Interactive Fly, Drosophila



Table of contents

Dictyostelium Shaggy homologs

Early during Dictyostelium development a fundamental cell-fate decision establishes the anteroposterior (prestalk/prespore) axis. Signaling via the 7-transmembrane cAMP receptor CAR4 is essential for creating and maintaining a normal pattern; car4-null alleles produce decreased levels of prestalk-specific mRNAs but show enhanced expression of prespore genes. car4- cells produce all of the signals required for prestalk differentiation but lack an extracellular factor necessary for prespore differentiation of wild-type cells. This secreted factor decreases the sensitivity of prespore cells to inhibition by the prestalk morphogen DIF-1. At the cell autonomous level, CAR4 is linked to intracellular circuits that activate prestalk but inhibit prespore differentiation. The autonomous action of CAR4 is antagonistic to the positive intracellular signals mediated by one or more other cAMP receptor, CAR1 and/or CAR3. These CAR-mediated pathways converge at the serine/threonine protein kinase GSK3, suggesting that the anterior (prestalk)/posterior (prespore) axis of Dictyostelium is regulated by the GSK3 pathway. GSK3 appears to be CAR regulated. gskA- cells (GSK3 null cells) have a cell phenotype that is substantially the opposite of the car4 null cells. CAR4 promotes prestalk differentiation and inhibits prespore differentiation by decreasing GSK3 activity. In context with the model for CAR control of prestalk and prespore differentiation, CAR4 and CAR1/CAR3 may be antagonistic and epistatic to GSK3 (Ginsburg, 1997).

Aardvark (Aar) is a Dictyostelium ß-catenin homolog with both cytoskeletal and signal transduction roles during development. Loss of aar causes a novel phenotype where multiple stalks appear during late development. Ectopic stalks are preceded by misexpression of the stalk marker ST-lacZ in the surrounding tissue. This process does not involve the kinase GSK-3. Mixing experiments show that ectopic ST-lacZ expression and stalk formation are cell non-autonomous. The protein-cellulose matrix surrounding the stalk of aar mutant fruiting bodies is defective, and damage to the stalk of wild-type fruiting bodies leads to ectopic ST-lacZ expression. It is postulated that poor synthesis of the stalk tube matrix allows diffusion of a stalk cell-inducing factor into the surrounding tissue (Coates, 2002).

Glycogen synthase kinase-3 (GSK-3) is required during metazoan development to mediate the effects of the extracellular signal wingless/Wnt-1 and hence is necessary for correct cell type specification. GSK-3 also regulates cell fate during Dictyostelium development, but in this case it appears to mediate the effects of extracellular cAMP. By direct measurement of GSK-3 kinase activity during Dictyostelium development, it has been found that there is a rise in activity at the initiation of multicellular development that can be induced by cAMP. The timing of the rise correlates with the requirement for the Dictyostelium homolog of GSK-3 (GSKA) to specify cell fate. Loss of the cAMP receptor cAR3 almost completely abolishes the rise in kinase activity and causes a mis-specification of cell fate that is equivalent to that seen in a gskA- mutant. The phenotype of a cAR3(-) mutant however is less severe than loss of gskA and ultimately gives rise to an apparently wild-type fruiting body. These results indicate that in Dictyostelium extracellular cAMP acts via cAR3 to cause a rise in GSKA kinase activity, which regulates cell type patterning during the initial stages of multicellularity. These findings contrast to those found for Wnt-1 signaling; here genetic evidence suggests that Wnt-1 stimulation leads to a decrease in kinase activity. This difference could mean the Dictyostelium and metaxoan GSK-3 proteins are regulated by different signaling pathways. A more interesting prospect is that these differences, reflect different aspects of a conserved regulatory network. There is no evidence in metazoa to preclude the exisence of an extracellular signal that increases GSK-3 activity (Plyte, 1999).

Inhibition of GSK3 by 7-TM Wnt/wg receptor signaling is critical for specifying embryonic cell fate patterns. In Dictyostelium, the 7-TM cAMP receptors regulate GSK3 by parallel, antagonistic pathways to establish a developmental body plan. A novel tyrosine kinase, ZAK1, downstream of 7-TM cAMP receptor signaling is described that is required for GSK3 activation during development. zak1-nulls have reduced GSK3 activity and are defective in GSK3-regulated developmental pathways. Moreover, recombinant ZAK1 phosphorylates and activates GSK3 in vitro. It is proposed that ZAK1 is a positive regulator of GSK3 activity required for cell pattern formation in Dictyostelium. It is speculated that similar mechanisms exist to antagonize Wnt/wg signaling for metazoan cell fate specification (Kim, 1999).

As the first identified tyrosine kinase required for cell fate determination in the facultative multicellular eukaryote Dictyostelium, ZAK1 function emphasizes the significance of phosphotyrosine signaling at the border of metazoan differentiation. While its tandem, dual kinase architecture is reminiscent of the JAKs, apart from their kinase domains, the proteins share no structural features. Like the JAKs, the C-terminal (DI) kinase of ZAK1 is a true protein tyrosine kinase (PTK) that has both auto- and transphosphorylation activity, but, unlike the pseudokinase character of the N-terminal (DII) kinase of the JAKs, the ZAK1 equivalent is a protein serine kinase (PSK) with autocatalytic activity, however limited. The in vitro data also argue that the DII PSK domain may be regulatory for the DI PTK activity, and it is interesting to speculate that the in vivo mechanism for PTK activation of ZAK1 during development may, in part, serve to repress an inhibitory role of the DII PSK (Kim, 1999).

ZAK1 function as an activating GSK3 kinase may yet provide novel insight into conserved regulatory mechanisms that establish embryonic body plans in diverse metazoa. The morphogen-mediated activation of ZAK1 leading to the phosphorylation and activation of GSK3 defines a novel pathway that regulates a component essential for Dictyostelium development, Wnt/wg 7-TM receptor signaling, and tumor suppression, although an activating pathway for GSK3 contrasts with the current paradigm for a constitutively active enzyme under negative control by Wnt/wg signaling. While active GSK3 in concert with APC, slimb/TrCP (see supernumerary limbs), and axin/conductin is suggested to destabilize cytosolic beta-catenin protein levels, thereby inhibiting beta-catenin-dependent gene expression, it must also be emphasized that not all Wnt cell specification pathways can be explained directly through inhibition of GSK3 and activation of beta-catenin-dependent pathways. This report provides evidence for an activation pathway for GSK3 in the context of receptor stimulation and cell fate patterning in Dictyostelium. It will be significant to determine whether ZAK1 orthologs or functionally equivalent PTKs induce the activation of GSK3 to regulate cell fate specification and tumor suppression in other organisms, perhaps by antagonizing Wnt/wg signaling (Kim, 1999).

Asymmetric body axis formation is central to metazoan development. Dictyostelium establishes an anterior/posterior axis utilizing seven-transmembrane cAMP morphogen receptors (CARs) and GSK3-mediated signal transductions that has a parallel with metazoan Wnt/Frizzled-GSK3 pathways. In Dictyostelium, GSK3 promotes posterior cell patterning but inhibits anterior cell differentiation. Tyrosine kinase ZAK1 mediates GSK3 activation. CAR4 regulates a tyrosine phosphatase that inhibits GSK3 activity. Essential phosphotyrosines have been identified in GSK3, confirming their role in activated/deactivated regulation and cell fate decisions, and they have been related to the predicted 3D structure of GSK3ß. CARs differentially regulate GSK3 activity by selectively activating a tyrosine phosphatase or kinase for pattern formation. The findings may provide a comparative understanding of CAR-GSK3 and Wnt/Frizzled-GSK3 pathways (Kim, 2002).

It is argued that the differential regulation of tyrosine phosphorylation of GSK3 by ZAK1 and a PTPase constitutes the core of regulatory machinery on GSK3 in the context of cell fate decision by the seven-transmembrane CARs. It is suggested that similar mechanistic activities may function during metazoan development. The precise conservation of the essential tyrosines between Dictyostelium and mammalian GSK3 supports the potential for a common regulatory network controlling GSK3 activity. Although CARs and Frizzled (Fz) seven-transmembrane receptors are activated by different ligands, cAMP and Wnt, respectively, and Fz had been considered a strictly metazoan receptor, the resemblance of their signaling pathways converging at GSK3 and ß-catenin for regulating cell fate decisions suggests an ancient origin for this essential pathway (Kim, 2002).

