptor to direct polarity and found that Fmi is not required in
any specific photoreceptor(s) for normal rotation; rather, if at least one
photoreceptor expresses fmi, the ommatidium can, but not necessarily
will, rotate correctly (data not shown). As with stbm, proper
rotation is only guaranteed when all photoreceptors within an ommatidium are
wild type for fmi. Das et al. (Das
et al., 2002) propose that Fmi function in R3 and R4 is necessary
and sufficient for polarity. While this may be true, we cannot rule out the
possibility that Fmi may also play a role in the remaining six photoreceptors
since we have seen a small fraction of mosaic ommatidia in which both R3 and
R4 are wild type for fmi, yet these ommatidia still rotate
incorrectly (data not shown).
To determine if Fmi is required in any specific photoreceptor for cell fate
specification, we examined developmental pairs of photoreceptors (R1/R6, R2/R5
and R3/R4) that were mosaic within the pair. If Fmi is required to specify the
R3 photoreceptor, for example, then there would be a trend such that in the
majority of mosaic R3/R4 pairs, the photoreceptor that expresses fmi
would become the R3. We do not see any such trends for any of the pairs,
consistent with the findings of Das et al.
(Das et al., 2002), suggesting
that Fmi is not required for binary photoreceptor fate decisions in developing
ommatidia.
Stbm localization is dynamic
Stbm is expressed in a dynamic pattern in the third larval instar. Anterior
to, in, and immediately posterior to the morphogenetic furrow, Stbm is
uniformly expressed on the apical membranes of all cells
(Fig. 4A-C). [In the discussion
that follows, row numbers are as defined by Wolff and Ready
(Wolff and Ready, 1993); each
row is equivalent to 1.5-2 hours of development.] Four to five rows posterior
to the furrow, at about the time ommatidial rotation first becomes apparent,
Stbm begins to undergo an intriguing change in its pattern of localization.
First, it becomes prominent at the membranes of photoreceptors R3 and R4,
except where they contact R2 and R5, respectively, while simultaneously
dropping to undetectable levels in photoreceptors R8, R2 and R5 [this stage is
also described by Strutt et al., using Stbm-YFP
(Strutt et al., 2002
)].
Second, no protein is detectable at the interfaces between photoreceptors
R3/R2 or R5/R4. A restricted region of Stbm staining is evident at the
posterior tip of R8 where it contacts R1, R7 and R6, and likely reflects the
presence of Stbm in R1, R7 and R6, but not in R8
(Fig. 4D-F). While the level of
resolution of these images does not reveal if Stbm is present in only R3 or
R4, or in both cells, studies of Stbm-YFP mosaic clones have demonstrated that
Stbm is present only in R4 at the R3/R4 boundary
(Strutt et al., 2002
).
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Later in development, Stbm is localized in the cone cells. Initially, it is most prominent at the points of contact between the cone cells. It continues to be expressed at high levels in the cone cells once they meet centrally (Fig. 4G-I). The functional relevance of Stbm at these sites is not obvious since cone cell assembly is virtually normal in stbm mutant eyes (a small percentage of ommatidia have only three cone cells, but this phenotype may be a secondary effect of improper recruitment by the underlying photoreceptors). No anti-Stbm staining is evident in eye discs that are null for stbm (stbm6cn) (data not shown).
Fmi and Stbm colocalize early, but not late, in ommatidial
development
Fmi co-localizes with Stbm anterior to, within and for several rows
posterior to the furrow (Fig. 5A, parts
a,b; 5B, parts a,b). [The pattern of Fmi localization was also
recently reported by others (Yang et al.,
2002; Das et al.,
2002
; Strutt et al.,
2002
); here, we extend these observations by providing a
developmental time-course for the dynamic distribution of Fmi.] The
co-localization persists until approximately seven rows posterior to the
furrow, at which point the patterns diverge in two intriguing ways. First,
approximately 7-8 rows behind the furrow, Fmi becomes diminished in
photoreceptor R3 and simultaneously becomes enhanced in photoreceptor R4
(Fig. 5Ac,Bc). As an
intermediate step in this change in protein distribution, Fmi becomes weaker
in the polar region of R3, resulting in a transient asymmetry in which Fmi is
more prominent in the equatorial region of R3. It is not clear why Fmi
undergoes a shift from expression in both R3 and R4 to expression in R4 alone.
