The Interactive Fly

Zygotically transcribed genes

Planar cell polarity/Tissue polarity genes

  • Signaling pathways involved in planar cell polarity
  • Jun mediates Frizzled-induced planar polarity determination in the Drosophila eye
  • Dissecting the molecular bridges that mediate the function of Frizzled in planar cell polarity
  • Prickle/spiny-legs isoforms control the polarity of the apical microtubule network in planar cell polarity
  • Clustering and negative feedback by endocytosis in planar cell polarity signaling is modulated by ubiquitinylation of Prickle
  • The proteins encoded by the Drosophila planar polarity effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of 'upstream' planar cell polarity proteins
  • The atypical cadherin Dachsous controls left-right asymmetry in Drosophila
  • Microtubules provide directional information for core PCP function
  • Symmetry breaking in an edgeless epithelium by Fat2-regulated microtubule polarity
  • The clathrin adaptor AP-1 complex and Arf1 regulate planar cell polarity in vivo
  • Positioning of centrioles is a conserved readout of Frizzled planar cell polarity signalling
  • The PCP pathway regulates Baz planar distribution in epithelial cells
  • SEGGA: a toolset for rapid automated analysis of epithelial cell polarity and dynamics
  • Planar polarized Rab35 functions as an oscillatory ratchet during cell intercalation in the Drosophila epithelium
  • Meru couples planar cell polarity with apical-basal polarity during asymmetric cell division
  • The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dach
  • Vamana couples fat signaling to the Hippo pathway
    Genes involved in tissue polarity

    Signaling pathways involved in planar cell polarity

    Frizzled (Fz) signaling regulates the establishment of planar cell polarity (PCP). The PCP genes prickle and strabismus are thought to antagonize Fz signaling. They act in the same cell, R4, adjacent to that in which the Fz/PCP pathway is required in the Drosophila eye. Stbm and Pk interact physically; Stbm recruits Pk to the cell membrane. Through this interaction, Pk affects Stbm membrane localization and can cause clustering of Stbm. Pk is also known to interact with Dsh and is thought to antagonize Dsh by affecting its membrane localization. Thus the data suggest that the Stbm/Pk complex modulates Fz/Dsh activity, resulting in a symmetry-breaking step during polarity signaling (Jenny, 2003).

    pk function is required in the R4 precursor, as opposed to fz PCP signaling in R3, for control of polarity establishment. Stbm, a transmembrane protein also required in R4, interacts genetically and physically with Pk. This interaction is important for the recruitment of Pk to the plasma membrane. In Xenopus animal-cap explants, Stbm and Pk relocalize each other to subdomains of the membrane. A model is proposed of how Pk/Stbm might regulate Fz/Dsh signaling activity (Jenny, 2003).

    The in vitro molecular interaction between Pk and Stbm and their mutual relocalization in Xenopus animal caps suggest that they form multiprotein complexes. Several pieces of evidence indicate that the physical interaction is physiologically important: (1) correct membrane localization of Pk depends on stbm function because in stbm mutant tissue Pk staining is diffuse and absent (or strongly reduced) at the membrane; (2) Pk and Stbm interact genetically by mutually enhancing each other's GOF and LOF phenotypes in the eye; (3) pk is necessary for PCP signaling in the R4 precursor, the same cell in which stbm is required; (4) expression of the interacting domains of Pk or Stbm interferes with polarity establishment. In particular, a subfragment of 131 amino acids of the C-terminus of Pk, required for the molecular interaction, is sufficient to affect polarity (Jenny, 2003).

    Both Pk and Stbm act as if they antagonize Fz signaling. (1) In zebrafish, Stbm overexpression can prevent Wnt11 from rescuing a wnt11 mutation. (2) In the Drosophila wing, overexpression of Pk leads to wing hairs pointing towards the source of the overexpressed protein, behaving like a fz LOF clone (whereas overexpression of Fz leads to hairs pointing away from the Fz source). stbm LOF clones show the opposite behavior to fz LOF clones: wing hairs point away from the mutant patch, consistent with the mutant tissue having a higher Fz-activity (Jenny, 2003).

    In the Drosophila eye, evidence that pk acts antagonistically to fz comes from the fact that the Notch-signaling-responsive R4-specific reporter mdelta0.5-lacZ is expressed for a prolonged period in both R3/R4 precursors in a pksple1 mutant. This is explained if Fz activity in the R4 precursor is increased, resulting in higher levels of Dl there. This in turn leads to N activation and concomitant mdelta0.5-lacZ reporter expression in both cells of the R3/R4 pair. Conversely, in fz and dsh mutant eye discs (where Fz signaling is absent or reduced and thus Dl should not be upregulated) N-signaling activity and mdelta-lacZ expression is initially reduced in both cells. Fz activity is also antagonized by stbm in the eye. Mosaic analysis of stbm shows that it has the capability to instruct a cell to become R4 as long as the other cell of the R3/R4 pair is mutant for stbm. Therefore, in such an all-or-nothing situation, Stbm in the R3 precursor can override a positive signal of Fz, resulting in a cell fate switch to R4 fate (Jenny, 2003).

    In a wild-type situation with all PCP components present in both cells, it is crucial that Stbm activity is higher in R4 than in R3 to ensure proper Fz-signaling regulation. Therefore it is an intriguing possibility that a Pk/Stbm complex in the R4 precursor ensures such higher Stbm activity, and the associated higher Fz repression there is important for a proper R3/R4 cell fate decision (Jenny, 2003).

    How does the Stbm/Pk complex regulate Fz-signaling activity? During PCP establishment in the wing, Fz, Dsh, Dgo, Fmi and Pk are initially localized uniformly around the apical circumference of wing cells. During and after PCP signaling, these proteins relocalize and become differentially enriched: Pk concentrates on the proximal side of the cell, whereas Fz and Dsh become enriched distally. Fmi becomes enriched at both sides (Jenny, 2003).

    In the eye, the situation is analogous. During PCP establishment, signaling components at the R3/R4 cell border are relocalized from a uniform to a more restricted pattern. Stbm-YFP is localized on the R4 but not on the R3 side, and Fz-GFP ends up on the R3 but not the R4 side. The analogy between the R4/R3 and proximal/distal cell borders is corroborated by the genetic requirements in R3 and R4: the distally localized factors Dsh and Fz are required in R3, while proximally localized Pk is required in R4. Fmi is localized on both poles of each wing cell and also required in both cells of the R3 and R4 pair. The function of fmi has been linked to both proposed complexes, the 'Fz/Dsh side' (fmi is required for apical localization of both Fz and Dsh) and the Stbm/Pk complex. In addition to the genetic interactions between fmi and stbm or pk, a reduced membrane staining of Pk in fmi- clones in wing cells has been shown (Jenny, 2003).

    How do these changes in localization occur? Localization studies in Drosophila and Xenopus suggest that Pk and Stbm influence each other's localization and form clusters in subdomains of the cell membrane. Interestingly, such Stbm/Pk complexes also affect Fz-dependent Dsh membrane localization. Thus it is an intriguing possibility that the patches observed in animal cap cells upon coinjection of Stbm with Pk represent the result of a similar, though unpolarized, symmetry-breaking step during PCP signaling (Jenny, 2003).

    The PET/LIM domain of Pk can interact with the DEP-domain/C-terminus of Dsh. This interaction has been suggested to prevent Dsh membrane recruitment. Also, the C-terminus of Stbm can interact with Dsh as long as the PDZ domain is present. Since the data suggest that Pk regulates the activity and localization of Stbm, this regulation might promote or stabilize the interactions of Dsh with Stbm and/or Pk, thereby helping to pull Dsh away from a Fz-signaling complex. The Stbm/Pk complexes could then cause active release of Dsh from the membrane or target it for degradation, resulting in low levels of Dsh (and by inference Fz) at places where Pk and Stbm are enriched. Furthermore, in the R3 cell (or distally in the wing) an unknown factor might act to prevent either the formation of the Stbm/Pk complex or its effect on Dsh (Jenny, 2003).

    In conclusion, Pk and Stbm form a functional complex during PCP signaling in Drosophila and during convergent extension in Xenopus. Interestingly, in zebrafish, in addition to its function in convergent extension, Stbm is also required for the caudal migration of hindbrain motor neurons. This function of Stbm is independent of Dsh and the PCP genes tested so far. It will be interesting to determine whether Stbm and Pk function together in this context as well (Jenny, 2003).

    Jun mediates Frizzled-induced planar polarity determination in the Drosophila eye

    Jun acts as a signal-regulated transcription factor in many cellular decisions, ranging from stress response to proliferation control and cell fate induction. Genetic interaction studies have suggested that Jun and JNK signaling are involved in Frizzled (Fz)-mediated planar polarity generation in the Drosophila eye. However, simple loss-of-function analysis of JNK signaling components does not show comparable planar polarity defects. To address the role of Jun and JNK in Fz signaling, a combination of loss- and gain-of-function studies has been used. Like Fz, Jun affects the bias between the R3/R4 photoreceptor pair that is critical for ommatidial polarity establishment. Detailed analysis of jun- clones reveals defects in R3 induction and planar polarity determination, whereas gain of Jun function induces the R3 fate and associated polarity phenotypes. Affecting the levels of JNK signaling by either reduction or overexpression leads to planar polarity defects. Similarly, hypomorphic allelic combinations and overexpression of the negative JNK regulator Puckered causes planar polarity eye phenotypes, establishing that JNK acts in planar polarity signaling. The observation that Delta transcription in the early R3/R4 precursor cells is deregulated by Jun or Hep/JNKK activation, reminiscent of the effects seen with Fz overexpression, suggests that Jun is one of the transcription factors that mediates the effects of fz in planar polarity generation (Weber, 2000).

    Jun, as a member of the AP-1 family, is activated by many distinct extracellular stimuli and acts downstream of several signaling pathways. Besides its involvement in stress response, Jun has been implicated in the control of proliferation, apoptosis, morphogenesis and cell fate induction. In Drosophila, Jun is critical for the process of dorsal closure in embryogenesis acting downstream of the JNK module. It has also been implicated in cell fate induction downstream of Ras/ERK signaling in the eye. This analysis has shown that Jun also acts downstream of Fz in planar polarity signaling in the eye. It is the first transcription factor implicated in Fz/planar polarity signaling. Fz signaling also requires a JNK (or related kinase) module, and thus in the eye imaginal disc Jun acts downstream of both ERK and JNK. How does Jun achieve a specific response in this context? The S/T residues that are phosphorylated in Jun are the same for both ERK and JNK. Thus, although differences in phosphorylation level and/or preference for any of the serine/threonine target residues cannot be excluded in vivo, differential phosphorylation is unlikely to create specificity. A potential mechanism for specificity might be provided by other transcription factors that cooperate with Jun in the different processes. This is supported by the observation that the sev-JunAsp (expression of a constitutively active Jun) phenotype is a composite of two events, photoreceptor recruitment and ommatidial polarity generation. These two effects can, however, be separated by the reduction of specific interacting partners. In the process of Ras/ERK signaling in photoreceptor induction Jun interacts and synergizes with the ETS domain transcription factor Pointed (Pnt). Pnt has been characterized as a target of the ERK/Rl kinase in Drosophila in all ERK-dependent processes analyzed. However, it has not been linked to any JNK-mediated process. Removing one dose of pnt strongly suppresses the Ras/ERK-related extra photoreceptor phenotype of sev-JunAsp, whereas the polarity defects persist and thus are more prominent. This observation indicates that, in the absence of normal Pnt levels, sev-JunAsp specifically affects polarity, suggesting that the interaction with Pnt is important for its role in the ERK-mediated induction. It is likely that for its planar polarity function other specific transcription factors provide the specificity cues (Weber, 2000).

    Although all components of the JNK module tested genetically interact with sev-Fz and sev-Dsh, analysis of existing loss-of-function mutants did not show defects in planar polarity establishment, suggesting a redundant role. Even null alleles of the Drosophila homolog of JNKK hep have no effects on planar polarity (Weber, 2000).

    However, expression of a dominant negative (kinase dead) isoform of Bsk interferes with planar polarity, giving rise to typical polarity phenotypes, implying that Bsk and JNK signaling are important in this process. Consistently, homozygous mutant clones of the deficiency Df(2R)flp170B that removes bsk and other neighboring loci (a deficiency considered to be a true null for bsk), show a mild polarity phenotype in the eye, including the presence of symmetrical ommatidia (Weber, 2000).

    What are the redundant kinases in this process? Genetic interaction analysis with sev-Msn (Misshapen expressed in a Sevenless pattern) has shown that, besides hep and bsk, deficiencies affecting other MKKs and the Drosophila p38a and p38b loci suppress the sev-Msn phenotype. This suggested that the p38 kinase module [related to JNK and has been shown to have (at least partially) overlapping phosphorylation targets] might be responsible for the redundancy in this process. The analysis with the dominant negative (DN) Bsk isoform and the respective deficiencies suggests that the p38 kinase(s) are contributing to this redundancy, because they enhance the DN-Bsk phenotype in a manner very similar to that of the bsk deficiency. The identification of specific mutant alleles of p38a/b and double mutant analysis with bsk will be necessary to further clarify this issue (Weber, 2000).

    The available results indicate that the level of JNK/p38 signaling in planar polarity establishment is important, but that the removal of a single kinase does not significantly affect this level. In support, the observation that an allelic combination of hep and puc hypomorphic alleles can give rise to planar polarity eye phenotypes suggests that the balance between negative and positive regulators of JNK and related kinases is critical. Similarly, overexpression of the negative JNK regulator Puc, a dual specificity phosphatase, causes typical polarity defects similar to those of fz or dsh mutants. It is likely that this phosphatase negatively regulates all JNK-related kinases and thus reduces the overall signaling more than the lack of a single kinase (Weber, 2000).

    In summary, these data indicate that the transcriptional events downstream of Fz in R3 specification and chirality establishment (e.g. regulation of Dl) are mediated by Jun. The factors with which Jun is redundant in the imaginal discs are not yet identified. It is possible that other members of the AP-1 family are also involved in planar polarity signaling, since they are related to Jun and could dimerize with it via the leucine-zipper motif. A potential candidate is Fos, because like Jun, Fos is required downstream of JNK in the process of dorsal closure in the embryo. Similarly, the ETS domain protein Yan acts as a negative regulator in dorsal closure and is inactivated by JNK in the process. However, these factors do not show informative planar polarity phenotypes in clones and thus their involvement in this process remains unclear. Although AP-1 and ETS family members are attractive candidates, transcription factors belonging to other families cannot be excluded in this context (Weber, 2000).