In Drosophila and vertebrates, canonical Wnt/Fz signaling is inhibitory to GSK3 function, in effective parallel with CAR4. Dominant-negative GSK3 redirects cell fate choice decision in Xenopus, as well as in Dictyostelium. In Xenopus, KI-GSK3 is suggested to compete for association of endogenous GSK3 with Axin-based scaffolding complexes. Potentially, such complexes are also essential for GSK3 signaling in Dictyostelium. CAR3-mediated activation of GSK3 finds equivalence in C. elegans, where the Fz receptor Mom-5 relays an activating Wnt (Mom-2) signal to GSK3 (Kim, 2002).

It will be very critical to determine whether cell fate decisions in these other systems are also regulated by differential tyrosine phosphorylation of GSK3. Although, to date, C. elegans has the only other positive GSK3 cascade yet identified, several mammalian tyrosine kinases, such as Fyn and Pyk2, are suggested to activate GSK3, raising the possibility that tyrosine phosphorylation of GSK3 may mediate antagonism to Wnt signaling to provide additional regulation for differentiation and tumor suppression and that an activated PTPase may be functionally equivalent to Wnt signaling to promote tumorigenesis (Kim, 2002).

Plant Shaggy homologs

Most signal transduction pathways central to development are not shared by plants and animals. Such is the case of the Wingless/Wnt signaling pathway, whose components play key roles in metazoan pattern formation and tumorigenesis, but are absent in plants, with the exception of SHAGGY/GSK3, a cytoplasmic protein kinase represented in the genome of Arabidopsis thaliana by a family of 10 AtSK genes for which mutational evidence is scarce. Mutant alleles of the Arabidopsis ULTRACURVATA1 (UCU1) gene have been characterized. The two strongest alleles dramatically reduce cell expansion along the proximodistal axis, dwarfing the mutant plants, whose cells expand properly across but not along most organs. Proximodistal expansion of adaxial (dorsal) and abaxial (ventral) leaf cells exhibits a differential dependence on UCU1 function, as suggested by the leaves of ucu1 mutants, which are rolled spirally downward in a circinate manner. The UCU1 gene, which encodes an AtSK protein involved in the cross-talk between auxin and brassinosteroid signaling pathways, as indicated by the responses of ucu1 mutants to plant hormones and the phenotypes of double mutants involving ucu1 alleles, has been positionally cloned (Pérez-Pérez, 2002).

Shaggy homologs in C. elegans

In a four-cell-stage Caenorhabditis elegans embryo, Wnt signaling polarizes an endoderm precursor called EMS. The polarization of this cell orients its mitotic spindle in addition to inducing endodermal fate in one daughter cell. Reducing the function of Wnt pathway genes, including a newly identified GSK-3beta homolog called gsk-3, disrupts endoderm induction, whereas only a subset of these genes (mom-1/porcupine, mom-5/frizzled and the Wnt pathway component gsk-3) are required to orient the EMS mitotic spindle. mom-1/porcupine (see Drosophila porcupine) is required specifically in the signaling cell P2. Wnt pathway genes thought to act downstream of gsk-3, including the Armadillo and APC homologs, appear not to be required for spindle orientation, suggesting that gsk-3 represents a branch point in the control of endoderm induction and spindle orientation. Orientation of the mitotic spindle does not require gene transcription in EMS, suggesting that Wnt signaling may directly target the cytoskeleton in a responding cell (Schlesinger, 1999).

Because rotational positioning of the EMS centrosomes appears to not require gene transcription, it is suggested that P2 to EMS signaling directly targets the cytoskeleton in EMS. Microtubules are the primary structural component of the mitotic spindle and are thought to interact with cortical microfilaments during rotation of the nucleus/centrosome complex in EMS. Either microtubules or microfilaments could be targets of Wnt signaling, as both are required for correct orientation of the mitotic spindle in EMS. Wnt pathway genes have been implicated in cytoskeleton-related processes in other systems. Mammalian GSK-3beta can phosphorylate the microtubule-associated protein tau, and this activity is regulated by Dishevelled, another Wnt pathway component (Wagner, 1997). Therefore P2 signaling might influence microtubule dynamics directly through gsk-3. Alternatively, P2 signaling might influence spindle orientation in EMS by locally activating microtubule motor complexes that directly interact with astral microtubules to orient the nucleus/centrosome complex before cell division. It seems likely that Wnt signaling directly targets the cytoskeleton to control other developmental processes. For example, the polarized organization of hair cells in the Drosophila epithelium appears to be controlled by the orientation of actin microfilaments, which in turn are regulated by tissue polarity genes including fz and dsh. Wnt signaling in C. elegans may also target the cytoskeleton to polarize endoderm potential in EMS, as the polarization of endoderm potential appears to require functional microfilaments and microtubules. Alternatively, P2 signaling may simply cause a localized activation of downstream Wnt pathway components, resulting in an asymmetric segregation of their activity to the daughters of EMS. In conclusion, it is emphasized that some developmental signals may influence cell fate by directly targeting cytoplasmic components in a responding cell and only indirectly regulate gene transcription. As the Wnt pathway is widely conserved, it will be interesting to learn if direct targeting of the cytoskeleton in cells responding to Wnt signaling proves to be of general importance during animal development (Schlesinger, 1999 and references).

The endoderm and much of the mesoderm arise from the EMS cell in the four-cell C. elegans embryo. MED-1 and -2 GATA factors specify the entire fate of EMS, which otherwise produces two C-like mesectodermal progenitors. The meds are direct targets of the maternal SKN-1 transcription factor; however, their forced expression can direct SKN-1-independent reprogramming of non-EMS cells into mesendodermal progenitors. SGG-1/GSK-3beta kinase acts both as a Wnt-dependent activator of endoderm in EMS and an apparently Wnt-independent repressor of the meds in the C lineage, indicating a dual role for this kinase in mesendoderm development. These results suggest that a broad tissue territory, mesendoderm, in vertebrates has been confined to a single cell in nematodes through a common gene regulatory network (Maduro, 2001).

In vertebrates, a broad set of cell types is generated from a single tissue territory, mesendoderm. The mesendoderm becomes subdivided into endoderm and a subset of the mesoderm ('splanchnopleural') that generates heart and blood. Similarly, in C. elegans, a broad set of cell types is generated from a single cell, EMS. The EMS lineage becomes subdivided into endoderm (E cell) and a subset of mesoderm (MS cell) that generates part of the heart-like pharynx and coelomocytes (putative primitive blood cells). Both zebrafish and C. elegans first express a GATA factor (Faust/GATA5 in zebrafish; MED-1,2 in C. elegans) throughout the mesendoderm prior to gastrulation that is sufficient to direct mesendoderm development in nonmesendodermal cells. In both C. elegans and vertebrates, a set of conserved regulators acts after each germ layer type has been segregated from the mesendoderm. These include HNF-3-like and HNF-4-like factors in the endoderm and the cardiac/pharynx-promoting Nkx2.5/CEH-22 factors in the mesoderm. These observations suggest that a conserved gene regulatory network may underly mesendoderm specification in all triploblastic metazoans. The generation of a common progenitor of endoderm and a subset of the mesoderm may reflect a decisive event in metazoan evolution that has been preserved in both a large group of cells in vertebrates and in a single cell in C. elegans (Maduro, 2001).

Axin, APC, and the kinase GSK3ß are part of a destruction complex that regulates the stability of the Wnt pathway effector ß-catenin. In C. elegans, several Wnt-controlled developmental processes have been described, but an Axin ortholog has not been found in the genome sequence and SGG-1/GSK3ß, and the APC-related protein APR-1 have been shown to act in a positive, rather than negative fashion in Wnt signaling. EGL-20/Wnt-dependent expression of the homeobox gene mab-5 in the Q neuroblast lineage requires BAR-1/ß-catenin and POP-1/Tcf. How BAR-1 is regulated by the EGL-20 pathway has been investigated. First, a negative regulator of the EGL-20 pathway, pry-1, has been characterized. pry-1 encodes an RGS and DIX domain-containing protein that is distantly related to Axin/Conductin. These results demonstrate that despite its sequence divergence, PRY-1 is a functional Axin homolog. PRY-1 interacts with BAR-1, SGG-1, and APR-1 and overexpression of PRY-1 inhibits mab-5 expression. Furthermore, pry-1 rescues the zebrafish axin1 mutation masterblind, showing that PRY-1 can functionally interact with vertebrate destruction complex components. Finally, SGG-1, in addition to its positive regulatory role in early embryonic Wnt signaling, may function as a negative regulator of the EGL-20 pathway. It is concluded that a highly divergent destruction complex consisting of PRY-1, SGG-1, and APR-1 regulates BAR-1/ß-catenin signaling in C. elegans (Korswagen, 2002).

