prickle
The expression patterns of the pk transcripts were
investigated on developmental Northern blots and by tissue in situs
using probes to the common exons and the unique 5' exons. Both the
temporal and spatial patterns of expression of the three transcripts
were indistinguishable, with the exception that the pkM
transcript was only detected during the embryonic stages. In 28- to
34-hr pupal wings, pk and sple transcripts are
expressed uniformly in intervein cells but leave the presumptive vein
regions unstained. At the same stage in
the pupal legs, pk and sple transcripts are expressed
in a similar pattern, uniform in most cells, but excluded from the
segmental boundaries. In third larval instar imaginal
discs, a low level of pk transcripts can be detected in restricted
domains that correlate with the places where pk is required.
In the eye disc, maximal expression is detected in a stripe of cells
behind the morphogenetic furrow (in the region where ommatidial
organization and polarity is being specified). In wing discs,
pk transcripts are expressed at higher levels along the
dorsoventral (D/V) compartment boundary, where the
bristles of the wing margin will form. In the embryo, pk probes show a dynamic expression pattern in cells engaged
in morphogenetic movements, such as invaginating mid-line cells, in the
cephalic fold, and at parasegmental boundaries (Gubb, 1999).
The integument of the Drosophila adult abdomen bears oriented hairs and bristles that indicate the planar polarity of the epidermal cells. Four polarity genes, frizzled (fz), prickle (pk), Van gogh/strabismus (Vang/stbm) and starry night/flamingo (stan/fmi) were examined in this study, and what happens when these genes are either removed or overexpressed in clones of cells was examined. The edges of the clones are interfaces between cells that carry different amounts of gene products, interfaces that can cause reversals of planar polarity in the clone and wild-type cells outside them. To explain, a model is presented that builds on an earlier picture of a gradient of X, the vector of which specifies planar polarity and depends on two cadherin proteins, Dachsous and Fat. It is conjectured that the X gradient is read out, cell by cell, as a scalar value of Fz activity, and that Pk acts in this process, possibly to determine the sign of the Fz activity gradient (Lawrence, 2004).
Evidence is discussed that cells compare their scalar readout of the level of X with that of their neighbors and set their own readout toward an average of these. This averaging, when it occurs near the edges of clones, changes the scalar response of cells inside and outside the clones, leading to new vectors that change polarity. The results argue that Stan must be present in both cells being compared and acts as a conduit between them for the transfer of information, and that Vang assists in the receipt of this information. The comparison between neighbors is crucial, because it gives the vector that orients hairs: these hairs point toward the neighbor cell that has the lowest level of Fz activity (Lawrence, 2004).
Recently, it has been shown that, for a limited period shortly before hair outgrowth in the wing, the four proteins studied, as well as others, become asymmetrically localised in the cell membrane, and this process is thought to be instrumental in the acquisition of cell polarity. However, some results do not fit with this view -- it is suggested that these localisations may be more a consequence than a cause of planar polarity (Lawrence, 2004).
There are a number of simple systems in which isolated cells orient to a
polarising signal. These include the localized outgrowth, or 'schmooing' of
yeast in response to mating pheromone and directed migration of Dictyostelium cells up a gradient of cyclic AMP. Small
differences (as little as 1%-5%) in receptor activation across single cells are
sufficient to polarise them, a response that, in yeast and elsewhere probably depends on localised exocytosis. It is not known whether the polarisation of single,
isolated cells is a model for planar polarity of cells in an epithelium, but
it is likely that they share at least some of the mechanisms (Lawrence, 2004).
It has been proposed that, in the abdomen of Drosophila, morphogen
gradients (Hh in the A compartment and Wg in the P compartment) organise a
secondary gradient ('X'); the vector of X specifying the polarity of each cell.
Although the composition of X is unknown, at least three proteins, Fj, Ds and
Ft, are implicated. All three may be expressed, or be active, in bell-shaped
distributions that peak near the A/P (Ds) or P/A (Fj, Ft) boundaries. Ds and
Ft are transmembrane proteins in the cadherin superfamily; Fj probably acts in
the Golgi. Ds and Ft are integrated into the membrane, suggesting that
the X gradient itself may not be diffusible but instead might depend on
information transfer from cell to cell (Lawrence, 2004).
How does Hh set up the X gradient? Although changing the real or perceived
level of Hh does affect polarity, many clones (for example clones that lack
Smo, an essential component of Hh reception) show there is no simple
correlation between Hh concentration and polarity. For instance, large
smo- clones in the center of the A compartment are
polarised normally, even though they are blind to Hh. Also, while
smo- clones in some regions of the A compartment do
affect polarity, both mutant and wild-type cells are repolarised. Both
these observations argue for some transfer of information about polarity
between cells, a process that would be at least partly Hh independent. This
paper explores this process and is concerned with four genes (stan, fz,
Vang and pk) that probably act downstream of ds, ft and
fj (Lawrence, 2004).
Perhaps normal cells could transfer information from one to another (this
might be particularly important for nascent cells following mitosis) to help
keep the readout of X as a smooth gradient? To do this they might make a
comparison of their neighbors and modify this readout of X toward an average
of those neighbors. X might be read by a receptor molecule and the results
point to Fz being the most likely candidate. The results indicate that the
comparison itself requires the cadherin Stan. Thus, a cell would need to read
and compare (using Stan) the levels of X (recorded in the activity of Fz) in
neighboring cells. Then, in a way analogous to how a Dictyostelium
amoeba reads the vector of a cAMP gradient, a cell would determine its
polarity from the vector of Fz activity. The results suggest that Vang also
acts in this step, helping cells to sense the level of Fz activity in
neighboring cells (Lawrence, 2004).
Some of the results are discussed in terms of the model (Lawrence, 2004).
Clones that lack, or overexpress Fz cause local and consistent repolarisations of cells that extend from within the clone and affect normal wild-type cells outside it. Because simply removing the fz gene
from all cells randomizes polarity in the ventral pleura, it is self-evident
that these organised polarity reversals must result from an interaction
between the clone and the surrounding cells. It has been argued that Stan and Fz
act in this process, but how? Note that stan and fz are the
only mutants that have randomised hairs in the pleura, and the results
indicate that neither Stan nor Fz can function properly without the other.
Averaging might depend on the capacity of Stan to form homophilic dimers as
bridges between neighboring cells, with such Stan:Stan dimers serving as a conduit for information about the relative level of Fz activity in each cell. However,
with respect to non-autonomy, the results with the two genes differ:
How far does the non-autonomy spread into wild-type cells? This process can be stimulated. According to the model this range would
depend on the value of a single adjustable parameter, a that relates to how much a cell's scalar is read from
X. At one extreme for this parameter (a=0), when the scalar of a cell
depends only on X, a wild-type cell just posterior to a clone of
fz- cells would reset its scalar as it was before;
there could be no averaging and only that cell and its
fz- neighbor will be repolarised. Thus the
non-autonomy would be limited to one cell. At the other extreme
(a=1), any local disturbance produced by a clone would decay rapidly
because of averaging, and the repolarisation will tend to be lost altogether.
In between these extremes, the non-autonomy spreads more than one cell, but
over diverse values for this parameter, the range is near the amount usually observed (2-4 cell diameters) (Lawrence, 2004).
It has been observed that fz- clones have effects
over longer range in backgrounds such as ds- where the X
gradient might be flatter than normal. Similarly, cells are normally polarised
in large smo- clones in the middle of the A
compartment, where, because there can be no input from Hh, the X gradient
could also be flat. Both these results are consistent with the model, because
the range affected by averaging will increase (Lawrence, 2004).
Many of the proteins required for normal cell polarity, including Fz, Dsh,
Dgo, Pk, Vang and Stan are found to be asymmetrically localised in the
proximodistal axis of wing cells. This localisation is restricted to a brief period of just a few hours shortly before the wing hairs grow out, but, nevertheless it is
assumed to be mechanistically important to planar polarity. For example, non-autonomy could be explained if localised proteins were components of one or more molecular complexes that propagate polarity from cell to cell. In support of this, note that loss of any of these proteins, including the removal of both Pk and Sple, prevents the asymmetric localisation of the others (Lawrence, 2004).
But the results do not seem to fit with such a mechanism, mainly
because they provide evidence that polarity can propagate into cells that
lack, or fail to localise all of these proteins. In particular,
pk- cells are normally polarised throughout the P
compartment and can be repolarised in both compartments by sharp
discontinuities in Fz activity even in the pleura (where polarity is
randomized in fz- and stan- animals). At a minimum, these findings challenge the hypothesis that Pk itself is an essential component of a feedback amplification mechanism responsible for polarising cells.
Furthermore, if it is assumed that the observed failure of Fz, Dsh, Vang and Stan
to localize in pk- wing cells reflects a general
property, these results also challenge the idea that Fz, Dsh, Pk, Vang, Diego
and Stan must be able to accumulate asymmetrically in order for cells to
detect, and be polarised by, the X gradient, or by disparities in Fz activity.
Indeed, Adler (2002) has already hinted that there is no convincing evidence that the asymmetric localisation of these proteins actually functions in planar
polarity: 'the preferential accumulation [of proteins] along the...edges
of wing cells is a process that intuitively seems likely to be part of a core
system...but perhaps it is not and if not...this would leave rather
little in the core' (Lawrence, 2004).
Are wing cells polarised only briefly just prior to the hair outgrowth? The
reason for raising this possibility is that the proteins are apparently only
asymmetrically localised at that time. If this localisation were not causal,
as it is now suggested, it could be that the cells are polarised for all or most of
development -- again arguing that the ephemeral localisation of the
proteins is more a consequence than a cause of polarisation (Lawrence, 2004).
