Dihydroceramide desaturase regulates the compartmentalization of Rac1 for neuronal oxidative stress.

Disruption of sphingolipid homeostasis is known to cause neurological disorders, but the mechanisms by which specific sphingolipid species modulate pathogenesis remain unclear. The last step of de novo sphingolipid synthesis is the conversion of dihydroceramide to ceramide by dihydroceramide desaturase (human DEGS1; Drosophila Ifc). Loss of ifc leads to dihydroceramide accumulation, oxidative stress, and photoreceptor degeneration, whereas human DEGS1 variants are associated with leukodystrophy and neuropathy. In this work, we demonstrate that DEGS1/ifc regulates Rac1 compartmentalization in neuronal cells and that dihydroceramide alters the association of active Rac1 with organelle-mimicking membranes. We further identify the Rac1-NADPH oxidase (NOX) complex as the major cause of reactive oxygen species (ROS) accumulation in ifc-knockout (ifc-KO) photoreceptors and in SH-SY5Y cells with the leukodystrophy-associated DEGS1H132R variant. Suppression of Rac1-NOX activity rescues degeneration of ifc-KO photoreceptors and ameliorates oxidative stress in DEGS1H132R-carrying cells. Therefore, we conclude that DEGS1/ifc deficiency causes dihydroceramide accumulation, resulting in Rac1 mislocalization and NOX-dependent neurodegeneration.

Thyroid hormone receptor beta mutations alter photoreceptor development and function in Danio rerio (zebrafish).

We investigate mutations in trß2, a splice variant of thrb, identifying changes in function, structure, and behavior in larval and adult zebrafish retinas. Two N-terminus CRISPR mutants were identified. The first is a 6BP+1 insertion deletion frameshift resulting in a truncated protein. The second is a 3BP in frame deletion with intact binding domains. ERG recordings of isolated cone signals showed that the 6BP+1 mutants did not respond to red wavelengths of light while the 3BP mutants did respond. 6BP+1 mutants lacked optomotor and optokinetic responses to red/black and green/black contrasts. Both larval and adult 6BP+1 mutants exhibit a loss of red-cone contribution to the ERG and an increase in UV-cone contribution. Transgenic reporters show loss of cone trß2 activation in the 6BP+1 mutant but increase in the density of cones with active blue, green, and UV opsin genes. Antibody reactivity for red-cone LWS1 and LWS2 opsin was absent in the 6BP+1 mutant, as was reactivity for arrestin3a. Our results confirm a critical role for trß2 in long-wavelength cone development.

Self-assembly of FUS through its low-complexity domain contributes to neurodegeneration.

Aggregation of fused in sarcoma (FUS) protein, and mutations in FUS gene, are causative to a range of neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. To gain insights into the molecular mechanism whereby FUS causes neurodegeneration, we generated transgenic Drosophila melanogaster overexpressing human FUS in the photoreceptor neurons, which exhibited mild retinal degeneration. Expression of familial ALS-mutant FUS aggravated the degeneration, which was associated with an increase in cytoplasmic localization of FUS. A carboxy-terminally truncated R495X mutant FUS also was localized in cytoplasm, whereas the degenerative phenotype was diminished. Double expression of R495X and wild-type FUS dramatically exacerbated degeneration, sequestrating wild-type FUS into cytoplasmic aggregates. Notably, replacement of all tyrosine residues within the low-complexity domain, which abolished self-assembly of FUS, completely eliminated the degenerative phenotypes. Taken together, we propose that self-assembly of FUS through its low-complexity domain contributes to FUS-induced neurodegeneration.

Glial lipid droplets and neurodegeneration in a Drosophila model of complex I deficiency.

