Developmental Biology

Notch signaling through tramtrack bypasses the mitosis promoting activity of the JNK pathway in the mitotic-to-endocycle transition of Drosophila follicle cells: Notch signaling is modulated by Shaggy

The follicle cells of the Drosophila egg chamber provide an excellent model in which to study modulation of the cell cycle. During mid-oogenesis, the follicle cells undergo a variation of the cell cycle, endocycle, in which the cells replicate their DNA, but do not go through mitosis. Previously, it was shown that Notch signaling is required for the mitotic-to-endocycle transition, through downregulating String/Cdc25, and Dacapo/p21 and upregulating Fizzy-related/Cdh1. In this paper, it is shown that Notch signaling is modulated by Shaggy and temporally induced by the ligand Delta, at the mitotic-to-endocycle transition. In addition, a downstream target of Notch, tramtrack, acts at the mitotic-to-endocycle transition. It is also demonstrated that the JNK pathway is required to promote mitosis prior to the transition, independent of the cell cycle components acted on by the Notch pathway. This work reveals new insights into the regulation of Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).

Notch controls the mitotic-to-endocycle transition in follicle epithelial cells; Notch pathway activity arrests mitotic cell cycle and promotes endocycles by downregulating string/cdc25 and dacapo/p21, and upregulating fzr/Cdh1. This study identified components regulating this transition, Delta, Shaggy, and Tramtrack. Shaggy and Delta are required for the activation of Notch protein. However, Delta is sufficient to activate Notch in this process, since premature expression of Delta in the germline stops mitotic division of the follicle cells. This study identified Tramtrack as a connection between Notch and the cell cycle regulators stg, fzr, and dap. Loss of Tramtrack function phenocopies the Notch and Su(H) phenotypes; overproliferation and misregulation of cell cycle components. However, high FAS3 expression, indicative of differentiation defects in Notch clones, is not observed in ttk clones, suggesting that Tramtrack might regulate a branch of the Notch pathway specific for cell cycle control. It was also shown that the JNK-pathway is a critical mitosis promoting pathway in follicle cells. Loss of JNK(bsk) or JNKK(hep) activities stop follicle cell mitotic cycles, while loss of JNK promotes premature endocycles. In addition, loss of the negative regulator of the pathway, the phosphatase Puckered, results in a lack of endocycles. However, the Notch-responsive cell cycle targets that, in combination, can induce the mitotic-to-endocycle transition, stg, fzr, and dap, are not regulated by the JNK-pathway (Jordan, 2006).

Notch signaling is highly regulated throughout development. The Notch receptor can be regulated by glycosylation of the extracellular domain, as well as by endocytosis and degradation of the intracellular domain, thus affecting the activity of the pathway. Shaggy has been shown to phosphorylate and thus affect the stability of Notch protein. Normal processing and clearing of Notch protein from the apical surface of follicle cells upon Notch activation does not occur in shaggy clones, indicating that Notch is not normally activated and therefore regulation of the downstream targets does not take place (Jordan, 2006).

In many organisms and tissues the Notch ligands are ubiquitously expressed and thus not likely to regulate Notch pathway activation. However, at the mitotic to endocycle transition, Delta is upregulated in the germline, making ligand expression a likely candidate for regulation of Notch activity. Premature expression of Delta in the germline can cause mitotic division to stop at least one stage earlier than in control ovarioles. Nonetheless, this effect is seen in only half of the ovarioles. Therefore, it is possible that yet another process is regulating Notch activity at the transition in addition to Delta expression. Further testing will determine if endocytosis of Notch might also regulate Notch activity at the mitotic-to-endocycle transition. One possible protein is Numb, which regulates Notch in human mammary carcinomas, indicating that Numb may have a more general role in cell cycle control than just the division of the sensory organ precursors (Jordan, 2006).

The fact that Notch overrides the mitotic activity of the JNK pathway by acting on cell cycle regulators that can induce the mitotic-to-endocycle transition puts further demand on understanding the connection between Su(H) and cell cycle regulators. One such component, the transcription factor Tramtrack, has been identified. Two Tramtrack proteins exist, Ttk69 and Ttk88, both of which are affected by the allele used in these studies. However, staining with antibodies specific to the two forms reveals that only Ttk69 is detectable in the follicle cells and downregulated in Notch clones (Jordan, 2006).

Ttk69 can control proliferation in glial cells, strengthening its candidacy for a critical component between Notch and cell cycle controllers in follicle epithelial cells. In addition, the Ttk-like BTB/POZ-domain zinc-finger transcription repressor in humans is Bcl-6, a protein associated with B-cell lymphomas (Jordan, 2006).

Ttk function in the follicle cell mitotic-to-endocycle transition was analyzed and it has been shown that the Notch-responsive cell cycle components stg, dap, and fzr are responsive to Ttk function. Interestingly, Ttk69 controls the string promoter in the Drosophila eye discs. In the future, it will be important to determine whether Ttk DNA binding sites are found in the Notch-responsive stg promoter as well. In addition, the binding sites of transcription factors that can interact with Ttk will be of interest, since Ttk can act as a DNA binding or non-binding repressor (Jordan, 2006).

Previous work revealed that the JNK pathway is closely connected to cell cycle control. For example, in fibroblasts the JNK pathway is critical for cdc2 expression and G2/M cell cycle progression. In the case of the follicle cell mitotic-to-endocycle transition, it was shown that the JNK pathway is a critical positive controller of the mitotic cycles. Lack of JNK activity leads to a block in mitosis and initiation of premature endocycles. Conversely, lack of the negative regulator of the JNK-pathway, the phosphatase Puckered, results in a loss of endocycles. However, puc mutant clones do not consistently support extra divisions but might induce apoptosis as shown recently in disc clones (Jordan, 2006).

These data are interesting in light of the results showing that the JNK pathway does not control the same cell cycle targets as the Notch pathway, and could be explained by the following hypothesis: the JNK-pathway positively regulates the mitotic cycles prior to stage 6 in follicle epithelial cells. This positive action on mitotic cycles is negatively short-circuited by the direct control of cell cycle regulators by the Notch pathway at stage 6 in oogenesis, resulting in the mitotic-to-endocycle transition. Premature termination of the JNK pathway is sufficient to induce mitotic-to-endocycle transition. However, prolonged JNK activity, while disrupting endocycles, cannot maintain mitotic cycling efficiently, due to Notch action on string, dacapo, and fzr (Jordan, 2006).

What then terminates JNK-pathway activity at stage 6 in oogenesis? Prolonged JNK activity (puc mutant clones) affects endocycles and the expression of pJNK and Puc subsides at stages 6-7; results that both suggest the downregulation of JNK activity at the mitotic-to-endocycle transition. One possibility is that Notch activity downregulates the JNK pathway. However, at least Su(H)-dependent Notch activity does not regulate the JNK pathway, since no effect on puckered expression was observed in Su(H) mutant clones. It is plausible that Su(H)-independent Notch activity regulates the JNK pathway in this context, as has been shown to be the case in dorsal closure. Interestingly, Deltex might play a role in this Su(H)-independent Notch activity (Jordan, 2006).

An important question in analyzing the developmental control of cell cycle is whether the same signaling pathways control both differentiation and cell cycle, and if so, how the labor is divided. The Notch-dependent mitotic-to-endocycle transition is an example of such a question; Notch action in stage 6 follicle cells is critical for the cell cycle switch and for at least some aspects of differentiation. This work reports the first component that separates Notch dependent cell cycle regulation from Fas3 marked differentiation; Ttk. In the ttk mutant clones, upregulation of FAS3, characteristic for Notch clones, is not observed. Therefore, Ttk constitutes a branch of Notch activity that might be solely required for cell cycle control in this context. However, Ttk's independent function cannot yet be rule out. In the future, it will be important to understand whether signaling pathways in general show a clear separation of differentiation and cell cycle control on the level of downstream transcription factors. Importantly, these and previous results have revealed the essential cell cycle regulators and their roles in controlling the Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).

Drosophila Syd-1, liprin-α, and protein phosphatase 2A B' subunit Wrd function in a linear pathway to prevent ectopic accumulation of synaptic materials in distal axons

During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1 or Rho GTPase activating protein at 100F) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, a proteomic screen was performed of Liprin-α-interacting proteins in Drosophila brains. Drosophila protein phosphatase 2A (PP2A; see MTS, the PP2A catalytic subunit) regulatory subunit B' [Wrd (Well Rounded) or PP2A-B'] was identified as a Liprin-α-interacting protein, and it was demonstrated that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3β kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, data is provided suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot) (Li, 2014).

During presynaptic development, small synaptic vesicle (SV) precursors, dense-core vesicles (DCVs), and synaptic cytomatrix proteins are generated in the soma, transported along the axon, and eventually incorporated into the nerve terminal. Within the nerve terminal, active zones (AZs) are specialized areas of plasma membrane containing a group of evolutionarily conserved proteins, including ELKS (glutamine, leucine, lysine, and serine-rich protein)[also called CAST (cytomatrix at the active zone-associated structural protein), Drosophila homologue is BRP (Bruchpilot)], Munc13 (mammalian uncoordinated homology 13), RIM (Rab3-interacting molecule), Syd-1 (synapse defective-1), and Liprin-α, in which the releasable pool of vesicles dock and are released on stimulation. Despite intensive studies of the proteins localized at the presynaptic density, the assembly and maintenance of AZs remains enigmatic. Studies conducted in invertebrate model organisms suggested that Syd-1, a putative RhoGAP, and Liprin-α are two master organizers of presynaptic differentiation. Genetic analyses in Caenorhabditis elegans demonstrated that Syd-1 works upstream of Liprin-α in synaptic assembly. Studies in Drosophila further confirmed this hierarchy by showing that Syd-1 regulates and retains proper localization of Liprin-α at the AZ. However, studies also found that Syd-1 regulates Liprin-α-independent processes, such as retention of Neurexin at the presynaptic side and glutamate receptor incorporation at the postsynaptic side. The morphology of the AZ is distinctly different in liprin-α and syd-1 mutants. Therefore, it is unclear how Syd-1- and Liprin-α-mediated signaling collaborate to achieve the complex regulation of presynaptic differentiation. Identifying novel Liprin-α-interacting proteins at the synapse holds the key to delineating the regulatory network mediated by these two genes (Li, 2014).