Many epithelial cells are polarized along the plane of the epithelium, a property termed planar cell polarity. The Drosophila wing and eye imaginal discs are the premier models of this process. Many proteins required for polarity establishment and its translation into cytoskeletal polarity were identified from studies of those tissues. More recently, several vertebrate tissues have been shown to exhibit planar cell polarity. Striking similarities and differences have been observed when different tissues exhibiting planar cell polarity are compared. This study describe a new tissue exhibiting planar cell polarity -- the denticles, hair-like projections of the Drosophila embryonic epidermis. the changes in the actin cytoskeleton that underlie denticle development are described in real time, and this is compared with the localization of microtubules, revealing new aspects of cytoskeletal dynamics that may have more general applicability. An initial characterization is presented of the localization of several actin regulators during denticle development. Several core planar cell polarity proteins are asymmetrically localized during the process. Finally, roles for the canonical Wingless and Hedgehog pathways and for core planar cell polarity proteins in denticle polarity are described (Price, 2006).
Among the hallmarks of PCP in structures as diverse as Drosophila wing hairs to stereocilia in the mammalian ear is polarization of the actin cytoskeleton. The polarized actin cytoskeleton underlying wing hair polarity has been described and defects in polarization in fz and dsh mutants have been documented. Microtubules (MTs) are also polarized in developing wing hairs, and disruption of either actin or MTs disrupts wing hair formation. The data suggest that basic features of cytoskeletal polarity in pupal wing hairs are also seen in denticles. Denticles, like wing hairs, arise from polarized actin accumulations – in denticles this occurs along the posterior cell margin. Further, like wing hairs, denticles all elongate in the same direction. The less detailed analysis of dorsal hairs suggests that they also arise from polarized actin accumulations, but these are more complex; different cell rows accumulate actin either along the anterior or posterior cell margin (Price, 2006).
The effect of Wg and Hh on denticle development is mediated in part by their regional activation of the Shaven-baby transcription factor (Ovo), which is necessary and sufficient for cells to generate actin-based denticles. Therefore genes that are targets of Shaven-baby are likely to be triggers for actin accumulation and cytoskeletal rearrangements. Wg and Hh signaling may also trigger polarization of cellular machinery that is not typically thought to be involved in PCP – e.g. the polarity of Arm that was observed. It will be useful in the future to examine whether proteins polarized during germband extension, such as Bazooka, are also polarized during denticle formation. Mutations in both hh and wg also affected the normal changes in cell shape accompanying denticle formation – rather than elongating along the dorsal-ventral axis, cells remain columnar. A similar failure of cells to polarize during dorsal closure is observed in wg mutants. These effects may reflect alterations in cell polarization or cytoskeletal regulation. It will be of interest to determine whether changes in cell shape are coupled to the establishment of cytoskeletal polarity (Price, 2006).
Thus far the analysis of actin in wild-type and mutant pupal wings has been restricted to snapshots in fixed tissue. This was extended by examining F-actin in developing denticles in real time, revealing features of polarization that have not been noted previously; these features may be shared with wing hairs or other polarized structures. The initial cytoskeletal change observed was actin accumulation all across the apical surface of the cell. This actin gradually 'condenses', becoming more restricted to the posterior cell margin and forming distinct condensations, which then brighten and sometimes merge. They then elongate, all in the posterior direction. It will be interesting to learn whether the dynamic aspects of condensation involve de novo actin polymerization and/or collection of preexisting actin filaments (Price, 2006).
It is only in late condensations that enrichment was seen of any of the actin regulators that were examined. Arp3 and Dia are weakly enriched in late condensations, with enrichment increasing as denticles elongate, and Ena is enriched even later. Of course, the localization of these actin regulators to developing denticles does not by itself demonstrate that they play an important role there, but it is consistent with the possibility that they have a role in actin remodeling associated with denticle elongation. To test this hypothesis, genetic analyses will be necessary. This presents significant obstacles, since Arp2/3 and Dia are required for much earlier events (syncytial stages and cellularization), while maternal Ena plays a role in oogenesis, complicating analysis of loss-of-function mutants. Surprisingly, none of these actin regulators localizes in an informative fashion during the initial formation of actin condensations (though APC2 localizes there during this time). Thus additional regulators functioning during early denticle development need to be identified. Studies of cytoskeletal regulation in the larger adult sensory bristles may guide this. EM studies, the use of cytoskeletal inhibitors, and FRAP, which has proved informative in studies of wing hairs and bristles, may reveal how actin in denticles is assembled. Finally, it will be important to study in denticles additional actin regulators that regulate bristle development (Price, 2006).
What signals regulate denticle polarity? As examples of PCP have proliferated, understanding of the signals that instruct cells about their orientation in epithelial sheets has evolved. Certain features are shared in many, if not all, tissues. Fz receptors play a key role. Other core polarity proteins including Dsh, Fmi, Van Gogh/Strabismus and Prickle act in many if not all places. The current data extend this analysis to the denticles. Intriguing differences were found between the phenotypes of loss of Wg or Hh signaling, in which polarity was severely altered or abolished and loss of proteins that play dedicated roles in PCP, such as embryos null for either fz or stbm, which exhibit more subtle defects. A strong polarity bias was retained in these latter mutants, with cells in the posterior denticle rows correctly polarized and only cells in the anterior two rows making frequent mistakes. Interestingly, occasional mistakes are also observed in wild-type embryos (albeit at much lower frequency) and these are also restricted to the anterior most rows. This is in strong contrast to the effects of these mutants in the wing disc, where they globally disrupt polarity (Price, 2006).
One possible reason for this difference is the different scales of the tissues. The embryonic segment is only 12 cells across, while the wing disc encompasses hundreds of cells. Many core polarity proteins help mediate a feedback loop that amplifies an initially small difference in signal strength between the two sides of a wing cell. Perhaps the small scale of the embryonic segment makes this reinforcement less essential. It is also intriguing that the polarity is most sensitive to disruption in the anterior two denticle rows. If signal emanated from the posterior, signal strength might be lower in the anteriormost cells, rendering the reinforcement process more important. The lower frequency of defects in pk1 mutants may also reflect the reduced role of the feedback loop, but this is subject to the caveat that pk is a complex locus with different mutations having different consequences. Future work will be needed to test these possibilities (Price, 2006).
Significant questions also remain about the signal(s) activating Fz receptors during PCP. Wnts were initial candidates, since Fz proteins are Wnt receptors. In vertebrates, this may be the case – Wnt11 regulates convergent extension and Wnt proteins can regulate PCP in the inner ear. By contrast, Drosophila Wnt proteins may not play a direct role. The Wg expression pattern in the eye and wing discs is not consistent with a role as the PCP ligand. Detailed studies of PCP in the eye and abdomen are most consistent with the idea that neither Wg nor other Wnt proteins are polarizing signals, but suggest that Wg regulates production of a secondary signal [dubbed `X'). Recent work suggests that Fj, Ds and Fat may be this elusive signal, with Drosophila Wg acting as an indirect cue of polarity. In fact, one cannot rule out the possibility Wnt11's role in vertebrate convergent extension is also indirect (Price, 2006).
Roles were found for Wg, Dsh and Arm in establishing denticle polarity. At face value, Arm's role is surprising, since the current view is that the Wg pathway diverges at Dsh, with a non-canonical branch mediating PCP and the canonical pathway playing no role in this. However, the data do not imply that Arm is required in denticle PCP per se. Wg acts in a paracrine feedback loop to maintain its own expression. In embryos maternally and zygotically mutant for arm alleles that cannot transduce Wg, Wg expression is lost by late stage 9. Thus, even though Arm is not in the non-canonical pathway, loss of Arm could still disrupt PCP indirectly due to the loss of Wg expression (Price, 2006).
While the data demonstrate that Wg is required for denticle PCP, two things suggest its role is indirect. wg mutants retain segmental periodicity in denticle orientation, suggesting that polarity is not totally disrupted, while in hh mutants there is no segmental periodicity. Second, when Wg signaling was reduced but did not eliminated, many cells retained normal polarity and there was segmental periodicity to which cells lost polarity or exhibited polarity reversals. This is consistent with the idea that Wg regulates production of another ligand. In fact, Wg's role may be even more indirect – given the more dramatic effect of hh, Wg's primary role in polarity may be to maintain Hh expression (this is also consistent with a requirement for canonical pathway components like Arm). Global activation of Hh signaling in the ptc mutant also disrupts polarity. Hh thus remains a possible directional cue. In the abdomen, Hh also plays an important role in polarity, but it does not seem to be the directional cue either but rather regulates its production; this may also be the case in the embryo. Thus the precise roles for canonical Wg and Hh signaling in denticle polarization must be addressed by future experiments. If neither Wnts nor Hh are directional signals, what is? Data from the eye, wing and abdomen suggest roles for Ds, Fj, Fat and Fmi but details differ in different tissues. It thus will also be useful to examine Ds, Fj and Fat's roles in embryonic PCP (Price, 2006).
To undertake a thorough analysis of the pk locus a
variety of genetic strategies were used to isolate new alleles. These alleles can
be divided into three phenotypic classes: Pk, Pk-Sple, and Sple. None of these
classes show any embryonic phenotype (even when homozygous mutant
embryos develop from homozygous mutant mothers). Consistent with this
lack of either a zygotic or maternal requirement, deletions of the
pk gene are fully viable and fertile. The defects of double
mutant pkpk-sple alleles are the same as those
seen with overlapping deletions that remove the entire gene and
eliminate all pk functions. Paradoxically, these
pkpk-sple alleles do not produce the most severe
phenotypes. Instead the single mutant alleles pkpk
and pksple give more extreme phenotypes, but in
reciprocal regions of the body; pkpk in the wing and
notum and pksple in the legs, abdomen, and eyes.