Perhaps there are Fmi-dependent qualities to being an R4 that cannot be
detected in a standard mosaic analysis, such as the placement or morphology of
the cell's rhabdomere.
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Fmi is processed through the endocytic pathway
The predominance of Fmi-containing vesicles at the junction of
photoreceptors R8, R2, R5, R3 and R4 suggested that this internalization may
be the means by which Fmi is removed from these cells, or at least from a
subset of these cells. In an effort to identify the process underlying this
internalization, we tested the efficiency of this process in two mutants that
interfere with the endocytic pathway: shibire (shi) and
hook (hk). We found that the vesicularization of Fmi is
altered in these mutants, indicating that Fmi is internalized via
endocytosis.
shi, which encodes the Drosophila dynamin, is required
early in the endocytic pathway for the budding of clathrin-coated pits from
the membrane upstream of the fusion of these structures with endosomes
(Chen et al., 1992) (reviewed
by Narayanan and Ramaswami,
2001
). Temperature-sensitive shi2ts larvae
were heat shocked for 1 hour at the restrictive temperature, sacrificed
immediately and immunostained with an antibody against Fmi. In
shi2ts larvae, the MVB-like, Fmi-containing vesicles
normally found in wild type (Fig.
6A) are abolished; Fmi is instead found in small puncta on cell
membranes (Fig. 6B). The large
Fmi-containing vesicles reappear in larvae allowed to recover for 1-6 hours at
room temperature (data not shown).
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The functional significance of Fmi endocytosis in the eye is not known.
Clearly, this internalization is taking place too late to initiate or mediate
rotation. Perhaps it is necessary for rotation to stop. It could also be
important for other aspects of development given that the endocytosis of
membrane-associated receptors is required for signaling in key developmental
pathways [for example, Notch, Dpp, and Wg
(Parks et al., 2000) (reviewed
by Narayanan and Ramaswami,
2001
)].
The asymmetric localization of Fmi requires Fz and Notch
activity
The regulation of Fmi localization in the larval eye disc shows a
dependency on fz (Fig.
5C,D) (Strutt et al.,
2002; Das et al.,
2002
) and Notch (N) (data not shown)
(Das et al., 2002
), genes
implicated in R3 and R4 cell fate determination, respectively. The dependency
of Fmi localization on fz has also been described for Fmi
localization in the wing (Usui et al.,
1999
; Chae et al.,
1999
).
The early pattern of Fmi localization is unaffected in the absence of Fz
it is still localized to all cell membranes anterior to the furrow
(data not shown) and in nascent photoreceptor clusters
(Fig. 5Ca,Da). Furthermore,
slightly later in development, Fmi is still abundant in photoreceptors R3 and
R4. However, whereas Fmi would ordinarily be removed from photoreceptors R8,
R2 and R5 at this stage in wild type, it is only partially removed from these
cells in the fzKD4A mutant
(Fig. 5Cb,Db). The most notable
change in Fmi localization is that it no longer accumulates asymmetrically in
R4 (Fig. 5Cc,Dc). The size,
number and location of Fmi-containing vesicles are also disrupted in
fzKD4A larvae: there are more vesicles, they are smaller
and they accumulate approximately four rows earlier in development
(Fig. 5Cd,Dd). We observe
similar defects in Fmi localization in Nts1 larvae
heat-shocked for 6 hours (data not shown). Additionally, Das et al.
(Das et al., 2002) show that
Fmi localization is also perturbed when N-mediated signaling is knocked down
via overexpression of the sev-Su(H)-EnR transgene. While these data
do suggest a role for N in the asymmetric localization of Fmi, one cannot yet
be assigned, given the abundance of roles for N throughout development.
The observations that fmi and stbm have similar phenotypes, that they interact genetically and that their products colocalize, suggested that they may act in the same pathway to specify tissue polarity. To explore the possibility that Stbm and Fmi define a complex, we investigated both the localization of Fmi in a null stbm background and the localization of Stbm in EGUF-fmi eyes. In neither case was the localization affected (data not shown), demonstrating that Stbm is not required for Fmi localization, nor is Fmi required for Stbm localization. Furthermore, we have been unable to demonstrate a physical interaction between Fmi and Stbm using co-immunoprecipitation assays (data not shown).
pk and stbm interact
In the deficiency screen described earlier, a second tissue polarity gene,
pk, was identified as a dominant genetic modifier of stbm.