In early C. elegans embryos, signaling between a posterior blastomere, P2, and a ventral blastomere, EMS, specifies endoderm and orients the division axis of the EMS cell. Although Wnt signaling contributes to this polarizing interaction, no mutants identified to date abolish P2/EMS signaling. Two tyrosine kinase-related genes, src-1 and mes-1, are required for the accumulation of phosphotyrosine between P2 and EMS. Moreover, src-1 and mes-1 mutants strongly enhance endoderm and EMS spindle rotation defects associated with Wnt pathway mutants. SRC-1 and MES-1 signal bidirectionally to control cell fate and division orientation in both EMS and P2. These findings suggest that Wnt and Src signaling function in parallel to control developmental outcomes within a single responding cell (Bei, 2002).

The mes-1 gene encodes a probable transmembrane protein with overall structural similarity to receptor tyrosine kinase and is a factor required for proper asymmetry and cell fate specification in embryonic germlineage. Null mutations in mes-1 cause a maternal-effect sterile phenotype in which the progeny of homozygous mothers are viable but mature without germcells. Most cell types are specified properly in mes-1 sterile animals, but the germline cell named P4 adopts the fate of its sister cell, a muscle precursor, called D and produces ectopic muscle at the expense of the germline. Interestingly, MES-1 protein is localized intensely at the contact site between the germline blastomere and intestinal precursor at each early developmental stage, starting from the four-cell stage where MES-1 is localized at the contact site between P2 and EMS. An intense phosphotyrosine signal that depends on mes-1(+) activity is correlated with MES-1 protein localization. MES-1 is required in both P2 and EMS and appears to act through a second gene, src-1, a homolog of the vertebrate protooncogene c-Srcpp60. A probable null mutant of src-1 is described that exhibits a fully penetrant maternal-effect embryonic lethal phenotype. The src-1 and mes-1 mutants exhibit similar germline defects and have a nearly identical set of genetic interactions with Wnt/Wg pathway components (Bei, 2002).

Double mutants between mes-1 or src-1 and each of several Wnt/Wg signaling components exhibit a complete loss of P2/EMS signaling, including a loss of the A/P division orientation in the EMS cell. Synergy was observed between mes-1 or src-1 mutants and each of the following previously described mutants: mom-1 (Porcupine), mom-2 (Wnt/Wg), mom-5 (Frizzled), sgg-1 (GSK-3), and mom-3 (uncloned). In addition, identical synergies were observed in the phenotypes of embryos produced by mes-1 or src-1 homozygotes after injection with a mixture of two double-stranded RNAs targeting the C. elegans Disheveled homologs dsh-2 and mig-5. RNAi targeting these Disheveled homologs individually does not induce visible defects in P2/EMS signaling. mes-1 functions in both EMS and P2 to direct MES-1 protein localization at EMS/P2 junction and to specify A/P cleavage orientation in the EMS cell, while src-1 is required cell autonomously in EMS for the induction of the EMS A/P division axis. These findings suggest that a homotypic interaction between MES-1-expressing cells, P2 and EMS, induces a SRC-1-mediated phosphotyrosine signaling pathway that functions in parallel with Wnt/Wg signaling to specify endoderm and to orient the division axis of EMS in early C. elegans embryos (Bei, 2002).

Recent work on dorsal closure in Drosophila has identified a possible convergence between Src and Wnt signaling at the level of regulation of the Jun N-terminal kinase (JNK). Dorsal closure is the process in which epithelial sheets spread over and enclose the dorsal region of the Drosophila embryo during morphogenesis. JNK signaling is essential for dorsal closure and mutants lacking JNK exhibit a dorsal-open phenotype and also exhibit loss of expression of a TGF-ß homolog decapentaplegic in the epithelial cells that lead the closure process. Recent genetic studies have implicated both Src-like kinases and Wnt signaling components in the dorsal closure process and in regulating the expression of dpp. Mutations in Src42A and Wnt signaling factors produce dorsal closure phenotypes similar to JNK mutants and activation of JNK signaling can partially suppress defects caused by these mutants. These findings suggest that Wnt and Src may converge to regulate JNK activity and dorsal closure in Drosophila and thus provide evidence from another system for interactions between these pathways in a developmental process (Bei, 2002).

At the onset of embryogenesis, key developmental regulators called determinants are activated asymmetrically to specify the body axes and tissue layers. In C. elegans, this process is regulated in part by a conserved family of CCCH-type zinc finger proteins that specify the fates of early embryonic cells. The asymmetric localization of these and other determinants is regulated in early embryos through motor-dependent physical translocation as well as selective proteolysis. This study shows that the CCCH-type zinc finger protein OMA-1 serves as a nexus for signals that regulate the transition from oogenesis to embryogenesis. While OMA-1 promotes oocyte maturation during meiosis, destruction of OMA-1 is needed during the first cell division for the initiation of ZIF-1-dependent proteolysis of cell-fate determinants. Mutations in four conserved protein kinase genes—mbk-2/Dyrk, kin-19/CK1α, gsk-3, and cdk-1/CDC2—cause stabilization of OMA-1 protein, and their phenotypes are partially suppressed by an oma-1 loss-of-function mutation. OMA-1 proteolysis also depends on Cyclin B3 and on a ZIF-1-independent CUL-2-based E3 ubiquitin ligase complex, as well as the CUL-2-interacting protein ZYG-11 and the Skp1-related proteins SKR-1 and SKR-2. These findings suggest that a CDK1/Cyclin B3-dependent activity links OMA-1 proteolysis to completion of the first cell cycle and support a model in which OMA-1 functions to prevent the premature activation of cell-fate determinants until after they are asymmetrically partitioned during the first mitosis (Shirayama, 2006).

Multiple Wnt signaling pathways converge to orient the mitotic spindle in early C. elegans embryos: GSK-3 functions downstream of three Dishevelled homologs

How cells integrate the input of multiple polarizing signals during division is poorly understood. Two distinct C. elegans Wnt pathways contribute to the polarization of the ABar blastomere by differentially regulating its duplicated centrosomes. Contact with the C blastomere orients the ABar spindle through a nontranscriptional Wnt spindle alignment pathway, while a Wnt/β-catenin pathway controls the timing of ABar spindle rotation. The three C. elegans Dishevelled homologs contribute to these processes in different ways, suggesting that functional distinctions may exist among them. CKI (KIN-19) plays a role not only in the Wnt/β-catenin pathway, but also in the Wnt spindle orientation pathway as well. Based on these findings, a model is established for the coordination of cell-cell interactions and distinct Wnt signaling pathways that ensures the robust timing and orientation of spindle rotation during a developmentally regulated cell division event (Walston, 2004).

During development, certain cell divisions must occur with a specific orientation to form complex structures and body plans. In many cases, the polarizing input for oriented divisions involves Wnt signaling. One example of such division involves neuroblasts in Drosophila, in which the first division of the pI sensory organ precursor cell is under the control of Frizzled (Fz) and Dishevelled (Dsh). The orientation of blastomere divisions in the early C. elegans embryo has also been shown to require Wnt signaling. In the 4-cell embryo, the EMS blastomere is induced by its posterior neighbor, the P2 blastomere. This induction has two consequences: it specifies the fates of EMS daughter cells and properly positions the mitotic spindle of EMS. Although both processes are under the control of Wnt signaling, they are controlled through divergent pathways. When EMS divides, the anterior daughter, MS, gives rise to progeny that are primarily mesodermal, and the posterior daughter, E, produces all of the endoderm. The fates of MS and E are controlled in part by a Wnt signaling pathway that regulates the activity of the Tcf/Lef transcription factor, POP-1, in conjunction with the β-catenin WRM-1. WRM-1 interacts with POP-1 through a cofactor, LIT-1, a NEMO-like kinase that is activated through a parallel mitogen-activated protein kinase (MAPK) pathway. Pathways that utilize a β-catenin to alter transcription are referred to as Wnt/β-catenin pathways. Removal of some components of the Wnt/β-catenin pathway alters the fates of the two EMS daughters. Although the fate of the EMS daughters is controlled by a Wnt/β-catenin pathway, the orientation of the EMS division is controlled by a different Wnt pathway (Walston, 2004).