Complementation between these classes of allele indicates two subtly
different functions at the pk locus (Gubb, 1999).
Complete lack of Pk function,
in pkpk-sple alleles, gives a weak polarity
phenotype in the wing, notum, abdomen, eye, and leg.
pkpk alleles cause an extreme polarity phenotype in
the wing and notum; pksple alleles affect eye,
abdomen, and leg. The pkpk wing phenotype shows a
characteristic reversal in the triple-row bristles along the anterior
margin; a whorl in the wing hairs near the tip of vein 2, and abrupt
discontinuities in hair polarity, e.g., pkpk1. The weak PkPk-sple phenotype shows a
slight effect on triple-row bristle orientation and gives gently curved
hair polarity vectors, e.g., pkpk-sple13. sple alleles are completely wild type, e.g.,
pksple1. The eye phenotype is wild-type in
pkpk1, showing a line of mirror
symmetry along the equator. On both sides of the equator the R3
photoreceptor cell is aligned toward the pole. In addition to being rotated through 180° ommatidia show
reversed chirality around the equator, so that both a rotation and a
reflection in the plane of the epithelium is required to superimpose
the ommatidial patterns. pksple1 eyes
contain a mixture of ommatidia with reversed polarity and chirality in
both hemispheres of the eye. These ommatidia remain aligned along the
polar axis, but with their R3 photoreceptors directed toward the
equator rather than the pole giving rise to D/V
mirror-image reversals of the normal rhabdomere pattern. In addition,
all the pksple alleles exhibit ~1%
anteroposterior (A/P) reversed ommatidia. pkpk-sple13 eyes contain a mixture of
chiral forms of ommatidia. Some ommatidia fail to rotate properly, and
the resulting imperfections in the hexagonal stacking give a slightly
rough eye phenotype. Some ommatidia are aligned at 60° to the
equator and some show A/P reversals, with the R3 rhabdomere anterior to R4. The tarsi
of pkpk1 are wild type; In pkpk-sple13, the T3 and T4
segments carry medial duplications of the proximal and distal joint
structures, with the middle of each segment deleted. This results in
alternating reversed-proximal and reversed-distal tarsal joint
structures with half the length of a normal segment. In
pksple1 the tarsal duplications affect T2, T3, and
T4 segments, with an occasional incipient ectopic joint in the distal
T1. The distal T5 segment remains unaffected in all mutant alleles (Gubb, 1999).
The duplicated wing hair phenotype typical of most type 1 tissue
polarity mutants (Gubb, 1982; Wong, 1993)
also affects pkpk alleles, but only 2%-3% of
cells show doubled hairs. Where the polarity vectors are changing
sharply, however, cells frequently show doubled hairs. After the last cell division in the pupal wing,
the cytoskeleton is reorganized. Cells become hexagonal, and prehairs
grow out from the distal vertex of each cell (for review, see Eaton,
1997). It is not possible, however, to fill an irregular shape such as
the wing blade with a perfect hexagonal array of cells, and occasional
defects, such as a distorted four-pentagon array, are seen.
The relationship between hair orientation and the cell shape, implied
by the localization of prehair initiation sites, is confirmed by the
doubled hairs near stacking flaws and the lack of
regular hexagonal packing in the vicinity of the anterior whorl (Gubb, 1999).
Mutations in the Van Gogh gene, shown to be allelic to strabismus, result in the altered polarity of adult Drosophila cuticular structures. The two original Vang alleles were recovered because of a dominant phenotype -- a swirl in the wing hair pattern in the C' region of the wing (this is the region that lies between the third and fourth veins proximal to the proximal cross vein). On the wing, Van Gogh mutations cause an altered polarity pattern that is typical of mutations that inactivate the frizzled signaling/signal transduction pathway. Flies homozygous for Van Gogh alleles show a tissue polarity bristle phenotype on the wing, thorax, leg and abdomen. On the abdomen, bristles point almost orthogonally to the midline instead of posteriorly. The tarsus joints are often duplicated as is typical for tissue polarity mutants. The phenotype differs from those seen previously in other polarity mutants, since the number of wing cells forming more than one hair is intermediate between that seen previously for typical frizzled-like or inturned-like mutations. Consistent with Van Gogh being involved in the function of the frizzled signaling/signal transduction pathway, Van Gogh mutations show strong interactions with mutations in frizzled and prickle.
pk is a slightly haploinsufficient gene. A deficiency for pk (and some pk point mutants) shows a weak, partially penetrant dominant tissue polarity phenotype. This effect is enhanced by several Vang alleles. Phenotypic data suggest that genetics of Vang and its interactions with pk are complex. Several, but not all, Vang alleles act as enhancers of the pk haploinsufficient tissue polarity phenotype, and the pkTBJ21 antimorphic phenotype. Mutations in pk can also act as suppressors of Vang dominant phenotypes. Mitotic clones of Van Gogh display domineering cell nonautonomy. In contrast to frizzled clones, Van Gogh clones alter the polarity of cells proximal (and in part anterior and posterior) but not distal to the clone. In further contrast to frizzled clones, Van Gogh clones cause neighboring wild-type hairs to point away from rather than toward the clone. This anti-frizzled type of domineering nonautonomy and the strong genetic interactions seen between frizzled and Van Gogh suggest the possibility that Van Gogh is required for the noncell autonomous function of frizzled. As a test of this possibility, frizzled clones were induced in a Van Gogh mutant background and Van Gogh clones were induced in a frizzled mutant background. In both cases the domineering nonautonomy is suppressed consistent with Van Gogh being essential for frizzled signaling (Taylor, 1998).
Coordinated morphogenesis of ommatidia during
Drosophila eye development establishes a mirror-image
symmetric pattern across the entire eye bisected by an
anteroposterior equator. The mechanisms
by which this pattern formation occurs have been investigated and the
results suggest that morphogenesis is coordinated by a
graded signal transmitted bidirectionally from the presumptive
equator to the dorsal and ventral poles. This
signal is mediated by frizzled, which encodes a cell surface
transmembrane protein. Mosaic analysis indicates that
frizzled acts non-autonomously in an equatorial to polar
direction. It also indicates that relative levels of frizzled in
photoreceptor cells R3 and R4 of each ommatidium affect
their positional fate choices such that the cell with greater
frizzled activity becomes an R3 cell and the cell with less
frizzled activity becomes an R4 cell. Moreover, this bias
affects the choice an ommatidium makes as to which
direction to rotate. Equator-outwards progression of elav
expression and expression of the nemo gene in the morphogenetic furrow are regulated by frizzled, which itself is dynamically expressed about the morphogenetic furrow. To
determine if nemo expression is regulated by fz, fz mutant flies were generated that carry an enhancer trap in the nemo gene.
Expression of beta-galactosidase from the enhancer trap
resembles the expression pattern of nemo transcripts. The expression of beta-galactosidase is greatly
reduced in fz eye imaginal discs, especially
in the morphogenetic furrow. It is proposed that frizzled mediates a bidirectional signal emanating from the equator (Zheng, 1995).
To gain further insight into the mechanism of mirror-image
symmetry formation, other polarity mutants were examined.
Mutations in the sple and dsh genes are seen weakly to
roughen the eye and disrupt ommatidial polarity. A third gene, pk, affects tissue polarity
but has no mutant eye phenotype (Gubb, 1993). However, a pk-sple double mutant gives rise
to a strongly roughened eye, suggesting that sple and pk act
redundantly in eye development. Sections of sple, pk-sple and dsh mutant eyes show
a disturbed ommatidial polarity with each ommatidium having
the normal arrangement of photoreceptor cells. Together with fz,
these polarity mutants can be categorized into two classes
based on the adult eye phenotypes. One class, which consists
of fz, pk-sple and dsh, exhibits all three aspects of polarity
phenotype seen in fz adult eyes. Among them, pk-sple had the
strongest eye phenotype. The other class, which
consists of sple, exhibits only one aspect of the polarity
phenotype seen in fz eyes. Although sple ommatidia are still
arranged in antiparallel arrays and the equator is still
detected, some of the ommatidia are oriented such that their
R7 cells are pointing away, rather than toward the equator. Anti-Elav staining of sple eye discs reveals that the
disorientation is due to incorrect direction of rotation as in
fz mutants (Zheng, 1995).
To determine which cells require the sple gene for ommatidial
polarity, mosaic analysis was performed by producing clones of
sple- cells. There was no evidence of the non-autonomous or
biasing effects that are observed in fz- clones. Since the sple
mutant phenotype is partially penetrant and photoreceptor identities could be assigned in mutant ommatidia, only
mosaic ommatidia that had rotated incorrectly were examined. Far
fewer mosaic ommatidia exhibit a mutant phenotype than
genotypically mutant ommatidia. At the borders of seven clones,
fourteen mosaic ommatidia with mutant polarity were scored. In
all cases, the presumptive R4 cell was sple- and, in almost all
cases, the presumptive R3 and R5 cells were sple-.
Thus, removal of sple from R3, R4 and R5 cells can lead to
incorrect rotational direction. If any one of these cells is sple+,
the ommatidium almost always rotates normally. Therefore, sple
function in either the R3, R4 or R5 cell appears to be sufficient to
drive an ommatidium in the right direction (Zheng, 1995).