This study identified protein phosphatase 2A (PP2A) as one prominent Liprin-α-interacting protein complex through an in vivo tandem affinity purification (TAP) approach. PP2A is an abundant heterotrimeric serine/threonine phosphatase that regulates a broad range of cellular processes. PP2A is highly enriched in neurons and is implicated in Tau-mediated neurodegeneration, regulation of long-term potentiation, and presynaptic and postsynaptic apposition. The diverse functions of PP2A are attributed primarily to its many interchangeable regulatory subunits (B, B', B'', or B'''), each showing specific spatial and temporal expression patterns. The Liprin-α-interacting PP2A holoenzyme that this study identified in the fly brain contains the B' regulatory subunit [also called Wrd (Well Rounded) in fly]. Wrd is highly expressed in synapses and regulates synaptic terminal growth at the Drosophila neuromuscular junction (NMJ). Interestingly, the Liprin-α-Wrd physical interaction may be evolutionarily conserved because PP2A B56γ, the human homolog of Wrd, can bind Liprin-α1 in HEK 293 cell. However, the function of the Liprin-α-Wrd/PP2A B56γ interaction in the nervous system is unexplored (Li, 2014).

This study shows that Syd-1, Liprin-α, and Wrd work in a linear pathway to restrain the localization of vesicles and presynaptic cytomatrix proteins at the nerve terminal. Disruption of such a pathway results in ectopic accumulation of SVs and presynaptic proteins at the distal, but not proximal, end of axons (Li, 2014).

Much progress toward understanding presynaptic differentiation has been made through unbiased forward genetic screens in invertebrates. These studies have led to the identification of several key factors for AZ formation, including two evolutionarily conserved master organizer proteins of AZ assembly: syd-1 and syd-2/liprin-α. However, how Syd-1/Liprin-α organize presynaptic sites remains unclear. This study identified a new synaptic player, the PP2A B′ regulatory subunit, that is localized to the synapse by Liprin-α and mediates Syd-1/Liprin-α signaling in stabilizing AZs and their associated vesicles at the nerve terminal (Li, 2014).

Liprin-α was first identified as a protein interacting with the LAR (leukocyte antigen-related-like) family of phosphatases. Studies during the past two decades demonstrate that Liprin-α regulates presynaptic and postsynaptic development, as well as neurotransmitter release through protein–protein interactions with a range of molecules, including CAST/ELKS/BRP, RIM, CASK (calcium/calmodulin-dependent serine protein kinase), GIT (G-protein-coupled receptor kinase-interacting ArfGAP), GRIP (glutamate receptor interacting protein), LAR, CaMKII, and Liprin-β. Proteomic data confirmed the interaction between Liprin-α and BRP/RIM in Drosophila. Another important Liprin-α binding partner was identified at the presynaptic sites, the B′ regulatory subunit of PP2A (Wrd), which depends on Liprin-α for it proper synaptic localization (Li, 2014).

Phenotypic analysis of syd-1, liprin-α, and wrd mutants demonstrate that they share a unique trafficking defect, in which SVs, DCVs, presynaptic scaffolding proteins, and voltage-gated Ca2+ channels ectopically accumulate at the distal, but not the proximal, region of the axon. Genetic rescue experiments define a linear pathway, from syd-1 to liprin-α to wrd, that works cell autonomously in the presynaptic neuron to ensure proper localization of presynaptic materials to the nerve terminal and prevents ectopic accumulation. Together, these biochemical and genetic data suggest that Wrd mediates a novel Syd-1/Liprin-α function at the presynaptic site. Such a Syd-1/Liprin-α function is likely independent of their well established roles in regulating the T-bar structure protein BRP/ELKS (Li, 2014).

Two lines of evidence suggest that a Wrd-containing PP2A mediates the function of Syd-1/Liprin-α in regulating AZ stability. First, two rounds of in vivo biochemical purification using either Liprin-α or Wrd as the bait copurified Liprin-α with Wrd and the other two core subunits of PP2A, indicating the presence of a Liprin-α/Wrd/PP2A protein complex in neurons. Second, loss of GSK-3β kinase [sgg (shaggy)] function suppresses the syd-1, liprin-α, and wrd mutant distal axon phenotype, suggesting that a Wrd/PP2A-mediated phosphatase activity normally functions to antagonize a GSK-3β kinase activity in neurons to stabilize AZ and clustering of SVs at the nerve terminal (Li, 2014).

What is the primary cause for the unique distal axon phenotype in syd-1/liprin-α/wrd mutant larvae? Liprin-α was shown to interact with KIF1A (kinesin family member 1A)/Unc-104, a neuron-specific kinesin motor known to transport SV precursors containing synaptophysin, Syt, and Rab1A. It was reported that Drosophila Liprin-α regulates the trafficking of SVs through its interaction with Kinesin-1 and that liprin-α mutant peripheral nerves show accumulation of clear-core vesicles similar to kinesin heavy chain (khc) mutants. However, when this study focused on the location of the phenotypes relative to the entire axonal length, liprin-α mutant accumulation of clear-core vesicles was found to be present exclusively in the distal end (the ventrolateral peripheral nerve bundles, as well as axonal regions proximal to NMJs), whereas khc mutant larvae show massive aggregation of SV-associated proteins in the proximal end (segmental nerve bundles), and very few SV precursors reach the distal of axon. The distribution pattern of the vesicle accumulation in syd-1 and wrd mutants is the same as liprin-α mutants. Such a pattern is distinct from that of typical trafficking defects induced by mutations in vesicle-transporting motors or cargos (Li, 2014).

Although a unique vesicle trafficking defect as the primary cause for the syd-1/liprin-α/wrd mutant axonal phenotype cannot be completely excluded, a number of lines of evidence suggest a plausible explanation: AZ materials at the nerve terminal become destabilized when the syd-1/liprin-α/wrd pathway is impaired, and the floating AZ materials diffuse back to the adjacent axonal regions as ectopic docking sites for vesicles. First, Syd-1, Liprin-α, and Wrd show clear synaptic localization, with little or no axonal localization detected, consistent with a collaborative function of the three at the AZs. Second, EM analysis detected floating AZ materials in the synaptic boutons and the connected axonal regions in syd-1 mutants. Some of the floating materials are very close to or touching the bouton plasma membrane, indicating a possible defect in AZ stabilization and subsequent back-diffusion of detached AZ materials to axonal regions. Third, AZ components such as BRP, RIM, and voltage-gated Ca2+ channels are identified in the mutant distal axons along with vesicles, including SVs and DCVs, but not vesicles that transport AZ scaffolding proteins, or other synaptically localized organelles, or transport machineries. This is consistent with an ectopic accumulation of vesicles attracted by ectopic floating AZ components. Fourth, live imaging analysis found that anterogradely transported DCVs accumulate at preferred spots at the mutant distal axons, consistent with the existence of static docking sites at these axonal regions. Fifth, ectopically accumulated vesicles do not participate in release or recycling, consistent with the notion that the vesicles do not dock on the axonal plasma membrane (Li, 2014).

The fact that knockdown of a kinase (GSK-3β) rescues the distal axonal defects of syd-1/liprin-α/wrd mutants indirectly suggests that a Wrd-dependent dephosphorylation event is antagonized by a phosphorylation event (mediated by GSK-3β) to regulate AZ stability. However, these data cannot exclude the possibility that PP2A-independent functions of Wrd are involved. One way to seek direct evidence that Wrd-containing PP2A is involved in regulating AZ stability is to study the loss of function of PP2A; however, this approach has its own set of complications. As a ubiquitous heterotrimetric enzyme, the substrate specificity and subcellular localization of PP2A are greatly dependent on its regulatory subunit (such as Wrd). Mutating the catalytic or structural domain blocks overall PP2A action mediated by all regulatory subunits, which precludes analysis of Wrd-specific PP2A action. For example, mutations in MTS (the PP2A catalytic subunit) cause early lethality. Overexpression of a dominant MTS protein causes massive axonal transport defects in the entire axon, as well as defects in AZ development. Therefore, identifying common substrates shared by Wrd/PP2A and GSK-3β and studying how their phosphorylation status regulates AZ stability and/or vesicle trafficking will ultimately unravel the mechanism by which a PP2A-dependent pathway regulates presynaptic development. In this context, this study set up a model to study how synapse scaffolding proteins can regulate localized phosphorylation/dephosphorylation through recruitment of specific phosphatases or kinases (Li, 2014).

A mammalian homolog of Syd-1 was identified recently as an important regulator of presynaptic differentiation at central synapses, at least partially through its interaction with mammalian Liprin-α2. Given that Liprin-α1 interacts with PP2A B56γ (mammalian homolog of Wrd) in HEK 293 cells, it will be of interest to investigate whether the function of Drosophila Liprin-α in mediating the signaling from Syd-1 to the PP2A B′ subunit is also evolutionarily conserved during vertebrate synapse development (Li, 2014).

Modulated Expression of the Protein Kinase GSK3 in Motor and Dopaminergic Neurons Increases Female Lifespan in Drosophila melanogaster
Most eukaryotic genes express multiple transcripts and proteins, and a sophisticated gene expression strategy plays a crucial role in ensuring the cell-specificity of genetic information and the correctness of phenotypes. The Drosophila melanogaster gene shaggy encodes several isoforms of the conserved glycogen synthase kinase 3 (GSK3), which is vitally important for multiple biological processes. To characterize the phenotypic effects of differential shaggy expression, this study explored how the multidirectional modulation of the expression of the main GSK3 isoform, Shaggy-PB, in different tissues and cells affects lifespan. To this end, lines with transgenic constructs were used that encode mutant variants of the protein. The effect of shaggy misexpression on lifespan depended on the direction of the presumed change in GSK3 activity and the type of tissue/cell. The modulation of GSK3 activity in motor and dopaminergic neurons improved female lifespan but caused seemingly negative changes in the structural (mitochondrial depletion; neuronal loss) and functional (perturbed locomotion) properties of the nervous system, indicating the importance of analyzing the relationship between lifespan and healthspan in invertebrate models. These findings provide new insights into the molecular and cellular bases of lifespan extension, demonstrating that the fine-tuning of transcript-specific shaggy expression in individual groups of neurons is sufficient to provide a sex-specific increase in survival and slow aging (Trostnikov, 2020).