    Dissecting the molecular bridges that mediate the function of Frizzled in planar cell polarity

    Many epithelia have a common planar cell polarity (PCP), as exemplified by the consistent orientation of hairs on mammalian skin and insect cuticle. One conserved system of PCP depends on Starry night (Stan, also called Flamingo), an atypical cadherin that forms homodimeric bridges between adjacent cells. Stan acts together with other transmembrane proteins, most notably Frizzled (Fz) and Van Gogh (Vang, also called Strabismus). In this study, using an in vivo assay for function, it was shown that the quintessential core of the Stan system is an asymmetric intercellular bridge between Stan in one cell and Stan acting together with Fz in its neighbour: such bridges are necessary and sufficient to polarise hairs in both cells, even in the absence of Vang. By contrast, Vang cannot polarise cells in the absence of Fz; instead, it appears to help Stan in each cell form effective bridges with Stan plus Fz in its neighbours. Finally, it was shown that cells containing Stan but lacking both Fz and Vang can be polarised to make hairs that point away from abutting cells that express Fz. It is deduced that each cell has a mechanism to estimate and compare the numbers of asymmetric bridges, made between Stan and Stan plus Fz, that link it with its neighbouring cells. It is proposed that cells normally use this mechanism to read the local slope of tissue-wide gradients of Fz activity, so that all cells come to point in the same direction (Struhl, 2012).

    In Drosophila and other animals, including vertebrates, there appear to be at least two conserved genetic systems responsible for planar cell polarity (PCP); this study is concerned with the Stan system. In Drosophila, epithelial cells become polarised by a multicellular gradient of Fz activity. To read this gradient, the Stan system builds intercellular bridges of Stan-Stan homodimers that allow neighbouring cells to compare their levels of Fz activity. Under this hypothesis, Fz and Stan are essential components, as without Fz there is nothing to compare and without Stan there is no means to make comparisons. The Stan system also depends on a third protein, Vang, which appears to act in a complementary way to Fz. This study has dissected the function of these proteins by confronting adjacent cells of different fz, Vang and stan genotypes and assaying the effects on PCP. The main finding is that, even in the absence of Vang, Fz can function to polarise cells if it is present in at least one of the two abutting cells. By contrast, Vang has no detectable function when Fz is absent. Based on these and on other results, it follows that, at the core of the Stan system, intercellular bridges form between Stan on its own and Stan complexed with Fz (StanFz), and these act to polarise cells on both sides. It is concluded that Vang acts as an auxiliary component, helping Stan bridge with StanFz. Furthermore, it is posited that the numbers and disposition of asymmetric Stan<<StanFz bridges linking each cell with its neighbours are the consequence of the Fz activity gradient and serve to polarise the cell (Struhl, 2012).

    A model has been built for how bridges between Stan and StanFz might determine the polarity of a cell (see The Stan system in PCP - a model). In the absence of Vang, expression of Fz in a sending cell can bias the polarity of a receiving cell that lacks Fz. Previous results indicate that within the receiving cell, Stan should accumulate only on the surface that faces the sending cell - because it is the only interface where it can form bridges with StanFz - and it is now proposed that it is this localised accumulation of Stan that biases the Vang- fz- receiving cell to make hairs on the other side, pointing away from the sending cell. A parsimonious hypothesis is that the apical membrane of each cell would have an unpolarised propensity to form hairs, and that an excess of Stan on one side locally inhibits this propensity, directing the production of hairs to the opposite side where there is least Stan. The response by a Vang- fz- cell eloquently suggests that the local accumulation of Stan bridged to StanFz in neighbouring cells is the main, and possibly the only, intracellular transducer of Stan system PCP (Struhl, 2012).

    Next consider the finding that Vang functions in receiving cells to help Stan interact productively with StanFz in sending cells. In the key experiment Vang is added to just the Vang- fz- receiving cell: this cell is now more strongly polarised by StanFz signal coming from the sending cell. Thus, Vang can act in the same cell as Stan to help it receive incoming StanFz signal. The model also explains why the polarising effect of the Fz-expressing cell propagates only one cell into the fz- surround, even when Vang activity is restored to the receiving cells - as Stan-Stan bridges do not form, and/or do not function, between neighbouring cells that lack Fz (Struhl, 2012).

    Last, consider the finding that cells lacking Fz can polarise cells devoid of Vang. In this case, only Stan<<StanFz and StanV<<StanFz bridges can form between the two cells, and as a consequence, only the StanFz form of Stan will accumulate on the surface of the Vang- receiving cell where it abuts the fz- sending cell. It is conjectured that Fz, when in a complex with Stan, acts to inhibit the normal action of Stan to block hair outgrowth. Therefore, the only place within the Vang- receiving cell where Stan can accumulate and block hair formation is on the far side, where it can form intercellular bridges with StanFz in the next Vang- cell. Accordingly, the receiving cell would be directed to make a hair on the near side, where it abuts the fz- sending cell. This reasoning also explains why the polarising effect of fz- sending cells on Vang- receiving cells appears to be limited mostly to the adjacent Vang- cell; because Stan<<StanFz and StanFz>>Stan bridges should form and/or function poorly between this cell and the next Vang- cell. Nevertheless, some imbalance between these two kinds of bridges probably does spread further than one cell; indeed fz- sending cells can polarise receiving cells up to two rows away in Vang- pupal wings (Struhl, 2012).

    All the many other experiments fit with the simple model, in which Stan accumulates at the cell surface only where it can form intercellular bridges with StanFz, and each cell is polarised by differences in the amounts of Stan that accumulate along each of its interfaces with adjacent cells. Vang is not essential for these bridges, but by acting on Stan it helps them form and/or makes them more effective (Struhl, 2012).

    How do wild-type cells acquire different numbers and dispositions of asymmetric bridges on opposite sides of the cell? In the Drosophila abdomen, in the anterior compartment of each segment, it has been argued that the Hh morphogen gradient drives a gradient of Fz activity. The slope of the vector of the Fz gradient would then be read by each cell via a comparison of the amount of Stan in its membranes. Within each cell, most Stan will accumulate on the cell surface that abuts the neighbour with most Fz activity, whereas most StanFz will accumulate on the opposite surface, where it confronts the neighbour with least Fz activity. This differential would then be amplified by feedback interactions both between and within cells. The result in each cell is a steep asymmetry in Stan activity that represses hair formation on one side, while allowing it at the other, directing all cells to make hairs that point 'down' the Fz gradient. The model differs in various and simplifying ways from the several and overlapping hypotheses published before. It makes Stan, rather than Fz, the main mediator of PCP, with differences in Fz activity between cells serving to regulate the local accumulation and transducing activity of Stan within cells (Struhl, 2012).

    A central premise of this model is that morphogen gradients do not act directly on each cell to polarise Fz activity, but rather indirectly, by first specifying stepwise differences in Fz activity between adjacent cells. Such an indirect mechanism is favored for two reasons. First, PCP in much of the abdominal epidermis is organised by Hh, which is transduced primarily by its effects on the transcription factor Cubitus interruptus (Ci). It is difficult to understand how graded extracellular Hh could act directly - without cell interactions and only through the regulation of transcription - to polarise Fz activity within each cell. In addition, previous studies used temperature to drive tissue-wide gradients of transcription of a fz transgene under the control of a heat shock promoter; these studies nicely establish that cell-by-cell differences in Fz activity generated by transcriptional regulation are sufficient to polarise cells. Second, it has been previously shown that the polarising action of Hh depends on the Stan system. Specifically, cells in which the Hh transduction pathway is autonomously activated by the removal of the negative regulator Patched require Stan to polarise neighbouring cells. That result adds to evidence that graded Hh creates differences in Fz activity between cells - presumably via transcriptional regulation - that lead to asymmetries in Fz and Stan activities within cells. The target gene could be either fz itself or any other gene whose activity might bias the formation of Stan<<StanFz versus StanFz>>Stan bridges (Struhl, 2012).

    Two previously developed staining experiments provide further support for this model with respect to Stan<<StanFz bridges. First, when Vang- clones are made in fz- flies (generating patches of Vang- fz- cells within fz- territory), a situation in which no Stan<<StanFz bridges can form, there is no accumulation of Stan near or at the border between the clone and the surround - and indeed this study now finds no polarisation of the fz- cells across the clone border. Second, and by contrast, when fz- clones are made in Vang- flies (generating patches of Vang- fz- cells within Vang- territory) Stan accumulates strongly along cell interfaces at the clone borders. Moreover, it is depleted from the cytoplasm of those cells of a clone that abut that border, indicating that Stan in Vang- fz- cells is accumulating at the apicolateral cell membrane where it can form stable intercellular Stan<<StanFz bridges. Previously, there was no evidence that this localisation of Stan within such Vang- fz- cells would polarise them. However, this study now shows that the Vang- fz- cells are polarised by their Fz-expressing neighbours and, also that the effect is reciprocal, their Fz-expressing neighbours are polarised in the same direction (Struhl, 2012).

    The molecular mechanisms by which Fz and Vang control the formation and activity of Stan bridges remain unknown. Consistent with a direct action of Fz on Stan, both in vivo and in vitro studies suggest a physical interaction between the two proteins. Thus, Fz might act in a StanFz complex to regulate both the bridging and transducing activities of Stan. There is no comparable evidence in Drosophila for direct interactions between Vang and Stan. However, their mammalian counterparts have been shown to interact with each other (Devenport, 2008). But Drosophila Vang does interact directly with Pk, while a different Pk-related protein, Espinas, appears to interact directly with Stan during Drosophila neuronal development. Hence, Vang and Pk might form a cis-complex with Stan in epidermal cells, allowing Vang to act directly on Stan and help it form intercellular bridges with StanFz. Intriguingly, there is some evidence that Vang in one cell can interact directly with Fz in adjacent cells. Such an interaction might enhance the capacity of Stan to bridge with StanFz by providing an additional binding surface between the two forms of Stan. Alternatively, Vang might affect the formation or stability of Stan<<StanFz bridges indirectly, consistent with evidence implicating it in the trafficking of proteins and lipids to the cell surface. For example, evidence has been presented that any Stan or StanFz on the cell surface that is not engaged in Stan<<StanFz bridges is rapidly endocytosed and recycled to other sites on the cell surface. Vang activity could bias this process in favour of Stan, thereby enhancing its capacity to form bridges with StanFz (Struhl, 2012).

    The results point to parallels between the Stan and Ds/Ft systems of PCP. First, both systems depend on the formation of asymmetric intercellular bridges between two distinct protocadherin-like molecules. For the Ds/Ft system, these are the Ds and Ft proteins themselves; for the Stan system, it is argued that these are two forms of Stan, either alone or in complex with Fz (StanFz). Second, morphogens may organise both systems by driving the graded transcription of target genes to create opposing gradients of bridging molecules. For the Ds/Ft system, at least two such target genes have been identified: ds itself and four-jointed (fj), a modulator of Ds/Ft interactions. For the Stan system, the existence of at least one such target gene induced by Hh is inferred. Third, for both systems, the two kinds of asymmetric bridges become distributed unequally on opposite faces of each cell, providing the information necessary to point all cells in the same direction. Thus for the Ds/Ft system, it is proposed that different amounts of Ds-Ft heterodimers would be distributed asymmetrically in the cell and this has been recently observed. Similarly, for the Stan system, there is plenty of evidence showing that Stan, Fz and Vang are unequally distributed within each cell. Finally, both systems have self-propagating properties: sharp disparities in Stan, Vang or Fz activity repolarise neighbouring cells over several cell diameters, even in the absence of the Ds/Ft syste, and the same is true of sharp disparities in Ds or Ft activity in the absence of the Stan system. Thus, the Stan and Ds/Ft systems may share a common logic that links morphogen gradients via the oriented assembly of asymmetric molecular bridges and feedback amplification, to cell polarisation (Struhl, 2012).

    Prickle/spiny-legs isoforms control the polarity of the apical microtubule network in planar cell polarity

    Microtubules (MTs) are substrates upon which plus- and minus-end directed motors control the directional movement of cargos that are essential for generating cell polarity. Although centrosomal MTs are organized with plus-ends away from the MT organizing center, the regulation of non-centrosomal MT polarity is poorly understood. Increasing evidence supports the model that directional information for planar polarization is derived from the alignment of a parallel apical network of MTs and the directional MT-dependent trafficking of downstream signaling components. The Fat/Dachsous/Four-jointed (Ft/Ds/Fj) signaling system contributes to orienting those MTs. In addition to previously defined functions in promoting asymmetric subcellular localization of 'core' planar cell polarity (PCP) proteins, this study found that alternative Prickle (Pk-Sple) protein isoforms control the polarity of this MT network. This function allows the isoforms of Pk-Sple to differentially determine the direction in which asymmetry is established and therefore, ultimately, the direction of tissue polarity. Oppositely oriented signals that are encoded by oppositely oriented Fj and Ds gradients produce the same polarity outcome in different tissues or compartments, and the tissue-specific activity of alternative Pk-Sple protein isoforms has been observed to rectify the interpretation of opposite upstream directional signals. The control of MT polarity, and thus the directionality of apical vesicle traffic, by Pk-Sple provides a mechanism for this rectification (Olofsson, 2014).

    A model is proposed for coupling Ft/Ds/Fj to the core module. Gradients of Fj and Ds, by promoting asymmetric distribution of Ft/Ds heterodimers, align a parallel network of apical MTs. Vesicles containing Dsh are transcytosed towards MT plus-ends. In the presence of Pk, MT plus-ends are biased towards the high end of the Fj gradient and the low end of the Ds gradient, whereas in the presence of Sple, the MT plus-ends are biased towards areas with low levels of Fj and high levels of Ds expression. Predominance of Pk or Sple, therefore, determines how tissues differentially interpret, or rectify, the Ft/Ds/Fj signal to the core module. It is hypothesized that this signal serves to both orient the breaking of initial symmetry and to provide continual directional bias throughout polarization. Additional validation of this model would require the measurement of Eb1::GFP comet directions while controlling Pk-Sple isoform expression in wings bearing ectopic Ds and Fj gradients, an experiment that is beyond the technical capabilities with currently available reagents. However, further evidence in support of this model is found in the observation that, in Pk-predominant wings, MT polarity and hair polarity point from regions with high toward low Ds expression both in wild-type wings and in wings with ectopic reversed Ds gradients (Olofsson, 2014).

    It is noted that the distal plus-end bias of MTs is seen in much of the wild-type wing, but this bias decreases to equal proximal-distal plus-end distribution near to the most distal region of the wing. Thus, the mechanism described in this study might not affect the entirety of the wing; in contrast, plus-end bias was observed across the entire A-abd compartment (Olofsson, 2014).