In wild-type embryos, the EMS spindle initially aligns along the left/right (L/R) axis and rotates to adopt an anterior/posterior (A/P) orientation during the initial stages of mitosis. In embryos that lack the function of certain Wnt signaling components, the EMS spindle often sets up in the proper orientation but fails to rotate along the A/P axis until the onset of anaphase. In some cases, the delayed spindle rotates dorsoventrally (D/V) before it adopts the proper A/P alignment. The Wnt spindle orientation pathway that controls EMS orientation involves a Wnt (MOM-2), Porcupine (Porc; MOM-1), and Fz (MOM-5). GSK-3, the C. elegans GSK-3β homolog, has been reported to act positively downstream of the Fz receptor to regulate EMS spindle positioning, rather than as a downregulator of β-catenin accumulation as observed with Wnt/β-catenin signaling. Indeed, Wnt/β-catenin signaling components downstream of GSK-3 are not involved in controlling EMS spindle alignment, and EMS spindle alignment occurs independently of gene transcription. Pathways such as the one that positions the spindle in EMS, which utilize GSK-3 but are independent of transcription, are referred to as Wnt spindle orientation pathways (Walston, 2004).

Although many Wnt signaling components have been identified that participate in spindle orientation, the role of the Dsh family has not been clearly characterized. The Dsh family proteins transmit Wnt signals received from Fz receptors. The Dshs use three domains (DIX, PDZ, and DEP) to interact with different downstream proteins and activate multiple Wnt pathways specifically. The C. elegans genome contains three Dsh family genes that possess the three conserved domains: dsh-1, dsh-2, and mig-5. Transcripts of dsh-2 and mig-5 are at similar, enriched levels in the 4- and 8-cell embryo based on microarray analysis, while dsh-1 levels are low (Walston, 2004).

Another molecule involved in Wnt signaling is Casein Kinase I (CKI). CKI has been shown to prime β-catenin for degradation by phosphorylating it at a specific serine residue. Once primed, the β-catenin can be further phosphorylated and targeted for destruction by GSK-3β. CKI has also been shown to bind and phosphorylate Dsh and may assist in inhibiting GSK-3β when Wnt signaling is active. Loss of function of the CKIα homolog, kin-19, causes defects in the fate of EMS daughter cells. Although the role of CKI in spindle alignment has not been examined, CKIα localizes to centrosomes and mitotic spindles in vertebrate systems (Walston, 2004).

A pathway involving MES-1, a receptor tyrosine kinase, and SRC-1, a Src family tyrosine kinase, acts redundantly with Wnt signaling with respect to the fate of EMS daughters and the orientation of the EMS spindle. When a Src pathway member and a member of the Wnt spindle orientation pathway are removed simultaneously, the EMS spindle fails to rotate into the proper A/P position prior to division and remains misaligned throughout division. Removal of Src pathway members also enhances endoderm fate specification defects observed following removal of Wnt/β-catenin pathway members. Spindle orientation defects in dsh-2(RNAi);mig-5(RNAi) embryos have not been reported unless the Src pathway is also removed; however, only defects in cell division orientation have been reported, as opposed to abnormalities in initial spindle positioning (Walston, 2004).

In addition to regulating the orientation of the EMS division, four of the mom (more mesoderm) genes, mom-1 (Porc), mom-2 (Wnt), mom-5 (Fz), and mom-3 (uncloned), cause spindle alignment defects in the ABar blastomere of the 8-cell embryo. Three of the four AB granddaughters, ABal, ABpl, and ABpr, divide with spindle orientations that are parallel to one another. ABar divides in an orientation that is roughly perpendicular to the other three, an event best viewed from the right side of the embryo, placing anterior to the right. When the function of one of the above mom genes is removed, ABar divides parallel to the other AB granddaughters, resulting in mispositioning of its daughter cells, such that ABarp, the wild-type posterior daughter cell, adopts a position that is anterior to its sister, ABara. The source of the polarizing cue(s) that orients the division of ABar is unclear. However, using blastomere isolations, it has been demonstrated that C, MS, and E are all competent to align the spindle and generate asymmetric expression of POP-1 within unidentified, dividing AB granddaughters, suggesting that one or more of these cells could produce signals that orient the division of ABar in vitro (Walston, 2004).

In this study, the roles of two Wnt signaling pathways involved in regulating the mitotic spindle are demonstrated. (1) The nontranscriptional Wnt spindle alignment pathway requires contact from the C blastomere to align the spindle of ABar. The three Dshs differentially participate in aligning the spindles of EMS and ABar and vary with respect to their interaction with the Src signaling pathway during spindle orientation. Moreover, while KIN-19 participates in endoderm induction through the Wnt/β-catenin pathway, it also acts in the Wnt spindle orientation pathway. (2) A Wnt/β-catenin pathway regulates the timing of spindle rotation in ABar, presumably by specifying the fate of neighboring blastomeres. Taken together, these studies indicate that spindle orientation during early development is a tightly regulated event, influenced by multiple cues transmitted via redundant pathways (Walston, 2004).

Wnt signals in the early embryo are transmitted from P2 to EMS to orient its spindle and to specify the fate of the EMS daughters. The orientation of the spindle relies on Wnt ligands, including MOM-2, that are secreted from P2 and activate MOM-5/Fz on the surface of EMS. This ultimately activates GSK-3, resulting in spindle alignment irrespective of gene transcription or other downstream Wnt/β-catenin components. The current analysis suggests that all three Dsh proteins are upstream of GSK-3 activation. Removal of the function of any of the dshs results in an incorrectly positioned EMS spindle, with varying penetrance. The strongest effect is seen in offspring of dsh-2 mutant mothers, suggesting that DSH-2 is primarily responsible for transducing the signal from MOM-5 to GSK-3 in EMS. Antibody staining shows an enrichment of DSH-2 at the area of cell-cell contact between EMS and P2, consistent with a MOM-2/Wnt signal activating DSH-2 at the cell cortex through the MOM-5/Fz receptor (Walston, 2004).

This analysis also shows that kin-19 contributes to the Wnt spindle orientation pathway in both EMS and ABar. Although KIN-19 participates in EMS fate specification, it has not been demonstrated to influence the orientation of the EMS spindle. Depletion of KIN-19 results in spindle misalignment in EMS and ABar. Additionally, KIN-19 localizes to centrosomes during mitosis: this has been shown to be important in establishing the initial polarization axis in the 1-cell embryo. How kin-19 operates within the pathway remains unclear. Because CKI family members have the ability to prime β-catenin for further phosphorylation by GSK-3, KIN-19 may act as a priming kinase for GSK-3-mediated phosphorylation of other unidentified target proteins. Based on the localization of KIN-19, these targets may be linked to the cytoskeleton, thereby affecting the physical alignment of the spindles of EMS and ABar (Walston, 2004).

This analysis shows that the same Wnt spindle orientation pathway that orients the EMS blastomere also aligns the spindle of the ABar blastomere. The results indicate that, as in EMS, this pathway does not require gene transcription to align the ABar spindle and that GSK-3 could be interacting directly or indirectly with the cytoskeleton (Walston, 2004).

All three dsh genes also act redundantly during ABar spindle orientation as well. Surprisingly, the data show that MIG-5 is the Dsh that is most important during ABar spindle orientation, contrary to the case for EMS spindle alignment, where DSH-2 is most important. The ABar spindle defects seen in dsh-2(or302) embryos suggest that DSH-2 also contributes significantly to ABar spindle orientation. DSH-1 seems to play only a minor role, since dsh-1(RNAi) does not result in ABar spindle defects unless performed along with mig-5(RNAi). This combination may remove enough total Dsh protein to prevent ABar from dividing correctly. In contrast, when dsh-1 function is removed in combination with that of dsh-2, the amount of MIG-5 present may be sufficient to maintain the total Dsh protein at a high enough level that the removal of dsh-1 function has no effect. Alternatively, the Dshs may have slightly different functions in regulating spindle orientation (Walston, 2004).