Effects of Mutation or Deletion

In embryos mutant for armadillo, dishevelled and porcupine, the changes in engrailed expression are identical to those in wingless mutant embryos, suggesting that their gene products act in the wingless pathway (van den Heuvel, 1993). dsh and porc act upstream of zw3, and arm acts downstream of zw3 (Siegfried, 1994).

The finding that Wingless (WG) and Decapentaplegic (DPP) suppress each others transcription provides a mechanism for creating developmental territories in fields of cells. What is the mechanism for that antagonism? The dishevelled and shaggy genes encode intracellular proteins generally thought of as downstream of WG signaling. The effects of changing either DSH or SGG activity were investigated on both cell fate and wg and dpp expression. At the level of cell fate in discs, DSH antagonizes SGG activity. At the level of gene expression, SGG positively regulates dpp expression and negatively regulates wg expression while DSH activity suppresses dpp expression and promotes wg expression. Sharp borders of gene expression correlating precisely with clone boundaries suggest that the effects of DSH and SGG on transcription of wg and dpp are not mediated by secreted factors but rather act through intracellular effectors. The interactions described here suggest a model for the antagonism between WG and DPP that is mediated via SGG. The model incorporates autoactivation and lateral inhibition, which are properties required for the production of stable patterns. In the Dorsal part of the leg disc, DPP signalling predominates; DPP together with SGG inhibit wg expression and the consequencent lack of inhibition of SGG promotes further dpp expression. In the ventral part of the disc, WG signaling predominates and WG acts through DSH to inhibit SGG activity thus removing the activator of dpp (SGG) and promoting its own expression by removing the combinatorial inhibition of SGG and DPP. The regulatory interactions described exhibit extensive ability to organize new pattern in response to manipulation or injury (Heslip, 1997).

To characterize Armadillo's ability to activate cell death, and further examine the role of APC-like in the Wingless pathway, effects brought about by other members of the Wingless signaling pathway on the apoptosis that is induced by Apc loss were also examined. One well-characterized negative regulator of Arm's signal transduction function is the serine/threonine kinase Zeste-white 3 (Zw3). Inactivation of Zw3 yields elevated levels of cytoplasmic Arm but has little effect on Arm's function in junctional complexes. Neuronal-specific overexpression of Zw3, directed by the elav-GAL4 transactivator, rescues many retinal neurons from apoptosis in the Apc mutant. Remarkably, the rescued cells are detected solely at the apical surface of the eye; more basal sections reveal no rescue. Thus, although the underlying differentiation defect persists, overexpression of Zw3 prevents retinal cell death in the Apc mutant, suggesting a role for cytoplasmic Arm in the activation of apoptosis (Ahmed, 1998).

naked cuticle (nkd) is an embryonic lethal recessive zygotic mutation that produces multiple segmentation defects, the most prominent of which is the replacement of denticles by excess naked cuticle. This phenotype is also seen in embryos exposed to excess Wg, as well as in embryos lacking both maternal and zygotic contributions from any of three genes that antagonize Wg: zeste-white3/glycogen synthase kinase 3beta (zw3/gsk3beta), D-axin and D-Apc2. In nkd embryos, hh and en transcripts initiate normally but accumulate in broad stripes, including cells further from the source of Wg, which suggests that these cells are hypersensitive to Wg. Next, a stripe of new wg transcription appears just posterior to the expanded Hh/En stripe. This extra wg stripe requires both wg and hh activity and is required for the excess naked cuticle seen in nkd mutants. The death of cells producing Hh/En contributes to the marked shortening of nkd mutant cuticles (Zeng, 2000).

In Drosophila embryos the protein Naked cuticle (Nkd) limits the effects of the Wnt signal Wingless (Wg) during early segmentation. nkd loss of function results in segment polarity defects and embryonic death, but how nkd affects Wnt signaling is unknown. Using ectopic expression, it has been found that Nkd affects, in a cell-autonomous manner, a transduction step between the Wnt signaling components Dishevelled (Dsh) and Zeste-white 3 kinase (Zw3). Zw3 is essential for repressing Wg target-gene transcription in the absence of a Wg signal, and the role of Wg is to relieve this inhibition. Double-mutant analysis shows that, in contrast to Zw3, Nkd acts to restrain signal transduction when the Wg pathway is active . Yeast two hybrid and in vitro experiments indicate that Nkd directly binds to the basic-PDZ region of Dsh. Specially timed Nkd overexpression is capable of abolishing Dsh function in a distinct signaling pathway that controls planar-cell polarity. These results suggest that Nkd acts directly through Dsh to limit Wg activity and thus determines how efficiently Wnt signals stabilize Armadillo (Arm)/ß-catenin and activate downstream genes (Rousset, 2001).

The relationship between Nkd and Zw3 could not be determined by a suppression test because both proteins are negative regulators of Wg. In addition, the subtlety of the nkd phenotype in the eye made this tissue unsuitable for analyzing the epistasis between nkd and zw3. Instead, Zw3/Gsk3ß was overproduced in nkd mutant embryos using genetic and mRNA injection methods: Heat shock promoter (hsp70)-controlled GAL4 was used to drive Zw3 production, or injections with Xenopus gsk3ß mRNA. nkd mutants lack ventral denticle belts and are considerably smaller than wild-type embryos. Overproduction of Gsk3ß or Zw3 in nkd mutants results in partial to almost complete restoration of denticle belts and restoration of more normal embryo size. Because Zw3 restores denticles to nkd mutants, Zw3 cannot act genetically upstream of the defect in nkd mutants (i.e., by stimulating nkd function) in the linear Wg pathway. Nkd therefore is likely to act upstream of, or in a pathway parallel to, Zw3 and downstream from, or at the level of, Dsh (Rousset, 2001).

Cuticles derived from embryos lacking wg activity (wg, dsh, or arm) have nearly continuous fields of denticles, whereas HS-wg embryos, or those mutant for the negative regulator zw3, secrete naked cuticle. Wg misexpression and double-mutant analyses show that Wg acts sequentially through Dsh, Zw3, and Arm. Embryos doubly mutant for wg and zw3 (zw3;wg), as well as zw3;dsh embryos, resemble zw3 embryos, whereas zw3;arm embryos resemble arm embryos, indicating that zw3 acts downstream from dsh and upstream of arm. Mutations in either nkd or zw3 give rise to a naked cuticle phenotype, with posterior expansion of en expression and ectopic wg expression in the developing embryo. However, in contrast to the naked cuticle phenotype of the zw3; wg embryo, the wg;nkd embryo has a wg-like phenotype, indicating a dependence on Wg for the naked cuticle phenotype of nkd mutants (Rousset, 2001 and references therein).

A role for the segment polarity gene shaggy/GSK-3 in the Drosophila circadian clock

Tissue-specific overexpression of the glycogen synthase kinase-3 (GSK-3) ortholog shaggy (sgg) shortens the period of the Drosophila circadian locomotor activity cycle. The short period phenotype has been attributed to premature nuclear translocation of the Period/Timeless heterodimer. Reducing Sgg/GSK-3 activity lengthens period, demonstrating an intrinsic role for the kinase in circadian rhythmicity. Lowered sgg activity decreases Timeless phosphorylation, and GSK-3ß specifically phosphorylates Timeless in vitro. Overexpression of sgg in vivo converts hypophosphorylated Timeless to a hyperphosphorylated protein whose electrophoretic mobility, and light and phosphatase sensitivity, are indistinguishable from the rhythmically produced hyperphosphorylated Timeless of wild-type flies. These results indicate a role for Sgg/GSK-3 in Timeless phosphorylation and in the regulated nuclear translocation of the Period/Timeless heterodimer (Martinek, 2001).

Two independent lines of evidence suggest that sgg regulates the period of molecular cycling primarily through effects on nuclear translocation of the Per/Tim heterodimer: (1) the transition point between delays and advances of the phase response curve, an indicator for nuclear entry of Per/Tim complexes, is advanced by 3 hr in flies overexpressing sgg; (2) nuclear Per is detected ~2 hr earlier in the lateral neurons of larvae overexpressing sgg than in wild-type LNs (Martinek, 2001).

sgg-induced shifts in the timing of nuclear translocation are likely to reflect changes in Tim phosphorylation that are in turn connected to altered levels of Per and Tim. Because Per and Tim are overproduced when sgg activity is low, it is suggested that sgg-dependent Tim phosphorylation accelerates Per/Tim heterodimerization or directly promotes nuclear translocation of Per/Tim complexes in wild-type flies. In this view, decreased Tim phosphorylation in sgg mutants would tend to retard nuclear transfer, and so require higher concentrations of the Per and Tim proteins at times of nuclear entry (Martinek, 2001).

Tim can be directly phosphorylated by GSK-3ß in vitro. Such experiments suggest a mechanism involving direct interaction of Sgg/GSK-3 and Tim in vivo, but do not exclude indirect regulation of Tim phosphorylation by this enzyme in the fly. Nor do these results rule out the involvement of additional protein kinases. For example, a tyrosine-linked phosphorylation of Tim has been implicated in the degradation of Tim by the proteasome. Because Sgg would not be expected to promote tyrosine phosphorylation, this kinase should not regulate all aspects of Tim function (Martinek, 2001).

Sgg/GSK-3 is well known for its central role in Wingless/Wnt signaling. Surprisingly, recent work has indicated that the vertebrate ortholog of Double-time, casein kinase Iepsilon, may also participate in this developmental pathway. For example, in Xenopus, inhibition of casein kinase Iepsilon produces developmental abnormalities closely corresponding to a loss of Wnt function. Casein kinase Iepsilon stabilizes ß-catenin and binds and phosphorylates Dishevelled, both established components of the Wnt signal transduction pathway. It is remarkable that two kinases that function together to provide specific developmental regulation may both act as controlling elements in a patently unrelated behavioral process. This could reflect an underlying synergism between Sgg/GSK-3 and casein kinase 1epsilon. Certainly the activities of both kinases must be integrated at some level for coherent transduction of Wnt signals. Because Dbt and Sgg appear to produce opposing effects on Per/Tim nuclear transfer, with Dbt retarding transfer and Sgg accelerating the process, the relative activities of these kinases could establish an important focus for stabilizing the period of Drosophila's circadian rhythms. For example, a control point composed of offsetting kinase activities might contribute to such homeostatic mechanisms as temperature compensation of the clock. In preliminary work, the effects on circadian rhythmicity of two other elements of the wg signal transduction pathway were examined. A temperature-sensitive allele of wg fails to show any effect on rhythmic locomotor activity, and a heat shock-dishevelled-rescued dsh mutant produces no circadian abnormalities. Thus, sgg's participation in the circadian oscillator may be unrelated to its function in wg signaling (Martinek, 2001).