    A model incorporating early Sple-dependent signaling and late Pk-dependent signaling has been proposed to explain PCP in the wing. The current observations and model are compatible with the data presented in support of that model; Sple expression, although always lower than Pk expression in wild-type wing, declined during pupal wing development, suggesting that, in pk mutants, polarity patterns might be set early in development, when Sple is still expressed and when Ds is present in a stripe through the central part of the wing, giving rise to anteroposterior oriented patterns (Olofsson, 2014).

    Pk (and presumably Sple, in Sple dependent compartments) is required for amplification of asymmetry by the core PCP mechanism (Tree, 2002; Amonlirdviman, 2005). These results indicate an additional, core module independent, function for these proteins in regulating the polarity of MTs. Furthermore, although the core function of Pk-Sple is not well defined, part of that function might include promoting the formation and movement along aligned apical microtubules of Fz-, Dsh- and Fmi-containing vesicles (Shimada, 2006). The relative abundance of transcytosing vesicles in Pk versus Sple tissues suggests that if Sple promotes MT-dependent trafficking, it does so less efficiently than Pk (Olofsson, 2014).

    These activities are remarkably similar to those that have been recently identified for Pk and Sple in fly axons, where Pk promotes or stabilizes MT minus-end orientation towards the cell body, and Sple promotes the orientation of minus-ends toward the synapse, which has effects on vesicle transport and neuronal activity. A common mechanism of differentially adapting the plus- and minus-ends of MT segments is proposed in both instances. In axons, similar to what was observed in this study, Pk also facilitates more robust cargo movement, whereas movement is less efficient when Sple is the dominantly expressed isoform. Furthermore, MT polarity defects might underlie the apical-basal polarity defects and early lethality of mouse prickle1 mutant embryos. As Ft and Ds are not known to regulate MTs in axons, these observations suggest that Pk and Sple are able to modify MT polarity independently of Ft/Ds. However, in wings, a consequence is only evident if MTs are first aligned by Ft/Ds activity (Olofsson, 2014).

    How Pk and Sple modulate the organization of MTs remains unknown, but possibilities include modifying the ability of Ft or Ds to capture or nucleate MTs, or altering plus-end dynamics to inhibit capture. These data also suggest the possibility of a more intimate link between the core PCP proteins and Ft/Ds than has been appreciated previously. Other concurrent signals, such as that proposed for Wnt4 and Wg at the wing margin, cannot be ruled out. However, the observations that (1) MTs correlate with the direction of core PCP polarization over space and time, (2) vesicle transcytosis is disrupted in ft clones in which MTs are randomized, (3) chemical disruption or stabilization of MTs disturbs polarity and (4) Pk and Sple isoform predominance rectifies signal interpretation by the core module in a fashion that follows both the wild-type and ectopic Ds gradients provide additional evidence for the model that a signal from the Ft/Ds/Fj system orients the core PCP system in substantial regions of the wing and abdomen (Olofsson, 2014).

    Clustering and negative feedback by endocytosis in planar cell polarity signaling is modulated by ubiquitinylation of Prickle

    The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. This study found that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. Pk was found to participate in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. these results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling (Cho, 2015).

    This study has shown that Cul1 complex-mediated ubiquitinylation of Pk is required for correct function of the core PCP signaling module, thereby ensuring proper alignment of hairs on the Drosophila wing. Ubiquitinylation by the Cul1 complex targets Pk for proteasome-dependent degradation, and in its absence, excess Pk accumulates, resulting in disruption of core PCP function. In several previous reports, ubiquitinylation has been recognized to regulate PCP signaling. In a mouse model, Smurf E3 ligases were shown to regulate PCP signaling by modulating Pk levels. However, mutation of Drosophila smurf failed to show PCP defects. In Drosophila, Cul3 E3 ligase-BTB protein-mediated regulation of Dsh ubiquitinylation modulates PCP signaling, as does the de-ubiqutinylating enzyme Faf, possibly acting on or upstream of Fmi, or more recently proposed to act on Pk. Loss of either activity shows subtle effects on final PCP outcomes in Drosophila. In no case is there a demonstrated mechanism for how these events impact the characteristic asymmetric subcellular localization of PCP proteins that underlies cell polarization (Cho, 2015).

    Slimb was found to be the F-box protein that mediates Pk and Cul1 complex association in vivo. It appears likely that the motif that mediates interaction between Pk and Slimb resides in the C-terminal half of the protein, as do the Vang interaction domain and the farnesylation (CaaX) motif. Of note, the amount of Slimb protein in the cell was also dependent on Pk. In previous cell culture studies, F-box proteins themselves were targeted for ubiquitinylation by their own Cul complexes when not bound by other substrates, and this appears to be the case here, as Slimb levels are increased in cul1 knock-down clones. Furthermore, this result supports the idea that Pk is the major target of the Cul1 complex during pupal wing development (Cho, 2015).

    If the Cul1-SkpA-Slimb complex targets Pk for degradation, why do Slimb and Pk accumulate together on the proximal side of wildtype cells? Pk is known to bind to Vang, and to localize with it in the proximal complex. Slimb adapts the Cul1 complex to Pk and is seen to colocalize with Vang on the proximal side, as well as with overexpressed Pk. However, this suggests that the Pk in this location is resistant to Cul1 complex-dependent degradation. Pk levels have long been known to be limited by a Vang-dependent activity. Recently, it has been shown that farnesylation of Pk is required for Pk to interact with Vang and promote its degradation, and that levels of Pk also depend on SkpA, leading to the suggestion that farnesylation-dependent Pk-Vang interaction results in SkpA-dependent Pk degradation. This study provides evidence suggesting that the Cul1-SkpA-Slimb E3 complex directly targets Pk for destruction, but in contrast, the finding that Pk with deleted CaaX domain accumulates to elevated levels in cul1 knock-down cells indicates that Cul1/SkpA/Slimb-dependent degradation is independent of farnesylation. Furthermore, the finding that Pk promotes internalization of Fmi-Vang-Pk during mutual exclusion of oppositely oriented core PCP complexes leads to a model, that is consistent with the shared observation that Pk associated with stable intercellular complexes ([Dsh-Fz-Fmi]-[Fmi-Vang-Pk]) is protected from degradation (Cho, 2015).

    In theory, generation of cell polarity requires the combination of a local self-enhancement of a cell polarity factor and a long range inhibition of the same factor. In isolated cells, likely the evolutionarily more ancient mechanism, intracellular local self-enhancement can arise through cooperativity among P proteins. Intracellular long range inhibition is most easily accomplished by limiting amounts of a component of the P complex, such that aggregation of P complexes in one location decreases the probability of aggregation elsewhere by depletion of that component (Cho, 2015).

    Cell polarization within a multicellular system introduces additional possible intercellular mechanisms for both the local self-enhancement and the long range inhibition (see Cell polarity establishment and the involvement of Pk-mediated endocytosis). If two polarity complexes, P and Q, exist, and can interact at junctions between adjacent cell boundaries, then both the local and long range effects can be mediated through these intercellular interactions. If P complexes recruit Q complexes to opposing sides of junctions, and if mutual antagonism between P and Q occurs, then long range inhibition can occur by P recruiting Q to the neighbor, where P is then excluded. Similarly, exclusion of P decreases Q in that region of the original cell, enabling the accretion of more P (in effect, cooperativity) (Cho, 2015).

    The peripheral membrane associated core PCP proteins Pk, Dsh and Dgo appear to mediate these polarization events, but how they do so is not known. They are not required for assembly of asymmetric [Fz-Fmi]-[Fmi-Vang] complexes, but were known to share the ability to induce clustering, and are all required for the feedback amplification that results in the asymmetric subcellular localization of PCP signaling complexes. While their action somehow promotes the assortment of proximal and distal core proteins to opposite sides of the cell, how they carry out this function, and in particular whether this is through intracellular or intercellular mechanisms, is unclear (Cho, 2015).

    To understand how excess Pk resulting from mutation of the Cul1 E3 complex disrupts PCP, Pk's role in establishment of core asymmetry was studied further. pk mutation causes symmetric distribution of other core proteins without substantially diminishing or enhancing their junctional recruitment. On the other hand, Pk overexpression causes both accumulation of higher levels of all proximal and distal core proteins and induces their clustering at apical membrane domains, generating discrete puncta. A Pk induced clustering of similarly oriented core complexes could explain both the aggregated punctate appearance and the increased levels of accumulated proteins if one assumes a steady state relationship between free asymmetric complexes and unassembled components as asymmetric complexes are sequestered into puncta (Cho, 2015).

    Pk over-expression study shows that Fz is not required for making Fmi clusters, but Vang is. This suggests an intracellular mechanism in which Pk interacts with Vang at the apical membrane to induce clustering. However, since Vang over-expression does not cause accumulation of other core proteins, a specific function for Pk beyond stabilization of Vang must be considered to explain the accumulation of other core proteins. Furthermore, the depletion of Fmi from the membrane achieved by the very high levels of Pk upon simultaneous Pk overexpression and Cul1 depletion argues for a function for Pk beyond clustering (Cho, 2015).

    Pk might stimulate amplification simply by promoting clustering, with long range inhibition mediated by other mechanisms, or perhaps by limiting amounts of Pk. However, the data suggest an alternative interpretation, as Pk-dependent mutual exclusion of oppositely oriented complexes is observed, forcing local accumulation of distal proteins induced the Pk-dependent removal of proximal proteins within the same cell. Exclusion is associated with Pk mediated internalization of Pk-Vang-Fmi complexes, suggesting that this exclusion involves endocytosis. The requirement for Vang in this internalization is consistent with a previous study showing that Vang contributes to Fmi internalization. It is therefore proposed that Pk is involved in an intercellular long range inhibition to promote feedback amplification (Cho, 2015).

    Like clustering, the Pk-induced routing of Fmi into intracellular vesicles was dependent on Vang, and Pk, Vang and Fmi colocalize in vesicles both apically and more basally, indicating that Fmi-Vang complex trafficking is regulated by associated Pk. However, unlike clustering, it is also dependent on Fz. This suggests a model for feedback inhibition in which oppositely oriented asymmetric complexes interact within clusters, leading to endocytosis and removal of Pk-Vang-Fmi. Competitive interaction between the proximal protein Pk and the distal protein Dgo for Dsh binding is known to occur, suggesting that these interactions might result in either of two alternative outcomes, one of which would be disruption of the proximal complex, and the other disruption of the distal complex. It is proposed that if the distal complex 'wins,' thus remaining stable, the proximal Pk-Vang-Fmi complex becomes internalized in a Pk-dependent step. Once there is a predominance of complex in a given orientation, Vang will be enriched on one side of the intercellular boundary with relatively little Fz present. Since Pk and Slimb associate with Vang, they too will be enriched, but the absence of competitive interactions from the Fz complex allows them to remain within clusters, accounting for the accumulation of Pk and Slimb on the proximal side of wildtype wing cells. According to this model, Pk and Slimb are observed primarily at sites where they are inactive and therefore not internalized (Cho, 2015).

    Modest levels of Pk overexpression both enhance accumulation of PCP protein complexes at the membrane and disrupt the normal orientation of polarization. This may be explained by enhanced feedback amplification that overwhelms the ability to interpret directional inputs. In contrast, the depletion of Fmi from the membrane observed with the very high levels of Pk induced by simultaneous Pk overexpression and Cul1 depletion suggests that sufficient Pk can induce internalization even without the competitive interactions from the Fz complex that normally stimulate internalization (Cho, 2015).

    The mechanism for Pk-dependent clustering is not known. As previously proposed, clustering may result from a scaffolding effect; the possibility of decreased endocytosis accounting for clustering was previously discounted. Whatever the mechanism, clustering by Pk must occur independent of Fz. Furthermore, Pk must enable the multimeric aggregation of complexes containing [Vang-Fmi]-[Fmi] or [Vang-Fmi]-[Fmi-Fz]. Induction of multimeric clustering would also provide a context for the dose-dependent competition that determines internalization of either the proximal or distal complex. Additional work will be required to determine how Pk facilitates clustering (Cho, 2015).

    Since Cul1 depletion increases the amount of Pk, and excess Pk produces clustering and amplification, how Cul1 might produce the observed phenotype is now considered. The simplest possibility is that in the Cul1 mutant, excess Pk produces excess clustering and amplification that overwhelms the directionality in the system. However, because Pk is associated with Slimb and yet stable in the polarized state, and because Pk degradation is dependent on Vang, the possibility is also entertained that Cul1-dependent degradation is somehow functionally coupled to Pk-mediated internalization. Additional studies will be required to distinguish these possibilities (Cho, 2015).

    In summary, a model is proposed in which Pk-dependent internalization of proximal complexes provides an intercellular long range inhibition that contributes to amplification of core protein asymmetric localization. At the same time, Pk provides a local cooperative effect by inducing clustering and accumulation of proximal complexes. The mechanism for clustering are not known, but a simple model is that Pk mediates closely related internalization events (Cho, 2015).

    It is noted that a similar intercellular long range inhibition was initially discussed long ago, except that [Vang-Pk] was proposed to disrupt [Fz-Dsh]. This interpretation was based largely on inference. The current study provides evidence that [Fz-Dsh] disrupts [Vang-Pk] (by promoting internalization). On theoretical grounds, either one would be sufficient to cause polarization, but the possibility cannot be excluded that both may occur. Indeed, vesicles containing Fz, Dsh and Fmi have been shown to be transcytosed in a microtubule-dependent fashion with a directional bias, and these vesicles appear to derive from apical junctions, where they may arise by exclusion (Cho, 2015).

    Although knock-down of smurf in flies reveals no function in PCP; the mechanism described in this study is similar to that inferred for Smurf in mouse PCP. Mice mutant for both Smurf1 and Smurf2 show PCP defects and lose asymmetric localization of core PCP proteins. Furthermore, biochemical evidence was provided that Smurfs, in the presence of the Dsh homolog Dvl2 (and Par6) mediated ubiquitinylation of mouse Pk1. From this, a model was proposed that proximal complexes containing Pk1, and presumably Vang and Celsr (Fmi), are disrupted upon proximity to distal complexes containing Fzd and Dvl2. This model is similar to the model of mutual exclusion, except that the mode of disruption was not directly addressed. While this study proposes disruption by internalization, perhaps coupled to degradation, the mouse stud was only able to address degradation. Furthermore, it is not known if, in mouse, Pk1 mediates clustering, perhaps by a related mechanism, as as is described in flies (Cho, 2015).