Mitochondrial defects associated with respiratory chain complex I deficiency lead to heterogeneous fatal syndromes. While the role of NDUFS8, an essential subunit of the core assembly of the complex I, is established in mitochondrial diseases, the mechanisms underlying neuropathology are poorly understood. We developed a Drosophila model of NDUFS8 deficiency by knocking down the expression of its fly homologue in neurons or in glial cells. Downregulating ND23 in neurons resulted in shortened lifespan, and decreased locomotion. Although total brain ATP levels were decreased, histological analysis did not reveal any signs of neurodegeneration except for photoreceptors of the retina. Interestingly, ND23 deficiency-associated phenotypes were rescued by overexpressing the glucose transporter hGluT3 demonstrating that boosting glucose metabolism in neurons was sufficient to bypass altered mitochondrial functions and to confer neuroprotection. We then analyzed the consequences of ND23 knockdown in glial cells. In contrast to neuronal knockdown, loss of ND23 in glia did not lead to significant behavioral defects nor to reduced lifespan, but induced brain degeneration, as visualized by numerous vacuoles found all over the nervous tissue. This phenotype was accompanied by the massive accumulation of lipid droplets at the cortex-neuropile boundaries, suggesting an alteration of lipid metabolism in glia. These results demonstrate that complex I deficiency triggers metabolic alterations both in neurons and glial cells which may contribute to the neuropathology.

Glial overexpression of Dube3a causes seizures and synaptic impairments in Drosophila concomitant with down regulation of the Na+/K+ pump ATPα.

Duplication 15q syndrome (Dup15q) is an autism-associated disorder co-incident with high rates of pediatric epilepsy. Additional copies of the E3 ubiquitin ligase UBE3A are thought to cause Dup15q phenotypes, yet models overexpressing UBE3A in neurons have not recapitulated the epilepsy phenotype. We show that Drosophila endogenously expresses Dube3a (fly UBE3A homolog) in glial cells and neurons, prompting an investigation into the consequences of glial Dube3a overexpression. Here we expand on previous work showing that the Na+/K+ pump ATPα is a direct ubiquitin ligase substrate of Dube3a. A robust seizure-like phenotype was observed in flies overexpressing Dube3a in glial cells, but not neurons. Glial-specific knockdown of ATPα also produced seizure-like behavior, and this phenotype was rescued by simultaneously overexpressing ATPα and Dube3a in glia. Our data provides the basis of a paradigm shift in Dup15q research given that clinical phenotypes have long been assumed to be due to neuronal UBE3A overexpression.

Lipophagy prevents activity-dependent neurodegeneration due to dihydroceramide accumulation in vivo.

Dihydroceramide desaturases are evolutionarily conserved enzymes that convert dihydroceramide (dhCer) to ceramide (Cer). While elevated Cer levels cause neurodegenerative diseases, the neuronal activity of its direct precursor, dhCer, remains unclear. We show that knockout of the fly dhCer desaturase gene, infertile crescent (ifc), results in larval lethality with increased dhCer and decreased Cer levels. Light stimulation leads to ROS increase and apoptotic cell death in ifc-KO photoreceptors, resulting in activity-dependent neurodegeneration. Lipid-containing Atg8/LC3-positive puncta accumulate in ifc-KO photoreceptors, suggesting lipophagy activation. Further enhancing lipophagy reduces lipid droplet accumulation and rescues ifc-KO defects, indicating that lipophagy plays a protective role. Reducing dhCer synthesis prevents photoreceptor degeneration and rescues ifc-KO lethality, while supplementing downstream sphingolipids does not. These results pinpoint that dhCer accumulation is responsible for ifc-KO defects. Human dhCer desaturase rescues ifc-KO larval lethality, and rapamycin reverses defects caused by dhCer accumulation in human neuroblastoma cells, suggesting evolutionarily conserved functions. This study demonstrates a novel requirement for dhCer desaturase in neuronal maintenance in vivo and shows that lipophagy activation prevents activity-dependent degeneration caused by dhCer accumulation.

Neurodegeneration caused by polyglutamine expansion is regulated by P-glycoprotein in Drosophila melanogaster.