The original deficiency, Roote 276, which uncovers 42E4-43E7, enhances the
sev-stbm phenotype from one in which 10% of ommatidia have defects in
polarity to one in which 27% have defects
(Table 1). The best candidate
interactor was pk, a tissue polarity gene that maps to 42F2-43A1. We
demonstrated that pk was the gene responsible for the dominant
enhancement of the sev-stbm phenotype: haploinsufficiency of
pkpk1, a pk allele with no eye phenotype,
enhances the sev-stbm phenotype to the same degree as the original
deficiency (Fig. 1F;
Table 2). We confirmed this
genetic interaction in a loss-of-function stbm background: the
percentage of symmetrical defects in stbm153,
pkpk1 double homozygotes is significantly enhanced relative to
stbm153 homozygous flies
(Fig. 2C,D;
Table 3).
The genetic interaction between stbm and pk may have its basis in a physical interaction that enhances or stabilizes these proteins at the R3/R4 boundary. To explore this possibility, we examined Stbm localization in a pk mutant background, and Pk localization in a stbm mutant background. Stbm localization does not appear to be affected in a pkeq background (a genetic null that fails to complement pkpk-sple alleles, data not shown). However, Pk localization is disrupted in a stbm6cn null background. We have characterized the distribution of Pk in wild-type eye imaginal discs (Fig. 7B-D) and find that it is indistinguishable from that of Stbm (Fig. 4). Pk is significantly reduced overall in the stbm6cn background. While some protein does accumulate at the boundary between R3 and R4, Pk is not detectable at the R8/R1/R7/R6 boundary (Fig. 7E-G). Physical interactions have not been demonstrated between either of these proteins, nor have genetic interactions between fmi and pk been shown. These data are consistent with the possibility that Stbm, Fmi and Pk may all function together in a complex.
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DISCUSSION |
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In an attempt to define more precisely the role of Stbm in the tissue polarity pathway, we have identified genetic interactions between stbm and two other tissue polarity genes, fmi and pk. Characterization of the fmi-stbm interaction revealed a requirement for Fmi in ommatidial polarity and a dynamic pattern of Fmi localization that depends on Fz and N. We have also raised an antibody against Stbm, characterized its subcellular localization, and shown that the localization of Fmi and Stbm differs in two ways: first, Fmi is enriched in R4, whereas Stbm is not, and second, Fmi, but not Stbm, is endocytosed. Characterization of the pk-stbm interaction showed that pk enhances the stbm phenotype and that Pk localization requires Stbm.
Pk localization requires stbm function
Three alternatively spliced transcripts are encoded by the pk
locus: pkpk, pkM and
pkpk-sple. Although these three isoforms differ in the
5' region, they all contain the single PET and three LIM domains
characteristic of the Pk protein (Gubb et
al., 1999). PET and LIM domains are thought to mediate
protein-protein interactions (Dawid et al.,
1998
). Isoform-specific mutations in the 5' region of the
transcript result in the pkpk phenotype, affecting only
the wing and notum, whereas mutations in the LIM- or PET-encoding domains
result in pkpk-sple alleles, null alleles that affect the
eye, legs and abdomen in addition to the wing and notum
(Gubb et al., 1999
).
Our observation that Pk distribution is altered in a null stbm background suggests that its localization is, at least in part, dependent on Stbm. The possibility that Pk localization is mediated directly by Stbm has not yet been explored, but the PET and LIM domains are candidates for domain-specific interactions with Stbm. Disruption of these domains would result in genetic null alleles, consistent with the pkpk-sple phenotype described above.
Although ommatidial polarity is not affected in individuals carrying the
pkpk1 allele, this allele enhances the stbm eye
phentoype. Functional redundancy could account for the ability of pk
to enhance the stbm phenotype such that there is no phenotype when
pk is knocked out but a reduction in pk gene dose can be
detected by Stbm. Furthermore, Gubb et al.
(Gubb et al., 1999) have
indicated that the balance of Pk isoforms contributes to the establishment of
tissue polarity. Perhaps this balance is also required for Stbm function.
Atypical cadherins in tissue polarity
Cadherins, or Ca2+-dependent cell adhesion molecules, have
traditionally been recognized for their role in adhesion and the resulting
tumorous phenotype. Fmi, Fat (Ft) and Dachsous (Ds), members of a class of
cadherins that contain a large number of extracellular cadherin domains
(atypical cadherins), have recently been shown to contribute to the
polarization of ommatidia (Fig.