Wg/Wnt signal can be transmitted through Arrow/LRP5,6 and Axin independently of Zw3/Gsk3ß activity

Activation of the Wnt signaling cascade provides key signals during development and in disease. By designing a Wnt receptor with ligand-independent signaling activity, evidence is provided that physical proximity of Arrow (LRP) to the Wnt receptor Frizzled-2 triggers the intracellular signaling cascade. A branch of the Wnt pathway has been uncovered in which Armadillo activity is regulated concomitantly with the levels of Axin protein. The intracellular pathway bypasses Gsk3ß/Zw3, the kinase normally required for controlling ß-catenin/Armadillo levels, suggesting that modulated degradation of Armadillo is not required for Wnt signaling. It is proposed that Arrow (LRP) recruits Axin to the membrane, and that this interaction leads to Axin degradation. As a consequence, Armadillo is no longer bound by Axin, resulting in nuclear signaling by Armadillo (Tolwinski, 2003).

The data argue for a different regulatory mechanism of Wg signal transduction, proceeding through the inhibition of the protein Axin, rather than through the inhibition of Zw3/GSK3β. Axin has been identified in both vertebrates and invertebrates as a negative component of the pathway. Later work established Axin as a critical scaffold protein required for the assembly and function of the degradation complex. This complex functions in the destruction of Arm/β-catenin by bringing the kinase Zw3 and Arm into close proximity, leading to the phosphorylation of Arm, and thereby targeting it to the proteasome for degradation. For efficient Arm degradation, both Axin and APC must be present in the complex. How Wg input controls activity of the degradation complex has never been properly established, although most models have focused on the inhibition of the kinase Zw3. It is also unclear whether Arm degradation always plays a central role in converting Wnt input into transcriptional responses. In sea urchins and mammals, the most obvious response to Wnt signaling is a relocalization of Arm protein from the cytoplasm to the nucleus; it has been shown that both Axin and APC have a profound effect on Arm localization that cannot be explained by their interaction with Zw3 or the degradation complex alone (Tolwinski, 2003).

Evidence is presented that the Wg signal can be transmitted through a posttranslational regulation of Axin accumulation. Despite uniform transcription of Axin using the UAS/GAL4 system, Axin accumulates to different levels in different cells across each parasegment. Cells with lower steady-state levels of Axin are those exposed to Wg input, and this was strictly dependent on Wg. Loss of Wg causes excess accumulation of Axin, whereas uniform Wg expression (and therefore signaling) lowers total Axin levels. The phenomena observed in embryos parallel earlier reports showing that Axin accumulation is affected by Wnt signaling in tissue culture cells. GSK3β phosphorylation of Axin leads to its stabilization. However, the actual role that phosphorylation plays appears to be more complex, since further work contradicted this finding. In the current experiments, the phosphorylation state of Axin was not examined in cells responding to Wg (those with low Axin levels), nor in those not exposed to Wg (high Axin levels). Therefore, whether modification may inactivate Axin or whether modification leads to removal of Axin by degradation cannot be distinguished. It was found, however, that Zw3 kinase activity is not necessary for the reduction in Axin accumulation that is observed; the Axin striping pattern is maintained in embryos that lack Zw3 function. These results argue for a link between Wg signaling and Axin accumulation that is independent of the Zw3-mediated degradation complex (Tolwinski, 2003).

In summary, Arrow and the Frizzled family of Wnt receptors function in a protein complex that triggers the intracellular signaling cascade. By binding to and causing a reduction in steady-state levels of Axin, Arrow provides a pivotal link between the receptor complex on the cell surface and the downstream events that control Arm activity. One consequence of Axin degradation may reflect its role as a scaffold for Zw3-mediated degradation of Arm. However, because zw3- embryos still respond to Wg input though they fail to degrade Arm, regulation of the degradation complex cannot be the only target of Wg signaling. A Zw3-independent branch in the Wg pathway is proposed, one that might regulate the release of Armadillo from Axin, resulting in nuclear accumulation and signaling (Tolwinski, 2003).

wingless signaling regulates the maintenance of ovarian somatic stem cells in Drosophila

Identifying the signals involved in maintaining stem cells is critical to understanding stem cell biology and to using stem cells in future regenerative medicine. In the Drosophila ovary, Hedgehog is the only known signal for maintaining somatic stem cells (SSCs). Wingless (Wg) signaling is also essential for SSC maintenance in the Drosophila ovary. Wg is expressed in terminal filament and cap cells, a few cells away from SSCs. Downregulation of Wg signaling in SSCs through removal of positive regulators of Wg signaling, dishevelled and armadillo, results in rapid SSC loss. Constitutive Wg signaling in SSCs through the removal of its negative regulators, Axin and shaggy, also causes SSC loss. Also, constitutive wg signaling causes over-proliferation and abnormal differentiation of somatic follicle cells. This work demonstrates that wg signaling regulates SSC maintenance and that its constitutive signaling influences follicle cell proliferation and differentiation. In mammals, constitutive ß-catenin causes over-proliferation and abnormal differentiation of skin cells, resulting in skin cancer formation. Possibly, mechanisms regulating proliferation and differentiation of epithelial cells, including epithelial stem cells, are conserved from Drosophila to man (Song, 2003).

Wg produced from terminal filament and cap cells may reach SSCs at a distance of a few cells by either diffusion or active transport, and then Wg directly controls SSC maintenance. Furthermore, correct intermediate levels of wg signaling seem to be important for maintaining SSCs in the Drosophila ovary. Reduction of wg signaling in SSCs by removal of positive regulators such as arm and dsh causes rapid SSC loss, as does constitutive wg signaling in SSCs by removal of negative regulators such as Axn and sgg. wg signaling maintains SSCs through several possible mechanisms: (1) wg signaling could be required for SSC self-renewal and/or survival; (2) it could maintain the association of SSCs with IGS cells, and/or (3) both mechanisms could work simultaneously. DE-cadherin-mediated cell adhesion has been shown to be important for keeping SSCs in their niche; it also shares arm as a common component with wg signaling. wg signaling is known to regulate levels of arm, which are also important for DE-cadherin-mediated cell adhesion. Thus, it is possible that wg signaling regulates cell adhesion between SSCs and their niches. In addition, arm mutant clonal analysis strongly argues that wg signaling must also directly regulate SSC self-renewal and/or survival. arm2 mutant SSC clones are lost very quickly over time in comparison with wild-type SSC clones, and the arm2 mutation primarily affects wg signaling but does not disrupt DE-cadherin-mediated cell adhesion. Therefore, wg signaling controls SSC maintenance through regulating SSC self-renewal/survival and/or cell adhesion between SSCs and their niche cells. The temperature-sensitive allele of wg gives very mild phenotypes in follicle cell production, however, removal of wg downstream components has a dramatic impact on SSC maintenance. In Drosophila, there are six other wg-related genes. This raises an interesting possibility that other wg-like molecules could also be involved in regulating SSC maintenance (Song, 2003).

In addition to wg signaling, hh signaling is also essential for SSC maintenance and proliferation. Hyperactive hh signaling causes follicle cell over-proliferation and abnormal differentiation of follicle cells. Disrupting hh signaling in SSCs by removing the function of hh downstream components such as Smoothened and Cubitus interruptus results in rapid SSC loss. Similarly, reduction or elimination of wg signaling also causes rapid SSC loss. Removal of patched, a negative regulator of the hh pathway, stabilizes SSCs. However, SSCs mutant for negative regulators for the wg pathway, sgg and Axn, are destabilized. All the evidence indicates that wg and hh may use different mechanisms to regulate SSCs in the Drosophila ovary (Song, 2003).

Constitutive wg signaling increases the division rates of early follicle cell progenitors in the germarium. When Fz2, dsh and activated arm are over-expressed, extra follicle cells accumulate in the ovarioles, suggesting that hyper-activation of wg signaling causes over-proliferation of follicle cells. Furthermore, sgg or Axn mutations cause over-proliferation of follicle cells, resulting in the formation of extra follicle cells that accumulate outside egg chambers. These cells are not mitotically active and usually assume some stalk cell characteristics. These results suggest that production of extra follicle cells by excessive wg signaling is because of higher mitotic activities of progenitors and/or SSCs in the germarium. It is important to note that sgg mutations are more potent than Axn in stimulating the proliferation of follicle cell progenitors. The different potencies may be because of differences in how these mutations affect wg signaling. Alternatively, because sgg negatively regulates hh signaling, sgg could be involved in negatively regulating both hh and wg signaling in the ovary. It has been demonstrated that excessive hh signaling causes extra follicle cells to accumulate outside egg chambers. Therefore, it might be probable that sgg is involved in regulating both hh and wg signaling pathways in follicle cells of the Drosophila ovary (Song, 2003).

This study also demonstrates that constitutive wg signaling disrupts the normal differentiation of somatic follicle cells. Mutant Axn or sgg follicle cells in and outside the germarium express higher levels of Hts in their membranes and tend to accumulate between egg chambers. In ovarioles that contain a majority of mutant follicle cells, germline cysts fail to undergo normal morphological changes necessary for proper encapsulation by follicle cells, although they are wild type, suggesting that the mutant follicle cells are defective in their interactions with germ cells. Although some of them are recruited to egg chambers, these mutant follicle cells have abnormal morphologies (e.g. smaller and irregular sizes). Huli tai shao is present not only on spectrosomes in GSCs, cystoblasts and fusomes in early germline cysts, but also on the membranes of somatic follicle cells. The abnormal follicle cell phenotype may be because of abnormal levels of Hts, which may prevent follicle cells from shape changes and growth. The extra mutant follicle cells accumulating outside egg chambers express Lamin C and do not divide similar to stalk cells. However, unlike stalk cells, they express high levels of Fas3. Similar to the mutant follicle cells in the germarium, the mutant follicle cells that are recruited to egg chambers also express high levels of Hts. Unlike the follicle cells in the germarium, the cells fail to express high levels of Fas3. These results indicate that constitutive wg signaling in follicle cells disrupts proper follicle cell differentiation (Song, 2003).