    The de-ubiquitinase USPX9 was recently identified as a regulator of Pk in the context of Pk's role in epilepsy in human, mouse, zebrafish and flies. Similarly, the orthologous Drosophila de-ubiquitinase Faf modulates the pksple dependent seizure phenotype in flies. These observations suggest that while the ubiquitinylating and de-ubiquitinylating activities of Smurf and USPX9 control the ubiquitinylation state of vertebrate Pk's, Cul1 and Faf may serve the analogous function to regulate ubiquitinylation of Drosophila Pk (Cho, 2015).

    The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of 'upstream' planar cell polarity proteins

    The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins, inturned, fuzzy and fritz, accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, this study has found that when over-expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. It was further shown that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. It was also found that over-expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group (Wang, 2014).

    The atypical cadherin Dachsous controls left-right asymmetry in Drosophila

    Left-right (LR) asymmetry is essential for organ development and function in metazoans, but how initial LR cue is relayed to tissues still remains unclear. This study proposes a mechanism by which the Drosophila LR determinant Myosin ID (MyoID) transfers LR information to neighboring cells through the planar cell polarity (PCP) atypical cadherin Dachsous (Ds). Molecular interaction between MyoID and Ds in a specific LR organizer controls dextral cell polarity of adjoining hindgut progenitors and is required for organ looping in adults. Loss of Ds blocks hindgut tissue polarization and looping, indicating that Ds is a crucial factor for both LR cue transmission and asymmetric morphogenesis. It was further shown that the Ds/Fat and Frizzled PCP pathways are required for the spreading of LR asymmetry throughout the hindgut progenitor tissue. These results identify a direct functional coupling between the LR determinant MyoID and PCP, essential for non-autonomous propagation of early LR asymmetry (Gonzalez-Morales, 2015).

    This work has revealed the existence of an hindgut-specific LR organizer having transient activity. LR information is transferred non-autonomously from this organizing center to the target tissue, involving a unique MyoID-Ds interaction taking place at a PCP signaling boundary (the H1/H2 boundary). Propagation of this initial LR information to the developing hindgut requires both Ds/Ft global and core Fz PCP signaling. Notably, these results suggest that MyoID can act as a directional cue to bias planar cell polarity (Gonzalez-Morales, 2015).

    So far, only a role for the core PCP pathway in cilia positioning and LR asymmetry had been reported in mouse, chick, and Xenopus. This study revealed a role of the Fat/Ds PCP pathway in LR asymmetry. The atypical cadherin Ds is essential for early LR planar polarization of hindgut precursors and later on for looping morphogenesis. Ds has a cell-non-autonomous function, allowing transfer of LR information from the H1 domain to H2 hindgut precursor cells. Ds, therefore, represents a critical relay factor acting at the boundary between, and linking, a LR organizer and its target tissue (Gonzalez-Morales, 2015).

    In addition to a MyoID-dependent function in H1, the mislooped phenotype induced upon Ds silencing in the H2 domain suggests that Ds also has a MyoID-independent activity in H2 cells, likely through interaction with other PCP genes. Indeed, reducing the activity of PCP global or core gene functions reveals that the two pathways are important in the H2 region for adult hindgut looping. However, the results reveal important differences in the way these pathways control hindgut asymmetry. First, although the adult phenotype is similar upon silencing of one or the other pathway, the early polarization of H2 cells in pupae (10 hr APF) is only affected when knocking down the activity of Ds, Ft, and Fj. These results show that the Ds/Ft pathway, but not the core pathway, is required for establishing early LR polarity. Second, the phenotype is quantitatively different, since silencing of the Ds, Ft, or Fj PCP gene led to a consistent and very strong phenotype, while reducing Fz PCP signaling had a significantly less penetrant one. These data suggest a partly overlapping function of both PCP signaling pathways for late hindgut morphogenesis. Therefore, the following sequential model is proposed: in H1 cells, MyoID interacts with the Ds intracellular domain, which becomes 'biased' toward dextral through a currently unknown mechanism. This initial LR bias is then transmitted across the H1/H2 boundary through Ds/Ft heterophilic interaction. Then, boundary H2 cells relay the initial bias and spread it to the remaining H2 cells through classical Ds/Ft PCP. It is interesting that the local signaling boundary suggested by this model is consistent with recent studies showing that Ds can propagate polarity information in a range of up to eight cells, a distance that is consistent with the size of the H2 domain at 10 hr APF. Once initial polarity has been set up through the Ds/Ft pathway, this is further relayed to and/or amplified by the core pathway. Notably, a similar two-step mechanism has also been proposed for the wing and could apply to other tissues (Gonzalez-Morales, 2015).

    The discovery of a coupling between the MyoID dextral factor and Ds is a nice example of crosstalk between existing signaling modules. In the simplest crosstalk model, the role of MyoID would just be to bias or tilt Ds function toward one side, possibly through Ds localization and/or activity polarization along the LR axis. Using both in vitro and in vivo assays, this study has shown that interaction between Ds and MyoID requires Ds intracellular domain, supporting a cytoplasmic interaction between the two proteins. These results, along with recent findings, suggest that Ds may represent a general platform for myosin function in different tissues. In particular, the intracellular domain of Ds was found to bind to the unconventional myosin Dachs, controlling Dachs polarized localization, which is important for subsequent cell rearrangements underlying thorax morphogenesis. However, in contrast to thoracic Dachs, MyoID is not obviously polarized in H1 cells, suggesting that the interaction between myosins and Ds may involve different mechanisms. Additionally, no LR polarized localization of MyoID or Ds was observed in H1 cells, although the existence of subtle asymmetries undetectable by available tools cannot be excluded. Nevertheless, alternative means to generate the LR bias in H1 include: (1) LR polarized expression of an unknown asymmetric factor or (2) LR asymmetric activity of Ds. These interesting possibilities are consistent with recent work showing that some type I myosins can generate directed spiral movement of actin filaments in vitro. It is tempting to speculate that, similarly, MyoID putative chiral activity could be translated into Ds asymmetrical function along the LR axis. Future work will explore this possibility as well as others to unravel the molecular basis of MyoID LR biasing activity in the H1 organizer (Gonzalez-Morales, 2015).

    The identification of the H1 domain as a specific adult tissue LR organizer demonstrates the existence of multiple independent tissue and stage-specific LR organizers in flies. This situation echoes what is known in other models, including vertebrates, in which at least two phases of asymmetry establishment can be distinguished. A first pre-gastrula phase, as early as the four-cell stage in Xenopus, involves the generation of asymmetric gradients of ions. Then, a second phase takes place at gastrulation and involves Nodal flow and asymmetric cell migration, eventually leading to asymmetric expression of the nodal gene in the left lateral plate mesoderm. In Drosophila, some interesting common and specific features can be drawn out by comparing the hindgut and terminalia organizers. The first major common feature is the fact that both organizers rely on MyoID function, showing the conserved role of this factor in Drosophila LR asymmetry. Second, the two organizers show temporal disconnection, acting much earlier than LR morphogenesis, which is expected of a structure providing directionality to tissues per se (24 hr for terminalia and ~72 hr for hindgut looping). Such temporal disconnection of MyoID function with late morphogenesis is also observed in the terminalia where a peak of MyoID activity precedes terminalia rotation by 24 hr. Time lag in MyoID function requires LR cue transmission and maintenance in developing tissues until directional morphogenesis. The finding of a role of Ds and PCP in hindgut LR asymmetry provides a simple mechanism by which initial LR information is maintained and transmitted across tissue through long-range PCP self-propagation (Gonzalez-Morales, 2015).

    Notably, the two organizers also show distinct features. In terminalia, MyoID has a cell-autonomous function in two adjacent domains. In addition, the terminalia organizer is permanent, developing as an integral component of the adult tissue. In contrast, MyoID in the imaginal ring has a cell-non-autonomous function. Indeed, a striking feature of the hindgut organizer is its transience as it detaches from the hindgut precursors 50 r before full looping morphogenesis prior to its degradation and elimination; hence, the need to transfer LR information to the H2 hindgut primordium. An interesting question then is whether the MyoID-Ds/PCP interaction is conserved in terminalia. This study has shown that the terminali rotation requires the activity of DE-cadherin; however, invalidation of the atypical cadherins Ds or Ft or core PCP signaling in the terminalia organizer did not affect asymmetry. The fact that PCP does not have a general role in Drosophila LR asymmetry is not altogether surprising, as MyoID cell-autonomous function in terminalia and organizer persistence does not require that LR information be transferred to and stored in other parts of the tissue, as is the case in the hindgut. Therefore, despite conservation of the MyoID-dependent upstream dextral cue, significant differences in downstream morphogenetic pathways imply alternative cellular mechanisms controlling cue transmission and maintenance (Gonzalez-Morales, 2015).

    The LR signaling module, comprising the dextral determinant MyoID and the still-unknown sinistral determinant, can therefore be coupled to distinct morphogenetic modules, including PCP, as shown in this study. It is suggested that coupling between LR asymmetry and PCP might be observed in processes requiring long-distance patterning of tissues and organ precursors, both in invertebrate and vertebrate models. Understanding organ LR morphogenesis clearly requires studying diverse and complementary models. In this context, the multiplicity of LR organizers discovered in Drosophila represents a powerful model to study the diversity in the coupling of LR organizers with downstream programs responsible for late tissue morphogenesis. In particular, the Drosophila hindgut represents an invaluable model for studying the genetic basis and molecular mechanisms coupling LR asymmetry with PCP patterning (Gonzalez-Morales, 2015).

    Microtubules provide directional information for core PCP function

    Planar cell polarity (PCP) signaling controls the polarization of cells within the plane of an epithelium. Two molecular modules composed of Fat(Ft) / Dachsous(Ds) / Four-jointed(Fj) and a 'PCP-core' including Frizzled(Fz) and Dishevelled(Dsh) contribute to polarization of individual cells. How polarity is globally coordinated with tissue axes is unresolved. Consistent with previous results, this study found that the Ft/Ds/Fj-module has an effect on a microtubule (MT)-cytoskeleton. Evidence is provided for the model that the Ft/Ds/Fj-module provides directional information to the core-module through this MT organizing function. Ft/Ds/Fj-dependent initial polarization of the apical MT-cytoskeleton is shown to occur prior to global alignment of the core-module, reveal that the anchoring of apical non-centrosomal MTs at apical junctions is polarized. Directional trafficking of vesicles containing Dsh was observed to depend on Ft. The feasibility of this model was demonstrated by mathematical simulation. Together, these results support the hypothesis that Ft/Ds/Fj provides a signal to orient core PCP function via MT polarization (Matis, 2014).

    Symmetry breaking in an edgeless epithelium by Fat2-regulated microtubule polarity

    Planar cell polarity (PCP) information is a critical determinant of organ morphogenesis. While PCP in bounded epithelial sheets is increasingly well understood, how PCP is organized in tubular and acinar tissues is not known. Drosophila egg chambers (follicles) are an acinus-like "edgeless epithelium" and exhibit a continuous, circumferential PCP that does not depend on pathways active in bounded epithelia; this follicle PCP directs formation of an ellipsoid rather than a spherical egg. This study uses an imaging algorithm to 'unroll' the entire 3D tissue surface and comprehensively analyze PCP onset. This approach traces chiral symmetry breaking to plus-end polarity of microtubules in the germarium, well before follicles form and rotate. PCP germarial microtubules provide chiral information that predicts the direction of whole-tissue rotation as soon as independent follicles form. Concordant microtubule polarity, but not microtubule alignment, requires the atypical cadherin Fat2, which acts at an early stage to translate plus-end bias into coordinated actin-mediated collective cell migration. Because microtubules are not required for PCP or migration after follicle rotation initiates, while dynamic actin and extracellular matrix are, polarized microtubules lie at the beginning of a handoff mechanism that passes early chiral PCP of the cytoskeleton to a supracellular planar polarized extracellular matrix and elongates the organ (Chen, 2016).

    This work identify a central symmetry-breaking role for microtubule polarity in PCP of an 'edgeless' epithelial organ. Microtubules are the earliest PCP molecule during follicle development, germarial microtubule polarity predicts the chirality of subsequent follicle PCP events, and disruption of either microtubule alignment or polarity in the germarium prevents all subsequent aspects of follicle PCP, including the coordinated cell motility that initiates follicle rotation. These requirements for microtubules are not due to secondary effects on actin, which retains its organization in germaria with disrupted microtubules. Importantly, unlike actin, which is required acutely and constantly for collective cell migration, microtubules are not strictly required for follicle cell motility once rotation has initiated. It is therefore proposed that microtubules provide the initial source of PCP information in the early follicle and that actin, through its role in promoting tissue rotation, serves to amplify and propagate PCP (Chen, 2016).

    The current data demonstrate that the microtubule polarity bias in the forming follicle predicts the chirality of PCP tissue rotation that initiates 10 hr later. It was recently reported that, in stage 4 follicles, microtubule plus-end orientation anticorrelates with rotation direction at stage 7. However, in that work, stage 4 and earlier follicles were thought to represent pre-rotation stages, contrary to the identification in this study of rotation initiation at stage 2 and that of another study that placed it at stage 1. Since both microtubule alignment and polarity are present in the germarium, the stage 5 correlation is not predictive and reflects pre-existing PCP information rather than revealing its source. In the absence of an independent and direct manipulation of microtubule plus-end orientation, it cannot be conclusively stated that the microtubule polarity that was document in the germarium is instructive for rotational direction. Nevertheless, the strong correlation between this chirality and the subsequent direction of follicle rotation, along with disruption of both in the absence of fat2, point to a model in which coordination of microtubule polarity in cells across the follicle is required for a unidirectional consensus among individual motile cells to initiate productive rotation (Chen, 2016).

    The data thus suggest that the atypical cadherin Fat2, a key regulator of follicle PCP, rotation, and elongation, acts through effects on early microtubule polarity. Through analyses of middle stages of follicle development, it has been argued that Fat2 regulates global PCP alignment of the cytoskeleton, as does the canonical PCP regulator Fat in the developing Drosophila wing. However, in fat2 germaria, actin and microtubule alignment are maintained; it is coordinated microtubule polarity that is lost. These phenotypes, along with its strong genetic interaction with CLASP, argue that Fat2 promotes rotation initiation and follicle PCP via its effects on microtubule polarity. An additional role in actin regulation other than polarized alignment has not been excluded; the requirement for Fat2 and actin, but not microtubules, to maintain rotation, as well as the direct binding of actin regulators by vertebrate Fat1, a possible ortholog of Drosophila Fat2, suggests such a role. Moreover, while this work was in revision, Squarr (2016) showed that Fat2 can directly influence the actin cytoskeleton via binding to the WAVE complex (Chen, 2016).