Trinucleotide CAG repeat disorders are caused by expansion of polyglutamine (polyQ) domains in certain proteins leading to fatal neurodegenerative disorders and are characterized by accumulation of inclusion bodies in the neurons. Clearance of these inclusion bodies holds the key to improve the disease phenotypes, which affects basic cellular processes such as transcription, protein degradation and cell signaling. In the present study, we show that P-glycoprotein (P-gp), originally identified as a causative agent of multidrug-resistant cancer cells, plays an important role in ameliorating the disease phenotype. Using a Drosophila transgenic strain that expresses a stretch of 127 glutamine repeats, we demonstrate that enhancing P-gp levels reduces eye degeneration caused by expression of polyQ, whereas reducing it increases the severity of the disease. Increase in polyQ inclusion bodies represses the expression of mdr genes, suggesting a functional link between P-gp and polyQ. P-gp up-regulation restores the defects in the actin organization and precise array of the neuronal connections caused by inclusion bodies. ß-Catenin homolog, Armadillo, also interacts with P-gp and regulates the accumulation of inclusion bodies. These results thus show that P-gp and polyQ interact with each other, and changing P-gp levels can directly affect neurodegeneration.

DLin-7 is required in postsynaptic lamina neurons to prevent light-induced photoreceptor degeneration in Drosophila.

Inherited retinal degeneration in humans is caused by mutations in a wide spectrum of genes that regulate photoreceptor development and homeostasis. Many of these genes are structurally and functionally conserved in Drosophila, making the fly eye an ideal system in which to study the cellular and molecular basis of blindness. DLin-7, the ortholog of vertebrate MALS/Veli, is a core component of the evolutionarily conserved Crumbs complex. Mutations in any core member of the Crb complex lead to retinal degeneration in Drosophila. Strikingly, mutations in the human ortholog, CRB1, result in retinitis pigmentosa 12 (RP12) and Leber congenital amaurosis, two severe retinal dystrophies. Unlike Crumbs, DLin-7 is expressed not only in photoreceptor cells but also in postsynaptic lamina neurons. Here, we show that DLin-7 is required in postsynaptic neurons, but not in photoreceptors such as Crumbs, to prevent light-dependent retinal degeneration. At the photoreceptor synapse, DLin-7 acts as part of a conserved DLin-7/CASK/DlgS97 complex required to control the number of capitate projections and active zones, important specializations in the photoreceptor synapse that are essential for proper neurotransmission. These results are the first to demonstrate that a postsynaptically acting protein prevents light-dependent photoreceptor degeneration and describe a novel, Crumbs-independent mechanism for photoreceptor degeneration.

Compartmentalization and Ca2+ buffering are essential for prevention of light-induced retinal degeneration.

Fly photoreceptors are polarized cells, each of which has an extended interface between its cell body and the light-signaling compartment, the rhabdomere. Upon intense illumination, rhabdomeric calcium concentration reaches millimolar levels that would be toxic if Ca(2+) diffusion between the rhabdomere and cell body was not robustly attenuated. Yet, it is not clear how such effective attenuation is obtained. Here we show that Ca(2+) homeostasis in the photoreceptor cell relies on the protein calphotin. This unique protein functions as an immobile Ca(2+) buffer localized along the base of the rhabdomere, separating the signaling compartment from the cell body. Generation and analyses of transgenic Drosophila strains, in which calphotin-expression levels were reduced in a graded manner, showed that moderately reduced calphotin expression impaired Ca(2+) homeostasis while calphotin elimination resulted in severe light-dependent photoreceptor degeneration. Electron microscopy, electrophysiology, and optical methods revealed that the degeneration was rescued by prevention of Ca(2+) overload via overexpression of CalX, the Na(+)-Ca(2+) exchanger. In addition, Ca(2+)-imaging experiments showed that reduced calphotin levels resulted in abnormally fast kinetics of Ca(2+) elevation in photoreceptor cells. Together, the data suggest that calphotin functions as a Ca(2+) buffer; a possibility that we directly demonstrate by expressing calphotin in a heterologous expression system. We propose that calphotin-mediated compartmentalization and Ca(2+) buffering constitute an effective strategy to protect cells from Ca(2+) overload and light-induced degeneration.

Crumbs regulates polarity and prevents light-induced degeneration of the simple eyes of Drosophila, the ocelli.