Trimeric G protein-dependent Frizzled signaling in Drosophila: G proteins act upstream of Dsh, Sgg, and Arm

Frizzled (Fz) proteins are serpentine receptors that transduce critical cellular signals during development. Serpentine receptors usually signal to downstream effectors through an associated trimeric G protein complex. However, clear evidence for the role of trimeric G protein complexes for the Fz family of receptors has hitherto been lacking. This study documents roles for the Galphao subunit (Go) in mediating the two distinct pathways transduced by Fz receptors in Drosophila: the Wnt and planar polarity pathways. Go is required for transduction of both pathways, and epistasis experiments suggest that it is an immediate transducer of Fz. While overexpression effects of the wild-type form are receptor dependent, the activated form (Go-GTP) can signal when the receptor is removed. Thus, Go is likely part of a trimeric G protein complex that directly tranduces Fz signals from the membrane to downstream components (Katanaev, 2005).

The evidence that Go transduces Wg signaling comes from the analysis of Go mutants, from overexpression studies, and from the epistasis experiments. These are addressed in the following discussion (Katanaev, 2005).

Further evidence for the role of Go in transducing Wg comes from the overexpression experiments. When Go is overexpressed in the wing disc, clear upregulation of Wg targets is evident. If Go achieves the upregulation of the target genes by hyperactivating the intracellular Wg transduction machinery, then abrogation of transduction downstream of Go should nullify its effects. To this end, it was shown that the upregulation of Wg targets is arm and dsh dependent and is abolished by overexpression of sgg. Furthermore, Go overexpression in embryos gives gain-of-function wg phenotypes that are arm dependent (Katanaev, 2005).

In arm and dsh clones (and fz, fz2 clones described below), residual Dll expression was sometimes found. This occurs in otherwise wild-type tissues and in both anterior and posterior domains of hh-Gal4; UAS-Go wing discs and is most noticeable with dsh known for strong perdurance. However, arm and dsh clones in the regions of Go overexpression lose Dll expression to a level comparable with clones in which Go is not overexpressed. Thus, it is inferred that the upregulation of Wg targets induced by overexpression of Go requires the Wg transduction pathway utilizing Dsh, Sgg, and Arm (Katanaev, 2005).

Cross-talk between Notch and Wingless pathway

A Drosophila homolog of the serine/threonine kinase GSK-3 beta, encoded by the zeste-white3/shaggy gene (zw3), has been implicated as a maternally provided antagonist of zygotic signaling by the secreted segmentation gene wingless (wg). The wg signal apparently causes a spatially localized inhibition of the ubiquitous repressor function of zw3. This double negative mechanism of signal transduction has been shown to mediate the patterning function of Wg in a number of developmental processes. Although wg is absolutely required for specifying the heart progenitors within the mesoderm of Drosophila, the role of zw3 in this process has been unclear. Evidence is presented that zw3 has a dual role in mesoderm development: (1) zw3 acts as an antagonist in cardiogenic wg signal transduction, and (2) zw3 also seems to be required to promote positively the formation of a larger mesodermal region, the tinman- and dpp-dependent "dorsal mesoderm," which is a prerequisite not only for cardiogenesis, but also for visceral mesoderm formation. A recently identified proximal component of the wg cascade, which is a transcription factor encoded by pangolin/dTCF (dTCF), also seems to mediate wg-dependent cardiogenesis. Evidence is presented that Notch (N), which opposes wg signaling in other situations, is unlikely to be directly involved in the cardiogenic wg pathway, but seems to have a number of other myogenic functions, one of which is to inhibit mesoderm differentiation altogether, when overexpressed as a constitutively active form (Park, 1998).

The Notch receptor triggers a wide range of cell fate choices in higher organisms. In Drosophila, segregation of neural from epidermal lineages results from competition among equivalent cells. These cells express achaete/scute genes, which confer neural potential. During lateral inhibition, a single neural precursor is selected, and neighboring cells are forced to adopt an epidermal fate. Lateral inhibition relies on proteolytic cleavage of Notch induced by the ligand Delta and translocation of the Notch intracellular domain (NICD) to the nuclei of inhibited cells. The activated NICD, interacting with Suppressor of Hairless [Su(H)], stimulates genes of the E(spl) complex, which in turn repress the proneural genes achaete/scute. New alleles of Notch are described that specifically display loss of microchaetae sensory precursors. This phenotype arises from a repression of neural fate, by a Notch signaling distinct from that involved in lateral inhibition. The loss of sensory organs associated with this phenotype results from a constitutive activation of a Deltex-dependent Notch-signaling event. These novel Notch alleles encode truncated receptors lacking the carboxy terminus of the NICD, which is the binding site for the repressor Dishevelled (Dsh). Dsh is known to be involved in crosstalk between Wingless and Notch pathways. These results reveal an antineural activity of Notch distinct from lateral inhibition mediated by Su(H). This activity, mediated by Deltex (Dx), represses neural fate and is antagonized by elements of the Wingless (Wg)-signaling cascade to allow alternative cell fate choices (Raiman, 2001).

In a screen for flies associated with the loss of microchaetae, a number of mutations in Notch were isolated that result in a dominant loss of thoracic microchaetae, which are called NMcd, where Mcd stands for microchaetae defective. These mutations are lethal, and, for this reason, their behavior was analyzed in mosaics in which clones of mutant cells are juxtaposed with wild-type territories. In these mosaics, mutant cells are recognized by the use of both bristle and epidermal markers. All mutants behave genetically in a similar manner, the strongest alleles, NMcd1 and NMcd5 (collectively NMcd1/5), were chosen for further analysis. In clones for NMcd1 and NMcd5, 99% of the microchaetae are absent, whereas macrochaetae are not affected (Raiman, 2001).

Genetic analysis indicates that the dominant effects of the NMcd alleles are due to antagonism of the wild-type function of Notch. The mutant phenotype of NMcd is enhanced when N+ is lowered and is partially suppressed when N+ is increased. Thus, these gain-of-function alleles of Notch do not induce an aberrant function of the receptor (neomorphism), but rather produce receptors that are more active on the normal function of Notch. NAx alleles exhibit a similar genetic behavior and a similar phenotype to the NMcd alleles. However, several differences distinguish NAx from NMcd. The NAx mutant exhibits a variable loss of both thoracic microchaetae and macrochaetae, leading to irregular patterns. In contrast, NMcd affects only microchaetae. Furthermore, the remaining microchaetae of the NMcd/+ flies are arranged in fewer rows, which are organized in a regular pattern. Finally, NAx/+ flies exhibit broader wings with shortened veins. In contrast, the wings of the NMcd/+ flies appear as those of wild-type flies. In this study of the NMcd alleles, focus was placed on the bristle pattern (Raiman, 2001).

Since clones of NMcd cells lack microchaetae, the development of their precursors was examined during pupal stages by means of neural-specific markers. The loss of microchaetae observed in NMcd1/5 is due to the loss of neural cells, as visualized by stainings using the neural-specific antibody 22C10, and to the loss of their precursors, as detected with the reporter neuA101. Since the proneural Ac activity is known to promote the development of the microchaetae precursors, Ac expression was examined in the NMcd mutants. The loss of microchaetae precursors is associated with a severe decrease in Ac expression (Raiman, 2001).

The NMcd phenotype is unlikely to be due to a lack of differentiation of the outer elements of the sensory organs, since 'escaped' microchaetae have a normal morphology. Thus, these results indicate that the NMcdmutations disrupt the early establishment of neural precursors rather than the late lineage that permits the differentiation of the sensory bristle (Raiman, 2001).

Different lines of work have suggested that the existence of Notch-signaling events are independent of the mechanism of lateral inhibition. Some of these experiments suggest that the adaptor protein Deltex (Dx) might be involved in some of these events (Raiman, 2001).

Dx is a cytoplasmic protein that regulates Notch through binding to the ankyrin repeats. Loss-of-function alleles of dx display an excess of microchaetae, whereas overexpression of Dx inhibits neurogenesis. It has been suggested that Dx is involved in a signal transduction event downstream of Notch. Loss-of-function dx alleles behave as dominant suppressors of all the NMcd alleles , and NMcd1/5 dx-clones display a fairly normal microchaetae pattern. The Dx effector, therefore, might represent an essential regulator of the antineural activity revealed by the NMcd receptors (Raiman, 2001).

In contrast, Shaggy, the Drosophila glycogen synthase kinase 3 (GSK3) is a central element in Wingless signal transduction and behaves genetically as a downstream element of the Notch pathway. Mutations in Sgg suppress the effects of NMcd mutants, like mutations in Dx. Altogether, these results indicate that both Dx and Sgg might be involved in the Notch-signaling event that is distinct from lateral inhibition (Raiman, 2001).

Since Achaete/Scute expression is required for the establishment of the neural fate, the novel Notch pathway revealed by the NMcd mutants must be repressed during wild-type neural development. One candidate to exert this repression is Dishevelled (Dsh), a component of the Wingless-signaling cascade, which has been shown to bind Notch and block some of its activities. Using a yeast two-hybrid assay, it has been found that Dsh does bind to the C-terminal 114 amino acids of the NICD that are absent in the truncated receptors. Therefore, the Dx-dependent repressive effect of the NMcd receptors appears as the consequence of the loss of the Dsh binding site (Raiman, 2001).

Therefore, Notch associates in vitro with Dsh through its C-terminal 114 amino acids. In order to test the functional significance of this C-terminal domain of Notch in vivo, the effect of overexpressed Dsh on the development of microchaetae was examined either in wild-type or in NMcd8 flies lacking the Dsh binding site. Flies carrying four copies of a hsp70-Dsh transgene were analyzed. One 15-min heat pulse (37°C) at the onset of pupariation leads to an increase of 5.8% of the number of microchaetae in a wild-type background. In contrast, the pulse has no effect on NMcd8 flies. These experiments suggest that Dsh binds the 114 amino acid C terminus of Notch in vivo to antagonize the Dx-dependent signaling of the receptor. The effects of overexpressed Dsh were examined in Notch mutant-carrying lesions in the extracellular EGF repeats (nd3; spl;Ax9B2; AxE2). In each case, an increase in the number of microchaetae was observed after heat treatment (Raiman, 2001).

