CKIepsilon/discs overgrown promotes both Wnt-Fz/beta-catenin and Fz/PCP signaling in Drosophila

The related Wnt-Frizzled(Fz)/beta-catenin and Fz/planar cell polarity (PCP) pathways are essential for the regulation of numerous developmental processes and are deregulated in many human diseases. Both pathways require members of the Dishevelled (Dsh or Dvl) family of cytoplasmic factors for signal transduction downstream of the Fz receptors. Dsh family members have been studied extensively, but their activation and regulation remains largely unknown. In particular, very little is known about how Dsh differentially signals to the two pathways. Recent work in cell culture has suggested that phosphorylation of Dsh by Casein Kinase I epsilon (CKIepsilon) may act as a molecular "switch," promoting Wnt/beta-catenin while inhibiting Fz/PCP signaling. Here, we demonstrate in vivo in Drosophila through a series of loss-of-function and coexpression assays that CKIepsilon acts positively for signaling in both pathways, rather than as a switch. Our data suggest that the kinase activity of CKIepsilon is required for peak levels of Wnt/beta-catenin signaling. In contrast, CKIepsilon is a mandatory signaling factor in the Fz/PCP pathway, possibly through a kinase-independent mechanism. Furthermore, we have identified the primary kinase target residue of CKIepsilon on Dsh. Thus, our data suggest that CKIepsilon modulates Wnt/beta-catenin and Fz/PCP signaling pathways via kinase-dependent and -independent mechanisms.     

CELLULAR LOCALIZATION AND EXPRESSION OF pygo DURING DROSOPHILA DEVELOPMENT

Abstract Wg/Wnt signaling is a key signaling pathway in Drosophila. Many genes involved in Wingless(wg) signal transduction pathway downstream of Wg, or it s vertebrate Wg homologue Wnt, have been identified. Transduction of the Wg signal downstream of Wg is mediated by nuclear TCF/LEF-1, through association with Armadillo (Arm)β-catenin. Pygopus (pygo) is a new identified component in this pathway. Cellular localization experiment showed that pygo was expressed specifically in the nucleus. The expression profile of pygo in embryos was examined using in situ hybridization. Although pygo expressed ubiquitously in the embryos, it expressed at relatively high level in pre-blastoderm embryos which indicate a high degree of maternally provided message, followed by a low level of ubiquitous zygotic expression. This continues into larval tissues (including wing disc, eye disc and leg disc), where pygo appears to be expressed at low level. Comparison of pygo expression levels, in the wing disc, eye disc and leg disc, showed pygo expression level in the wing disc pouch and leg disc were relative higher.     

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.     

A mutational analysis of dishevelled in Drosophila defines novel domains in the dishevelled protein as well as novel suppressing alleles of axin

Drosophila dishevelled (dsh) functions in two pathways: it is necessary to transduce Wingless (Wg) signaling and it is required in planar cell polarity. To learn more about how Dsh can discriminate between these functions, we performed genetic screens to isolate additional dsh alleles and we examined the potential role of protein phosphorylation by site-directed mutagenesis. We identified two alleles with point mutations in the Dsh DEP domain that specifically disrupt planar polarity signaling. When positioned in the structure of the DEP domain, these mutations are located close to each other and to a previously identified planar polarity mutation. In addition to the requirement for the DEP domain, we found that a cluster of potential phosphorylation sites in a binding domain for the protein kinase PAR-1 is also essential for planar polarity signaling. To identify regions of dsh that are necessary for Wg signaling, we screened for mutations that modified a GMR-GAL4;UAS-dsh overexpression phenotype in the eye. We recovered many alleles of the transgene containing missense mutations, including mutations in the DIX domain and in the DEP domain, the latter group mapping separately from the planar polarity mutations. In addition, several transgenes had mutations within a domain containing a consensus sequence for an SH3-binding protein. We also recovered second-site-suppressing mutations in axin, mapping at a region that may specifically interact with overexpressed Dsh.     

Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling

Frizzled (Fz) signaling regulates the establishment of planar cell polarity (PCP). The PCP genes prickle (pk) and strabismus (stbm) are thought to antagonize Fz signaling. We show that they act in the same cell, R4, adjacent to that in which the Fz/PCP pathway is required in the Drosophila eye. We demonstrate that Stbm and Pk interact physically and that 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 our data suggest that the Stbm/Pk complex modulates Fz/Dsh activity, resulting in a symmetry-breaking step during polarity signaling.     

Nonautonomous planar polarity patterning in Drosophila: dishevelled-independent functions of frizzled

The frizzled (fz) gene of Drosophila is required for planar polarity establishment in the adult cuticle, acting both cell autonomously and nonautonomously. We demonstrate that these two activities of fz in planar polarity are temporally separable in both the eye and wing. The nonautonomous function is dishevelled (dsh) independent, and its loss results in polarity phenotypes that resemble those seen for mutations in dachsous (ds). Genetic interactions and epistasis analysis suggest that fz, ds, and fat (ft) act together in the long-range propagation of polarity signals in the eye and wing. We also find evidence that polarity information may be propagated by modulation of the binding affinities of the cadherins encoded by the ds and ft loci.     