In each facet of the Drosophila compound eye, a cluster of photoreceptor cells assumes an asymmetric trapezoidal pattern. These clusters have opposite orientations
above and below an equator, showing global dorsoventral mirror symmetry. However, in the mutant spiny legs, the polarization of each cluster appears to be
random, so that no equator is evident. The apparent lack of an equator suggests that spiny legs+ may be involved in the establishment of global dorsoventral identity
that might be essential for proper polarization of the photoreceptor clusters. Alternatively, a global dorsoventral pattern could be present, but spiny legs+ may be
required for local polarization of individual clusters. Using an enhancer trap strain in which white+ gene expression is restricted to the dorsal field, it has been shown that
white+ expression in spiny legs correctly respects dorsoventral position even in facets with inappropriate polarizations; the dorsoventral boundary is indeed present,
whereas the mechanism for polarization is perturbed. It is suggested that the boundary is established before the action of spiny legs+ by an independent mechanism (Choi, 1996).
Presented here is a cytogenetic analysis of the 43A-E region of chromosome 2 in Drosophila melanogaster. Within this interval, 27 complementation groups have been
identified by extensive F2 screens and ordered by deletion mapping. The region includes the cellular polarity genes prickle and spiny-legs, the segmentation genes
costa and torso, the morphogenetic locus sine oculis and is bounded on its distal side by the eye-color gene cinnabar. In addition 19 novel lethal complementation
groups and two semi-lethal complementation groups with morphogenetic escaper phenotypes are described (Heitzler, 1993).
In wild-type flies, the body surface is covered with cuticular
structures that reflect the polarity of the underlying epithelial cells. In most regions, cuticular bristles and hairs are aligned approximately along the long axis of the structures that they cover. A set of mutants causing discrete changes in the orientations
of bristles and hairs was described by Gubb and
Garcia-Bellido (1982). Mutations of different genes affect
different regions of the body, but different mutant alleles at
each locus are very similar to each other and have the same
regional specificity. Most of these polarity mutants; dishevelled (dsh), frazzled (frz), fritz (frtz), frizzled (fz), fuzzy (fy),
inturned (in), multiple wing hairs (mwh) and prickle (pk)
affect the wing disc. In addition, the abdomen
is affected by dsh, frtz, fz, fy, in and spiny-legs (sple); the legs
by dsh, frz, frtz, fz, fy, in and sple; while three of the mutants
give a rough eye phenotype: dsh, frz and fz. Taken together,
these mutants affect all regions of the body. Two of the
mutants, dsh and fz, affect the whole body surface although
a class of fz alleles have been recovered that do not
affect the eye. The pk and sple mutants are unusual in that
they affect reciprocal regions of the body and represent
discrete classes of lesion in a complex genetic locus. Double
mutant pk-sple alleles affect the whole body surface and
give a rough eye phenotype (Heitzler, 1993 and Gubb, 1993).
In general, the topography of the regions affected by these
mutants remains wild-type as does the distribution of specialized
cell types (Gubb, 1982). In this
sense, cellular polarity is affected independent of other
elements of the pattern. In addition to altering the orientation
of hairs, the polarity mutations cause additional hairs to
be secreted by wing cells. This phenotype appears to be
common to all the mutations that affect the wing blade
although the effect is very slight with pk1 (Gubb,
1982) and fz1, with only occasional
wing cells giving duplicated hairs. Some
combinations of pk mutations and some fz alleles,
however, give a significant frequency of cells with doubled
hairs. At the other end of the range, mwh expresses several
hairs in every wing cell. The duplicated hair
phenotype is associated with abnormal organization of the
F-actin bundles that are present at the normal site of hair
initiation. This
suggests a link between polarity and cytoskeletal architecture
as in yeast cells (Gubb, 1993 and references).
As a general rule, the hair and bristle polarity of these
mutants is cell autonomous in clones. Mosaics of mutant
tissue within a wild-type background express the pattern
typical of that region in a homozygous wing. Both pk and fz
clones, however, can cause a disruption in the polarity of
neighboring cells. In the case of fz, this disruption may be
quite extensive, but is restricted to cells distal to the fz clone.
In addition to altering the orientation and number of wing
hairs, the imaginal polarity mutants cause fine-scale rotations
and mirror-image reversals. In the tibia, the bristles, together
with their associated socket cells and bracts, are rotated as a
unit.
Similar rotations are shown between the ommatidial units of
the compound eye in dsh, fz, frz and pk-sple. In these mutants the overall
shape of the eye remains normal, but the surface becomes
roughened as the corneal lenses fail to pack into an hexagonal
array. The internal structure of the ommatidia shows mirror-image
reflections as well as rotations. In sple1 flies,
ommatidia show reflections, but no rotations and the surface
of the eye remains smooth (Gubb, 1993 and references).
The different classes of mutants at the pk-sple locus
give a phenotypic series. In pk eyes, the ommatidial organization
is wild-type; sple mutations show mirror-image reflections
while pk-sple13 eyes show both rotations and reflections. This last phenotype is particularly informative in that
even when the ommatidial units are rotated, their spacing
remains relatively uniform. In this sense, the hexagonal array
is retained, although the orientation and handedness of
adjacent units is no longer co-ordinated.
The idea that the ommatidial units in the eye and the bristle
sense organs behave as discrete fields fits the known development
of these structures, which has been well studied. In contrast, very little is known about the
process of segmentation in the adult leg of Drosophila. Classical experiments in the cockroach,
however, show that individual tarsi behave as discrete developmental
units. The tarsal
segments of the legs of dsh, pk-sple, sple, in, fz, and frz flies show mirror-image duplications. The tarsal pattern
transformation is similar in all the mutants and corresponds
to a mirror-image duplication of both the proximal and distal
regions of each segment with loss of medial pattern elements. In addition, frtz tarsi show a weak transformation
consisting of occasional ectopic joints. The tarsi, therefore,
are regions in which these polarity mutants alter both topographical
shape and the distribution pattern of specialized cell
types, in addition to the polarity of individual cells. These
mirror-image duplications, together with the small size of the
tarsi, suggest that the tarsal segments represent discrete fields,
within which the fate of a cell is dependent on its immediate
neighbor. In the more proximal regions of the legs of dsh,
pk-sple, sple, in, fz, frz and frtz mutants, the orientation of
bristles and hairs is affected across large regions, but mirror-image
duplications are not seen.
The different classes of morphogenetic change caused by
the imaginal polarity mutants can be rationalized as follows:
the primary effect of the mutations is to alter cellular polarity.
In regions of the imaginal discs where fields of specialized
cell types are specified by interactions between adjacent
cells, mirror-image duplications and rotations of the fields
can occur. The fields are separated by regions of 'background'
cells that are not visibly differentiated with respect
to one another. In these regions, the polarity of individual cells
is altered, as indicated by the polarity of cuticular hairs. What
is not observed are distortions in the shape of imaginal structures,
frizzled A surprising result in the initial study of the imaginal
polarity mutants (Gubb, 1982) was the
demonstration of a large class of mutations that are viable,
but which cause discrete alterations in the polarity of adult
structures. The mutations correspond to complete lack of
function, at least by genetic tests, and yet the adult patterns
are uniform within a population, but more complex than
the wild-type pattern. In addition, most of the genes affect
different pathways, by the criterion that double mutants have
intermediate phenotypes. The exceptions to this are that
mwh and in are epistatic to pk, in that pk;mwh and pk;in
flies express the mwh and in phenotypes, respectively. An
indication that fy might affect a pathway related to both pk
and mwh is that fy;mwh and fy;pk flies give a weak tarsal
duplication phenotype showing ectopic tarsal joints, which are
not found in the fy, pk or mwh single mutant stocks, although
fy affects bristle and hair polarity and mwh causes multiple
hairs in the legs. Similarly, dsh and sple mutations together
give a more extreme tarsal joint pattern transformation than
either mutation alone and the wing polarity of dsh;sple flies
is much less extreme than dsh flies, although sple wings
have wild-type polarity (Gubb, 1993 and references).
It seemed unlikely that such a large set of genes should
be required for the correct orientation of bristles and hairs,
particularly as lack of function of these gene products
produces not random polarity but discrete alternative
patterns. The demonstration of fine-scale mirror-image
duplications in the tarsi and ommatidia shows that the
imaginal polarity mutants are affecting cellular polarity at a
fundamental level. This makes it all the more surprising that
the gene functions should be non-essential. One possibility
is that these polarity mutants control related products that
are functionally redundant. While this may well be true for
some of these products, it would predict that some of the
double mutant combinations of viable mutations might be
lethal. This is not the case for any of the double mutant combinations of dsh, pk, sple, frz, frtz, fz, fy, in and mwh, with the possible exception of frtz;pk and frtz;fz flies, which eclose at less than 1% the expected frequency.
The alternative hypothesis is that these gene products are
only required during the last few cell divisions in the
imaginal discs, so that even severe perturbations in cell
polarity do not cause a major developmental crisis (Gubb, 1993 and references).