In Wnt signaling mutants, defective EMS spindle orientation is eventually corrected to the proper orientation, which is presumably due to the activity of the parallel src-1 pathway. In contrast, the Src pathway does not rescue spindle defects in ABar, although the src-1 pathway does influence ABar division. At this time, targets of SRC-1 in spindle orientation are unknown. It is possible that one or more of the Dshs are SRC-1 targets; however, the more severe phenotype of src-1 mutants in EMS suggests that other targets are also affected. Interestingly, in EMS and ABar, removal of src-1 function along with the function of either dsh-1 or mig-5 has very little additional effect on spindle polarity; however, when src-1 function is removed in dsh-2(or302) mutants, spindle misalignment is enhanced to nearly complete penetrance in EMS and ABar. Thus, while the three Dsh proteins act partially redundantly, there may be differences in how they impinge on other pathways (Walston, 2004).

In the 8-cell embryo, ABar contacts the C and MS blastomeres. Blastomere isolations have been used to demonstrate that C and MS can orient the spindle of unidentified AB granddaughters. They also demonstrate that AB granddaughters have random spindle orientation when presented with a mom-2 mutant C blastomere, but not with a mom-2 mutant MS blastomere. Using pal-1(RNAi) to alter the fate of C and laser killing of blastomeres to create steric hindrance within the embryo, ABar has been unambiguously identified. These results show that a loss of contact between C and ABar results in misalignment of its spindle in virtually all cases. Thus, contact with C is not only sufficient to align the spindle of an AB granddaughter but is also necessary to properly orient the ABar spindle through the Wnt spindle alignment pathway. These results further suggest that the polarizing activity of C is mediated by MOM-2/Wnt (Walston, 2004).

The orientation of the EMS spindle is not affected when Wnt/β-catenin signaling is abrogated through disruption of transcription or removal of WRM-1/β-catenin or POP-1/Tcf/Lef. In contrast, when wrm-1, lit-1, pop-1, or ama-1 function is removed, the ABar spindle is delayed in rotating into position. All of these treatments are known to affect the differentiation of the progeny of EMS. Moreover, MS has been shown to be capable of orienting the spindle of AB granddaughters in isolated blastomeres independent of MOM-2 function. Given the physical proximity of the blastomeres to ABar in the wild-type embryo, MS may produce a MOM-2-independent signal that ultimately affects positioning of the ABar centrosome further from C. The data further suggest that abnormalities in the fate of EMS daughters result in rotation defects. In wrm-1(RNAi) embryos, both EMS daughters become MS-like, and β-tubulin::GFP analysis reveals that the centrosomes of ABar do not rotate properly in many cases. If a signal that aids orientation of the spindle of ABar is normally secreted by MS, the two MS-like daughter cells specified in wrm-1(RNAi) embryos could produce competing signals that result in spindle rotation defects in ABar. Similarly, when both of the EMS daughters adopt an E-like fate, as in pop-1(RNAi), altered signaling from EMS daughters could again lead to a similar phenotype. In these cases, the centrosomal positioning presumably relies solely on the Wnt signal from C to eventually position the spindle in the correct orientation (Walston, 2004).

In conclusion, spindle orientation in the early C. elegans embryo is regulated through a Wnt spindle alignment pathway involving the Dshs and KIN-19 but independent of gene transcription. In addition, in ABar, the Wnt/β-catenin pathway regulates the timing of spindle rotation in a transcription-dependent manner, presumably indirectly by altering the fates of E and MS. The components of the Wnt spindle orientation pathway downstream of KIN-19 and GSK-3 are unknown; future work should be aimed at identifying these components and determining which Wnts are involved in specific inductive events (Walston, 2004).

Other invertebrate Shaggy homologs

Stabilization of ß-catenin by inhibiting the activity of glycogen synthase kinase-3ß has been shown to initiate axis formation or axial patterning processes in many bilaterians. In hydra, the head organizer is located in the hypostome, the apical portion of the head. Treatment of hydra with alsterpaullone, a specific inhibitor of glycogen synthase kinase-3ß, results in the body column acquiring characteristics of the head organizer, as measured by transplantation experiments, and by the expression of genes associated with the head organizer. Hence, the role of the canonical Wnt pathway for the initiation of axis formation was established early in metazoan evolution (Broun, 2005).

Among the diploblasts (organisms develop from two germ layers), axis formation and axial patterning processes have been extensively investigated in the cnidarian hydra. The hydra polyp consists of a single axis with radial symmetry. The regions along the axis are the head, body column and foot. Because of the tissue dynamics of an adult hydra, the processes governing axial patterning are continuously active. Similarly, since bud formation, a form of asexual reproduction, also occurs continuously in hydra, the processes governing the initiation of axis formation are also constantly active. Axial patterning, especially of the head, is well understood in hydra at the tissue level. The head organizer located in the hypostome, the upper portion of the head in a hydra, produces a signal and transmits it to the body column. The signal sets up a morphogenetic gradient that decreases down the body column. This gradient, referred to as a positional value gradient, a source density gradient or the head activation gradient, confers head formation capacity on tissue of the body column. The head organizer also produces a second signal, head inhibition, which is transmitted to and graded down the body column. Head inhibition prevents body column tissue from forming heads (Broun, 2005 and references therein).

The molecular basis of the head organizer and these gradients is not well understood. Homologs of a number of genes that affect axial patterning in bilaterians have been isolated from hydra, and appear to play similar roles. For example, the expression patterns of Cnox-3, a homolog of the Hox gene labial/Hox-1, and of the parahox gene Cnox-2, a homolog of Gsx, in several experimental situations suggest that these two genes are involved in the control of head formation (Broun, 2005).

With respect to the head organizer, genes of the canonical Wnt pathway have been identified in hydra, and their expression patterns suggest that this pathway plays a role in this structure. HyWnt is expressed exclusively in the hypostome, where the head organizer is located. The gene is also expressed very early in the apical tip of the developing head during head regeneration and bud formation as the head organizer is developing. HyTcf has a similar, although slightly more extended, range of expression, and Hyß-Cat is expressed strongly during early stages of head formation. In addition, there are data implicating glycogen synthase kinase-3ß in the activity of the head organizer. Treatment with LiCl, which inhibits GSK-3ß, results in the formation of ectopic tentacles along the body column. Exposure to diacylglycerol, which activates protein kinase-C (PKC), which in turn blocks GSK-3ß activity, causes the formation of individual ectopic tentacles or complete heads along the body column. However, both reagents have other effects. For example, Li+ blocks, and diacylglyercol catalyzes, the traverse of the phosphoinositol pathway (Broun, 2005).

To gain more direct evidence for the role of the canonical Wnt pathway in the formation of the head organizer, use was made of alsterpaullone, which specifically blocks the activity of GSK-3ß. Treatment with this inhibitor blocked GSK-3ß activity throughout the animal as well as elevating the level of ß-catenin in the nuclei of body column cells. The treatment also conferred characteristics of the head organizer on the body column as well as inducing the expression of genes of the Wnt pathway in the body column. These results provide direct evidence for the role of the canonical Wnt pathway in the formation and maintenance of the head organizer in hydra (Broun, 2005).

GSK3 is a central regulator of metazoan development and the Dictyostelium GSK3 homologue, GskA, also controls cellular differentiation. The originally derived gskA-null mutant exhibits a severe pattern formation defect. It forms very large numbers of pre-basal disc cells at the expense of the prespore population. This defect arises early during multicellular development, making it impossible to examine later functions of GskA. The analysis is reported of a gskA-null mutant, generated in a different parental strain, that proceeds through development to form mature fruiting bodies. In this strain, Ax2/gskA-, early development is accelerated and slug migration greatly curtailed. In a monolayer assay of stalk cell formation, the Ax2/gskA- strain is hypersensitive to the stalk cell-inducing action of DIF-1 but largely refractory to the repressive effect exerted by extracellular cAMP. During normal development, apically situated prestalk cells express the ecmB gene just as they commit themselves to stalk cell differentiation. In the Ax2/gskA- mutant, ecmB is expressed throughout the prestalk region of the slug, suggesting that GskA forms part of the repressive signalling pathway that prevents premature commitment to stalk cell differentiation. GskA may also play an inductive developmental role, because microarray analysis identifies a large gene family, the 2C family, that require gskA for optimal expression. These observations show that GskA functions throughout Dictyostelium development, to regulate several key aspects of cellular patterning (Schilde, 2004).