Mtl interacts with members of Egfr signaling and cell adhesion genes in the Drosophila eye

Mtl is a member of the Rho family of small GTPases in Drosophila. It was shown that Mtl is involved in planar cell polarity (PCP) establishment, together with other members of the same family like Cdc42, Rac1, Rac2 and RhoA. However, while Rac1, Rac2 and RhoA function downstream of Dsh in Fz/PCP signaling and upstream of a JNK cassette, Mtl and Cdc42 do not. To determine the functional context of Mtl during PCP establishment in the Drosophila eye, we performed a loss-of-function screen to search for dominant modifiers of a sev>Mtl rough eye phenotype. In addition, genetic interaction assays with candidate genes were also carried out. Our results show that Mtl interacts genetically with members and effectors of Egfr signaling, with components and/or regulators of other signal transduction pathways, and with genes involved in cell adhesion and cytoskeleton organization. One of these genes is hibris (hbs), which encodes a member of the immunoglobulin superfamily in Drosophila. Phenotypic analyses and genetic interaction assays suggest that it may have a role during PCP establishment, interacting with both Egfr and Fz/PCP signaling during this process. Taken together, our results indicate that Mtl is functionally related to the Egfr pathway regulating ommatidial rotation during PCP establishment in the eye, being a positive regulator of this pathway. Since Egfr signaling is linked to cytoskeletal and cell junctional elements, it is likely that Mtl may be regulating cytoskeleton dynamics and thus cell adhesion during ommatidial rotation in the context of that pathway.     

Control of planar cell polarity by interaction of DWnt4 and four-jointed

The Drosophila eye and the wing display specific planar cell polarity. Although Frizzled (Fz) signaling has been implicated in the establishment of ommatidial and wing hair polarity, evidence for the Wnt gene function has been limited. Here we examined the function of a Drosophila homolog of Wnt4 (DWnt4) in the control of planar polarity. We show that DWnt4 mRNA and protein are preferentially expressed in the ventral region of eye disc. DWnt4 mutant eyes show polarity reversals mostly in the ventral domain, consistent with the ventral expression of DWnt4. Ectopic expression of DWnt4 in the dorsoventral (DV) polar margins is insufficient to induce ommatidial polarity but becomes inductive when coexpressed with Four-jointed (Fj). Similarly, DWnt4 and Fj result in synergistic induction of hair polarity toward the source of expression in the wing. Consistent with genetic interaction, we provide evidence for direct interaction of DWnt4 and Fj transmembrane protein. The extracellular domain of Fj is required for direct binding to DWnt4 and for the induction of hair polarity. In contrast to the synergy between DWnt4 and Fj, DWnt4 antagonizes the polarizing effect of Fz. Our results suggest that DWnt4 is involved in ommatidial polarity signaling in the ventral region of the eye and its function is mediated by interacting with Fj.     

Asymmetric colocalization of Flamingo, a seven-pass transmembrane cadherin, and Dishevelled in planar cell polarization

The Drosophila wing provides an appropriate model system for studying genetic programming of planar cell polarity (PCP) [1-4]. Each wing cell respects the proximodistal (PD) axis; i.e., it localizes an assembly of actin bundles to its distalmost vertex and produces a single prehair. This PD polarization requires the redistribution of Flamingo (Fmi), a seven-pass transmembrane cadherin, to proximal/distal cell boundaries; otherwise, the cell mislocalizes the prehair [5]. Achievement of the biased Fmi pattern depends on two upstream components in the PCP signaling pathway: Frizzled (Fz), a receptor for a hypothetical polarity signal, and an intracellular protein, Dishevelled (Dsh) [6-8]. Here, we visualized endogenous Dsh in the developing wing. A portion of Dsh colocalized with Fmi, and the distributions of both proteins were interdependent. Furthermore, Fz controlled the association of Dsh with cell boundaries, which association was correlated with the presence of hyperphosphorylated forms of Dsh. Our results, together with a recent study on Fz distribution [9], support the possibility that Fz, Dsh, and Fmi constitute a signaling complex and that its restricted localization directs cytoskeletal reorganization only at the distal cell edge.     

The prickle-related gene in vertebrates is essential for gastrulation cell movements

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis [1,2]. Recently, it was reported that a noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila [3,4], participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo [5-8]. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal [6-8]. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK [9,10] and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, we show that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene [11-13], is an essential component for gastrulation cell movement. Both gain-of-function and loss-of-function of Xpk severely perturbed gastrulation and caused spina bifida embryos without affecting mesodermal differentiation. We also demonstrate that XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation.     