The pattern of connections between R1-R6 neurons and their targets in the lamina is one of the most extraordinary examples of connection specificity known. An
interwoven set of connections precisely maps R cells in different ommatidia that 'see' the same point in space onto the same group of postsynaptic cells, the lamina
cartridge. R1-R6 cells that see the same point in space are distributed over six neighboring ommatidia as a consequence of the curvature of the eye and the angular
placement of their light-sensing organelles. Conversely, each of the R1-R6 axons from a single ommatidium sees a different point in
space and connects to a different set of lamina target neurons arranged in an invariant pattern. Each cartridge is innervated by a complete set of R1-R6 neurons from six different
ommatidia (i.e., an R1 from one ommatidium, an R2 from another, and so on). By superimposing multiple inputs from the same point in visual space upon a single
synaptic unit, the signal-to-noise ratio of the response to a signal in the visual field is enhanced. This phenomenon is called neural
superposition (Clandinin, 2000 and references therein).
The R1-R6 projection pattern develops in two temporally distinct stages. During the third larval stage, R cells extend axons into the brain, where they terminate
between two layers of glia, forming the lamina plexus. These glia act as intermediate targets for R1-R6 neurons. R cell axons induce the differentiation and organization of lamina target neurons and glia. At this stage of
development, R cell axons from the same ommatidium form a single fascicle. A column of lamina neurons forms above the lamina plexus, in tight association with a
single R cell axon fascicle. By the sequential addition of ommatidial bundles and their associated columns of lamina neurons, a precise retinotopic map forms in which
fascicles from neighboring ommatidia terminate adjacent to each other. As lamina neurons differentiate, they send axons along the surface of R cell axons through the
plexus and fasciculate with R7 and R8 as they project into the medulla. Although lamina neurons are in close association with R cell axons at this early stage, no
synaptic contacts are formed (Clandinin, 2000).
In the second phase of development, ~30 hr after reaching the lamina plexus, R cell axons defasciculate from each ommatidial bundle and project across the surface
of the lamina to their synaptic partners, making the pattern of connections characteristic of neural superposition. Growth of R cell axons toward their targets occurs
approximately simultaneously in all ommatidial bundles and is presaged by an invariant sequence of contacts between R cell growth cones. This reorganization of terminals converts a strictly anatomical retinotopic map that reflects neighbor relationships between ommatidia into a new
topographic map that reflects R cell visual response and reconstructs visual space in the first layer of the optic ganglion (Clandinin, 2000 and references therein).
R cell projections from a single ommatidium display two prominent features. (1) Each R cell axon terminates in an invariant
position relative to the other axons from the same ommatidial fascicle. (2) The projection is oriented with respect to the dorsoventral
midline of the eye (i.e., the equator), with the R3 axon extending toward the equator -- as a result, the projection patterns on opposite sides
of the dorsoventral midline of the eye are mirror images. Using mutations that eliminate specific subsets of R cells or alter ommatidial polarity, tests were performed to see whether R cell
synaptic specificity requires interactions among neighboring afferent axons or reflects independent navigation of each axon to its target. It has been demonstrated that
interactions between specific R cells are required for target selection, and it is proposed that the precise composition of R cell axons within a fascicle plays a critical role in
target specificity (Clandinin, 2000).
Neural superposition was first noted 90 years ago and the R1-R6 connection pattern in the lamina was first described using serial reconstruction of electron microscopic images in 1965. This pattern is cited as a classic example of extreme connection specificity. However, mechanistic analysis of this pattern was prevented by the absence of a rapid method for assessing R cell projections. In particular, the complexity of the pattern precludes conventional approaches based on visualizing all R cell axons in the target region, yet the assessment of connection specificity requires visualization of all R cell axons from one ommatidium. A method has been developed to label individual ommatidia with DiI and visualize the projection pattern using confocal microscopy. R1-R6 axons form a single bundle as they project into the brain. They defasciculate, project across the surface of the lamina, and then turn 90° and extend into the lamina cartridge. R cell axons elaborate a complex en passant presynaptic structure with lamina interneurons within the lamina cartridge. The axons of R7 and R8 project through the lamina, into the medulla. The relative positions of lamina targets chosen by each R1-R6 growth cone are invariant between ommatidia. This labeling method facilitates analysis of R1-R6 projections in various genetic backgrounds and creates a unique experimental system in which synaptic partner choices made by identified neurons can be directly assessed (Clandinin, 2000).
Serial electron microscopic reconstruction studies have revealed that, during pupal development, individual R cell axons leave their original bundle and migrate outward, in the precise direction of their final targets. This process was visualized using confocal microscopy. Early in pupal development, each ommatidial bundle forms a compact mass of expanded growth cones in the lamina plexus. This spherical mass then flattens, as distinct filopodial extensions corresponding to individual R cell axons become visible. This pattern of connections forms within a spatially patterned environment containing lamina target neurons and glial cells, as well as R cell axons. Since extension from the bundle is not preceded by extensive filopodial exploration, interactions between axons within ommatidial bundles may specify the initial trajectory of each growth cone. To address whether cell intrinsic mechanisms or interactions between R cell growth cones or both control target specificity, R cell projections were examined in mutant animals lacking specific subsets of R1-R6 cells. R cell axons from single ommatidia were labeled with DiI and visualized by confocal microscopy. In this series of experiments, animals were analyzed in which the eye was genetically mutant and the lamina neurons and glia in the target were wild type. Three mutant backgrounds were examined: (1) phyllopod, in which R1, R6, and R7 are transformed into nonneuronal cone cells; (2) lozengesprite, in which R3 and R4 are transformed into R7 cells; and (3) seven-up, in which R1, R3, R4, and R6 are transformed into R7 cells (Clandinin, 2000).
The first step of lamina target innervation is the coordinated defasciculation of R cell axons from bundles comprising axons from the same ommatidium. To determine whether interactions between specific subsets of R1-R6 axons are necessary for this defasciculation, R cell projections were assessed in phyllopod, seven-up, and lozengesprite mutants. In all three of the R cell transformation mutants examined, R cell axons migrated outward from the bundle. In particular, 4 R cell fibers in the lamina of 14/15 phyllopod mutant animals (missing R1, R6, and R7) and 20/24 lozengesprite mutants (missing R3 and R4) defasciculated from the bundle and projected to local targets. Similarly, in 17/23 seven-up mutants (missing R1, R3, R4, and R6), it was observed that the two remaining R cell axons defasciculated from the ommatidial bundle and innervated separate cartridges. In some cases, additional R cell axons also defasciculated, consistent with the reported incomplete expressivity of cell fate transformations in these mutants. In each case, axons projected to lamina targets in the local environment of the fascicle terminus. It is concluded that each R cell subtype is programmed to initiate a search for targets in a local region of the lamina target, independent of interactions between other R cell subtypes. In the following sections, whether interactions between specific R1-R6 cells regulate target specificity is assessed (Clandinin, 2000).
Two models could explain the mechanisms that determine the precise projection of R3 and R4 axons toward the dorsoventral midline and, by extension, the relative orientations of the other R cell axons. The growth cones of R3 and R4 may respond to an orienting cue in the lamina that promotes extension toward the dorsoventral midline. Alternatively, the orientation of R cell bodies in the retina may determine the orientation of R cell growth cones in the lamina, independent of any environmental cues. To assess the role of ommatidial polarity on projection specificity, projections from misoriented ommatidia were assessed (Clandinin, 2000).
If a lamina cue can promote equatorial extension of the R3 and R4 axons, ommatidia that rotate incorrectly should project their axons normally, toward the equator. Alternatively, if ommatidial orientation determines the direction of axon projection in the lamina, incorrectly oriented ommatidia should project their R3 and R4 axons away from the equator (Clandinin, 2000).
In wild-type animals, ommatidia are mirror image reflected about the dorsoventral equator of the eye. R cell projections are also mirror image symmetric about the equator but are rotated 180° with respect to the retina. That is, while the R3 cell body is oriented toward the pole in each ommatidium, its axon projects toward the equator in the lamina. This rotation is generated by a twist in the axon fascicle that occurs between the retina and the lamina (Clandinin, 2000).
To test the effects of large changes in ommatidial orientation, two mutations, spiny legs (in homozygous animals) and frizzled (in somatic mosaic animals in which a mutant eye projects to a wild-type target), were examined. In these mutants, ommatidia frequently adopt orientations that are 180° rotated; that is, the R3 cell body is frequently oriented toward the equator in the eye. In these two mutant backgrounds, the orientation of projections from ommatidia that were correctly oriented was normal. Therefore, neither gene is required for R cell axons to respond to orienting cues in the target. However, almost 90% of the ommatidia that were ~180° misoriented in the eye made projections that were also 180° misoriented in the lamina. Rare, abnormal projections of single R cell axons in both of these mutant backgrounds were observed, irrespective of ommatidial orientation. Therefore, the orientation of R cell projections along the dorsoventral axis of the lamina is largely determined by the orientation of ommatidia in the retina (Clandinin, 2000).
Three exceptional cases, in which misoriented ommatidia projected axons toward the equator, were observed. Thus, a cue in the lamina may reinforce the ommatidial orientation cue to ensure the correct direction of outgrowth along the dorsoventral axis. To test whether such a cue contributes to directionality of R cell projections, a mutation that causes a more moderate defect in ommatidial orientation was examined. In nemo mutant animals, ommatidia are misoriented up to 45°. If ommatidial orientation directly determines the directionality of R cell projections, they would be misoriented 45° with respect to the equator; the angle between ommatidial orientation and the axon projection pattern would remain 180°. However, while ommatidial orientation was disrupted in nemo, R cell projections were normal with respect to the equator. This observation suggests that in addition to ommatidial polarity, a cue in the lamina can influence R cell projection orientation (Clandinin, 2000).