In Saccharomyces cerevisiae, many meiotic genes are activated by a heteromeric transcription factor composed of Ime1p and Ume6p. Ime1p-Ume6p complex formation depends on the protein kinase Rim11p, which interacts with and phosphorylates both Ime1p and Ume6p in vitro. Rim11p (also called Mds1p and ScGSK3) is required for meiosis. Rim11p promotes formation of the Ime1p-Ume6p complex, which activates transcription of early meiotic genes. Fusion of a transcriptional activation domain to Ume6p permits meiosis in the absence of Rim11p and Ime1p; apparently the only essential role in meiosis of Rim11p and Ime1p is to modify Ume6p. Rim11p is similar to members of the eukaryotic glycogen synthase kinase-3beta (GSK3beta)/shaggy family in both structure and function. GSK3beta family members have catalytic regions with >80% amino acid sequence identity and share 55%-60% identity with Rim11p. (For comparison, the GSK3beta and protein kinase A catalytic domains share <25% identity.) Several of the GSK3beta family members act in the Wnt/wingless signaling pathway to promote formation of a beta-catenin-adenomatous polyposis coli (APC) complex. The Ime1p-Ume6p and beta-catenin-APC complexes have no structural similarity, but their relationships to Rim11p and GSK3beta share a common feature: the protein kinases bind to and phosphorylate both subunits of their target complex. This finding is consistent with two distinct models for Rim11p and GSK3beta function. One is that the kinases have a catalytic role in complex formation; for example, Rim11p phosphorylates both Ime1p and Ume6p, and phospho-Ime1p then interacts with phospho-Ume6p. The other model is that the kinases have a structural role; for example, a Rim11p-Ime1p complex interacts with Ume6p, and the functional transcriptional activator may be a ternary Ime1p-Rim11p-Ume6p complex (Malathi, 1999).

Rim11p may promote complex formation through its phosphorylation of Ime1p and Ume6p or simply through its interaction with both proteins. Mutant Ime1p derivatives that interact with Rim11p but are not phosphorylated in vitro have been characterized. These mutant proteins are also defective in interaction with Ume6p. These results argue that Ime1p must be phosphorylated to interact with Ume6p. Genetic observations suggest that Ime1p tyrosine residues are among the Rim11p phosphoacceptors. Ime1p reacts with an anti-phosphotyrosine antibody. Ime1p and Rim11p have been thought to act only through Ume6p; Ime1p and Rim11p promote meiosis at a very low level in the absence of Ume6p. A nonphosphorylatable mutant Ime1p derivative promotes sporulation through this Ume6p-independent pathway, as does a mutant Rim11p derivative that fails to interact with Ime1p. Therefore, Ime1p and Rim11p have two genetically separable functions in the sporulation program. However, catalytic activity of Rim11p is required for sporulation in the presence or absence of Ume6p (Malathi, 1999).

In the sea urchin embryo, the animal-vegetal axis is defined before fertilization and different embryonic territories are established along this axis by mechanisms which are largely unknown. Significantly, the boundaries of these territories can be shifted by treatment with various reagents including zinc and lithium. Reported here is the isolation and characterization of a sea urchin homolog of GSK3beta/shaggy, a lithium-sensitive kinase, which is a component of the Wnt pathway and known to be involved in axial patterning in other embryos, including Xenopus. Sea urchin GSK3beta is about 75% identical with vertebrate GSK3beta. In the kinase domain, which covers more than 2/3 of the protein, the sea urchin and human GSK3beta share 87% identity. The effects of overexpressing the normal and mutant forms of GSK3beta derived either from sea urchin or Xenopus were analyzed by observation of the morphology of 48 hour embryos (pluteus stage) and by monitoring spatial expression of the hatching enzyme (HE) gene, a very early gene whose expression is restricted to an animal domain with a sharp border roughly coinciding with the future ectoderm / endoderm boundary. Inactive forms of GSK3beta predicted to have a dominant-negative activity, vegetalize the embryo and decrease the size of the HE expression domain, apparently by shifting the boundary towards the animal pole. These effects are similar to, but even stronger than, those of lithium. Conversely, overexpression of wild type GSK3beta animalizes the embryo and causes the HE domain to enlarge towards the vegetal pole. Unlike zinc treatment, GSK3beta overexpression thus appears to provoke a true animalization, through extension of the presumptive ectoderm territory. These results indicate that in sea urchin embryos the level of GSKbeta activity controls the position of the boundary between the presumptive ectoderm and endoderm territories and thus, the relative extent of these tissue layers in late embryos. GSK3beta and probably other downstream components of the Wnt pathway thus mediate patterning both along the primary AV axis of the sea urchin embryo and along the dorsal-ventral axis in Xenopus, suggesting a conserved basis for axial patterning between invertebrate and vertebrate in deuterostomes (Emily-Fenouil, 1998).

Beta-catenin is thought to mediate cell fate specification events by localizing to the nucleus where it modulates gene expression. To ask whether beta-catenin is involved in cell fate specification during sea urchin embryogenesis, the distribution of nuclear beta-catenin was analyzed in both normal and experimentally manipulated embryos. In unperturbed embryos, beta-catenin accumulates in nuclei that include the precursors of the endoderm and mesoderm, suggesting that it plays a role in vegetal specification. Using pharmacological, embryological and molecular approaches, the function of beta-catenin in vegetal development was determined by examining the relationship between the pattern of nuclear beta-catenin and the formation of endodermal and mesodermal tissues. Treatment of embryos with LiCl, a known vegetalizing agent, causes both an enhancement in the levels of nuclear beta-catenin and an expansion in the pattern of nuclear beta-catenin that coincides with an increase in endoderm and mesoderm. Conversely, overexpression of a sea urchin cadherin blocks the accumulation of nuclear beta-catenin and consequently inhibits the formation of endodermal and mesodermal tissues, including micromere-derived skeletogenic mesenchyme. In addition, nuclear beta-catenin-deficient micromeres fail to induce a secondary axis when transplanted to the animal pole of uninjected host embryos, indicating that nuclear beta-catenin also plays a role in the production of micromere-derived signals. To examine further the relationship between nuclear beta-catenin in vegetal nuclei and micromere signaling, both transplantations and deletions of micromeres at the 16-cell stage were performed and it was demonstrated that the accumulation of beta-catenin in vegetal nuclei does not require micromere-derived cues. Moreover, cell autonomous signals appear to regulate the pattern of nuclear beta-catenin since dissociated blastomeres possess nuclear beta-catenin in approximately the same proportion as that seen in intact embryos. Together, these data show that the accumulation of beta-catenin in nuclei of vegetal cells is regulated cell autonomously and that this localization is required for the establishment of all vegetal cell fates and the production of micromere-derived signals (Logan, 1999).

Localization of nuclear beta-catenin initiates specification of vegetal fates in sea urchin embryos. SpKrl, a gene that is activated upon nuclear entry of beta-catenin, is upregulated when nuclear beta-catenin activity is increased with LiCl and downregulated in embryos injected with molecules that inhibit beta-catenin nuclear function. LiCl-mediated SpKrl activation is independent of protein synthesis, indicating that SpKrl is a direct target of beta-catenin and TCF. Database searches reveal a conserved motif between adjacent zinc fingers, called the H-C link, which places SpKrl in the Kruppel-like family of transcription factors. The most closely related proteins found using a Blast search were the products of the Drosophila genes, Glass and Kruppel. In common with many Kruppel-related factors, sequence similarity is limited to the zinc finger regions of the DNA-binding domain, and SpKrl has no significant match to known proteins outside this region. Embryos in which SpKrl translation is inhibited with morpholino antisense oligonucleotides lack endoderm. Conversely, SpKrl mRNA injection rescues some vegetal structures in beta-catenin-deficient embryos. SpKrl negatively regulates expression of the animalizing transcription factor, SpSoxB1. It is proposed that SpKrl functions in patterning the vegetal domain by suppressing animal regulatory activities (Howard, 2001).