3) (Das et al.,
2002; Strutt et al.,
2002
; Rawls et al.,
2002
; Yang et al.,
2002
). While the ability of cells to adhere to one another is
clearly essential for the establishment of polarity within epithelia, recent
work suggests the role of cadherins extends beyond adhesion.
Several lines of evidence suggest atypical cadherins may be involved in
signaling. For example, Ft is required in the haltere to inhibit DV signaling
and ft mutants display haltere to wing transformations
(Shashidhara et al., 1999). In
the fly eye, Ft and Ds have been proposed to be required for the transduction
of a dorsal-ventral positional signal via cell-cell relay
(Rawls et al., 2002
). In
addition, Yang et al. (Yang et al.,
2002
) have shown that gradients of Ds and Four-jointed (Fj)
activity may regulate Ft to establish this dorsal-ventral cue. It has been
suggested that the combined activities of Ds, Fj and Ft, which appear to be
functionally conserved in the wing, leg and abdomen
(Ziedler et al., 2000
),
constitute the `elusive' factor `X' in the morphogen model for tissue polarity
(Casal et al., 2002
).
The data described here are consistent with the notion that Fmi also plays
a role in the intracellular signaling required for the establishment of tissue
polarity. Given that Fmi is capable of mediating homophilic association
between S2 cells (Usui et al.,
1999), its role in signal transduction may be indirect and a
consequence of a primary role in cell adhesion. However, fmi clones
in the eye do not give rise to tumors, nor is the tissue grossly disrupted as
has been noted in clones of genes that maintain the integrity of tissue [for
example, epithelial phenotypes described for shg mutant embryos
(Tepass et al., 1996
;
Uemura et al., 1996
)].
Therefore, it is possible that the primary role of fmi is not to
maintain the integrity of tissue via cell adhesion, but rather to maintain
sufficient contact between cells to mediate signaling, or even to signal
directly.
Model for the regulation of N activity by Stbm, Fmi and Pk
Ommatidial polarization is thought to rely heavily upon the proper
specification of two photoreceptors: R3 and R4. Although these two
photoreceptors are recruited into the growing ommatidium as a pair and they
morphologically resemble one another in early stages of development, they have
long been known to be distinct from one another based on their adoption of
distinct sets of contacts early in development
(Tomlinson, 1985). Recent work
on a number of tissue polarity genes provides genetic and molecular evidence
that the complexes of tissue polarity proteins are not identical in
photoreceptors R3 and R4. The asymmetric regulation of N by these complexes
may ultimately lead to low levels of N activity in R3 and high levels in R4,
the combination of which is thought to be essential for the specification of
the R3 and R4 cell fates.
Fmi has been shown to interact homophilically, and while current data do
not establish that Fmi is present in both R3 and R4 at the junction between R3
and R4, in the model that follows, we assume homophilic interactions between
the extracellular cadherin domains of Fmi help to anchor Fmi in R3 and R4 on
both sides of the R3/R4 interface (Fig.
8). Furthermore, we suggest the intracellular tail of Fmi is
involved in signaling, and that it signals through a complex that is made up
of at least three proteins: Fmi, Diego (Diego localization depends on Fmi) and
Dsh (Dsh co-localizes with Fmi) (Das et
al., 2002). Dsh has also been shown to interact physically with
two proteins required for R4 specification, N and Stbm
(Axelrod et al., 1996
;
Strutt et al., 2002
; Park and
Moon, 2001) and with Pk (Tree et al.,
2002
). Finally, our stbm-pk genetic and protein
localization data suggest Pk and Stbm could physically interact within a
complex.
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We propose that the direct interaction between N and Dsh blocks N signaling, and that the different subset of proteins bound to Dsh is the basis of the asymmetry of the complex. In the future photoreceptor R3, N binds Dsh (which is part of the Fmi/Diego/Dsh scaffold) thereby inhibiting N activity in R3 (Fig. 8). In the future R4 cell, where Stbm and perhaps Pk are localized, Fmi, Diego and Dsh also form a complex. However, in this case, the re-organization of the Fmi/Diego/Dsh complex to include Stbm and Pk bound to Dsh may prevent N from binding to Dsh, leading to high levels of N-mediated signaling in R4 (Fig. 8). Ultimately, these differences in gene activity in the R3 and R4 precursors direct the fate specification of these cells.
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ACKNOWLEDGMENTS |
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REFERENCES |
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