Oroshigane, a New Segment Polarity Gene of Drosophila Melanogaster, Functions in Hedgehog Signal Transduction

Here we describe a new segment polarity gene of Drosophila melanogaster, oroshigane (oro). Identified as a dominant enhancer of Bar (B), oro is also recessive embryonic lethal, and homozygous oro embryos show variable substitution of naked cuticle with denticles. These patterns are distinctly similar to those of hedgehog (hh) and wingless (wg) embryos, which indicates that oro functions in determining embryonic segment polarity. Evidence that oro function is involved in Hh signal transduction during embryogenesis is provided by its genetic interactions with the segment polarity genes patched (ptc) and fused (fu). Furthermore, ptc(IN) is a dominant suppressor of the oro embryonic lethal phenotype, suggesting a close and dose-dependent relationship between oro and ptc in Hh signal transduction. oro function is also required in imaginal development. The oro(1) allele significantly reduces decapentaplegic (dpp), but not hh, expression in the eye imaginal disc. Furthermore, oro enhances the fu(1) wing phenotype in a dominant manner. Based upon the interactions of oro with hh, ptc, and fu, we propose that the oro gene plays important roles in Hh signal transduction.     

JAK／STAT signaling regulates tissue outgrowth and male germline stem cell fate in Drosophila

In multicellular organisms, biological activities are regulated by cell signaling. The various signal transduction pathways regulate cell fate, proliferation, migration, and polarity. Miscoordination of the communicative signals will lead to disasters like cancer and other fatal diseases. The JAK/STAT signal transduction pathway is one of the pathways, which was first identified in vertebrates and is highly conserved throughout evolution. Studying the JAK/STAT signal transduction pathway in Drosophila provides an excellent opportunity to understand the molecular mechanism of the cell regulation during development and tumor formation. In this review, we discuss the general overview of JAK/STAT signaling in Drosophila with respect to its functions in the eye development and stem cell fate determination.     

Dishevelled activates Ca2+ flux,PKC, and CamKII in vertebrate embryos

Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt-beta-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt-Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt-beta-catenin and the PCP pathways, its potential involvement in the Wnt-Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.     

Dissecting the PCP pathway: One or more pathways?

Planar cell polarity (PCP), the alignment of cells within 2D tissue planes, involves a set of core molecular regulators highly conserved between animals and cell types. These include the transmembrane proteins Frizzled (Fz) and VanGogh and the cytoplasmic regulators Dishevelled (Dsh) and Prickle. It is widely accepted that this core forms part of a 'PCP pathway' for signal transduction, which can affect cell morphology through activation of an evolutionary ancient regulatory module involving Rho family GTPases and Myosin II, and/or the JNK kinase cascade. We have re-examined the evidence for interactions between the proposed PCP pathway components, and question the placing of the cell morphology regulators in the same pathway as the PCP core. While Fz and Dsh are clearly involved in both PCP and Rho-based cell morphology regulation, available evidence cannot currently discriminate whether these processes are linked mechanistically by a shared Fz/Dsh population, or pass by two distinct pathways.     

Stage-dependent Dishevelled-1 expression during mouse spermatogenesis suggests a role in regulating spermatid morphological changes

Dishevelled (Dsh in Drosophila or DVL in mice) is a member of the highly conserved Wg/Wnt signaling pathway, which regulates important processes such as cell proliferation, polarity, and specification of cell fate. Three orthologous genes of Dishevelled (Dvl-1, Dvl-2, and Dvl-3) have been found in both humans and mice. They play pivotal roles in regulating cell morphology and a variety of changes in cell behaviors. In the present study, we show that the expression of Dvl-1 is stage-dependent during mouse spermatogenesis, although Dvl-2 and Dvl-3 show relative consistent expression. The expression of Dvl-1 mRNA first appears in pachytene spermatocytes, increases in round and elongating spermatids, and then turns to an undetectable level in mature sperm cells. Analyses of immunohistochemistry and immunofluorescence staining show that DVL-1 is present diffusely in the cytoplasm of pachytene spermatocytes and exhibits mainly a vesicular pattern and perinuclear distribution and a weak diffusely cytoplasmic signal in round and elongating spermatids. The vesicular pattern of DVL-1 has been observed by previous studies in somatic cells, and suggested to play roles in signal transduction. Immunoprecipitation experiments show that DVL-1 coimmunprecipitates with spermatogenic cells beta-actin rather than alpha-tubulin. These results indicate that DVL-1 may be involved in spermatid morphological changes during mouse spermiogenesis through mediating signal transduction and/or regulating actin cytoskeleton organization.     

Frizzled-Dishevelled signaling specificity outcome can be modulated by Diego in Drosophila

Members of the Frizzled (Fz) family of seven-pass transmembrane receptors are required for the transduction of both Wnt-Fz/beta-catenin and Fz/planar cell polarity (PCP) signals. Although both pathways transduce signals via interactions between Fz and the cytoplasmic protein Dishevelled (Dsh), each pathway has specific and distinct effectors. One explanation for the pathway specificity is that signal-induced conformational changes result in unique Fz-Dsh interactions. Our mutational analyses of Fz-Dsh activities in vivo do however not support this model, since both pathways are affected by all mutations tested. Alternatively, the interaction of Fz or Dsh with other proteins could modulate the signaling outcome. We examined the role of a Dsh-binding PCP molecule, Diego (Dgo), in both Wnt-Fz/beta-catenin and Fz/PCP signaling. Both loss-of-function and gain-of-function results suggest that Dgo promotes Fz-Dsh/PCP signaling at the expense of Wnt-Fz/beta-catenin signaling. Our data suggest that Dgo sequesters Dsh to a functionally distinct Fz/PCP signaling compartment within the cell.