The evolutionary conserved transmembrane protein Crumbs (Crb) regulates morphogenesis of photoreceptor cells in the compound eye of Drosophila and prevents light-dependent retinal degeneration. Here we examine the role of Crb in the ocelli, the simple eyes of Drosophila. We show that Crb is expressed in ocellar photoreceptor cells, where it defines a stalk membrane apical to the adherens junctions, similar as in photoreceptor cells of the compound eyes. Loss of function of crb disrupts polarity of ocellar photoreceptor cells, and results in mislocalisation of adherens junction proteins. This phenotype is more severe than that observed in mutant photoreceptor cells of the compound eye, and resembles more that of embryonic epithelia lacking crb. Similar as in compound eyes, crb protects ocellar photoreceptors from light induced degeneration, a function that depends on the extracellular portion of the Crb protein. Our data demonstrate that the function of crb in photoreceptor development and homeostasis is conserved in compound eyes and ocelli and underscores the evolutionarily relationship between these visual sense organs of Drosophila. The data will be discussed with respect to the difference in apico-basal organisation of these two cell types.

Cathepsin proteases mediate photoreceptor cell degeneration in Drosophila.

Endocytosis-mediated cell death is a form of degeneration displayed in several Drosophila mutants. This form of degeneration is displayed in several Drosophila mutant lines including flies lacking the eye-specific PLC (norpA). The cell death pathway is initiated by the stabilization of complexes between rhodopsin and arrestin which undergo massive endocytosis into the cell body. The internalized rhodopsin becomes insoluble and builds up in the late endosomal system, wherein it triggers cell death. Cathepsins are resident late endosome/lysosome proteases that have been shown to mediate apoptosis in many disease models. Therefore we sought to test the involvement of cathepsins in endocytosis-mediated retinal degeneration. Here we show that cathepsins mediate cell death in light-exposed norpA eyes. Moreover, we show that the cathepsin L-like cysteine protease, CP1, specifically mediates retinal degeneration, while the aspartyl protease, cathepsin D, does not. Furthermore, eye-specific expression of pan-cathepsin inhibitors also blocks cell death. Western blot analysis demonstrates that cathepsin L levels remain unchanged during retinal degeneration. However, whole mount immunohistochemistry performed on light-exposed retinas revealed a decrease in cathepsin L levels and a loss of rhodopsin/ CP1 colocalization, suggesting that cathepsin L translocates during the degeneration process. Lastly, we show that the retinal degeneration can be enhanced by the overexpression of cathepsin L in the sensitized norpA background. Together these data show that cathepsins play a crucial role in endocytosis-mediated retinal degeneration and are consistent with a model where rhodopsin internalization and accumulation in the endosomal/lysosomal system triggers cathepsin translocation to the cytosol.

Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster.

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.

Expression of human FUS protein in Drosophila leads to progressive neurodegeneration.

Mutations in the Fused in sarcoma/Translated in liposarcoma gene (FUS/TLS, FUS) have been identified among patients with amyotrophic lateral sclerosis (ALS). FUS protein aggregation is a major pathological hallmark of FUS proteinopathy, a group of neurodegenerative diseases characterized by FUS-immunoreactive inclusion bodies. We prepared transgenic Drosophila expressing either the wild type (Wt) or ALS-mutant human FUS protein (hFUS) using the UAS-Gal4 system. When expressing Wt, R524S or P525L mutant FUS in photoreceptors, mushroom bodies (MBs) or motor neurons (MNs), transgenic flies show age-dependent progressive neural damages, including axonal loss in MB neurons, morphological changes and functional impairment in MNs. The transgenic flies expressing the hFUS gene recapitulate key features of FUS proteinopathy, representing the first stable animal model for this group of devastating diseases.

Cystamine and intrabody co-treatment confers additional benefits in a fly model of Huntington's disease.