Dsh and Dx display antagonistic activities. Overexpressed Dx inhibits neurogenesis, whereas overexpressed Dsh increases the number of microchaetae in wild-type flies. Furthermore, this latter excess of microchaetae is accentuated when the dosage of Dx is lowered (Raiman, 2001).

Potentially, Dsh could exert its repressing effect by modulating the proteasome-dependent proteolysis of Notch or the phosphorylation state versus cytoplasmic/nuclear distribution of the NICD. Interestingly, Dsh contains two proline-rich sequences, PPLP and PPXY, putative binding sites for Su(dx), a cytoplasmic ubiquitin ligase involved in ubiquitinylation/turnover of proteins. When binding to Notch, Dsh could serve as a docking protein for Su(Dx) and could regulate the activity of Dx in targeting the proteasome activity to the C terminus of Notch (Raiman, 2001).

How the Dx-dependent transduction is achieved in the cells is poorly understood. One could speculate that the repressing activity of Dsh may also rely on a direct effect on the Dx-dependent signaling. Thus, Dsh and Dx antagonistically regulate a common target, JNK (JUN N-terminal kinase), and Sgg antagonizes JNK-dependent activation of the JUN transcription factor. dJUN might therefore represent an element mediating the antineural activity of Dx (Raiman, 2001).

The Dx-dependent antineural activity of Notch is regulated by elements of the Wingless-signaling cascade, e.g., the cytoplasmic protein Dsh or the kinase Sgg. Overexpression of Dsh generates extrasensory organs in wild-type flies and fails to elicite ectopic bristles in the NMcdmutants lacking the Dsh binding site. The kinase Sgg is negatively regulated by Dsh in the Wingless-signaling cascade. Dsh and Sgg have opposite effects on the Dx-dependent Notch pathway. Loss-of-function alleles of sgg lead to a constitutive derepression of Wingless signaling and elicit the same number of ectopic bristles in wild-type and NMcd mutant flies (Raiman, 2001).

This analysis of the NMcd mutants supports the idea that Dsh, an effector of the Wingless pathway, directly interacts with Notch in wild-type flies in order to maintain the neural potential. Dsh antagonizes the cytoplasmic activity of Dx and then represses the antineural Dx-dependent function of Notch. In wild-type flies, crosstalks between Wingless and Notch allow stimulation of the ac/sc expression in the equivalent cells of the proneural clusters until a given threshold. It has been reported that Su(H) functions as the core of a molecular switch, acting as a repressor of Notch target genes in the absence of nuclear NICD. Thus, prior to the onset of lateral signaling, the repressive activity of Su(H) is compatible with the activation of ac/sc by the Wingless-dependent pathway. When a given level is reached, ac/sc can activate the Dl gene, and cells can compete with each other for the choice of the neural precursor via lateral signaling. At this stage, the Wg and the Su(H)-dependent Notch signalings have opposite effects on the expression of ac/sc. ac/sc is repressed in the inhibited cells, suggesting that the Su(H)-dependent Notch signaling overrides the Wingless pathway (Raiman, 2001).

Though the NMcd5 allele shares the same loss-of-microchaetae phenotype as other NMcd and affects the same developmental pathway, the NMcd5 mutant receptor carries a single point mutation, leading to the C739Y substitution that disrupts the 18th EGF repeat of the extracellular domain, whereas the other NMcdalleles encode truncated receptors lacking the C terminus of the intracellular domain. Experiments with NMcd5 suggest that the region of the 18th EGF is instrumental for the regulation of alternative Notch signaling. The extracellular EGF domain is known to physically bind Wingless. Further experiments are necessary to determine whether the NMcd5 lesion in the 18th EGF repeat specifically alters the binding of Wingless, Fringe, or other unknown effector(s) (Raiman, 2001).

The present study of NMcd alleles demonstrates that a Deltex-mediated function of Notch represses the proneural activity during establishment of the neural precursors of the thoracic microchaetae. This repressive activity precedes and is distinct from that which mediates lateral inhibition and is constitutively active in NMcd mutants. The NMcd alleles encode truncated receptors that lack the binding domain of the repressor Dishevelled, which is involved in functional interactions between Notch and Wingless signalings. The results suggest a model in which Dishevelled is used to alleviate this initial repressive function of Notch in wild-type development, thereby permitting lateral inhibition to generate the regularly spaced sensory microchaetae. In the absence of ligands or effectors, the repressive function of the Dx-dependent activity of Notch could therefore maintain the cells in an uncommited state. In the presence of effectors like Dsh (Wingless signaling) that repress this antineural activity, cells become competent for further choice between two alternative fates (lateral inhibition). It is proposed that Notch acts during development either as a repressor preventing cell differentiation or as a receptor involved in the choice of cell fate during lateral signaling. This dual function is likely to be regulated in a ligand-dependent manner by crosstalk between the Notch and Wingless pathways. It will be important to find out the different components of this new Dx-dependent repressive cascade of Notch (Raiman, 2001).

Shaggy, the homolog of Glycogen synthase kinase 3, controls neuromuscular junction growth in Drosophila

A protein-trap screen using the Drosophila neuromuscular junction (NMJ) as a model synapse was performed to identify genes that control synaptic structure or plasticity. In this approach, a green fluorescent protein (GFP) exon is inserted within genes, leading to fusion fluorescent proteins. Shaggy (Sgg), the Drosophila homolog of the mammalian glycogen synthase kinases 3alpha and ß, two serine-threonine kinases, was found to be concentrated at this synapse. Using various combinations of mutant alleles of shaggy, it was found that Shaggy negatively controlled the NMJ growth. Moreover, tissue-specific expression of a dominant-negative Sgg indicated that this kinase is required in the motoneuron, but not in the muscle, to control NMJ growth. Finally, it was shown that Sgg controls the microtubule cytoskeleton dynamics in the motoneuron and that Futsch, a microtubule-associated protein, is required for Shaggy function on synaptic growth (Franco, 2004).

Using the mutant sggK22 and a dominant-negative construct of Sgg, it was shown that Shaggy negatively controls the NMJ growth during the larval stages. These data are the first to reveal a role of this kinase in the growth of a differentiated and functional synapse. Using different presynaptic and postsynaptic markers, no obvious defect could be detected in synapse differentiation. However, the sggK22 allele is not a null allele. Thus, it cannot be excluded that shaggy plays other roles than growth control at the NMJ (Franco, 2004).

The function of GSK-3 ß in neuronal development, and notably in synapse differentiation had been studied previously on neuronal cultures using lithium chloride (LiCl) as a GSK-3 ß inhibitor. More synapsin-positive clusters were found along the axons in the presence of LiCl. This last observation was interpreted as an increase in accumulation of synapsin at the synapses (i.e., a modification in the differentiation of synapses). However, this result can be interpreted as an increase in the number of synapses. This in vitro result would then be in accordance with in vivo data at the Drosophila NMJ, where inhibition of Shaggy increases the number of synaptic boutons (Franco, 2004).

Targeted expression of SggDN shows that the function of Shaggy on the growth of the NMJ is required presynaptically and that it requires the kinase function of Sgg. Some major presynaptic targets of GSK-3 ß known in vertebrates are microtubule-associated proteins like tau, MAP1B, and MAP2. When Shaggy is inhibited in the motoneuron, there is an increase in the number of microtubule loops. This change in the dynamics of the microtubule skeleton suggests that some microtubule-associated proteins like the Drosophila tau homolog or the MAP1B homolog, Futsch, may also be substrates of Shaggy. Of interest, consensus sites for GSK-3 like phosphorylation [SXXX(ST)(PR)XXS] could be identified in the sequence of these proteins (96 for Futsch; 1 for tau) but no evidence is yet available that these proteins are phosphorylated by Shaggy. Recently, an effect of LiCl treatment was observed on axonal transport defects resulting from human Tau overexpression in Drosophila larvae, suggesting a functional interaction between Shaggy and Tau. This study demonstrated that the protein Futsch is required for the overgrowth phenotype observed in sgg loss-of-function conditions (Franco, 2004).

How is the activity of Sgg controlled at the NMJ? The kinase Sgg is known to be inhibited in both the insulin and the WNT/Wingless (Wg) signaling pathways. There is little evidence of the presence of insulin-like peptides or insulin receptor-like proteins at the NMJ in Drosophila. Wg has been shown to be released by larval motoneurons in Drosophila and to control the NMJ growth. The control of Sgg activity via different signaling pathways at the NMJ is still an open question (Franco, 2004).

Overexpression screen in Drosophila identifies neuronal roles of GSK-3 beta/shaggy as a regulator of AP-1-dependent developmental plasticity

AP-1, an immediate-early transcription factor comprising heterodimers of the Fos and Jun proteins, has been shown in several animal models, including Drosophila, to control neuronal development and plasticity. In spite of this important role, very little is known about additional proteins that regulate, cooperate with, or are downstream targets of AP-1 in neurons. This paper outlines results from an overexpression/misexpression screen in Drosophila to identify potential regulators of AP-1 function at third instar larval neuromuscular junction (NMJ) synapses. First, >4000 enhancer and promoter (EP) and EPgy2 lines were used to screen a large subset of Drosophila genes for their ability to modify an AP-1-dependent eye-growth phenotype. Of 303 initially identified genes, a set of selection criteria were used to arrive at 25 prioritized genes from the resulting collection of putative interactors. Of these, perturbations in 13 genes result in synaptic phenotypes. Finally, one candidate, the GSK-3α-kinase homolog, shaggy, negatively influences AP-1-dependent synaptic growth, by modulating the Jun-N-terminal kinase pathway, and also regulates presynaptic neurotransmitter release at the larval neuromuscular junction. Other candidates identified in this screen provide a useful starting point to investigate genes that interact with AP-1 in vivo to regulate neuronal development and plasticity (Franciscovich, 2008).