    As with PCP in the developing Drosophila wing disc, the initial PCP bias provided by microtubule polarity within the early follicle precursors is mild but becomes more robust as organogenesis progresses. A mechanism for amplification in the follicle involves whole-tissue rotation. Preventing rotation by disrupting actin or integrins causes a rapid loss of all PCP organization primed in the germarium. Interestingly, just as microtubules are largely dispensable for PCP after actin PCP becomes established, actin PCP is dispensable after circumferentially aligned ECM becomes established. Hence, PCP transitions from highly dynamic and intracellular microtubules, to longer-lasting and sometimes juxtacellular actin filaments, and then finally to the durable ECM fibrils that span multiple cells. PCP information in the follicle is therefore passed along by a 'handoff' mechanism to increasingly stable as well as larger-scale components that can ultimately biophysically influence the shape of the organ (Chen, 2016).

    Non-centrosomal microtubule arrays, and in particular their regulated polarized organization, have previously been implicated as central governors of global PCP in tissues such as Drosophila wings, zebrafish gastrulae, and mammalian airway epithelia. In 'edged' PCP tissues in Drosophila, a 'global PCP' module molecularly controlled by Fat is thought to use gradients of positional information from specific sources to bring individual cell PCP in alignment with overall body axes. In the circumferential 'edgeless' PCP axis of the follicle epithelium, where no such graded information is known, Fat2 seems to similarly coordinate the PCP of individual cells. That both contexts involve important roles for polarized microtubules and are controlled by related atypical cadherins raises the possibility of ancient links between the modes of epithelial PCP organization (Chen, 2016).

    The clathrin adaptor AP-1 complex and Arf1 regulate planar cell polarity in vivo

    A key step in generating a planar cell polarity (PCP) is the formation of restricted junctional domains containing Frizzled/Dishevelled/Diego (Fz/Dsh/Dgo) or Van Gogh/Prickle (Vang/Pk) complexes within the same cell, stabilized via Flamingo (Fmi) across cell membranes. Although models have been proposed for how these complexes acquire and maintain their polarized localization, the machinery involved in moving core PCP proteins around cells remains unknown. This study describes the AP-1 adaptor complex and Arf1 as major regulators of PCP protein trafficking in vivo. AP-1 and Arf1 disruption affects the accumulation of Fz/Fmi and Vang/Fmi complexes in the proximo-distal axis, producing severe PCP phenotypes. Using novel tools, a direct and specific Arf1 involvement was detected in Fz trafficking in vivo. Moreover, a conserved Arf1 PCP function was uncovered in vertebrates. These data support a model whereby the trafficking machinery plays an important part during PCP establishment, promoting formation of polarized PCP-core complexes in vivo (Carvajal-Gonzalez, 2015).

    This study demonstrates that the AP-1 complex and Arf1 are critical for PCP-core protein membrane localization during PCP establishment in multiple Drosophila tissues, and that it likely serves as a conserved function in zebrafish. Detailed analysis in Drosophila wings and zebrafish cells during gastrulation revealed that Arf1 function is required for polarized accumulation of core PCP components during PCP axis establishment. Defects in correct planar-polarized localization resulted in classical PCP defects in both animal models, and thus this study reveals a conserved function of the Arf1 protein network (Carvajal-Gonzalez, 2015).

    Arf1 and AP-1 act as the trafficking machinery for the core PCP components in epithelial cells during PCP establishment. A few studies suggested connections between PCP and trafficking before. First Rabenosyn, an endocytic-related protein, was found polarized in a PCP-type fashion following Fmi localization. Rabenosyn mutant cells display defects in cellular packing and accumulation of PCP-core components at the PM50. Second, the GTPase Rab23, which, similar to Rabenosyn, causes packing defects and multiple cellular hairs (mch), interacts with the cytosolic PCP-core protein Pk. And third, Gish/Rab11/nuf were recently shown to affect hair formation by either increasing the numbers of trichomes per cell (mch) or shortening the hairs (stunted hair phenotype). However, none of these proteins affected PCP-core component localization, including Fmi or Fz, or hair orientation in the adult, similar to how the Arf1/AP-1 network does (Carvajal-Gonzalez, 2015).

    Arf1 is known to act during trafficking from the Golgi in Drosophila and vertebrate cells. In the TGN, Arf1 aids in sorting of cargo proteins into carrier vesicles, regulating the binding of clathrin adaptor proteins such as GGAs (Golgi-localized, γ-adaptin ear-containing and ARF-binding proteins) or the clathrin adaptor complex AP-1. It was demonstrated that perturbation of Arf1 and AP-1 function reduced polarized accumulation of Fmi, Fz and Vang with minimal effects on non-planar-polarized membrane proteins (for example, DE-cadherin and FasIII). A distinct subpopulation of PCP-core component complexes with different turnover dynamics from unpolarized membrane junctional domains has recently been identified. These complexes are polarized, stable and restricted to large puncta, and also insensitive to manipulations of the endocytic/recycling machinery. Together with established Arf1/AP-1 functions at the Golgi, the current data support the notion that stable PCP complexes originate mainly from newly synthesized PM proteins. A hypothesis that is further supported by using the DmrD4-Fz-GFP fusion and manipulations to synchronize its ER release. Inhibition of Arf1 with BFA treatment prevented the arrival of Fz to the membrane in S2 cells. Importantly, Arf1 inhibition also inhibited Fz delivery to the apical junctional regions in vivo, where it must localize to participate in PCP establishment. A model is proposed whereby Arf1/AP-1 are involved in the specific transport of the PCP-core factor Fz, and most likely also Vang and Fmi, to the PM during PCP establishment, a process required for enrichment of these proteins in polarized complexes. Recently, it was shown in a mammalian non-polarized culture system that Vangl2 trafficking out of the Golgi depends on Arfaptin and the AP-1 complex, which is consistent with the current data and supports the evolutionary conserved task for these proteins in promoting PCP-core component localization (Carvajal-Gonzalez, 2015).

    Arf1 can also act through the COPI complex, and, for example, in flies, at the cis-Golgi, Arf1 together with its guanine nucleotide exchange factor/GEF Gartenzwerg regulate COPI retrograde transport. Thus attempts were made to test this possibility. However, the dsRNA-based KD in vivo of all COPI complex components is cell lethal and thus not informative in this context. Similarly, in cell culture COPI KD, depending on the level, was either too toxic or failed to show an effect on Fz trafficking. As such, although it cannot be completely excluded that COPI is also involved in the process, the comparable results of Arf1 and AP-1 indicate that Arf1 acts at least in part on AP-1 in PCP trafficking (Carvajal-Gonzalez, 2015).

    Several studies have identified genes that affect localized actin hair formation in Drosophila wing cells. Interestingly, apart from the PCP-core components, these usually fall into two cellular machinery categories: actin polymerization-related proteins and trafficking-related proteins. Importantly, both sets of factors have been linked to each other in diverse cellular environments, including carrier formation at the Golgi, yeast budding, formation of phagocytic cups or lamellipodia formation in migrating cells. Silencing of Arf1, Rab11, PI4KIIIβ and AP-1 leads to defects in actin-based hair formation in wing cells, notably leading to the formation of mch. Using Drosophila S2 cells, Arf1 is found not only in the Golgi but also at actin-rich cell edges, and that it is essential for lamellipodium biogenesis. In vertebrates, Arf1 is also required for actin polymerization, for example, in neuronal tissues during plasticity Arf1 regulates Arp2/3 through PICK1. Based on these data, a direct/parallel involvement of the Arf1 protein network is likely during hair formation, and indeed an enrichment of these proteins was observed in the growing actin hairs. Thus, in summary, the data on Arf1/AP-1 and its network suggest that it functions repeatedly in different PCP establishment processes, ranging from the initial PCP-core component localization to later restricting the foci of actin polymerization (Carvajal-Gonzalez, 2015).

    Positioning of centrioles is a conserved readout of Frizzled planar cell polarity signalling

    Planar cell polarity (PCP) signalling is a well-conserved developmental pathway regulating cellular orientation during development. An evolutionarily conserved pathway readout is not established and, moreover, it is thought that PCP mediated cellular responses are tissue-specific. A key PCP function in vertebrates is to regulate coordinated centriole/cilia positioning, a function that has not been associated with PCP in Drosophila. This study reports instructive input of Frizzled-PCP (Fz/PCP) signalling into polarized centriole positioning in Drosophila wings. It was shown that centrioles are polarized in pupal wing cells as a readout of PCP signalling, with both gain and loss-of-function Fz/PCP signalling affecting centriole polarization. Importantly, loss or gain of centrioles does not affect Fz/PCP establishment, implicating centriolar positioning as a conserved PCP-readout, likely downstream of PCP-regulated actin polymerization. Together with vertebrate data, these results suggest a unifying model of centriole/cilia positioning as a common downstream effect of PCP signalling from flies to mammals (Carvajal-Gonzalez, 2016).

    Taken together with observations that Fz/PCP signalling regulates basal body and cilia positioning in vertebrates (Song, 2010; Gray; 2011; Borovina, 2010), the current data on centriole positioning as a Fz/PCP readout in non-ciliated Drosophila wing cells indicate that centriole/MTOC (MT organizing centre)/basal body positioning is an evolutionarily conserved downstream effect of Fz/PCP signalling. Its link with actin polymerization (hair formation in Drosophila wing cells) suggests that actin polymerization effectors also affect cilia positioning, possibly through docking of the basal bodies to the apical membranes. Inturned, Fuzzy and Rho GTPases regulate apical actin assembly necessary for the docking of basal bodies to the apical membrane (Park, 2006; Pan, 2007) and this apical actin membrane accumulation is lost in Dvl1-3-depleted cells (Carvajal-Gonzalez, 2016).

    In left-right asymmetry establishment of the Drosophila hindgut, which is not a Fz/PCP-dependent process, asymmetric centriole positioning is observed. During this so-called planar cell shape chirality process, which affects gut-looping and thus embryonic left/right asymmetry, centriole positioning is however still dependent upon actin polymerization downstream of Rho GTPases (Rac and Rho), via MyoD and DE-cadherin control. As Rho GTPases (Rac, Cdc42 and Rho) are downstream effectors of Fz-Dsh/PCP complexes, and their mutants cause PCP-like phenotypes including multiple cellular hairs or loss of hairs in wing cells. It is thus tempting to infer that both processes, planar cell shape chirality and Fz/PCP, regulate centriole positioning through a common Rho GTPase-mediated actin polymerization pathway, initiated by an upstream cellular communication system, although this assumption will require experimental confirmation. In the mouse, Fz/PCP signalling regulates cilia movement/positioning in cochlear sensory cells via Rho GTPase-mediated processes, suggesting a similar mechanism in a representative mammalian PCP model system. In conclusion, the positioning of centrioles appears to be a key and an evolutionary conserved downstream readout of Fz/PCP signalling, ranging from flies to mammals in both ciliated and non-ciliated cells (Carvajal-Gonzalez, 2016).

    The PCP pathway regulates Baz planar distribution in epithelial cells

    The localisation of apico-basal polarity proteins along the Z-axis of epithelial cells is well understood while their distribution in the plane of the epithelium is poorly characterised. This study provides a systematic description of the planar localisation of apico-basal polarity proteins in the Drosophila ommatidial epithelium. The adherens junction proteins Shotgun and Armadillo, as well as the baso-lateral complexes, are bilateral, i.e. present on both sides of cell interfaces. In contrast, it is reported that other key adherens junction proteins, Bazooka and the myosin regulatory light chain (Spaghetti squash) are unilateral, i.e. present on one side of cell interfaces. Furthermore, planar cell polarity (PCP) and not the apical determinants Crumbs and Par-6 control Bazooka unilaterality in cone cells. Altogether, this work unravels an unexpected organisation and combination of apico-basal, cytoskeletal and planar polarity proteins that is different on either side of cell-cell interfaces and unique for the different contacts of the same cell (Aigouy, 2016).

    SEGGA: a toolset for rapid automated analysis of epithelial cell polarity and dynamics

    Epithelial remodeling determines the structure of many organs in the body through changes in cell shape, polarity and behavior and is a major area of study in developmental biology. Accurate and high-throughput methods are necessary to systematically analyze epithelial organization and dynamics at single-cell resolution. This study developed SEGGA, an easy-to-use software for automated image segmentation, cell tracking and quantitative analysis of cell shape, polarity and behavior in epithelial tissues. SEGGA is free, open source, and provides a full suite of tools that allow users with no prior computational expertise to independently perform all steps of automated image segmentation, semi-automated user-guided error correction, and data analysis. This study uses SEGGA to analyze changes in cell shape, cell interactions and planar polarity during convergent extension in the Drosophila embryo. These studies demonstrate that planar polarity is rapidly established in a spatiotemporally regulated pattern that is dynamically remodeled in response to changes in cell orientation. These findings reveal an unexpected plasticity that maintains coordinated planar polarity in actively moving populations through the continual realignment of cell polarity with the tissue axes (Farrell, 2017).

    Planar polarized Rab35 functions as an oscillatory ratchet during cell intercalation in the Drosophila epithelium

    The coordination between membrane trafficking and actomyosin networks is essential to the regulation of cell and tissue shape. This study examined Rab protein distributions during Drosophila epithelial tissue remodeling and show that Rab35 is dynamically planar polarized. Rab35 compartments are enriched at contractile interfaces of intercae

    The Rab family of small GTPase proteins is key mediators of membrane trafficking and cytoskeletal dynamics. Rab proteins regulate membrane compartment behaviors through their association with tethering and trafficking effectors, and mutations in Rab proteins are associated with a variety of diseases and developmental disorders. The Rab trafficking pathways that operate during cell intercalation in the early Drosophila gastrula have remained undefined, although the function of classic Clathrin and Dynamin-dependent early endocytic pathways has been explored. Indeed, it has been demonstrated that Formin and Myosin II proteins direct the endocytic uptake of dextran through specialized CIV (cortical immobile vesicle) structures. Additionally, in Drosophila, Rab35 has been shown to play a critical role in directing the morphogenesis of tracheal tube growth as well as synaptic vesicle sorting at the neuromuscular junction. In tissue culture models, Rab35 functions early in endosomal pathways to drive the generation of newborn endosomes and is essential for the terminal steps of cytokinesis and neurite outgrowth (Jewett, 2017).

