Regulation of Frizzled by fat-like cadherins during planar polarity signaling in the Drosophila compound eye

Planar polarity is evident in the coordinated orientation of ommatidia in the Drosophila eye. This process requires that the R3 photoreceptor precursor of each ommatidium have a higher level of Frizzled signaling than its neighboring R4 precursor. We show that two cadherin superfamily members, Fat and Dachsous, and the transmembrane/secreted protein Four-jointed play important roles in this process. Our data support a model in which the bias of Frizzled signaling between the R3/R4 precursors results from higher Fat function in the precursor cell closer to the equator, which becomes R3. We also provide evidence that positional information regulating Fat action is provided by graded expression of Dachsous across the eye and the action of Four-jointed, which is expressed in an opposing expression gradient and appears to modulate Dachsous function.     

Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity under the control of Frizzled.

We identified a seven-pass transmembrane receptor of the cadherin superfamily, designated Flamingo (Fmi), localized at cell-cell boundaries in the Drosophila wing. In the absence of Fmi, planar polarity was distorted. Before morphological polarization of wing cells along the proximal-distal (P-D) axis, Fmi was redistributed predominantly to proximal and distal cell edges. This biased localization of Fmi appears to be driven by an imbalance of the activity of Frizzled (Fz) across the proximal/distal cell boundary. These results, together with phenotypes caused by ectopic expression of fz and fmi, suggest that cells acquire the P-D polarity by way of the Fz-dependent boundary localization of Fmi.     

Frizzled signaling and cell-cell interactions in planar polarity.

The function of the Frizzled pathway is essential for the formation of the array of distally pointing hairs found on the Drosophila wing. Previous research found that regulating the subcellular location for hair initiation controlled hair polarity. Recent work argues a graded Frizzled-dependent signal results in the accumulation of the Frizzled, Dishevelled and Flamingo proteins along the distal edge of the wing cells. This cortical mark leads to the local activation of downstream gene products and the subsequent activation of the cytoskeleton to form a hair.     

Decoding vectorial information from a gradient: sequential roles of the receptors Frizzled and Notch in establishing planar polarity in the Drosophila eye

The Drosophila eye is composed of several hundred ommatidia that can exist in either of two chiral forms, depending on position: ommatidia in the dorsal half of the eye adopt one chiral form, whereas ommatidia in the ventral half adopt the other. Chirality appears to be specified by a polarizing signal with a high activity at the interface between the two halves (the 'equator'), which declines in opposite directions towards the dorsal and ventral poles. Here, using genetic mosaics, we show that this polarizing signal is decoded by the sequential use of two receptor systems. The first depends on the seven-transmembrane receptor Frizzled (Fz) and distinguishes between the two members of the R3/R4 pair of presumptive photoreceptor cells, predisposing the cell that is located closer to the equator and having higher Fz activity towards the R3 photoreceptor fate and the cell further away towards the R4 fate. This bias is then amplified by subsequent interactions between the two cells mediated by the receptor Notch (N) and its ligand Delta (Dl), ensuring that the equatorial cell becomes the R3 photoreceptor while the polar cell becomes the R4 photoreceptor. As a consequence of this reciprocal cell fate decision, the R4 cell moves asymmetrically relative to the R3 cell, initiating the appropriate chiral pattern of the remaining cells of the ommatidium.     

The Drosophila STE20-like kinase misshapen is required downstream of the Frizzled receptor in planar polarity signaling.

The Drosophila misshapen (msn) gene is a member of the STE20 kinase family. We show that msn acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings. Both msn loss- and gain-of-function result in defective ommatidial polarity and wing hair formation. Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes. We also show that Msn acts in EPP signaling through the JNK (Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, we provide evidence for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn.     