Huntington's disease (HD) is a lethal, neurodegenerative disorder caused by expansion of the polyglutamine repeat in the Huntingtin gene (HTT), leading to mutant protein misfolding, aggregation, and neuronal death. Feeding a Drosophila HD model cystamine, or expressing a transgene encoding the anti-htt intracellular antibody (intrabody) C4-scFv in the nervous system, demonstrated therapeutic potential, but suppression of pathology was incomplete. We hypothesized that a combinatorial approach entailing drug and intrabody administration could enhance rescue of HD pathology in flies and that timing of treatment would affect outcomes. Feeding cystamine to adult HD flies expressing the intrabody resulted in a significant, additional rescue of photoreceptor neurodegeneration, but no additional benefit in longevity. Feeding cystamine during both larval and adult stages produced the converse result: longevity was significantly improved, but increased photoreceptor survival was not. We conclude that cystamine-intrabody combination therapies can be effective, reducing neurodegeneration and prolonging survival, depending on administration protocols.

Retinoblastoma family protein promotes normal R8-photoreceptor differentiation in the absence of rhinoceros by inhibiting dE2F1 activity.

The retinoblastoma gene Rb is a prototype tumor suppressor which is conserved in Drosophila. Although much is known about the roles of Rb in cell proliferation and apoptosis, much less is known about how Rb regulates cell differentiation. Inactivation of Drosophila Rb (rbf) exhibited subtle differentiation defects similar to inactivation of Rb in mice, suggesting the existence of redundant mechanisms in the control of cell differentiation. To test this possibility and to characterize the role of Rbf in cell differentiation during retinal development, we carried out a genetic screen and identified a mutation in rhinoceros (rno), which leads to synergistic differentiation defects in conjunction with rbf inactivation. Characterization of an early differentiation defect, the multiple-R8 phenotype, revealed that this phenotype was caused by limiting amounts of Notch signaling due to reduced expression of the Notch ligand, Delta (Dl). Decreasing the gene dosage of Dl enhanced the multiple-R8 phenotype, while increasing the level of Dl suppressed this phenotype. Interestingly, removal of the transcriptional activation of dE2F1 partially restores Dl expression in rbf,rno mutant clones and suppresses the associated differentiation defects, indicating that this differentiation function of RBF is mediated by its regulation of dE2F1 activity.

Central projections of photoreceptor axons originating from ectopic eyes in Drosophila.

Ectopic expression of the retinal determination gene eyeless (ey) induces the formation of supernumerary eyes on antennae, legs, wings, and halteres. These ectopic eyes form ommatidia that contain photoreceptors and accessory cells and respond to light. Here, we demonstrate that ectopic eyes on antennae and legs extend axonal projections to the central nervous system. Furthermore, electroretinograms and morphological evidence indicate that the photoreceptor axons of at least the antennal ectopic eyes can form completely constituted ectopic synapses with foreign postsynaptic elements and suggest that transmission at these sites may be functional. However, the ectopic axons do not connect to their correct optic lobe targets and do not project deeply into the neuropile, but rather form synapses at superficial positions in the neuropils. By means of confocal and electron microscopy we show that these ectopic synapses resemble normal synapses, albeit with some distinct morphological differences. Our data strongly suggest that the developmental programs controlling photoreceptor synaptogenesis and visual map formation depend to a considerable extent on presynaptic and thus photoreceptor-autonomous steps. Our data also suggest that photoreceptor axon projections and the establishment of the highly stereotypical neural circuitry in the optic lobe, the normal target neuropil, may depend on target-specific cues that appear to be absent from the antennal lobe and thoracic ganglion.

Catalase, Bax and p53 expression in the visual system of the crab Ucides cordatus following exposure to ultraviolet radiation.

In invertebrates, a few studies have suggested apoptosis as the mechanism of choice to protect the retina after exposure to ultraviolet (UV) radiation. We demonstrated previously, by electron microscopy, that the retina and lamina ganglionaris (or lamina) cells of the crab Ucides cordatus displayed subcellular signs of apoptosis after exposure to UVB and UVC. Here, we first ascertained, by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) technique, that UV irradiation indeed produced the previously reported results. We next tested, in the visual system of U. cordatus, whether the expression (as analyzed by immunohistochemistry and observed with laser scanning microscopy) and levels (as examined by Western blotting) of catalase, Bax, and p53 were affected by the same dose of UV radiation as that used previously. Our data revealed that the intensity of catalase, Bax, and p53 labeling was stronger in irradiated retina and lamina cells than in non-irradiated retina and lamina. However, no significant difference was observed in the concentrations of these proteins isolated from the whole optic lobe. The results thus suggest that UVB and UVC induce apoptosis in the crustacean retina and lamina by increasing catalase expression and activating the Bax- and p53-mediated apoptosis pathways.