    This study shows that Rab35 demonstrates remarkable compartmental behaviors at the plasma membrane. Rab35 compartments are initially contiguous with the cell surface and form dynamic structures that grow and shrink on the minute time scale. In the absence of Rab35 function, cell interfaces undergo contractile steps, but these steps rapidly reverse themselves, consistent with Rab35 mediating an essential 'ratcheting' function that directs progressive interface contraction. Rab35 compartments form independently of Myosin II function, but require actomyosin forces to terminate. These compartments further function as endocytic hubs that have transient interactions late in their formation with Rab5 and Rab11 endosomes (Jewett, 2017).

    The current results have shown that a trafficking network centered on Rab35 acts as a ratcheting mechanism to ensure that interface contraction is progressive and irreversible. Rab35 functions upstream of junctional actomyosin forces during interface contraction to provide a membranous ratchet, and suggests that cytoskeletal-derived forces are required to terminate compartmental behaviors. Rab35 compartments are more numerous and possess longer compartmental times at contractile interfaces of actively intercalating cells. Rab35 compartments form at the plasma membrane and rapidly grow in size during periods of interface contraction, before shrinking through endocytic-dependent processes. Rab35 compartmental behaviors therefore represent a critical point of convergence at which cytoskeletal and membrane trafficking pathways function to drive changes in cell shape (Jewett, 2017).

    Changes in cell shape in many systems are driven by pulsatile processes that initiate directed movement before cycling through periods in which the force generating network reforms. Previous work on ratcheting function has concentrated on different actomyosin regimes governed by Myosin II regulators such as Rho1 and Rok that trigger contraction and cell ratcheting. Importantly, when Rab35 function is disrupted, apical cell areas maintain oscillatory behaviors and AP interface lengths still undergo brief periods of contraction. However, contractile periods are followed by reversals and interfaces re-lengthen, producing a failure in interface shortening. This 'wobble' behavior is consistent with Rab35 functioning as a ratchet ensuring unidirectional movement during interface contraction. These results also suggest that junctional actomyosin-driven behaviors may be responding to an upstream trafficking apparatus centered on Rab35. Indeed, junctional Myosin II localization is disrupted in Rab35 knockdown embryos, and accumulates intracellularly along with integral membrane proteins (Neurotactin) and F-actin. In tissue culture cells, it has been shown that disruption of Rab35 similarly leads to an accumulation of F-actin during abscission, which provides another link between Rab35 and cytoskeletal dynamics. Finally, and again consistent with expectations for a compartment-driven ratcheting function, it is also striking that Rab35 compartments are not present uniformly during cell intercalation, but begin to form specifically as interface contraction occurs (Jewett, 2017).

    The AP patterning system is responsible for directing Rab35 compartment formation away from apical/medial sites and functionally engaging Rab35 at AP interfaces. It is interesting to note that high levels of anterior patterning information, such as is present in the head region, inhibit planar polarity, cell intercalation, and Rab35 compartment dynamics, while maintaining interfacial Rab35. The common appearance of apically localized Rab35 compartments in the ventral furrow and in bnt mutant embryos (embryos derived from mothers homozygous for the genes bicoid, nanos and torso-like, 'bnt') suggests a model in which ventral fate genes could mediate apical constriction by re-directing Rab35 compartment formation through the inhibition of AP patterning cues (Jewett, 2017).

    Myosin II is found in several different populations within intercalating cells. Transient, web-like Myosin II localizations are found in highly dynamic structures in the apical/medial regions of epithelial cells, while more stable, cable-like structures are present at cell junctions. Junctional Myosin II enrichment is dependent on Rab35 function, and is required for the termination of Rab35 compartments. However, the role of apical/medial actomyosin forces and cell oscillations on Rab35 compartmental behaviors is less clear. It appears that Rab35 compartments may require Myosin II medial 'flows' to function as a polarizing cue, as Rab35 compartments become less planar polarized in the absence of Myosin II function. It is hypothesize that cell oscillations produce cycles of high and low tension during which Rab35 compartments can form as infoldings of slackened plasma membrane that, when internalized, prevents interface length from rebounding to the same length as was present prior to area contraction. It is important to note that the termination of Rab35 compartments and the internalization of membrane is critical to ratcheting and productive interface contraction, as merely driving more plasma membrane into Rab35 compartments (as is observed in Y-27632, PitStop2, or chlorpromazine-injected embryos) in the absence of compartment termination does not direct lasting changes in cell topologies (Jewett, 2017).

    These results also suggest how oscillatory area contractions can be linked to productive movements. In worm and fly embryos14, 47, actomyosin contractions show both productive and non-productive periods. The functional engagement of area oscillations may depend on the presence of the Rab35 ratchet, as immediately prior to intercalation Rab35 compartments are absent from cell interfaces. However, it is intriguing that Rab35 compartments can form when Myosin II function is compromised in Y-27632-injected embryos. It may be that the small amount of Myosin II function left after Y-27632 injection is enough to drive small oscillations in area, or that thermal fluctuations in cell area are enough for compartment formation. Regardless, this will be an interesting area for further studies (Jewett, 2017).

    What is the nature of Rab35 compartments? While there are punctate Rab35 compartments present in the apical cytoplasm that partially colocalize with endosomal markers, the major population of Rab35 during early gastrulation and cell intercalation is present in tubular compartments that are contiguous with the cell surface. The extremely elongated Rab35 plasma membrane tubules observed in either Myosin II-disrupted or endocytosis-inhibited embryos, as well as the immediate filling of Rab35 compartments at the cell surface, are consistent with Rab35 marking endocytic infoldings of the plasma membrane. Additionally, Rab35 compartments are positive for a plasma membrane PIP2 species, PtdIns(4,5)P2, and Rab35 immunogold TEM imaging and the quantification of immunogold particle localization demonstrates that Rab35 is largely present in compartments at the cell surface. Indeed, open infoldings marked by Rab35 immunogold form in subapical zones near the adherens junctions. It is interesting to note that similar open, tubular structures have also been observed by TEM near electron dense junctions during cell intercalation18. When Rab35 function is compromised, there is a shift of endocytic, dextran uptake away from AP interfaces and a dramatic increase in failed endocytic events. In tissue culture, Rab35 has been shown to function early in endosomal pathways and immediately after vesicle scission, to drive the generation of newborn endosomes. The data suggest a subtle shift in Rab35 function to an earlier time point occurs during early Drosophila morphogenesis, but is broadly consistent with a Rab35-driven function in directing the delivery of membrane from the cell surface to endosomal pathways. However, the results do not exclude an added role for Rab35 at endosomal compartments, as F-actin and Myosin II accumulate intracellularly in Rab35 compromised embryos. This accumulation is at substantially lower levels than occurs at the cell cortex in wild-type embryos, but could contribute to the observed cell intercalation dynamics, especially the ~17% decrease in oscillatory area amplitudes (Jewett, 2017).

    The association of Rab35 compartments with markers for early (Rab5) or recycling (Rab11) endosomes was also examined. While the large majority of Rab35 compartments do not localize with early or recycling endosomes, there was a small fraction of compartments that do (15%). Interestingly, when observed under live imaging conditions, it became apparent that Rab35 compartments do often have transient interactions with endosomal compartments (98%) that occur specifically toward the end of Rab35 lifetimes, consistent with Rab35 feeding endocytosed membrane through a specialized plasma membrane contiguous compartment. These results are consistent with the broader conclusion of this work that endocytic uptake of plasma membrane components is essential to ensuring irreversible changes in interface length that drive cell neighbor exchange. This endocytic uptake could function through two potential mechanisms: (1) E-cadherin adhesion complex removal, or (2) general membrane removal, which would require that individual interfaces possess isolated membrane domains. Embryos that expressed fluorescently labeled Rab35 and E-cadherin did not express E-cadherin at levels sufficient to be resolvable by confocal microscopy, thus making it difficult to distinguish between these models, but this will be an important open question going forward. Previous work has proposed that MyoII function clusters E-cad complexes for endocytosis, while the current results show that Rab35 functions upstream of junctional Myosin. Thus, Rab35 compartments may serve to direct Myosin II clustering of E-cadherin adhesion complexes for endocytosis and junctional remodeling (Jewett, 2017).

    An interesting feature of Rab35 behaviors is the absence of paired compartments at contracting interfaces. Approximately 50% of Rab35 compartments form without the presence of any Rab35 compartments on the opposing side of an interface, and 93% of compartments are present without a matching, synchronous Rab35 compartment. This monopolarity of Rab35 function is the first evidence of asymmetric behaviors across a common, shared interface during early germ-band extension (GBE). As Rab35 compartmental behaviors reflect Myosin II activity, this further suggests that there are likely anisotropic forces on opposing sides of individual interfaces. This anisotropy could drive the generation of shear forces that would cause the dissociation of homophilic extracellular E-cadherin bonds. It is also intriguing that, in terms of area oscillations, Rab35 compartmental formation is best correlated with cycles of area contractions in the opposing cell at a shared interface. This would be consistent with the above model in which changes in apical cell area are transmitted through E-cadherin junctions to a neighboring cell that may be in a differentially tensioned state. This may then again permit slackened plasma membrane to be taken up by Rab35-dependent endocytic processes (Jewett, 2017).

    Meru couples planar cell polarity with apical-basal polarity during asymmetric cell division

    Polarity is a shared feature of most cells. In epithelia, apical-basal polarity often coexists, and sometimes intersects with planar cell polarity (PCP), which orients cells in the epithelial plane. From a limited set of core building blocks (e.g. the Par complexes for apical-basal polarity and the Frizzled/Dishevelled complex for PCP), a diverse array of polarized cells and tissues are generated. This suggests the existence of little-studied tissue-specific factors that rewire the core polarity modules to the appropriate conformation. In Drosophila sensory organ precursors (SOPs), the core PCP components initiate the planar polarization of apical-basal determinants, ensuring asymmetric division into daughter cells of different fates. This study shows that Meru, a RASSF9/RASSF10 homologue, is expressed specifically in SOPs, recruited to the posterior cortex by Frizzled/Dishevelled, and in turn polarizes the apical-basal polarity factor Bazooka (Par3). Thus, Meru belongs to a class of proteins that act cell/tissue-specifically to remodel the core polarity machinery (Banerjee, 2017).

    Polarity is a fundamental feature of most cells and tissues. It is evident both at the level of individual cells and groups of cells (e.g. planar cell polarity (PCP) in epithelia. However, despite the fact that different cell types use a common set of molecules to establish and maintain polarity (Par complexes, Fz-PCP pathway), the organization of polarized cells and cell assemblies varies dramatically across different species and tissues. This implies the existence of factors that act in a cell or tissue-specific manner to modulate/rewire the core polarity machinery into the appropriate organization. Despite many advances in understanding of polarity in unicellular and multicellular contexts, little is known about the identity or function of such factors (Banerjee, 2017).

    An example of polarity remodeling is the process of asymmetric cell division (ACD), where cells need to rearrange their polarity determinants into a machinery capable of asymmetrically segregating cell fate determinants, vesicles and organelles, as well as controlling the orientation of the mitotic spindle. ACDs result in two daughter cells of different fates and occur in numerous cell types and across species. Well-studied examples include budding in Saccharomyces cerevisiae, ACD in the early embryo of Caenorhabditis elegans, or ACD of progenitor cells in the mammalian stratified epidermis and neural stem cells in the mammalian neocortex. In Drosophila melanogaster, the study of germline stem cells, neuroblasts (neural stem cells) and sensory organ precursors (SOPs) has greatly contributed to understanding of the cell biology and molecular mechanisms of ACD (Banerjee, 2017).

    SOPs (or pI cells) divide asymmetrically within the plane of the epithelium into pIIa and pIIb daughter cells. pIIa and pIIb themselves divide asymmetrically to give rise to the different cell types of the external sensory organs (bristles), which are part of the peripheral nervous system and allow the adult fly to sense mechanical or chemical stimuli. Individual SOPs are selected by Notch-dependent lateral inhibition from multicellular clusters of epithelial cells expressing proneural genes (proneural clusters) (Banerjee, 2017).

    The unequal segregation of cell fate determinants (the Notch pathway modulators Numb and Neuralized), which specifies the different fates of the daughter cells, requires their asymmetric localization on one side of the cell cortex prior to mitosis. This is achieved by remodeling the PCP and apical-basal polarity systems in the SOP, and by orienting the spindle relative to the tissue axis. The epithelial sheet that forms the pupal notum (dorsal thorax), where the best-studied SOPs are located, is planar polarized along the anterior-posterior tissue axis, with the transmembrane receptor Frizzled (Fz) and its effector Dishevelled (Dsh) localizing to the posterior side of the cell cortex, while the transmembrane protein Van Gogh (Vang, also known as Strabismus) and its interactor Prickle (Pk) are found anteriorly. The apical-basal polarity determinants central to SOP polarity are the PDZ domain-containing scaffold protein Bazooka (Baz, or Par3), atypical Protein Kinase C (aPKC) and Partitioning defective 6 (Par6), which localize apically in epithelial cells and the basolaterally localized membrane-associated guanylate kinase homologues (MAGUK) protein Discs-large (Dlg). In most epithelial cells, these proteins localize uniformly around the cell cortex, whereas in SOPs they show a striking asymmetric localization during mitosis: the Baz-aPKC-Par6 complex is found at the posterior cell cortex, opposite an anterior complex consisting of Dlg, Partner of Inscuteable (Pins) and the G-protein subunit Gαi. The Fz-Dsh complex provides the spatial information for the Baz-aPKC-Par6 complex, while Vang-Pk positions the Dlg-Pins-Gαi complex (likely through direct interaction between Vang and Dlg). The asymmetric distribution of the polarity determinants then directs the positioning of cell fate determinants at the anterior cell cortex. Additionally, Fz-Dsh and Pins orient the spindle along the anterior-posterior axis by anchoring it on both sides of the cell via Mushroom body defective (Mud, mammalian NuMA) and Dynein (Banerjee, 2017).

    The planar symmetry of the Baz-aPKC-Par6 complex in SOPs is initially broken in interphase via Fz-Dsh, and is independent of the Dlg-Pins-Gαi complex. Once this initial asymmetry is established, the core PCP components become dispensable for Par complex polarization at metaphase due to the mutual antagonism between the opposing polarity complexes, which then maintains asymmetry during cell division. Indeed, Baz is still polarized in fz mutants during mitosis, but losing both pins and fz results in Baz spreading uniformly around the cortex. Crucially, it is unclear how Fz-Dsh can transmit planar information to the Baz-aPKC-Par6 complex in SOPs but not in neighboring epithelial cells. The cell-type dependent coupling between PCP and apical-basal polarity suggests the involvement of unknown SOP-specific factors in this process (Banerjee, 2017).

