TI - Discussion . AB - Our results indicate that Wg and Hh act as instructive cues in the Drosophila embryonic epidermis to establish planar cell polarity . Though the complete molecular mechanisms that control the complex system of PCP in the ventral epidermis remain to be determined , this process appears to occur in part through the asymmetric localization of Arm at the membrane . Further , proper polarity signaling is abolished if specific PHOSphorylation sites within the alpha-catenin binding domain of Arm are mutated . These sites were originally found to increase the affinity of beta-catenin for alpha-catenin when PHOSphorylated by Casein Kinase II in vitro , suggesting one mechanism for stabilizing junctions[28] . Our findings provide in vivo support for this hypothesis , as low levels of ArmAA rescued cellular junction defects to a similar extent as expression of an alpha-catenin/E-cadherin fusion protein , a protein that makes overly stable junctions [30] . Higher levels of ArmAA expression lead to apparent polarity defects . As ArmAA does not localize asymmetrically the way that wild-type Arm does , we inferred that CKII PHOSphorylation may be required for the accumulation of junctions in specific regions of cells implying that stable junctions at specific sites in a cell are required for proper planar cell polarity . Further , our findings revealed that when all signaling activity is abolished through null mutations in the Wg or Hh signaling pathways , both cell identity and polarity determination was disrupted . It remains to be determined how Wg and Arm proteins function in polarity signaling , specifically whether they work through known PCP components , function similarly to their role in dorsal closure , or perhaps through novel signaling mechanisms like the interaction with Notch or Axin[36]-[39] . The wg and hh genes are required for the proper establishment of cell identities within segments[12] . Several studies have suggested that there are multiple roles for Wg and Hh during embryogenesis[40]-[44] . Uniform expression of Wg in the embryo leads to a completely naked cuticle[45] , but short early bursts of expression establish what appears to be relatively normal patterning[44] . Upon closer inspection , however , the denticle orientations of these early expression rescue experiments do not entirely resemble the wild-type patterning[44] , [46] . This suggests that early expression of Wg can rescue several aspects of cell identity , including development of naked cuticle , but Wg is also required in the later stages when denticles form to specify proper orientations . Expression of ectopic Wg has been observed to correlate with denticles pointing toward the source of Wg[47] and expression of ectopic Hh also leads to denticles pointing away from its source[48] . These studies , however could not distinguish between cell fate transformation and changes in cell polarity since the sources of both ligands were in the normal orientation . Our observations argue that Hh and Wg can have direct effects on cell polarity since denticles and their precursors ( the Actin foci ) are rotated 90deg away from the anterior-posterior axis corresponding to the direction of ligand expression . In the early embryo , expression of Wg and Hh is determined by pair-rule genes , but this effect is transient and requires mutually reinforcing positive activation loops to form between cells expressing Wg and En/Hh[12] . This is the early signaling event that establishes an organizer region in each parasegment[48] . Therefore , if either Hh or Wg is missing , expression of both is lost . The early effects of Hh and Wg expression are important for the establishment of segment boundaries[47] , [49] , and these boundaries function in limiting Wg function , giving this morphogen an asymmetric range[33] . Our findings agree with these observations , because we observe that the Wg effect is best observed when hh is absent , suggesting that when the hh gene is present a boundary may be formed , thus preventing Wg from orienting the denticles to the same extent . It also appears that the distance over which Wg can act is longer in the absence of hh as expected from previous observations[33] . According to the proposed boundary model , the extent of Wg influence is to the first denticle-secreting cell , . This finding , along with our discovery that denticles orient toward the source of Wg , may explain why the first row of denticles in wild-type larvae points toward the anterior of the embryo . Only this row of cells receives Wg signal as the segment boundary blocks further action by Wg to the next row of cells[33] . On the other hand , Hh can and does affect the next two rows of cells . We found that expression of Hh causes a rotation away from the source , and could explain why the next two rows of denticles point toward the posterior of the embryo . Our results do not explain the final orientation of all rows of denticles , and one likely complication is that in late embryonic stages the Notch and EGFR signaling pathways affect the identities of cells within the denticle belt[12] , [20] , [21] . It will be interesting to test what effects these signals have on the final orientation of the orientation of denticles , and whether the Notch pathway functions in polarity as well . The PCP signaling pathway determines planar polarity in a variety of tissues[4] . In vertebrate