CHIP protects from the neurotoxicity of expanded and wild-type ataxin-1 and promotes their ubiquitination and degradation.

CHIP (C terminus of Hsc-70 interacting protein) is an E3 ligase that links the protein folding machinery with the ubiquitin-proteasome system and has been implicated in disorders characterized by protein misfolding and aggregation. Here we investigate the role of CHIP in protecting from ataxin-1-induced neurodegeneration. Ataxin-1 is a polyglutamine protein whose expansion causes spinocerebellar ataxia type-1 (SCA1) and triggers the formation of nuclear inclusions (NIs). We find that CHIP and ataxin-1 proteins directly interact and co-localize in NIs both in cell culture and SCA1 postmortem neurons. CHIP promotes ubiquitination of expanded ataxin-1 both in vitro and in cell culture. The Hsp70 chaperone increases CHIP-mediated ubiquitination of ataxin-1 in vitro, and the tetratricopeptide repeat domain, which mediates CHIP interactions with chaperones, is required for ataxin-1 ubitiquination in cell culture. Interestingly, CHIP also interacts with and ubiquitinates unexpanded ataxin-1. Overexpression of CHIP in a Drosophila model of SCA1 decreases the protein steady-state levels of both expanded and unexpanded ataxin-1 and suppresses their toxicity. Finally we investigate the ability of CHIP to protect against toxicity caused by expanded polyglutamine tracts in different protein contexts. We find that CHIP is not effective in suppressing the toxicity caused by a bare 127Q tract with only a short hemagglutinin tag, but it is very efficient in suppressing toxicity caused by a 128Q tract in the context of an N-terminal huntingtin backbone. These data underscore the importance of the protein framework for modulating the effects of polyglutamine-induced neurodegeneration.

A rapid cellular FRET assay of polyglutamine aggregation identifies a novel inhibitor.

Many neurodegenerative diseases, including tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, and the polyglutamine diseases, are characterized by intracellular aggregation of pathogenic proteins. It is difficult to study modifiers of this process in intact cells in a high-throughput and quantitative manner, although this could facilitate molecular insights into disease pathogenesis. Here we introduce a high-throughput assay to measure intracellular polyglutamine protein aggregation using fluorescence resonance energy transfer (FRET). We screened over 2800 biologically active small molecules for inhibitory activity and have characterized one lead compound in detail. Y-27632, an inhibitor of the Rho-associated kinase p160ROCK, diminished polyglutamine protein aggregation (EC(50) congruent with 5 microM) and reduced neurodegeneration in a Drosophila model of polyglutamine disease. This establishes a novel high-throughput approach to study protein misfolding and aggregation associated with neurodegenerative diseases and implicates a signaling pathway of previously unrecognized importance in polyglutamine protein processing.

Splits ends is a tissue/promoter specific regulator of Wingless signaling.

Wingless directs many developmental processes in Drosophila by regulating expression of specific target genes through a conserved signaling pathway. Although many nuclear factors have been implicated in mediating Wingless-induced transcription, the mechanism of how Wingless regulates different targets in different tissues remains poorly understood. We report here that the split ends gene is required for Wingless signaling in the eye, wing and leg imaginal discs. Expression of a dominant-negative version of split ends resulted in more dramatic reductions in Wingless signaling than split ends-null alleles, suggesting that it may have a redundant partner. However, removal of split ends or expression of the dominant-negative had no effect on several Wingless signaling readouts in the embryo. The expression pattern of Split ends cannot explain this tissue-specific requirement, as the protein is predominantly nuclear and present throughout embryogenesis and larval tissues. Consistent with its nuclear location, the split ends dominant-negative acts downstream of Armadillo stabilization. Our data indicate that Split ends is an important positive regulator of Wingless signaling in larval tissues. However, it has no detectable role in the embryonic Wingless pathway, suggesting that it is a tissue or promoter-specific factor.