    The four N-terminal RASSFs (Ras association domain family) in humans (RASSF7-10) have been associated with various forms of cancer, but the exact processes in which these scaffolding proteins act remain mostly elusive. Drosophila RASSF8, the homologue of human RASSF7 and RASSF8, is required for junctional integrity via Baz. Interestingly, human RASSF9 and RASSF10 were found in an interaction network with Par3 (the mammalian Baz homologue) and with several PCP proteins. The Drosophila genes CG13875 and CG32150 are believed to be homologues of human RASSF9 and RASSF10, respectively and remarkably, CG32150 mRNA is highly enriched in SOPs (Banerjee, 2017).

    This study shows that Meru, encoded by CG32150, is an SOP-specific factor, capable of linking PCP and apical-basal polarity. Meru localizes asymmetrically in SOPs based on the polarity information provided by Fz/Dsh, and is able to recruit Baz to the posterior cortex (Banerjee, 2017).

    PCP provides the spatial information for the initial polarization of SOPs at interphase, resulting in the planar polarization of Baz, which is uniformly localized prior to SOP differentiation. How Fz/Dsh communicate with Baz and enable its asymmetric enrichment was unknown. Based on the current results and previous findings, the following model is proposed for the role of Meru in SOP polarization. Upon selection and specification of SOPs, Meru expression is transcriptionally activated by the AS-C transcription factors (Reeves and Posakony, 2005). At interphase, planar-polarized Fz/Dsh recruit Meru to the membrane and hence direct its polarization. Meru in turn positions and asymmetrically enriches Baz, promoting the asymmetry of aPKC-Par6. Upon entry into mitosis, Meru is also required to retain laterally localized Baz, thus supporting the antagonism between the opposing Dlg-Pins-Gαi and Baz-aPKC-Par6 complexes, ultimately enabling the correct positioning of cell fate determinants (Banerjee, 2017).

    The meru mutant cell fate phenotype (bristle duplication or loss) is weaker than the baz loss-of-function phenotype, which results in loss of entire SOPs. This is likely due to two factors: (1) unlike meru mutants, the full baz mutant phenotype is the result of a complete loss of Baz in all cells of the SOP lineage, which is known to cause multiple defects including apoptosis of many sensory organ cells as well as cell fate transformations; (2) since a small amount of Baz is retained at the cortex of some meru mutant cells, it is likely that this residual Baz can still be polarized through the antagonistic activity of Pins at metaphase and thus partially rescues SOP polarization. Indeed, it was observed that reduction of pins or baz levels by RNAi strongly enhanced the meru cell specification phenotype. Conversely, supplying excess levels of Baz in a meru mutant background presumably restores sufficient Baz at the cortex to rescue the meru specification defect, as long as Pins is present to drive asymmetry at mitosis. (Banerjee, 2017).

    While a decrease in cortical Baz can account for the cell specification defects in meru mutants, it does not explain the spindle orientation phenotypee. This abnormal spindle alignment could either be due to a decrease in Fz/Dsh levels/activity, or a decrease in the ability of Dsh to recruit the spindle-tethering factor Mud. No gross abnormalities were detected in Fz levels in meru mutants, though the presence of Fz in all neighboring cells would make it difficult to detect subtle decreases in SOPs. Further work will be required to understand Meru's role in spindle orientation (Banerjee, 2017).

    Analysis of Meru in Drosophila is in agreement with the association of human RASSF9 and RASSF10 with both Par3 and PCP proteins previously reported. However, while the interaction with Dsh is conserved between the fly and human proteins, the transmembrane protein Vangl1 (the mammalian homologue of Vang), rather than its antagonist Fz was recovered in the mammalian proteomic analysis. This could reflect species-specific differences or altered polarity in the transformed human embryonic kidney 293 cells used for the mammalian work. Although Meru (CG32150) was classified as a potential homologue of RASSF10, alignment of the protein sequences showed similar sequence identities for both human RASSF9 (31%) and RASSF10 (26%). Thus, further functional work on Meru, its Drosophila paralogue CG13875, as well as mammalian RASSF9 and RASSF10 is required to understand the evolutionary and functional relationships between these proteins (Banerjee, 2017).

    Little is known about the in vivo functions of either RASSF9 or RASSF10 in other species. Xenopus RASSF10 is prominently expressed in the brain and other neural tissues of tadpoles, potentially indicating a function in neurogenesis, a process where ACDs are known to take place. Interestingly, mouse RASSF9 shows a cell-specific expression in keratinocytes of the skin and loss of RASSF9 results in differentiation defects of the stratified epidermis. Considering that Par3 is required for ACD of basal layer progenitors of the stratified epidermis this raises the exciting prospect that RASSF9 might regulate ACD in the mammalian skin (Banerjee, 2017).

    The polarization of cells and tissues is essential for their architecture and ultimately allows them to fulfill their function. The polarity machinery can be considered as a series of modules that are combined in a cell or tissue-specific manner, and hence requires specific factors that can create a polarity network appropriate to each tissue and cell type. This study has identified Meru as an SOP-specific factor, which is able to link PCP (Fz-Dsh) with apical-basal polarity (Baz). The PCP proteins Vang and Pk promote the positioning of the opposing Dlg-Pins-Gαi complex. Although Vang can directly bind to Dlg, the SOP and neuroblast-specific factor, Banderuola (aka Wide Awake) was recently shown to be required for Dlg localization and could thus constitute a link between the two polarity systems on the opposite side of the cortex (Banerjee, 2017).

    There is increasing evidence that cell-type specific rewiring of the polarity modules may be a widespread phenomenon. For instance, in different parts of the embryonic epidermis, Baz is planar polarized by Rho-kinase or by the Fat-PCP pathway, while in the retina, Vang is responsible for Baz polarization. Apical-basal polarity can also operate upstream of PCP in some systems, as in Drosophila photoreceptor specification, where aPKC restricts Fz activity by inhibitory phosphorylation in a subset of photoreceptor precursors. Thus, tissue-specific factors are likely to operate in a number of different contexts (Banerjee, 2017).

    The interplay between PCP and apical-basal polarity is also evident in other species, as Dishevelled has been reported to promote axon differentiation in rat hippocampal neurons by stabilizing aPKC, while Xenopus Dishevelled is required for Lethal giant larvae (Lgl) basal localization in the ectoderm. Interestingly, both mammalian Par3 and the Vang homologue Vangl2 are required for progenitor cell ACD in the developing mouse neocortex, raising the question as to whether PCP and apical-basal polarity are also connected in mammalian ACDs. It is therefore proposed that tissue-specific factors such as Meru might enable the diversity and plasticity observed across different polarized cells and tissues by rewiring the core polarity systems (Banerjee, 2017).

    The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dach

    Much of the Hippo and planar cell polarity (PCP) signaling mediated by the Drosophila protocadherin Fat depends on its ability to change the subcellular localization, levels and activity of the unconventional myosin Dachs. To better understand this process, a structure-function analysis of Dachs was performed, and this was used to identify a novel and important mediator of Fat and Dachs activities, a Dachs-binding SH3 protein that has been named Dlish (Dachs ligand with SH3s). Dlish was found to be regulated by Fat and Dachs. Dlish also binds Fat and the Dachs regulator Approximated, and Dlish is required for Dachs localization, levels and activity in both wild type and fat mutant tissue. The evidence supports dual roles for Dlish. Dlish tethers Dachs to the subapical cell cortex, an effect partly mediated by the palmitoyltransferase Approximated under the control of Fat. Conversely, Dlish promotes the Fat-mediated degradation of Dachs (Zhang, 2016).

    Heterophilic binding between the giant Drosophila protocadherins Fat and Dachsous (Ds) both limits organ growth, via regulation of the Hippo pathway, and orients planar cell polarity (PCP), through cell-by-cell polarization of Fat, Ds and their downstream effectors. Loss of Fat and, to a lesser extent, Ds, leads to the profound overgrowth of the Drosophila imaginal discs that give rise to adult appendages, and loss of either disorders the polarity of cell divisions, hairs and other morphological features in a variety of Drosophila tissues. But while players and pathways have been defined that are genetically downstream of Fat-Ds binding, only a little is known about the biochemical links between these and their most powerful regulator, the intracellular domain (ICD) of Fat (Zhang, 2016).

    A good deal of the recent work on Fat effectors has focused on the regulation of unconventional type XX myosin Dachs. Dachs is critical first because it provides the only known marker specifically sensitive to changes in the Fat/Ds branches of both the Hippo and PCP pathways. Dachs is normally concentrated in the subapical cell cortex, overlapping subapically-concentrated Fat and Ds. Loss of Fat greatly increases subapical Dachs levels, and polarization of Fat and Ds to opposite cell faces can in turn polarize Dachs to the face with less Fat. Fat thus inhibits or destabilizes subapical Dachs, while Ds may do the opposite. Downstream changes in Hippo or PCP activities do not affect Dachs (Zhang, 2016).

    Dachs changes are also critical because they play a major role downstream of Fat. Dachs binds to and inhibits the activity of the kinase Warts (the Drosophila Lats1/2 ortholog), both reducing Warts levels and changing its conformation. Warts is concentrated in the subapical cell cortex, and thus the increased cortical Dachs of fat mutants should reduce the phosphorylation of Yorkie by Warts, allowing Yorkie to move into the nucleus to drive the transcription of growth-promoting target genes. Indeed, Dachs is necessary for the overgrowth and increased Yorkie target gene expression of fat mutants. Dachs overexpression also causes overgrowth, although more weakly than the overgrowth caused by the loss of Fat, indicating that Dachs is partly sufficient (Zhang, 2016).

    Dachs can also bind to the core PCP pathway component Spiny legs (Sple) and alter its localization, thus influencing PCP in the subset of tissues that rely on Sple. The increased levels of unpolarized Dachs in fat mutants may misdirect Sple, accounting for at least some of the PCP defects; fat mutant hair PCP defects are improved, although not eliminated, by loss of dachs (Zhang, 2016).

    Dachs has not been shown to interact directly with Fat's ICD, and only three other proteins are known to affect Dachs accumulation in the subapical cell cortex, the casein kinase ε Discs overgrown (Dco), Approximated (App) and F-box-like 7 (Fbxl7). Dco may act through Fat itself: Dco binds and phosphorylates the Fat ICD, and loss of Dco function causes strong overgrowth and increases subapical Dachs, similar to loss of Fat (Zhang, 2016).

    App suggests a mechanism in which Fat inhibits the tethering of Dachs to protein complexes in the subapical domain. app mutants decrease subapical Dachs levels and reduce Dachs activity. Thus, like dachs mutants, app mutants reverse the overgrowth and increased Yorkie target gene expression normally observed in fat mutants, and improve hair PCP. App is one of 20 Drosophila DHHC palmitoyltransferases, transmembrane proteins responsible for adding palmitates to cytoplasmic proteins and thereby anchoring them to cell membranes. App is also concentrated in the subapical cell membrane and can bind both Dachs and the Fat ICD. Thus, in the simplest model App palmitoylates or tethers Dachs, concentrating it in the cell cortex, and Fat works in part by sequestering or inhibiting App. However, Dachs is not detectably palmitoylated (Zhang, 2016).

    This study describes the function of a novel Dachs binding protein, and shows that its effects provide strong evidence for both the palmitoylation-dependent and degradation-dependent regulation of Dachs. Structure-function analysis of Dachs found regions required for its normal subapical localization, and this information was used as the basis for a screen for novel Dachs binding partners. A direct binding partner was found for the Dachs C-terminus, the previously uncharacterized SH3 domain protein CG10933, which has been renamed Dachs ligand with SH3s, or Dlish. The activity and subapical concentration of Dlish are regulated by Fat, Dco and Dachs, and Dlish in turn is required for the subapical concentration and full activity of Dachs in both wild type and fat mutant cells. Dlish localization also depends on App; furthermore Dlish binds to and is palmitoylated by App, and palmitoylation can be suppressed by Fat. Loss of Dlish also increases the total levels of Dachs, likely by blocking Fat-mediated destabilization of Dachs. It is proposed that Dlish targets Dachs to subapical protein complexes in part via Fat-regulated, App-mediated palmitoylation. Dlish thereby concentrates Dachs where it can efficiently inhibit subapical Warts, and conversely links Dachs to the machinery for Fat-dependent destabilization (Zhang, 2016).

    The unconventional myosin Dachs is an important effector Fat/Ds-regulated Hippo signaling, as its heightened subapical levels in fat mutants inhibit and destabilize Warts, freeing Yorkie to increase the expression of growth-promoting genes. A structure-function analysis of Dachs was used as a springboard to search for new binding partners that are critical for Dachs localization and function, and have found Dlish (CG10933), a novel SH3 domain protein. Dlish binds directly to the Dachs C-terminus; loss of Dlish disrupts Dachs localization, levels and function: subapical accumulation of Dachs is reduced and cytoplasmic and total levels increase, both in wild type and fat mutant tissue, while activity is lost. Importantly, Dlish is regulated by Fat, as loss of Fat greatly increases Dlish levels in the subapical cell cortex and, like Dachs, Dlish is needed for much of the fat mutant overgrowth (Zhang, 2016).

    Dlish also binds the ICD of Fat and other Fat-binding proteins, including two that likely mediate part of its function: the palmitoyltransferase App and the F-box protein Fbxl7. Thus Dlish provides a new biochemical link from the Fat ICD to Dachs regulation. Evidence indicates that Dlish plays two different and opposing roles (see Models of Fat-mediated regulation of subapical Dachs by Fat-inhibited subapical tethering and Fat-stimulated destabilization). First, it helps tether Dachs in the subapical cell membrane, in part via Fat-regulated, App-dependent palmitoylation, so that Dachs can more efficiently inhibit Warts. Second, it links Dachs to Fat-organized machinery for Dachs destabilization, including Fbxl7, and thus helps reduce Dachs levels (Zhang, 2016).

    Dlish and Dachs cooperate to target or tether the Dlish-Dachs complex, as each is necessary, and to a weaker extent sufficient, for the subapical concentration of the other. The Dachs contribution is likely through tethering the complex to the cortical cytoskeleton, as this study found that loss of the F-Actin-binding myosin head blocks the subapical localization of Dachs. This would agree with recent biochemical analyses that suggest that Dachs has no motor function, acting rather as an F-Actin-binding scaffolding protein (Zhang, 2016).

    The Dlish contribution, on the other hand, depends at least in part on its ability to bind the transmembrane DHHC palmitoyltransferase App. Loss of App and Dlish have very similar effects on Dachs localization and activity. This study found that loss of App disrupts the subapical accumulation of Dlish in vivo and that App can stimulate palmitoylation of Dlish in vitro. Thus, palmitoylation of Dlish likely stimulates membrane association of both Dlish and its binding partner Dachs (Zhang, 2016).