and C.elegans studies , Wnts have been implicated in the establishment of polarity , but only one study in Drosophila suggested a role for Wg in PCP[14] . In fact , the present model excludes the known morphogens , and suggests that PCP is established through cell cell interactions involving atypical cadherins like Flamingo or through an as yet unidentified factor X[50]-[57] . Though our study does not address the function of the known components of PCP signaling in the embryo , it is interesting that mutants in PCP signaling pathway components affect the polarity of the first two rows of denticles[14] , [58] . Our findings support the possibility that Wg and Hh lead to the expression of an unknown factor affecting the polarization of denticles , because blocking the transcriptional readout of either Wg or Hh with tcf or ci mutations respectively prevents the polarizing activity of both pathways . This is similar to the PCP disruptions found in the Drosophila eye model for Wg signaling components[50] . Our observations do , however , offer a further possibility , namely that by blocking all Wg signaling with null mutations the underlying polarity organizing function of Wg may be obscured . We find that in the weak armF1A mutant the orientation of denticles can be affected by the expression of Wg without affecting the cell -fates , suggesting that perhaps Wg can affect polarity directly . This effect of Wg was not observed in stronger arm mutant embryos suggesting that Arm protein is required for the Wg effect on denticle orientation . Interestingly , cell culture work has recently implicated Wg in controlling adherens junction strength[59] . The use of the embryonic epidermis allowed us to observe the interesting possibility that Arm functions in cell polarity . Since some of the molecules involved in the PCP signaling pathway are similar to Cadherins[57] , it seems logical that adhesion is involved in the establishment of polarity . However , adherens junctions have not been implicated so far . This is likely due to the difficulty of working with adherens junction component mutations that are often cell -lethal in the systems that have been used to study PCP . Here we have used the embryo , a system that allows relatively simple perturbation of arm function , and efficient ubiquitous or directional ectopic expression . Unfortunately , the major limitation of the ventral midline expression assay is that it only works for secreted , diffusible ligands . Thus , cell -autonomous activation of Hh or Wg pathway components ( such as with activated Arm or Smo ) along the ventral midline cannot be observed , since these cells invaginate and do not become a part of the external epidermis . We are currently working on ways to overcome this technical limitation . The fact that beta-catenin is both an oncogene and a component of adherens junctions has led to many studies attempting to link the PHOSphorylation state of beta-catenin in adherens junctions to the epithelial to mesenchymal transition ( EMT ) in cancer cells and during development . PHOSphorylation of tyrosine residues in beta-catenin is thought to lead to disassembly of adherens junctions[25] , [26] , [60] , but recent studies both in vivo and in vitro have challenged this[27] , [61] . Certainly these discrepancies will have to be resolved , but here we provide evidence for a different mechanism for regulating junctions , and perhaps EMT , through threonine phosphorylaTION-based stabilization or dePHOSphorylation-based destabilization of junctions . It will be crucial to establish which is the regulated step , and whether there are any phosphatases involved in this process in addition to the known kinase CKII[28] . Interestingly , the recent findings that alpha-catenin and beta-catenin do not form a stable complex in junctions [23] , [24] , suggests a possible explanation for our findings . We speculate that expression of ArmAA can rescue the basic activity of junctions lost in strong arm mutant embryos , which is to hold a tissue together . However , its reduced affinity for alpha-catenin does not cause a local increase in alpha-catenin levels and therefore Actin levels do not become asymmetric . This leads to a skewing of the normal polarization of the Actin cytoskeleton . It will be crucial to determine how junctions are localized asymmetrically in the first place , and whether this is dependent on extracellular signaling . These findings , and the effects of alpha-catenin mutations on inflammation and tumor progression in the mouse epidermis[62] make analysis of the interaction between alpha - and beta-catenin particularly important . These experiments provide some of the first evidence that the Hh signaling pathway is involved in polarity . It is particularly interesting that Hh expression leads to the reorganization of Actin structures within epithelial cells , since this suggests that Hh can affect the polarity of the Actin cytoskeleton . This finding is also relevant to cancer biology , because during metastasis , cancer cells lose polarity and essentially ignore their environment . Our results show that Wnts and Hh can affect cell polarity , in addition to their well-known effects on cell proliferation[16] , [63] , [64] . Along with the recent report that TGFbeta signaling affects polarity and EMT[65] , our findings imply that this dual role may be a general feature of oncogenic signaling pathways .