Strabismus requires Flamingo and Prickle function to regulate tissue polarity in the Drosophila eye

Tissue polarity in Drosophila is regulated by a number of genes that are thought to function in a complex, many of which interact genetically and/or physically, co-localize, and require other tissue polarity proteins for their localization. We report the enhancement of the strabismus tissue polarity phenotype by mutations in two other tissue polarity genes, flamingo and prickle. Flamingo is autonomously required for the establishment of ommatidial polarity. Its localization is dynamic throughout ommatidial development and is dependent on Frizzled and Notch. Flamingo and Strabismus co-localize for several rows posterior to the morphogenetic furrow and subsequently diverge. While neither of these proteins is required for the other's localization, Prickle localization is influenced by Strabismus function. Our data suggest that Strabismus, Flamingo and Prickle function together to regulate the establishment of tissue polarity in the Drosophila eye.     

Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton

Frizzled (Fz) and Dishevelled (Dsh) are components of an evolutionarily conserved signaling pathway that regulates planar cell polarity. How this signaling pathway directs asymmetric cytoskeletal reorganization and polarized cell morphology remains unknown. Here, we show that Drosophila Rho-associated kinase (Drok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Drok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain, and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton.     

Asymmetric homotypic interactions of the atypical cadherin flamingo mediate intercellular polarity signaling

Acquisition of planar cell polarity (PCP) in epithelia involves intercellular communication, during which cells align their polarity with that of their neighbors. The transmembrane proteins Frizzled (Fz) and Van Gogh (Vang) are essential components of the intercellular communication mechanism, as loss of either strongly perturbs the polarity of neighboring cells. How Fz and Vang communicate polarity information between neighboring cells is poorly understood. The atypical cadherin, Flamingo (Fmi), is implicated in this process, yet whether Fmi acts permissively as a scaffold or instructively as a signal is unclear. Here, we provide evidence that Fmi functions instructively to mediate Fz-Vang intercellular signal relay, recruiting Fz and Vang to opposite sides of cell boundaries. We propose that two functional forms of Fmi, one of which is induced by and physically interacts with Fz, bind each other to create cadherin homodimers that signal bidirectionally and asymmetrically, instructing unequal responses in adjacent cell membranes to establish molecular asymmetry.     

Planar cell polarity controls directional Notch signaling in the Drosophila leg

The generation of functional structures during development requires tight spatial regulation of signaling pathways. Thus, in Drosophila legs, in which Notch pathway activity is required to specify joints, only cells distal to ligand-producing cells are capable of responding. Here, we show that the asymmetric distribution of planar cell polarity (PCP) proteins correlates with this spatial restriction of Notch activation. Frizzled and Dishevelled are enriched at distal sides of each cell and hence localize at the interface with ligand-expressing cells in the non-responding cells. Elimination of PCP gene function in cells proximal to ligand-expressing cells is sufficient to alleviate the repression, resulting in ectopic Notch activity and ectopic joint formation. Mutations that compromise a direct interaction between Dishevelled and Notch reduce the efficacy of repression. Likewise, increased Rab5 levels or dominant-negative Deltex can suppress the ectopic joints. Together, these results suggest that PCP coordinates the spatial activity of the Notch pathway by regulating endocytic trafficking of the receptor.     

Flamingo controls the planar polarity of sensory bristles and asymmetric division of sensory organ precursors in Drosophila.

The sensory bristles of the fruit fly Drosophila are organized in a polarized fashion such that bristles on the thorax point posteriorly. These bristles are derived from asymmetric division of sensory organ precursors (SOPs). The Numb protein, which is localized asymmetrically in a cortical crescent in each SOP, segregates into only one of the two daughter cells during cell division, thereby conferring distinct fates to the daughter cells [1] [2]. In neuroblasts, establishment of apical-basal polarity by the protein Inscuteable is crucial for orienting asymmetric division, but this is not the case for division of SOPs [3]. Instead, the Frizzled (Fz) protein mediates a planar polarity signal that controls the anteroposteriorly oriented first division (pl) of SOPs [4]. Here, we report that Flamingo (Fmi), a seven-transmembrane cadherin [5], controls the planar polarity of sensory bristles and the orientation of the SOP pl division. Both the loss of function and overexpression of fmi disrupted bristle polarity. During mitosis of the SOP, the axis of the pl division and the positioning of the Numb crescent were randomized in the absence of Fmi activity. Overexpression of Fmi and Fz caused similar effects. The dependence of proper Fmi localization on Fz activity suggests that Fmi functions downstream of Fz in controlling planar polarity. We also present evidence suggesting that Fz also functions in the Wingless pathway to pattern sensory organs.     