It is concluded that interactions between R cell afferents play a crucial role in target specification, and it is proposed that the spatial relationships between axons within a fascicle influence synaptic specificity. It is hypothesized that the interactions between R cell subtypes that are required for target specificity are mediated by direct contacts between specific growth cones. R3 and R4 are required for the remaining R cell axons to choose their normal targets. R1 and R6 are required for R2 and R5 projections but are not required for the projections of R3 and R4. These interactions could occur between growth cones from the same or neighboring ommatidial bundles. The characteristic morphological changes of these growth cones as revealed through electron microscopic reconstruction studies are consistent with the notion that precise spatial relationships between specific growth cones within the lamina plexus are required for these critical interactions to occur. This sequence of interactions determines the relative positions of targets chosen by R cell axons from the same ommatidium (Clandinin, 2000).
R cell transformation mutants could disrupt these interactions in two ways. First, transformation of specific R cells could directly disrupt the instructive signals between R cell growth cones within the plexus that determine growth cone trajectories. Alternatively, these mutations could affect the interactions indirectly, by disrupting the spatial relationships between the remaining R cell axons. That is, outgrowth trajectory could be determined passively by the position each growth cone occupies as it leaves the ommatidial fascicle. In this view, these mutant backgrounds alter the composition of axons within each ommatidial bundle and, hence, disrupt the precise packing of axons within the fascicle. The differential requirements for particular R cell subtypes would reflect their specific roles in directing the spatial relationships between growth cones within the fascicle, rather than interactions between specific growth cones in the target region (Clandinin, 2000).
Ommatidial polarity is defined by the relative positions of R cells within an ommatidium. Each R cell occupies an invariant position; R1-R6 cells within each ommatidium create a pattern that is mirror-image symmetric about the dorsoventral midline of the eye. The observation that ommatidial polarity determines projection orientation requires that the spatial relationships between R cell bodies be maintained in ommatidial axon fascicles. Indeed, a striking feature of Drosophila visual system connectivity is the perfect conservation of spatial relationships between R cell axons, both within each bundle and with respect to the dorsoventral axis of the eye. It is hypothesized that the developmental mechanisms that determine where each R cell differentiates in the retina also control where each R cell axon lies within the fascicle and how the fascicle is oriented along the dorsoventral axis (Clandinin, 2000).
Control of projection orientation by ommatidial polarity also requires that the relative positions of R cell axons within a fascicle, as well as the dorsoventral orientation of the fascicle itself, be 'read out' in the lamina. In this view, the relative positions of axons within the fascicle allows the specific interactions between growth cones that control synaptic specificity to 'self-organize' the pattern of targets. Since axons from both correctly oriented and misoriented ommatidia choose targets arranged in a normal pattern, these interactions between growth cones must occur independent of orientation along the dorsoventral axis. In this model, fascicle orientation determines whether the pattern of targets chosen is oriented either dorsally or ventrally but does not determine the relative positions of the targets within the pattern. This approach of 'encoding' the spatial arrangement of sensory neuron cell bodies within an axon fascicle followed by 'reading out' the preserved orientation cues within the target may provide a general mechanism to generate highly precise patterns of connections (Clandinin, 2000).
Projection specificity in the lamina is not solely controlled by interactions between R cell axons. The observation that the R cell projections are correctly oriented in nemo mutant animals provides evidence that a cue(s) in the target can reorient R cell axons. Such a cue need only orient a subset of R cell axons, likely R3 and R4; these axons could then organize the remaining R1-R6 projections. This cue could be a weak signal that directs R3 and R4 axon outgrowth toward the equator. Alternatively, this cue could simply confine the outgrowth of R3 and R4 to the dorsoventral axis, without determining whether outgrowth is either dorsal or ventral. These target-derived cues could correct for small variations in ommatidial orientation (and fascicle orientation) that exist in the pupal eye prior to programmed cell death in the retina (Clandinin, 2000).
Previous studies suggest that connection specificity can be generated by activity-dependent refinement of synaptic contacts. Indeed, interactions between synapses mediated by electrical activity are clearly important for several aspects of neuronal connectivity in the vertebrate visual system, including the maintenance of ocular dominance columns and formation of eye-specific layers in the lateral geniculate nucleus. In the Drosophila retina-lamina projection, the role of neuronal activity is unclear. These connections likely develop independent of visual input since the projections form when R cells display little light-evoked response. Moreover, phototransduction-defective mutants display normal numbers of R cell termini in each cartridge, suggesting that R cell target selection occurs normally in these backgrounds. These connections also form prior to the development of morphologically distinct synaptic contacts between R cells and lamina neurons (Clandinin, 2000).
To test the effects of changes in neuronal activity and synaptic transmission on R cell targeting, known mutations affecting both sodium and potassium channel subunits, as well as synaptotagmin were examined. None of these backgrounds displays defects in R cell synaptic specificity. However, these genetic approaches are confounded by the significant molecular redundancy present in these protein families within the Drosophila genome. Synaptobrevin-mediated vesicle release was disrupted by expressing tetanus toxin specifically in R cells. Synaptobrevin blockade, however, affects expression of cell adhesion molecules in the Drosophila visual system and disrupts axonal morphology. Hence, it remains unclear whether disruption reflects a role for synaptic transmission in R cell target specificity or is an indirect result of effects on other cellular processes. In summary, while these experiments demonstrate that developmental mechanisms that are likely to be independent of neuronal activity are sufficient to generate the precise pattern of retina-lamina connections, a role for neuronal activity cannot be excluded (Clandinin, 2000).
The cellular mechanisms described here provide a conceptual framework for understanding the molecular basis of synaptic specificity. While the DiI method facilitates the analysis of R1-R6 specificity on a scale sufficient to analyze many mutants, it is too laborious to accommodate large-scale screening. Hence, a genetic screen based on visual behavior driven specifically by R1-R6 is required to extend these studies to the molecular level. A wealth of visual behaviors have been described in Drosophila, one of which, the optomoter response, is mediated by these cells. Techniques that generate mosaic flies in which only R cells are made homozygous for randomly induced mutations, while the rest of the fly is heterozygous, have recently been described. Currently, projects are underway, combining this specific behavioral screen with genetic mosaics, in order to screen for genes controlling R1-R6 synaptic specificity (Clandinin, 2000).
The frizzled gene is required for the development of distally pointing hairs on the Drosophila wing. It has been suggested that fz is
needed for the propagation of a signal along the proximal distal axis of the wing. The directional domineering non-autonomy of fz clones
could be a consequence of a failure in the propagation of this signal. This hypothesis was tested in two ways. In one set of experiments the domineering non-autonomy of fz and Vang Gogh (Vang) clones was used to assess the direction of planar polarity signaling in the wing.
prickle (pk) mutations alter wing hair polarity in a cell autonomous way, so pk cannot be altering a global polarity signal. However, pk mutations alter the direction of the domineering non-autonomy of fz and Vang clones, arguing that this domineering non-autonomy
is not due to an alteration in a global signal. In a second series of experiments, cells in the pupal wing were ablated. A lack of cells
that could be propagating a long-range signal does not alter hair polarity. It is suggested that fz and Vang clones result in altered levels of a locally
acting signal and the domineering non-autonomy results from wild-type cells responding to this abnormal signal (Adler, 2000).
The directional domineering non-autonomy of fz clones
in the wing was originally suggested to be due to a failure in
the proximal to distal propagation or transmission of a
polarity signal. This model
predicted a special population of cells that serve as a
source (or origin) of the signal. If this model is correct
how could mutations in pk and dachsous (ds) result in an altered direction of fz domineering non-autonomy? An obvious possibility is that these mutations could change the fate of some
cells so that an ectopic source of signal was produced. This
hypothesis is inconsistent with the cell autonomy/non-autonomy of pk clones. Consider the possibility that pk
produces a tissue polarity phenotype by causing the formation of an ectopic source of signal at a new location in the
wing. If a pk clone is located in such a region, then the
clone would be predicted to show domineering non-autonomy. This is infrequent in pk clones, but is not restricted
to clones in one or a few regions of the wing. If a pk clone
is located elsewhere, it would be expected to have no consequences for polarity. However, it was observed that cells inside
of all pk clones show altered hair polarity.
The source and directional transmission model also fails
to explain the results of temperature shift experiments with a cold-sensitive fz allele. If fz
function is required for the propagation of a signal along
the proximal distal axis of the wing it is predicted that a
temperature shift from the permissive to the restrictive
temperature during the middle of the temperature sensitive
period would result in a wing with a permissive phenotype
proximally and a restrictive phenotype distally. This is not
what was seen. Instead an intermediate phenotype
in all regions of the wing is found. This result argues that fz functions
in all regions of the wing at the same time and is not consistent with fz functioning in the propagation of a signal down
the wing (Adler, 2000).
The complementary nature of the domineering nonautonomy of fz and Vang clones is striking. It is true for
the anatomical direction of the non-autonomy (i.e. distal vs.
proximal); the relationship of the domineering non-autonomy to the clone (i.e. affected wild-type hairs pointing toward or away form the clone), and
for the interactions with pk mutations. It is suggested that the
domineering non-autonomy of fz clones is a consequence of
a failure of the clone cells to send a locally acting polarity
signal. The domineering non-autonomy of Vang clones
could be due to the Vang clone sending excess signal
(models that reverse this arrangement are also possible) (Adler, 2000).
A model for tissue polarity signaling in the wing is presented.