The transcription factor AP-1 is a key regulator of neuronal growth, development, and plasticity, and in addition to cAMP response element binding (CREB) protein, it controls transcriptional responses in neurons during plasticity. Acute inhibition of Fos attenuates learning in mice and in invertebrate models such as Drosophila; AP-1 positively regulates developmental plasticity of motor neurons. Essential to the understanding of AP-1 activity in neurons is the knowledge of other proteins that influence AP-1 function or are downstream transcriptional targets. This study describes a forward genetic screen for modifiers of AP-1 in Drosophila (Franciscovich, 2008).

Using a conveniently scored AP-1-dependent adult-eye phenotype, 4307 EP and EPgy2 lines were screened for genes that modified this phenotype. Several advantages of this screen include: (1) the ease and rapidity of screening as compared to the neuromuscular junction, (2) immediate gene identification, (3) the potential to analyze in vivo phenotypes that arise from overexpression/misexpression, and finally (4) the scope for rapidly generating loss-of-function mutations through imprecise excision of the same P-element. A total of 249 known genes were isolated of which 73 can be directly implicated in eye development. The selection was prioritized using several criteria, to derive a short list of 13 final candidates that were then tested at the NMJ. Future work will focus on other predicted but as yet unstudied genes that are likely to have important functions at the NMJ (Franciscovich, 2008).

The prescreening strategy using the adult eye was successful because (1) almost all the genes selected did not result in eye phenotypes when expressed on their own, but selectively modified a Fbz dependent phenotype (Fbz is a dominant-negative transgenic construct that expresses the Bzip domain of Drosophila Fos); (2) several genes were identified that are known to interact with AP-1 in regulating synaptic phenotypes (these include ras and bsk); (3) multiple alleles of some genes were recovered confirming the sensitivity of the screening technique; (4) several genes involved in eye development were isolated (including cyclinB, which has been shown to be a downstream target of Fos in the regulation of G2/M transition in the developing eye); (5) a large number of putative interactors have connections with neural physiology and/or AP-1 function in other cell types; (6) some candidates with strong phenotypes have previously been shown to play important roles in motor neurons; and finally (7) the majority of candidates (but not all) isolated as enhancers or suppressors of Fbz in the eye exerted a similar effect on AP-1 at the synapse (Franciscovich, 2008).

Although the relative success and merits of a functional screen are considerable, there are a few disadvantages. First, the use of P-element transposons naturally excludes a large fraction of genes that are refractory to P-element transposition events. Second, insertions of EP elements within or in inverse orientation to the gene make it difficult to assign phenotypes to specific genes. Even in instances where overexpression was predicted, it has to be verified that this is indeed the case and also the phenotypes derive from hypomorphic mutations that result from the insertion of the P-element close to the target gene have to be tested. Third, although recover genes that play conserved roles in AP-1 biology is to be expected, those genes that specifically affect synaptic physiology and play no role in the eye will be excluded by this scheme. Finally, this screen will not discriminate between genes that function upstream or downstream of AP-1 in neurons. In spite of these deficiencies, it is believed that candidates identified in this screen provide strong impetus for the investigation of additional factors that are involved in the regulation of synaptic plasticity and development by AP-1 (Franciscovich, 2008).

Following their identification, it was found that several candidates had synaptic functions since several of these genes resulted in significant differences in synaptic size when compared to appropriate controls. This provided the first confirmation of the screening strategy. Next, experiments to determine genetic interaction with AP-1 showed that expression of four genes (pigeon, lbm, Cnx99A, and sty) suppressed the Fbz-dependent small synapse phenotype. Of these, sty had been isolated as an enhancer while the other three similarly suppressed the Fbz-derived eye phenotype, suggesting potentially conserved functions of these genes in the two tissues (Franciscovich, 2008).

Four genes isolated as enhancers, similarly enhanced an Fbz-mediated small synapse (cnk, pde8, fkbp13, and sgg). Notably, expression of these genes also suppressed an AP-1-dependent synapse expansion at the NMJ. These two lines of evidence indicate that these genes are negative regulators of AP-1 function in these neurons. Together with the fact that all four have previously described functions in the nervous system, these observations confirm the validity of the screen and highlight the utility of genetic screens to uncover novel molecular interactions. Further studies will provide a more comprehensive understanding of the interplay between these genes and AP-1 in the regulation of neuronal development and plasticity. For instance, more careful analysis needs to be carried out to discern whether synaptic phenotypes in each of these cases are due to overexpression or potential insertional mutagenesis of specific genes (Franciscovich, 2008).

Although GSK-3β-signaling has been implicated in several neurological disorders such as Alzheimer's disease, it is only recently that neuronal roles for this important kinase have come to light. For instance, several studies have demonstrated the role of GSK-3β in the regulation of long-term potentiation (LTP) in vertebrate hippocampal synapses (Hooper, 2007; Peineau, 2007; Zhu, 2007). In particular, these reports highlight the negative regulatory role of GSK-3β in the induction of LTP or in one case, the switching of long-term depression (LTD) into LTP. Interestingly, LTP induction leads to GSK-3β-inhibition thus precluding LTD induction in the same neurons. In flies, sgg mutations have defects in olfactory habituation, circadian rhythms and synaptic growth. These observations point to a conserved and central role for GSK-3β in neuronal physiology (Franciscovich, 2008).

GSK-3β-dependent modulation of transcriptional responses is widely acknowledged. Among several transcription factors that are known to be regulated by this kinase, are AP-1, CREB, NFAT, c/EBP, and NF-kappaB. In the context of neuronal function, for instance, RNA interference-based experiments in cultured rat cortical neurons have shown that GSK-3β-activity influences CREB and NF-kappaB-dependent transcription. Additionally, two other transcription factors, early growth response 1 and Smad3/4 have been identified in DNA profiling experiments in the same study. Significantly, GSK-3β is also a primary target of lithium, a drug used extensively to treat mood disorders. Lithium treatment has been reported to result in an upregulation of AP-1-dependent transcription, though a role for GSK-3β in this phenomenon has not been tested directly (Franciscovich, 2008).

In Drosophila, recent experiments have described the negative regulation of synaptic growth by the GSK3β-homolog shaggy (Franco, 2004). These studies demonstrate that sgg controls synaptic growth through the phosphorylation of the Drosophila MAP1B homolog futsch. The current studies suggest that Sgg-dependent regulation of synapse size occurs through the immediate-early transcription factor AP-1. GSK-3β is believed to inhibit transcriptional activity of AP-1 in cultured cells by direct inhibitory phosphorylation of c-Jun. Circumstantial evidence also suggests that GSK-3β provides an inhibitory input into AP-1 function in neurons (Franciscovich, 2008).

It was intriguing to find that Sgg inhibition leads to an expanded synapse with reduced presynaptic transmitter release, similar to highwire mutants. Given that in several instances, Sgg-dependent phosphorylation targets a protein for ubiquitination, and that Highwire encodes an E3 ubiquitin ligase, it is conceivable that sgg and hiw function in the same signaling pathway. Consistent with this hypothesis, both hiw and sgg function at the synapse seem to impinge on AP-1-dependent transcription through modulation of the JNK signaling pathway. Considering previous reports of GSK-3β-involvement in multiple signaling cascades, it will be interesting to study how sgg controls multiple aspects of cellular physiology to regulate neural development and plasticity, particularly in the context of brain function and action of widely used drugs such as lithium (Franciscovich, 2008).

Glycogen synthase kinase-3/Shaggy mediates ethanol-induced excitotoxic cell death of Drosophila olfactory neurons

It has long been known that heavy alcohol consumption leads to neuropathology and neuronal death. While the response of neurons to an ethanol insult is strongly influenced by genetic background, the underlying mechanisms are poorly understood. This study shows that even a single intoxicating exposure to ethanol causes non-cell-autonomous apoptotic death specifically of Drosophila olfactory neurons, which is accompanied by a loss of a behavioral response to the smell of ethanol and a blackening of the third antennal segment. The Drosophila homolog of glycogen synthase kinase-3 (GSK-3)β, Shaggy, is required for ethanol-induced apoptosis. Consistent with this requirement, the GSK-3β inhibitor lithium protects against the neurotoxic effects of ethanol, indicating the possibility for pharmacological intervention in cases of alcohol-induced neurodegeneration. Ethanol-induced death of olfactory neurons requires both their neural activity and functional NMDA receptors. This system will allow the investigation of the genetic and molecular basis of ethanol-induced apoptosis in general and provide an understanding of the molecular role of GSK-3β in programmed cell death (French, 2009).

Identification of domains responsible for ubiquitin-dependent degradation of dMyc by glycogen synthase kinase 3beta and casein kinase 1 kinases

Ubiquitin-mediated degradation of dMyc, the Drosophila homologue of the human c-myc proto-oncogene, is regulated in vitro and in vivo by members of the casein kinase 1 (CK1) family and by glycogen synthase kinase 3beta (GSK3beta). Using Drosophila S2 cells, it was demonstrated that CK1alpha promotes dMyc ubiquitination and degradation with a mechanism similar to the one mediated by GSK3beta in vertebrates. Mutation of ck1alpha or ck1epsilon (discs overgrown) or sgg/gsk3beta in Drosophila wing imaginal discs results in the accumulation of dMyc protein, suggesting a physiological role for these kinases in vivo. Analysis of the dMyc amino acid sequence reveals the presence of conserved domains containing potential phosphorylation sites for mitogen kinases, GSK3beta, and members of the CK1 family. Mutations of specific residues within these phosphorylation domains regulate dMyc protein stability and confer resistance to degradation by CK1alpha and GSK3beta kinases. Expression of the dMyc mutants in the compound eye of the adult fly results in a visible defect that is attributed to the effect of dMyc on growth, cell death, and inhibition of ommatidial differentiation (Galletti, 2009).

In vivo downregulation of GSK3β and CK1α or CK1ɛ kinases in wing imaginal discs results in the accumulation of dMyc protein, an effect particularly visible in the hinge and notum regions but not in cells adjacent to the zone of nonproliferative cells (ZNC). Reduction of GSK3β and CK1α activates Wingless (Wg) signaling, which in turns negatively regulates dmyc RNA in the ZNC. This functional relationship might explain the lack of expression of dMyc protein in clones falling in the wing pouch area and in the ZNC. This positional effect also suggests that dMyc activity is regulated by patterning signals active during the development of the wing imaginal discs (Galletti, 2009).