Flamingo,a cadherin-type receptor involved in the Drosophila planar polarity pathway,can block signaling via the canonical wnt pathway in Xenopus laevis

The Flamingo gene encodes a seven-pass transmembrane receptor of the cadherin super family and is one of a growing number of components identified as being necessary for the establishment of planar polarity in the Drosophila wing. Although vertebrate homologues of Flamingo have been identified in both man and mice, no function has as yet been ascribed to them. Here, we report the cloning of the Xenopus homologue of Flamingo (XFmi). XFmi is expressed in the dorsal ectoderm during gastrulation and in the forebrain and midbrain subsequently. We show that ectopic expression of the murine Flamingo gene can prevent the wnt mediated posteriorisation of the neural plate by interfering with the canonical wnt signalling pathway.     

The apical determinants aPKC and dPatj regulate Frizzled-dependent planar cell polarity in the Drosophila eye

Planar cell polarity (PCP) is a common feature of many vertebrate and invertebrate epithelia and is perpendicular to their apical/basal (A/B) polarity axis. While apical localization of PCP determinants such as Frizzled (Fz1) is critical for their function, the link between A/B polarity and PCP is poorly understood. Here, we describe a direct molecular link between A/B determinants and Fz1-mediated PCP establishment in the Drosophila eye. We demonstrate that dPatj binds the cytoplasmic tail of Fz1 and propose that it recruits aPKC, which in turn phosphorylates and inhibits Fz1. Accordingly, components of the aPKC complex and dPatj produce PCP defects in the eye. We also show that during PCP signaling, aPKC and dPatj are downregulated, while Bazooka is upregulated, suggesting an antagonistic effect of Bazooka on dPatj/aPKC. We propose a model whereby the dPatj/aPKC complex regulates PCP by inhibiting Fz1 in cells where it should not be active.     

The cadherin Flamingo mediates level-dependent interactions that guide photoreceptor target choice in Drosophila

Quantitative differences in cadherin activity have been proposed to play important roles in patterning connections between pre- and postsynaptic neurons. However, no examples of such a function have yet been described, and the mechanisms that would allow such differences to direct growth cones to specific synaptic targets are unknown. In the Drosophila visual system, photoreceptors are genetically programmed to make a complex, stereotypic set of synaptic connections. Here we show that the atypical cadherin Flamingo functions as a short-range, homophilic signal, passing between specific R cell growth cones to influence their choice of postsynaptic partners. We find that individual growth cones are sensitive to differences in Flamingo activity through opposing interactions between neighboring cells and require these interactions to be balanced in order to extend along the appropriate trajectory.     

The Notch regulator Numb links the Notch and TCR signaling pathways

Both the Notch and TCR signaling pathways play an important role in T cell development, but the links between these signaling pathways are largely unexplored. The adapter protein Numb is a well-characterized inhibitor of Notch and also contains a phosphotyrosine binding domain, suggesting that Numb could provide a link between these pathways. We explored this possibility by investigating the physical interactions among Notch, Numb, and the TCR signaling apparatus and by examining the consequences of a Numb mutation on T cell development. We found that Notch and Numb cocluster with the TCR at the APC contact during Ag-driven T cell-APC interactions in both immature and mature T cells. Furthermore, Numb coimmunoprecipitates with components of the TCR signaling apparatus. Despite this association, T cell development and T cell activation occur normally in the absence of Numb, perhaps due to the expression of the related protein, Numblike. Together our data suggest that Notch and TCR signals may be integrated at the cell membrane, and that Numb may be an important adapter in this process.     

Temporal regulation of planar cell polarity: insights from the Drosophila eye

In this issue of Cell, identify a first regulatory link between planar cell polarity (PCP) signaling and apical-basal polarity. The authors propose that a component of the apical Crumbs complex regulates the phosphorylation of the Frizzled (Fz) PCP receptor, thus modulating PCP in the Drosophila eye.