Early models to explain planar polarity in the insect
epidermis suggested it could be a reflection of the vector
of a concentration gradient
and this idea has remained popular. It is suggested
that a distal/proximal gradient of fz activity is produced in
the early prepupal wing (or wing disc). One way this could
be achieved is by a gradient of a Wnt (or other type of
ligand) resulting in a gradient of ligand bound Fz. Later in development cells would produce a
locally acting second signal in amounts proportional to Fz
activity. This hypothetical signal is referred to as Z and it is
suggested that ligand bound Fz activates more Z production
than unbound Fz. In this way a gradient of Fz
activity would be translated into a gradient of Z. Cells
would respond by initiating prehair morphogenesis on the
side of the cell where Z level was lowest. This would result
in hair polarity being oriented in the same direction as the
vector of the Z concentration gradient. This is consistent
with previous results showing that a directed gradient of fz
expression results in cells with higher Fz levels producing
hairs that point toward cells of lower levels. The absence of fz activity in clone cells would result
in no Z being produced by the clone and a local decrease in
Z levels that would cause surrounding cells to produce hairs
that point toward the clone as is observed. Such a model
can effectively incorporate the affects of pk and ds mutations
on the direction of fz domineering non-autonomy. Mutations
in these genes could alter the relationship between the
ligand bound state of fz and Z production. For example, in
a new antimorphic dominant pk allele, pkD wing unbound Fz receptor could act as a super-activator of Z production. This would lead to a reversed gradient of Z and to the reversal of both polarity and the direction
of fz domineering non-autonomy. This model can
also explain the observation that cells inside of a pk clone
display the same polarity as do cells in a similar position in
an entirely pk wing, since the alternative polarity caused by pk
mutations would be due to abnormal amounts of Z. Such a
model can also explain some of the results seen with Vang. The domineering non-autonomy of Vang could be due to Vang cells being constitutive for the
production of high levels of Z. This would lead to locally
elevated Z levels and cells surrounding Vang clones producing hairs that point away from the clone, as is observed.
The model can also explain the ability of pkD to enhance
the extent of fz domineering non-autonomy and suppress the
extent of Vang domineering non-autonomy. In the model
the level of Z will be higher in all regions of a pkD wing
since now both bound and unbound Fz receptor will be strongly
activating the production of Z. Thus, when a clone
of cells lacking functional Fz protein is produced, the difference between the amount of Z produced by the clone cells and their neighbors will be increased over that seen in an otherwise wild-type wing. The ability of pkD to inhibit the
extent of domineering non-autonomy of Vang clones can be
explained by the reduced difference in the level of Z
produced by the clone and neighboring cells (Adler, 2000).
At first glance the model cannot explain the suppression
of Vang domineering non-autonomy in a fz mutant background, because the clone should produce high levels of Z in a background where there is little or no
Z produced. One possibility is that in the absence of functional Fz no Z can be produced. A second possibility is that fz has multiple functions in wing tissue polarity and that an additional function is what suppresses the domineering non-autonomy of Vang. The model can also explain the relatively weak and
poorly penetrant domineering non-autonomy of pk clones.
The cells in such clones would produce aberrant amounts of
Z, however the difference between the normal and mutant
levels would be less than is seen in a fz mutant clone (that
produces no Z) or in a Vang mutant clone (that produces
constitutive high levels of Z). Thus, it is reasonable that pk
(and ds) clones would show weak domineering non-autonomy (Adler, 2000).
The frizzled gene of Drosophila encodes a transmembrane receptor molecule required for cell polarity
decisions in the adult cuticle. In the wing, a single trichome is produced by each cell, which normally points
distally. In the absence of frizzled function, the trichomes no longer point uniformly distalward. During cell polarization, the Frizzled receptor (visualized using Frizzled-Green fluorescent protein) is localized to the distal cell edge, probably resulting in asymmetric
Frizzled activity across the axis of the cell. Furthermore, Frizzled localization correlates with subsequent
trichome polarity, suggesting that it may be an instructive cue in the determination of cell polarity. This differential
receptor distribution may represent a novel mechanism for amplifying small differences in signaling activity across
the axis of a cell (Strutt, 2001).
To understand the asymmetric distribution of Fz-GFP, the distribution was studied in flies mutant for other genes involved in trichome polarity establishment. In clones of cells lacking starry night (stan) function, both the apical and PD localization of Fz-GFP is completely abolished. However, in cells lacking dsh function, in which Fz signal transduction is compromised, Fz-GFP apical localization is preserved, but there is no proximodistal (PD) localization, with a splotchy irregular distribution being seen instead. The same phenotype is observed for mutations in the prickle-spiny-legs (pkpk-sple) and Van Gogh (Vang) genes. This would be consistent with the trichome polarity phenotypes of these mutations being due to a failure of Fz localization (Strutt, 2001).
Genetic data indicate that the polarity genes in, fy, and mwh act downstream of Fz/Dsh, inhibiting trichome formation where Fz is not active. In agreement with this, mutations in these loci do not alter Fz-GFP distribution despite trichome polarity being disrupted (Strutt, 2001).
The following model is put forward for Fz function in the polarization of single cells in the developing wing. Initially, unlocalized Fz is required for the long-range propagation of a polarity signal. Fz is then recruited apically in a Stan-dependent manner and becomes stably localized at the distal cell edge in a process requiring Fz signaling and the activities of Stan, Dsh, Pkpk-sple, and Vang. This Fz localization then restricts the site of trichome initiation to the distal cell vertex. It is possible that Fz signaling activates Stan molecules to bind both to Fz (in the same cell) and to Stan molecules in the adjacent cell, and so anchors Fz at the distal edge of the cell. Localization of Fz may lead to further increased Fz signaling (possibly through the effects of receptor clustering), which could, in turn, recruit more Stan and Fz. Over time, increased activity of clustered Fz receptors at the distal cell edge would lead to the majority of the Fz in the cell being recruited to this location. In heterologous systems, Fz activity leads to recruitment of Dsh to the cell membrane, so it is likely that Dsh is also present at the distal cell boundary. A precedent for Fz-dependent localization of a cytoplasmic protein during planar polarity establishment is provided by the observation that the Numb protein requires Fz activity for correct asymmetric subcellular localization during sense organ precursor cell divisions (Strutt, 2001).
The stable PD localization of Fz also requires Pk-Sple and Vang activity, with their loss having a similar effect on Fz-GFP localization as loss of Dsh activity. It is possible that, like Dsh, they are required for the transduction of the Fz signal, or they may be involved in the function of Dsh itself. Interestingly, Vang activity on only one side of the PD boundary is sufficient for Fz-GFP localization to occur. Further investigations of the biochemical activities of these proteins will be required to fully elucidate their roles in planar polarity establishment (Strutt, 2001).
Tissue polarity in Drosophila is regulated by a number of genes
that are thought to function in a complex, many of which interact genetically
and/or physically, co-localize, and require other tissue polarity proteins for
their localization. The enhancement of the strabismus
tissue polarity phenotype by mutations in two other tissue polarity genes,
flamingo and prickle, is reported. Flamingo is autonomously required for
the establishment of ommatidial polarity. Its localization is dynamic
throughout ommatidial development and is dependent on Frizzled and Notch.
Flamingo and Strabismus co-localize for several rows posterior to the
morphogenetic furrow and subsequently diverge. While neither of these proteins
is required for the other's localization, Prickle localization is influenced
by Strabismus function. The data suggest that Strabismus, Flamingo and Prickle
function together to regulate the establishment of tissue polarity in the Drosophila eye (Rawls, 2003).
In an attempt to define more precisely the role of Stbm in the tissue
polarity pathway, genetic interactions were identifed between
stbm and two other tissue polarity genes, fmi and
pk. Characterization of the fmi-stbm interaction reveals a
requirement for Fmi in ommatidial polarity and a dynamic pattern of Fmi
localization that depends on Fz and N. An antibody was raised against
Stbm, its subcellular localization was characterized, and the
localization of Fmi and Stbm was shown to differ 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 shows that pk
enhances the stbm phenotype and that Pk localization requires
Stbm (Rawls, 2003).
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. PET and LIM domains are thought to mediate
protein-protein interactions. 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 (Rawls, 2003).
The 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 in this study (Rawls, 2003).
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, the balance of Pk isoforms contributes to the establishment of tissue polarity. Perhaps this balance is also required for Stbm function (Rawls, 2003).
In a deficiency screen,
pk was identified as a dominant genetic modifier of stbm.
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, Stbm localization was examined
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). However, Pk
localization is disrupted in a stbm6cn null background. The distribution of Pk was characterized in wild-type eye imaginal discs; it is
indistinguishable from that of Stbm. 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. 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 (Rawls, 2003).
In order to differentially affect signal transduction
through the N pathway, the assembly and/or activity of proteins that set up polarity must be different in R3 and R4. The model presented below requires that Stbm and Pk be restricted to the R4 cell to properly modulate N signaling. Stbm has been shown to be restricted to R4 at the R3/R4 boundary; the subcellular location of Pk in the eye has not yet been determined (Rawls, 2003).
It is proposed 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. 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. Ultimately, these differences in gene activity in the R3 and R4 precursors direct the fate specification of these cells (Rawls, 2003).
The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but they are poorly understood. This problem was investigated in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented by the planar cell polarity pathway. They are constructed by a hexagonal array of wing epithelial cells. Wing epithelial cells are irregularly arranged throughout most of development, but they become hexagonally packed shortly before hair formation. During the process, individual cell boundaries grow and shrink, resulting in local neighbor exchanges, and Cadherin is actively endocytosed and recycled through Rab11 endosomes. Hexagonal packing depends on the activity of the planar cell polarity proteins. It is proposed that these proteins polarize trafficking of Cadherin-containing exocyst vesicles during junction remodeling. This may be a common mechanism for the action of planar cell polarity proteins in diverse systems (Classen, 2005).