ß-Catenin has a central role in the early axial patterning of metazoan embryos. In the sea urchin, ß-catenin accumulates in the nuclei of vegetal blastomeres and controls endomesoderm specification. In-vivo measurements of the half-life of fluorescently tagged ß-catenin in specific blastomeres has been used to demonstrate a gradient in ß-catenin stability along the animal-vegetal axis during early cleavage. This gradient is dependent on GSK3ß-mediated phosphorylation of ß-catenin. Calculations show that the difference in ß-catenin half-life at the animal and vegetal poles of the early embryo is sufficient to produce a difference of more than 100-fold in levels of the protein in less than 2 hours. Dishevelled (Dsh), a key signaling protein, is required for the stabilization of ß-catenin in vegetal cells; evidence is provided that Dsh undergoes a local activation in the vegetal region of the embryo. GFP-tagged Dsh is targeted specifically to the vegetal cortex of the fertilized egg. During cleavage, Dsh-GFP is partitioned predominantly into vegetal blastomeres. An extensive mutational analysis of Dsh identifies several regions of the protein that are required for vegetal cortical targeting, including a phospholipid-binding motif near the N-terminus (Weitzel, 2004).

Regulation of GSK3ß

The inactivation of glycogen synthase kinase (GSK)3 has been proposed to play important roles in insulin and Wnt signalling. To define the role that inactivation of GSK3 plays, homozygous knockin mice were generated in which the protein kinase B phosphorylation sites on GSK3alpha (Ser21) and GSK3beta (Ser9) were changed to Ala. The knockin mice were viable and were not diabetic. Using these mice it was shown that inactivation of GSK3beta rather than GSK3alpha is the major route by which insulin activates muscle glycogen synthase. In contrast, the activation of muscle glycogen synthase by contraction, the stimulation of muscle glucose uptake by insulin, or the activation of hepatic glycogen synthase by glucose do not require GSK3 phosphorylation on Ser21/Ser9. GSK3 also becomes inhibited in the Wnt-signalling pathway, by a poorly defined mechanism. In GSK3alpha/GSK3beta homozygous knockin cells, Wnt3a induces normal inactivation of GSK3, as judged by the stabilisation of beta-catenin and stimulation of Wnt-dependent transcription. These results establish the function of Ser21/Ser9 phosphorylation in several processes in which GSK3 inactivation has been implicated (McManus, 2005).

Plexins are receptors for the axonal guidance molecules known as semaphorins, and the semaphorin 4D (Sema4D) receptor plexin-B1 induces repulsive responses by functioning as an R-Ras GTPase-activating protein (GAP). This study characterized the downstream signalling of plexin-B1-mediated R-Ras GAP activity, inducing growth cone collapse. Sema4D suppresses R-Ras activity in hippocampal neurons, in parallel with dephosphorylation of Akt and activation of glycogen synthase kinase (GSK)-3beta. Ectopic expression of the constitutively active mutant of Akt or treatment with GSK-3 inhibitors suppressea the Sema4D-induced growth cone collapse. Constitutive activation of phosphatidylinositol-3-OH kinase (PI(3)K), an upstream kinase of Akt and GSK-3beta, also blocka the growth cone collapse. The R-Ras GAP activity is necessary for plexin-B1-induced dephosphorylation of Akt and activation of GSK-3beta and is also required for phosphorylation of a downstream kinase of GSK-3beta, collapsin response mediator protein-2. Plexin-A1 also induces dephosphorylation of Akt and GSK-3beta through its R-Ras GAP activity. It is concluded that plexin-B1 inactivates PI(3)K and dephosphorylates Akt and GSK-3beta through R-Ras GAP activity, inducing growth cone collapse (Ito, 2006).

GSK-3beta position in the Wnt pathway

Wnt signaling involves inhibition of glycogen synthase kinase-3beta (GSK-3beta) and elevation of cytoplasmic beta-catenin. This pathway is essential during embryonic development and oncogenesis. Previous studies on both Xenopus and mammalian cells indicate that lithium mimics Wnt signaling by inactivating GSK-3beta. Serum enhances accumulation of cytoplasmic beta-catenin induced by lithium in both 293 and C57MG cell lines and growth factors are responsible for this enhancing activity. Growth factors mediate this effect through activation of protein kinase C (PKC), not through Ras or phosphatidylinositol 3-kinase. In addition, Wnt-induced accumulation of cytoplasmic beta-catenin is partially inhibited by PKC inhibitors and by chronic treatment of cells with phorbol ester. Both calphostin C, a PKC inhibitor, and a dominant negative PKC exhibit partial inhibition on Wnt-mediated transcriptional activation. It is proposed that Wnt signaling to beta-catenin consists of two interactive components: one involves inhibition of GSK-3beta and is mimicked by lithium, and the other involves PKC and serves to augment the effects of GSK-3beta inhibition (Chen, 2000).

The Wnt pathway controls numerous developmental processes via the ß-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of ß-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic ß-catenin associates with APC and axin and is continuously phosphorylated by GSK-3ß, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3ß from phosphorylating ß-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. The regulation of ß-catenin phosphorylation and degradation by the Wnt pathway has been studied. Using mass spectrometry and phosphopeptide-specific antibodies, it has been shown that a complex of axin and casein kinase I (CKI) induces ß-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate ß-catenin in vitro at S45; CKI inhibition suppresses this phosphorylation in vivo. CKI phosphorylation creates a priming site for GSK-3ß and is both necessary and sufficient to initiate the ß-catenin phosphorylation-degradation cascade. Wnt3A signaling and Dvl overexpression suppress S45 phosphorylation, thereby precluding the initiation of the cascade. Thus, a single, CKI-dependent phosphorylation event serves as a molecular switch for the Wnt pathway (Amit, 2002).

β-catenin-dependent or canonical Wnt signals are fundamental in animal development and tumor progression. Using Xenopus laevis, it is reported that the BTB/POZ zinc finger family member Kaiso directly represses canonical Wnt gene targets (Siamois, c-Fos, Cyclin-D1, and c-Myc) in conjunction with TCF/LEF (TCF). Analogous to β-catenin relief of TCF repressive activity, it is shown that p120-catenin relieves Kaiso-mediated repression of Siamois. Furthermore, Kaiso and TCF coassociate, and the combination of Kaiso and TCF derepression results in pronounced Siamois expression and increased β-catenin coprecipitation with the Siamois promoter. The functional interdependency is underlined by Kaiso suppression of β-catenin-induced axis duplication and by TCF-3 rescue of Kaiso depletion phenotypes. These studies point to convergence of parallel p120-catenin/Kaiso and β-catenin/TCF signaling pathways to regulate gene expression in vertebrate development and possibly carcinogenesis (Par, 2005).

In mammalian cells, glycogen synthase kinase-3 (GSK-3) exists as two homologs, GSK-3α and GSK-3β, encoded by independent genes, which share similar kinase domains but differ substantially in their termini. This study describes the generation of an allelic series of mouse embryonic stem cell (ESC) lines with 0-4 functional GSK-3 alleles and examines GSK-3-isoform function in Wnt/β-catenin signaling. No compensatory upregulation in GSK-3 protein levels or activity was detected in cells lacking either GSK-3α or GSK-3β, and Wnt/β-catenin signaling was normal. Only in cells lacking three or all four of the alleles was a gene-dosage effect on β-catenin/TCF-mediated transcription observed. Indeed, GSK-3α/β double-knockout ESCs displayed hyperactivated Wnt/β-catenin signaling and were severely compromised in their ability to differentiate, but could be rescued to normality by re-expression of functional GSK-3. The rheostatic regulation of GSK-3 highlights the importance of considering the contributions of both homologs when studying GSK-3 functions in mammalian systems (Doble, 2007).

The Wnt-β-catenin canonical signaling pathway is crucial for normal embryonic development, and aberrant expression of components of this pathway results in oncogenesis. Upon scanning for the mitogen-activated protein kinase (MAPK) pathways that might intersect with the canonical Wnt-β-catenin signaling pathway in response to Wnt3a, a strong activation of p38 MAPK was observed in mouse F9 teratocarcinoma cells. Wnt3a-induced p38 MAPK activation was sensitive to siRNAs against Gαq or Gαs, but not against either Gαo or Gα11. Activation of p38 MAPK is critical for canonical Wnt-β-catenin signaling. Chemical inhibitors of p38 MAPK (SB203580 or SB239063) and expression of a dominant negative-version of p38 MAPK attenuate Wnt3a-induced accumulation of β-catenin, Lef/Tcf-sensitive gene activation, and primitive endoderm formation. Furthermore, epistasis experiments pinpoint p38 MAPK as operating downstream of Dishevelleds. It was also demonstrated that chemical inhibition of p38 MAPK restores Wnt3a-attenuated GSK3β kinase activity. The involvement of G-proteins and Dishevelleds in Wnt3a-induced p38 MAPK activation was demonstrated, highlighting a critical role for p38 MAPK in canonical Wnt-β-catenin signaling (Bikkavilli, 2008).