This analysis of the dMyc amino acid sequence uncovered novel conserved domains, which serve as potential phosphorylation substrates for CK1s or GSK3β kinases. Biochemical characterization of these domains indicated that a combination of amino acid substitutions (S201A, S205A, and S207A) in the dMyc-PI sequence produces a protein with a shorter half-life than dMyc-WT. In vivo expression of the dMyc-MPI mutant did not confer the typical ommatidial roughness that is induced by the expression of dMyc-WT. Moreover, expression of dMyc-PI failed to induce apoptosis in the eye imaginal discs, an effect normally associated with dMyc-WT overexpression. In conclusion, the data suggest that dMyc-PI produces a protein that is less stable than dMyc-WT. In vertebrates, phosphorylation of c-Myc on Ser-62 by MAPK/ERK, JNK N-terminal kinase, or CDK4 increases its stability. The dMyc-PI sequence does not contain a bona fide ERK phosphorylation site (PXSP). However, Ser-201 lies in a favorable context for phosphorylation by the ribosomal S6 kinase-p90 RSK. RSK-p90 belongs to a class of Ser/Thr kinases, activated by ERK and insulin signaling, that phosphorylates the S6 protein component of the 40S ribosomal subunit in response to mitogenic stimulation, resulting in enhanced translation. Interestingly, it has been reported that RSK-p90 activation by ERK is capable of switching on mTOR signaling via inactivation of the TSC1/2 complex, suggesting a role for this kinase in protein synthesis and mass accumulation. No evidence for this regulatory mechanism has been described thus far in Drosophila. It is hypothesized that growth factors may stabilize Myc protein, possibly through phosphorylation by the RSK-p90 kinase, and promote ribosomal biogenesis, in accordance with the prominent role played by dMyc in the production of mass and growth regulation (Galletti, 2009).

Biochemical analysis of the protein stability of dMyc-PII, dMyc-PV, and dMyc-AB showed an increased half-life of these mutants compared to dMyc-WT. The sequence within the dMyc-PII domain (S324A-T328A-S330A) contains potential targets for phosphorylation by GSK3β at Ser-324 [324-S/T-XXX-S/T-(PO4)+4], which requires a priming event of phosphorylation at the +4 position (Thr-330). This phosphorylation event also acts as priming for other kinases (i.e., CK1s) and creates an optimum consensus site for phosphorylation by CK1s at Thr-330 [S/T-(PO4)-XX-330-S/T]. This study found that alanine substitutions of amino acids 324, 328, and 330 conferred resistance to dMyc protein degradation upon phosphorylation by the CK1α and GSK3β kinases. These experiments show that mutation of the residues S324, T328, and S330 confers to the dMyc-PII mutant a resistance to degradation mediated by the ubiquitin ligase Ago. Moreover, it was found that dMyc-MPV, which is degraded by CK1α and GSK3β kinases, is somewhat resistant to degradation by Ago, suggesting that CK1α- and GSK3β-mediated phosphorylation of dMyc is not sufficient to induce its degradation by Ago but perhaps by another unknown ubiquitin ligase (Galletti, 2009).

These data also demonstrate that the dMyc-AB plays an important role in the regulation of dMyc protein stability. Mutation of acidic amino acids imparted to dMyc resistance to degradation primed by CK1α and GSK3β kinases. It has been proposed that acidic domains act as docking sites for the CK1 and CK2, enabling proper positioning of the kinases to recognize their substrates. It is speculated that the conserved acidic amino acid stretch in Myc protein helps the binding of CK1 and CK2 kinases and favors Myc phosphorylation. In support of this hypothesis, the dMyc-PV amino acid sequence (residues 405, 407, and 409), located within the AB (amino acids 404 to 414), was found to be highly homologous to the PEST domain of c-Myc (amino acids 226 to 270). This domain was previously demonstrated to be relevant for c-Myc stability and to act as a potential substrate for CK2 phosphorylation. These biochemical data show that mutations of the dMyc-PV and the AB domains confer increased stability to dMyc protein and suggest that the acidic sequence functions similarly to the PEST domain to control dMyc stability. Notably, Ser-407 constitutes an optimum consensus site for phosphorylation by CK2 (S/T-407-XX-D/E). This observation agrees with the hypothesis that in mammals CK2 is involved in the regulation of c-Myc degradation by targeting the PEST domain (Galletti, 2009).

In vivo expression of the stable mutants dMyc-PII, dMyc-PV, and dMyc-AB resulted in a visible eye defect, accompanied by a reduction of the head capsule and a diminution of the number of the ommatidia. This was particularly visible for dMyc-PV and -AB. Cellular analysis of third-instar larvae eye imaginal discs revealed that expression of these mutants induced apoptosis during disc development. Apoptosis was detected not only within the compartment of dMyc expression (cell autonomous) but also in the neighboring cells (non-cell autonomous). This is a well-documented phenomenon and illustrates the role of dMyc in cell competition, where cells expressing high dMyc kill slower-proliferating neighboring cells nonautonomously through an unidentified mechanism (Galletti, 2009).

In conclusion, multiple phosphorylation events may work hierarchically to prime Myc phosphoamino acids for binding by multiple kinases. It is proposed that different kinases respond to a 'phosphorylation code' that is required to properly control Myc protein stability. This code will depend on an upstream program that in turn activates these kinases. The identification of other phosphorylation residues in dMyc will help in drawing a complete map of phosphorylation activities and will elucidate the events necessary for robust regulation of Myc protein stability. For example, it is speculated that components of growth signaling pathways, such as ras or insulin, may influence the activities of different combinations of kinases, thus affecting phosphorylation at different amino acids to control dMyc protein stability. In support of this hypothesis, preliminary data was produced showing that activation of the DILP (for Drosophila insulinlike peptides) pathway increases dMyc protein stability in vivo through the inactivation of GSK3β kinase, suggesting that the metabolic and nutrient pathways affect growth by partially controlling dMyc protein expression (Galletti, 2009).

Regulation of Hedgehog ligand expression by the N-end rule ubiquitin-protein ligase Hyperplastic discs and the Drosophila GSK3β homologue, Shaggy

Hedgehog (Hh) morphogen signalling plays an essential role in tissue development and homeostasis. While much is known about the Hh signal transduction pathway, far less is known about the molecules that regulate the expression of the hedgehog (hh) ligand itself. This study revealed that Shaggy (Sgg), the Drosophila melanogaster orthologue of GSK3β, and the N-end Rule Ubiquitin-protein ligase Hyperplastic Discs (Hyd) act together to co-ordinate Hedgehog signalling through regulating hh ligand expression and Cubitus interruptus (Ci) expression. Increased hh and Ci expression within hyd mutant clones was effectively suppressed by sgg RNAi, placing sgg downstream of hyd. Functionally, sgg RNAi also rescued the adult hyd mutant head phenotype. Consistent with the genetic interactions, Hyd esd found to physically interact with Sgg and Ci. Taken together it iw proposed that Hyd and Sgg function to co-ordinate hh ligand and Ci expression, which in turn influences important developmental signalling pathways during imaginal disc development. These findings are important as tight temporal/spatial regulation of hh ligand expression underlies its important roles in animal development and tissue homeostasis. When deregulated, hh ligand family misexpression underlies numerous human diseases (e.g., colorectal, lung, pancreatic and haematological cancers) and developmental defects (e.g., cyclopia and polydactyly). In summary, these Drosophila-based findings highlight an apical role for Hyd and Sgg in initiating Hedgehog signalling, which could also be evolutionarily conserved in mammals (Moncrieff, 2015).

Ethanol stimulates the in vivo axonal movement of neuropeptide dense core vesicles in Drosophila motor neurons

Proper neuronal function requires essential biological cargoes to be packaged within membranous vesicles and transported, intracellularly, through the extensive outgrowth of axonal and dendritic fibers. The precise spatiotemporal movement of these cargoes is vital for neuronal survival and, thus, is highly regulated. This study tested how the axonal movement of a neuropeptide containing dense core vesicle (DCV) responds to alcohol stressors. Ethanol was found to induce a strong anterograde bias in vesicle movement. Low doses of ethanol stimulate the anterograde movement of neuropeptide-DCV while high doses inhibit bidirectional movement. This process required the presence of functional kinesin-1 motors as reduction of kinesin prevented the ethanol-induced stimulation of the anterograde movement of neuropeptide-DCV. Furthermore, expression of inactive GSK-3beta also prevented ethanol-induced stimulation of neuropeptide-DCV movement, similar to pharmacological inhibition of GSK-3beta with lithium. Conversely, inhibition of PI3K/AKT signaling with wortmannin led to a partial prevention of ethanol-stimulated transport of neuropeptide-DCV. Taken together, it is concluded that GSK-3beta signaling mediates the stimulatory effects of ethanol. Therefore, this study provides new insight into the physiological response of the axonal movement of neuropeptide-DCV to exogenous stressors (Iacobucci, 2017).

>Disordered expression of shaggy, the Drosophila gene encoding a serine-threonine protein kinase GSK3, affects the lifespan in a transcript-, stage-, and tissue-specific manner

GSK3 (glycogen synthase kinase 3) is a conserved protein kinase governing numerous regulatory pathways. In Drosophila melanogaster, GSK3 is encoded by shaggy (sgg), which forms 17 annotated transcripts corresponding to 10 protein isoforms. The goal was to demonstrate how differential sgg transcription affects lifespan, which GSK3 isoforms are important for the nervous system, and which changes in the nervous system accompany accelerated aging. Overexpression of three sgg transcripts affected the lifespan in a stage- and tissue-specific way: sgg-RA and sgg-RO affected the lifespan only when overexpressed in muscles and in embryos, respectively; the essential sgg-RB transcript affected lifespan when overexpressed in all tissues tested. In the nervous system, only sgg-RB overexpression affected lifespan, causing accelerated aging in a neuron-specific way, with the strongest effects in dopaminergic neurons and the weakest effects in GABAergic neurons. Pan-neuronal sgg-RB overexpression violated the properties of the nervous system, including the integrity of neuron bodies; the number, distribution, and structure of mitochondria; cytoskeletal characteristics; and synaptic activity. Such changes observed in young individuals indicated premature aging of their nervous system, which paralleled a decline in survival. These findings demonstrated the key role of GSK3 in ensuring the link between the pathology of neurons and lifespan (Trostnikov, 2019).


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shaggy: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 12 February 2023

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