A link between the PCP pathway and epithelial repacking is suspected, because repacking occurs at the time that these proteins are thought to polarize. Therefore neighbor number and junction length variability was quantified at the time of hair outgrowth in different PCP mutants. For prickle (pk-sple13/26), neighbor number was quantitated over time (Classen, 2005).
pk-sple13/26 wings begin repacking at the same time as wild-type; however, the process is less successful. Whereas wild-type wings reduce the percentage of pentagonal cells from 34% to 13% by the time that hairs begin to emerge, pk-sple13/26 wings retain 21%. Thus, about 40% of the pentagonal cells that normally assemble boundaries with new neighbors (and become hexagonal) fail to do so in pk-sple mutants. Consistent with this, pk-sple wing epithelia contain abnormally high numbers of four-way vertices between cells. pk1 mutant wings are even more irregularly packed than pk-sple13/26 wings. A total of 62% of the pentagonal cells that would normally become hexagonal fail to assemble boundaries with new neighbors in pk1 wings. Even four-sided cells accumulate significantly in pk1 mutant wings. Individual cell contact lengths are also much more variable; while pk-sple13/26 boundary lengths were 9% more variable than wild-type, those of pk1 were 42% more variable. These data are consistent with the earlier observation that adult pk wings frequently contain pentagonal cells. These data suggest that the assembly of new cell boundaries and regularization of junction length do not occur efficiently in the absence of products of the Pk-Sple locus (Classen, 2005).
Packing defects of the hypomorphic Flamingo (fmi) allele, fmi(stan)3, are mild but significant. The null allele fmiE59 produces much stronger defects. The variability of individual junctional lengths in these cells is more than twice that of wild-type, and only 69% of fmiE59 mutant cells become hexagonal, compared with 78% in wild-type. Pentagonal cells persisted in fmiE59 mutants (27% compared with 13% in wild-type). This suggests that the majority of pentagonal cells fail to assemble boundaries with new neighbors when Fmi is missing (Classen, 2005).
The packing geometry was examined of two different frizzled (fz) alleles, fzR52 and fzP21. fzP21 mutant wings fall into two classes. While the majority of wild-type and PCP mutant wings initiate hair formation by 42 hr after puparium formation (APF) (at 22°C), a subset of fzP21 mutant wings does not. Since these wings were not apoptotic (as indicated by Caspase staining), they were included in the analysis and quantified separately. Even at 50 hr APF, their packing is much more irregular than that of wild-type . Defects in fzP21 mutant wings that do initiate hair formation by 42 hr APF are milder but still significant. fzR52 homozygotes do not produce viable pupae in these experiments, and homozygous mutant clones are small. These clones have even stronger packing defects than those of fzP21, suggesting that little repacking occurs in fzR52 homozygous tissue. Thus, Fz is needed to develop regular hexagonal packing (Classen, 2005).
stbm6 and dgo380 mutant wings have milder, but significant, alterations in the ratio of pentagons, hexagons, and heptagons and of four-way vertices. Both mutants, however, affect junction length variability more strongly than pk-sple13/26. Taken together, these data indicate that PCP mutant cells fail to efficiently assemble boundaries with new neighbors and cannot regularize their packing geometry (Classen, 2005).
To ask whether interfering with PCP polarity could alter the geometry of packing in wild-type cells, cells were examined surrounding PCP mutant clones with either autonomous (fmiE59) or nonautonomous (fzR52) effects on polarity. The frequency of pentagons, hexagons, and heptagons was examined in fzR52 and fmiE59 mutant clones, and in the areas of disturbed and normal Fmi polarity surrounding both. The mutant cells within both fzR52 and fmiE59 clones are abnormally packed. However, whereas the packing defects caused by Fmi clones are predominantly restricted to the clone and directly adjacent cells, Fz clones alter packing over long distances in wild-type tissue in the same regions where Fmi polarity is disturbed. The abnormal packing of wild-type cells surrounding fzR52 clones is unlikely to be a consequence of altered cell packing within the mutant clone, because fmiE59 mutant clones pack just as abnormally, but do not perturb packing in the surrounding tissue. This suggests that dominant reorientation of Fmi polarity by frizzled mutant clones disturbs the repacking of wild-type cells (Classen, 2005).
To investigate how the PCP proteins were localized during repacking, pupal wings were imaged for Fmi before, during, and after hexagonal packing. Since it is thought that PCP proteins do not polarize until shortly before hair formation, it was surprised to find that the subcellular distribution of Fmi is polarized in many areas of the wing before junction remodeling is initiated, even in late third instar wing discs and prepupal wings. Fz-GFP is distributed similarly. This polarity may have been missed because it exhibits less long-range coherence in imaginal discs and prepupal wings than it does later (Classen, 2005).
In prepupal wings, Fmi polarity is roughly proximal-distal in the region surrounding L3. Coherent Fmi polarity is lost at the beginning of the pupal period: this is exactly the time at which junction remodeling initiates. Although polarity is not coherent, Fmi is not uniformly distributed along cell boundaries. This can be clearly seen when Fmi localization is compared to that of E-Cadherin (Classen, 2005).
At pupal time TP1, Fmi polarization begins in vein cells as they contract their apical cross-section. Intervein regions contain only small groups of cells with coherent polarity, and the axes of these groups are not always proximal-distal. By TP2, Fmi polarity is coherent between larger groups of cells, although the axis of polarity is still mixed. Fmi polarity is aligned in large coherent domains along the proximal-distal axis by TP4, when hexagonal packing is completed, and it remains unchanged at TP5 when hairs emerge. In summary, PCP proteins polarize during larval and prepupal stages, alignment of polarity between cells is disturbed when junction remodeling begins, and long-range polarity is reestablished as hexagonal packing is completed. Early polarization of PCP proteins is consistent with the genetic requirement for fz and ds activity at this time to determine the axis of polarity, and it suggests that the feedback loop that organizes coupled proximal and distal domains probably acts during these early stages (Classen, 2005).
It was asked whether PCP proteins might affect packing by influencing recycling of junctional components. Therefore, it was asked whether PCP mutants enhance the hole formation caused by shi loss of function. Double mutant pupae were shifted to a subrestrictive temperature that never causes holes to form in shi mutants or in PCP mutants. When shi is combined with dgo380, stbm6, stbm153, stbmD, stan3, pk-spl1, or pk1, hole formation occurs even under these mild conditions. This raises the possibility that PCP proteins may worsen Cadherin recycling defects in dynamin mutant cells. Consistent with this, gaps in Cadherin arise more frequently in double shi;pk1 or shi;dgo380 mutant wings than in wings mutant for shi alone. This suggests that Cadherin is recycled less efficiently in the absence of PCP proteins (Classen, 2005).
Despite this enhancement, no striking abnormalities in Cadherin distribution were seen in most PCP mutants. fzP21 mutant cells sometimes show gaps in E-Cadherin that are similar to, but much less frequent than, those of shi mutants. In fmiE59 mutant cells, E-Cadherin levels are elevated, but no gaps in localization are observed. These observations suggest that PCP proteins are not required for delivery of Cadherin to cell contacts during remodeling. Nevertheless, the PCP mutants enhance Cadherin recycling defects caused by loss of Dynamin. One model consistent with this shows that PCP proteins bias Cadherin recycling to specific places on the cortex. Reducing both the rate of recycling and its elevation at a particular site could exacerbate the failure of Cadherin delivery to growing cell boundaries (Classen, 2005).
To test whether exocyst components were polarized by PCP proteins, Sec5 localization was examined during repacking of the wing epithelium. At this time, cell shapes are irregular, and Fmi polarity is not coherent between cells. Nevertheless, Fmi accumulates preferentially on specific regions of the cortex. Although Sec5 vesicles are seen throughout the cell, they are particularly enriched near Fmi-positive cell boundaries. Enrichment persists as Fmi polarity becomes aligned (Classen, 2005).
To test whether Fmi plays an active role in recruiting Sec5, Fmi was overexpressed and Sec5 localization was examined. Overexpressed Fmi is present uniformly around the cortex and in large punctate structures within the cell. Sec5 dramatically accumulates in cells overexpressing Fmi and is recruited to sites of Fmi localization. Large internal structures positive for Fmi and Sec5 also contain Cadherin. These observations indicate that Fmi can recruit Sec5-positive vesicles containing E-Cadherin, and they suggest that PCP proteins may promote hexagonal packing by polarizing membrane trafficking (Classen, 2005).
The conserved cassette of PCP proteins controls a variety of seemingly different developmental processes, and no common cell biological mechanism has ever been proposed for their action. Polarizing membrane trafficking by recruiting Sec5 is a basic function that could be utilized in many different contexts, and it may help explain the requirement of PCP proteins in a divergent set of processes. Both rotation of photoreceptor clusters and convergent extension movements depend on the ability of cells to make and break intercellular contacts, as they do during hexagonal packing in the wing. Consistent with this, Silberblick (Wnt-11) acts through the PCP pathway and appears to affect endocytic trafficking of Cadherin during zebrafish gastrulation. Recruitment of exocyst components might also be a plausible mechanism to explain the ability of PCP proteins to bias Notch Delta signaling between R3 and R4 photoreceptors, since Delta delivery is dependent on the exocyst. In the future, identifying the chain of events that leads from PCP protein localization to exocyst recruitment may increase the understanding of these important processes (Classen, 2005).
prickle:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| References
date revised: 17 October 99
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