    App also has additional effects on Fat pathway activity. First, App has palmitoyltransferase-independent activity and can co-IP Dachs in vitro. Thus, while palmitoylation of Dlish may mediate some of App's activity, subapical App may simultaneously help localize the Dlish-Dachs complex by physical tethering. And while both palmitoylation and tethering of the Dlish-Dachs complex is likely critical for the fat mutant phenotype, App also has a function that depends on the presence of Fat, as App can bind, palmitoylate and inhibit the activity of Fat's ICD (Zhang, 2016).

    An important question is whether the absence of Fat regulates the App-dependent tethering of the Dlish-Dachs complex. The Fat ICD can complex with both Dlish and App. Dlish and App can bind not only the C-terminal region of the Fat ICD where Fat is palmitoylated, but also the PH and Hippo domains which was shown to played the strongest role in Dachs regulation. An attractive mechanism is that Fat inhibits the interaction between App and Dlish, reducing App's ability to palmitoylate and tether the Dlish-Dachs complex. In the absence of Fat, App and Dlish are freed to tether Dachs, and Dachs now inhibits and destabilizes Warts, causing overgrowth. In support of this model, it was found that overexpression of Fat's ICD in vitro can reduce App-stimulated palmitoylation of Dlish (Zhang, 2016).

    The evidence further indicates that Dlish targets Dachs for Fat-dependent destabilization. Loss of Fat increases not only subapical Dachs, but also total Dachs levels. In the presence of Dlish the increased Dachs remains subapical. Loss of Dlish also increases the total levels of Dachs, but now that increase is cytoplasmic, and much less effective at inhibiting Warts. These Dachs increases are unlikely to have independent causes, as they are not additive; total Dachs levels are similar after loss of Fat, Dlish or both (Zhang, 2016).

    It is proposed that in wild type cells there is a flux of the Dachs-Dlish complex from the cytoplasm to the subapical cell cortex, where a Fat-dependent complex destabilizes Dachs. Normally the tethering effects of Dlish predominate over the Fat-dependent destabilization, and moderate levels of subapical Dachs are maintained. Destabilization is lost without Fat; this combines with Dlish-mediated tethering to increase subapical Dachs. Without Dlish the subapical tethering of Dachs is disrupted, access to Fat-dependent destabilization is lost and the now cytoplasmic Dachs increases. The model thus explains why the excess, largely cytoplasmic Dachs caused by reduced Dlish function is not greatly influenced by the presence or absence of Fat (Zhang, 2016).

    In addition to any effects caused by changing the subcellular localization of Dachs, Dlish may also provide a direct link to the machinery for protein ubiquitination, as Dlish can co-IP with the E3 ubiquitin ligase Fbxl7, as well as the related F-box ubiquitin ligase Slimb. Fbxl7 is particularly intriguing, as it binds to and is regulated by Fat's ICD, and reduces subapical Dachs, perhaps via ubiquitination. Slimb can bind and ubiquitinate Expanded, a subapical regulator of Hippo signaling with links to Fat and Dachs function. But while it was found that loss of Fbxl7 or Slimb increases subapical Dachs and Dlish, these effects are weak, and the large increase in total Dachs levels caused by loss of Dlish or Fat must involve additional partners (Zhang, 2016).

    Mutations in Fat’s closest mammalian homolog Fat4 (FatJ) and its Ds-like ligands strongly disrupt PCP-like processes, and have in humans been associated with the multisystem defects of Hennekam and Van Maldergem syndromes. There has been some debate, however, about whether the mammalian proteins retain direct regulation of Hippo activity. Nonetheless, Fat4 has been linked to Hippo changes in both normal development and tumors, mutations in Fat4 or Dachsous1 change the balance of precursors and mature neurons in the developing neuroepithelium of both humans and mice, and the mouse defect can be reversed by knockdown of the Yki homolog Yap. But the mechanisms underlying these effects are unknown, and Fat4 cannot regulate Hippo signaling in Drosophila (Zhang, 2016).

    It is therefore important to note that while homologs of Dachs and Dlish are found throughout the animal kingdom, they are apparently absent from vertebrates. This suggests that the Dachs-Dlish branch of the Fat-Ds pathway, with its powerful effect on Warts activity, is also lacking. Nonetheless, it has been suggested that Drosophila Fat and Ds can affect Hippo pathway activity in a Dachs-independent manner. It is also clear that Drosophila Fat has Dachs-independent effects on PCP; indeed the N-terminal 'PCP' domain of the Fat ICD that did not affect Dachs in this study is sufficient to improve the PCP defects of fat mutants. These or alternative pathways may still be present in mammals (Zhang, 2016).

    Vamana couples fat signaling to the Hippo pathway

    The protocadherins Dachsous and Fat initiate a signaling pathway that controls growth and planar cell polarity by regulating the membrane localization of the atypical myosin Dachs. How Dachs is regulated by Fat signaling has remained unclear. This study identified the vamana gene (CG10933: Dachs ligand with SH3s or Dlish) as playing a crucial role in regulating membrane localization of Dachs and in linking Fat and Dachsous to Dachs regulation. Vamana, an SH3-domain-containing protein, physically associates with and co-localizes with Dachs and promotes its membrane localization. Vamana also associates with the Dachsous intracellular domain and with a region of the Fat intracellular domain that is essential for controlling Hippo signaling and levels of Dachs. Epistasis experiments, structure-function analysis, and physical interaction experiments argue that Fat negatively regulates Dachs in a Vamana-dependent process. These findings establish Vamana as a crucial component of the Dachsous-Fat pathway that transmits Fat signaling by regulating Dachs (Misra, 2016).

    Coordinated growth and morphogenesis is critical to the development of tissues of specific size and shape. Dachsous (Ds)-Fat signaling (henceforth, Fat signaling) controls both growth, through regulation of Hippo signaling, and morphogenesis, through regulation of planar cell polarity (PCP). Fat signaling regulates Hippo signaling and PCP by controlling the membrane localization of the atypical myosin protein Dachs. Many studies have provided important insights into both how Dachs influences Hippo signaling, and how it influences PCP. In contrast, the mechanism by which Fat signaling actually controls Dachs has remained less well understood (Misra, 2016).

    Fat and Ds are atypical cadherins with novel intracellular domains (ICD), which localize to the plasma membrane just apical to the adherens junctions. Fat and Ds bind to each other in a heterophilic manner, and this interaction is modulated by the Golgi-resident kinase, Four-jointed (Fj), which phosphorylates their extracellular domains. This heterophilic binding, together with the graded expression of Ds and Fj, contribute to polarization of Ds and Fat localization within cells. Three different ways by which Fat signaling influences Hippo signaling have been described: Fat signaling influences the membrane localization of Expanded (Ex) , the levels of Wts protein, and the interaction of Wts with its cofactor Mats. Each of these effects on Hippo signaling depends upon Dachs. Fat signaling affects PCP in at least two ways: through an influence on junctional tension, and by regulating the Spiny-legs (Sple) isoform of the prickle locus. Both of these effects also involve Dachs (Misra, 2016).

    Dachs was identified as a key downstream effector of Fat signaling because mutations in dachs completely suppress the overgrowth induced by fat mutations, and partially suppress the PCP defects induced by fat mutations. Dachs localizes to the cell membrane just apical to the adherens junction in a polarized manner; in the developing wing Dachs is localized to the distal sides of the cell, in response to the proximal-distal gradients of Ds and Fj expression. Dachs membrane localization requires a palmitoyltransferase encoded by approximated (app), but how App influences Dachs localization is unknown. In fat or ds mutants increased levels of Dachs are observed at the apical membrane and Dachs is no longer polarized. Forcing Dachs membrane localization by fusing it to Zyxin phenocopies fat mutants. Conversely, overexpression of full-length Fat or even just the Fat intracellular domain (ICD) displaces Dachs from the membrane into the cytoplasm. These and other observations have indicated that Fat regulates growth by modulating the levels of Dachs at apical membranes, and regulates Dachs-dependent PCP by directing Dachs asymmetry (Misra, 2016).

    To understand how Fat functions, several studies have examined the roles of different regions of the Fat ICD. These studies identified two regions that mediate its growth-suppressive function. One, the D region, around amino acids 4,975 to 4,993, makes a modest contribution to Hippo pathway regulation, as when this region is deleted flies are viable but their wings are approximately 30% larger than normal, and also rounder than normal. The D region is required for interaction with the ubiquitin ligase, Fbxl7, which reduces Dachs membrane levels, and mutation of which results in phenotypes similar to deletion of the D region. A second region, which has been referred to as HM, Hpo, or H2, is defined by observations that deletions within this region block the ability of Fat to activate Hippo signaling. Two alleles of fat, fat61 and fatsum, have also been identified that harbor mutations within this region, and are associated with tissue overgrowth comparable with that caused by fat null mutations. However, the mechanism by which this region, which for simplicity is referred to as the H region, regulates the Hippo pathway, and whether it affects Dachs, are unknown (Misra, 2016).

    This study reports the isolation and characterization of the Src homology 3 (SH3)-domain-containing protein encoded by vamana (vam). Loss of vam function decreases growth, whereas overexpression of vam promotes growth. These effects are mediated through regulation of the Hippo pathway, and vam functions genetically downstream of fat, as vam mutations can suppress both growth and PCP phenotypes of fat. Vam localizes to the apical region of epithelial cells in a polarized manner, co-localizing with Dachs, and is required for normal membrane localization of Dachs. Vam physically associates with the carboxy-terminal domain of Dachs and the ICDs of Ds and Fat, and is regulated by the H region of the Fat ICD. These observations identify Vam as a key mediator of signaling from Fat to Dachs (Misra, 2016).

    These studies identified the C-terminal region of Dachs as sufficient to mediate its interaction with Vam. Interestingly, the original dachs allele, described almost a century ago by Bridges and Morgan (1919), is a hypomorphic allele associated with insertion of a blood transposon just upstream of the C-terminal region. Hence this allele likely encodes a truncated protein that lacks the Vam-interaction domain. Consistent with this inference, the vam null phenotype appears similar to the dachs1 phenotype. Thus, a requirement for interaction with Vam can explain the basis for the original identification of dachs (Misra, 2016).

    Vam is evolutionarily conserved among insects but with no close homologs in vertebrates. This is consistent with the fact that Dachs is also only found in insects, and the sequence of the H region is not conserved in vertebrate Fat genes. Nonetheless, Vam is structurally related to a broad family of SH2- and SH3-domain-containing proteins exemplified by CRK, Grb2, Myd88, and NCK. These proteins are referred to as signal-transducing adapter proteins and facilitate formation of protein complexes that play key roles in signal transduction. Vam is composed of just three SH3 domains; this domain organization is most similar to that of the NCK family of adapters, which contain three SH3 domains along with one SH2 domain. The finding that Vam uses both SH3-1 and SH3-3 to interact with Fat and Ds is also reminiscent of NCK family adapters, as they engage effectors using multiple SH3 domains. The Drosophila ortholog of NCK, dreadlocks (dock), interacts with cell-adhesion molecules encoded by hibris, kirre, roughest, and sticks and stones (sns) to regulate actin polymerization and growth cone migration, and functional redundancy of SH3 domains has been observed for dock. Multiple SH3 domains are also commonly observed in proteins involved in vesicular trafficking. The observation that in vam mutants Dachs accumulates in cytoplasmic puncta that could be vesicular structures suggests that Vam might influence the trafficking of Dachs (Misra, 2016).

    Fat and Ds proteins are conserved in vertebrates, where they play important roles in controlling PCP, and have also been proposed to influence Hippo signaling. In the absence of a Dachs homolog, however, it has been unclear how downstream signaling is mediated in vertebrate Ds-Fat pathways. The discovery that Vam links Ds and Fat to downstream signaling raises the possibility that a different member of the signal-transducing adapter proteins could mediate downstream Ds-Fat signaling in vertebrates (Misra, 2016).

    The H region of the Fat ICD plays a crucial role in Hippo pathway regulation. This analysis of Fat ICD truncations revealed that the H region inhibits Vam and Dachs membrane accumulation, the influence of Fat ICD deletions on Hippo signaling correlates with their influence on Vam and Dachs membrane localization, and the H region of Fat can associate with Vam. Together with observations that Vam associates with and regulates Dachs, these observations lead to the inferrence that the H region normally functions to promote Hippo signaling through its association with, and regulation of, Vam. Fat also influences growth and Dachs accumulation through a second region of the ICD, the D region, which interacts with Fbxl7. Because mutation of the D region, or mutations in Fbxl7, have weaker phenotypes than mutations in the H region, the H region appears to play the larger role in Dachs regulation, but nonetheless it is expected that both regions normally act in parallel to regulate membrane levels of Dachs and thus, ultimately, Hippo signaling (Misra, 2016).

    The localization of Vam in different genotypes, together with its physical interactions, suggests models for how Vam regulates Dachs localization. Since Vam and Dachs are reciprocally required for each other's membrane localization, it is inferred that a complex between these two proteins is required for their stable localization to apical junctions, where Dachs regulates PCP (via interactions with Sple) and Hippo signaling (via interactions with Zyxin and Warts). The observations that Fat promotes removal of Vam and Dachs from the subapical membrane, associates with Vam, yet does not visibly co-localize with Vam at apical junctions, suggests that Fat normally removes Vam-Dachs complexes from the subapical membrane. One mechanism by which this might occur is through binding of Fat to Vam, followed by endocytosis of Fat-Vam-Dachs complexes. Alternatively, Fat binding might disrupt Vam-Dachs binding, as these proteins normally do not localize to the membrane in isolation (Misra, 2016).

    It was also observed that Vam can interact with the Ds ICD, and that it does so through the same SH3 domains as it uses to interact with the Fat ICD. This suggests that these interactions are likely to be competitive. In this case, interaction of Vam with the Ds ICD could promote Vam and Dachs membrane localization by opposing the influence of Fat on Vam. For example, by competing with Fat for binding to Vam, Ds could prevent Fat from disrupting Vam-Dachs interactions, or promoting endocytosis of a Vam-Dachs complex. Consistent with this suggestion that the Ds ICD stabilizes Vam and Dachs at apical junctions, Vam, Ds, and Dachs normally all co-localize in puncta on the distal side of wing cells. The ability of Vam to associate with the ICDs of both Fat and Ds could thus provide a simple mechanism explaining how the ICD of Ds seems to promote Dachs membrane localization, whereas the ICD of Fat inhibits it (Misra, 2016).


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    genes expressed in tissue polarity

    Zygotically transcribed genes

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