A Smad action turnover switch operated by WW domain readers of a phosphoserine code

When directed to the nucleus by TGF-β or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. This study demonstrates that there is an order of events-Smad activation first and destruction later-and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-β pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers (see A Smad action turnover switch operated by WW domain readers of a phosphoserine code) provide an efficient solution to the problem of coupling TGF-β signal delivery to turnover of the Smad signal transducers (Aragón, 2011).

Axin and GSK3-β control Smad3 protein stability and modulate TGF-β signaling

The broad range of biological responses elicited by transforming growth factor-β (TGF-β) in various types of tissues and cells is mainly determined by the expression level and activity of the effector proteins Smad2 and Smad3. It is not fully understood how the baseline properties of Smad3 are regulated, although this molecule is in complex with many other proteins at the steady state. This stud shows that nonactivated Smad3, but not Smad2, undergoes proteasome-dependent degradation due to the concerted action of the scaffolding protein Axin and its associated kinase, glycogen synthase kinase 3-β (GSK3-β). Smad3 physically interacts with Axin and GSK3-β only in the absence of TGF-β. Reduction in the expression or activity of Axin/GSK3-β leads to increased Smad3 stability and transcriptional activity without affecting TGF-β receptors or Smad2, whereas overexpression of these proteins promotes Smad3 basal degradation and desensitizes cells to TGF-β. Mechanistically, Axin facilitates GSK3-β-mediated phosphorylation of Smad3 at Thr66, which triggers Smad3 ubiquitination and degradation. Thr66 mutants of Smad3 show altered protein stability and hence transcriptional activity. These results indicate that the steady-state stability of Smad3 is an important determinant of cellular sensitivity to TGF-β, and suggest a new function of the Axin/GSK3-β complex in modulating critical TGF-β/Smad3-regulated processes during development and tumor progression (Guo, 2008).

GSK-3beta and the insulin pathway

Glycogen synthase kinase-3 (GSK3) is implicated in the regulation of several physiological processes, including the control of glycogen and protein synthesis by insulin; modulation of the transcription factors AP-1 and CREB; the specification of cell fate in Drosophila, and dorsoventral patterning in Xenopus embryos. GSK3 is inhibited by serine phosphorylation in response to insulin or growth factors and in vitro by either MAP kinase-activated protein (MAPKAP) kinase-1 (also known as p90rsk) or p70 ribosomal S6 kinase (p70S6k). Agents that prevent the activation of both MAPKAP kinase-1 and p70S6k by insulin in vivo do not block the phosphorylation and inhibition of GSK3. Another insulin-stimulated protein kinase inactivates GSK3 under these conditions: this kinase has been shown to be the product of the proto-oncogene protein kinase B (PKB, also known as Akt/RAC). Like the inhibition of GSK3, the activation of PKB is prevented by inhibitors of phosphatidylinositol (PI) 3-kinase (Cross, 1995).

Glycogen synthase kinase 3beta (GSK3beta) is a key component in many biological processes including insulin and Wnt signaling. Since the activation of each signaling pathway results in a decrease in GSK3beta activity, the specificity of their downstream effects were examined in the same cell type. Insulin induces an increased activity of glycogen synthase but has no influence on the protein level of beta-catenin. In contrast, Wnt increases the cytosolic pool of beta-catenin but not glycogen synthase activity. Unlike insulin, neither the phosphorylation status of the serine9 residue of GSK3beta nor the activity of protein kinase B is regulated by Wnt. Although the decrease in GSK3beta activity is required, GSK3beta may not be the limiting component for Wnt signaling in the cells that were examined. These results suggest that the axin-conductin complexed GSK3beta may be dedicated to Wnt rather than insulin signaling. Insulin and Wnt pathways regulate GSK3beta through different mechanisms, and therefore lead to distinct downstream events (Ding, 2000).

The inhibition of GSK3 is required for the stimulation of glycogen and protein synthesis by insulin and the specification of cell fate during development. The insulin-induced inhibition of GSK3 and its unique substrate specificity are explained by the existence of a phosphate binding site in which Arg-96 is critical. Thus, mutation of Arg-96 abolishes the phosphorylation of 'primed' glycogen synthase as well as inhibition by PKB-mediated phosphorylation of Ser-9 (See Drosophila Akt1). Hence, the phosphorylated N terminus acts as a pseudosubstrate, occupying the same phosphate binding site used by primed substrates. Significantly, this mutation does not affect phosphorylation of 'nonprimed' substrates in the Wnt-signaling pathway (Axin and ß-catenin), suggesting new approaches to design more selective GSK3 inhibitors for the treatment of diabetes (Frame, 2001).

GSK3 is phylogenetically most closely related to the cyclin-dependent protein kinases (CDKs), such as CDK1 (also called cdc2) and CDK2. However, the specificity of GSK3 is unique in that it requires a priming phosphate located at n + 4 (where n is the site of phosphorylation) in order to phosphorylate many of its substrates, such as glycogen synthase. In contrast, the phosphorylation of Axin and ß-catenin in the Wnt signaling pathway is not known to require a priming phosphate and may rely on high-affinity interactions in a multiprotein complex with GSK3. Thus, Axin binds to both GSK3 and ß-catenin, bringing these proteins into close proximity to facilitate their phosphorylation by GSK3. This study presents evidence for a specific site of interaction between the phosphate of the primed substrate and Arg-96 of GSK3. This same phosphate binding site is also occupied by Ser-9 once it becomes phosphorylated by PKB. The existence of this site helps to explain several features of GSK3, such as its unusual substrate specificity requirements and the mechanism by which it becomes inhibited in response to insulin and growth factors. These findings have important implications for drug development in this area (Frame, 2001).

The endosomal protein Appl1 mediates Akt substrate specificity towards GSK3-β

During development of multicellular organisms, cells respond to extracellular cues through nonlinear signal transduction cascades whose principal components have been identified. Nevertheless, the molecular mechanisms underlying specificity of cellular responses remain poorly understood. Spatial distribution of signaling proteins may contribute to signaling specificity. This hypothesis by investigating the role of the Rab5 effector Appl1 (adaptor protein containing pH domain, PTB domain, and Leucine zipper motif, also termed DIP13α and β), an endosomal protein that interacts with transmembrane receptors and Akt. In zebrafish, Appl1 regulates Akt activity and substrate specificity, controlling GSK-3β but not TSC2. Consistent with this pattern, Appl1 is selectively required for cell survival, most critically in highly expressing tissues. Remarkably, Appl1 function requires its endosomal localization. Indeed, Akt and GSK-3β, but not TSC2, dynamically associate with Appl1 endosomes upon growth factor stimulation. It is proposed that partitioning of Akt and selected effectors onto endosomal compartments represents a key mechanism contributing to the specificity of signal transduction in vertebrate development (Schenck, 2008).

Based on these data, the current model of Akt regulation and specificity needs to be refined to take into account the contribution of APPL endosomes to the signaling mechanisms. Previously, Akt phosphorylation at Thr308 has been reported to uncouple signaling to FoxO1/3 transcription factors from other Akt effectors. With APPL1, a crucial missing link has been provided between the dependence on Rab5 for the activity of Akt (Hunker, 2006; Su, 2006) and its functional specification. It is proposed that receptors internalized into APPL endosomes are exposed to a molecular membrane environment enriched in selected signaling factors, thus 'channeling' signaling flow downstream of Akt to evoke cell survival uncoupled from growth and proliferation. A platform for selective recruitment and activation of signaling components is envisioned. The protein and lipid composition of such platform needs to be thoroughly established, but it is likely to depend on a combinatorial use of protein-protein and protein-lipid interactions. For example, APPL proteins utilize coincidence detection of membrane curvature and presence of Rab5 to achieve proper organelle targeting. Rab5 alone is not sufficient since APPL localization also crucially depends on its BAR domain. A similar combinatorial use of binding sites is likely to ensure the recruitment of downstream signaling components such as Akt and GSK-3β. However, such recruitment appears to be transient, suggesting that dynamic interactions rather than stable signaling complexes on the Appl endosomes account for signal propagation via Akt and GSK-3β. The precise kinetics and biochemical features of these interactions need to be evaluated using ad hoc developed quantitative live cell imaging techniques (Schenck, 2008).

Table of contents

shaggy: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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