Rough Eye Research Articles

Drosophila Mcm10 Is Required for DNA Replication and Differentiation in the Compound Eye

Mini chromosome maintenance 10 (Mcm10) is an essential protein, which is conserved from S. cerevisiae to Drosophila and human, and is required for the initiation of DNA replication. Knockdown of Drosophila Mcm10 (dMcm10) by RNA interference in eye imaginal discs induces abnormal eye morphology (rough eye phenotype), and the number of ommatidia is decreased in adult eyes. We also observed a delay in the S phase and M phase in eye discs of dMcm10 knockdown fly lines. These results show important roles for dMcm10 in the progression of S and M phases. Furthermore, genome damage and apoptosis were induced by dMcm10 knockdown in eye imaginal discs. Surprisingly, when we used deadpan-lacZ and klingon-lacZ enhancer trap lines to monitor the photoreceptor cells in eye discs, knockdown of dMcm10 by the GMR-GAL4 driver reduced the signals of R7 photoreceptor cells. These data suggest an involvement of dMcm10 in R7 cell differentiation. This involvement appears to be independent of the apoptosis induced by dMcm10 knockdown. Together, these results suggest that dMcm10 knockdown has an effect on DNA replication and R7 cell differentiation.

Interactions between Tau and α-synuclein augment neurotoxicity in a Drosophila model of Parkinson's disease

Clinical and pathological studies have suggested considerable overlap between tauopathies and synucleinopathies. Several genome-wide association studies have identified alpha-Synuclein (SNCA) and Tau (MAPT) polymorphisms as common risk factors for sporadic Parkinson's disease (PD). However, the mechanisms by which subtle variations in the expression of wild-type SNCA and MAPT influence risk for PD and the underlying cellular events that effect neurotoxicity remain unclear. To examine causes of neurotoxicity associated with the α-Syn/Tau interaction, we used the fruit fly as a model. We utilized misexpression paradigms in three different tissues to probe the α-Syn/Tau interaction: the retina, dopaminergic neurons and the larval neuromuscular junction. Misexpression of Tau and α-Syn enhanced a rough eye phenotype and loss of dopaminergic neurons in fly tauopathy and synucleinopathy models, respectively. Our findings suggest that interactions between α-Syn and Tau at the cellular level cause disruption of cytoskeletal organization, axonal transport defects and aberrant synaptic organization that contribute to neuronal dysfunction and death associated with sporadic PD. α-Syn did not alter levels of Tau phosphorylated at the AT8 epitope. However, α-Syn and Tau colocalized in ubiquitin-positive aggregates in eye imaginal discs. The presence of Tau also led to an increase in urea soluble α-Syn. Our findings have important implications in understanding the cellular and molecular mechanisms underlying α-Syn/Tau-mediated synaptic dysfunction, which likely arise in the early asymptomatic phase of sporadic PD.

Lack of evidence of the interaction of the Aß peptide with the Wnt signaling cascade in Drosophila models of Alzheimer¿s disease

BackgroundAlzheimer's disease (AD) is the leading form of dementia worldwide. The Aβ-peptide is believed to be the major pathogenic compound of the disease. Since several years it is hypothesized that Aβ impacts the Wnt signaling cascade and therefore activation of this signaling pathway is proposed to rescue the neurotoxic effect of Aβ.FindingsExpression of the human Aβ42 in the Drosophila nervous system leads to a drastically shortened life span. We found that the action of Aβ42 specifically in the glutamatergic motoneurons is responsible for the reduced survival. However, we find that the morphology of the glutamatergic larval neuromuscular junctions, which are widely used as the model for mammalian central nervous system synapses, is not affected by Aβ42 expression. We furthermore demonstrate that genetic activation of the Wnt signal transduction pathway in the nervous system is not able to rescue the shortened life span or a rough eye phenotype in Drosophila.ConclusionsOur data confirm that the life span is a useful readout of Aβ42 induced neurotoxicity in Drosophila; the neuromuscular junction seems however not to be an appropriate model to study AD in flies. Additionally, our results challenge the hypothesis that Wnt signaling might be implicated in Aβ42 toxicity and might serve as a drug target against AD.

Drosophila Dyrk2 Plays a Role in the Development of the Visual System

The DYRKs (dual-specificity tyrosine phosphorylation-regulated kinases) are a conserved family of protein kinases that are associated with a number of neurological disorders, but whose biological targets are poorly understood. Drosophila encodes three Dyrks: minibrain/Dyrk1A, DmDyrk2, and DmDyrk3. Here we describe the creation and characterization of a DmDyrk2 null allele, DmDyrk21w17. We provide evidence that the smell impaired allele smi35A1, is likely to encode DmDyrk2. We also demonstrate that DmDyrk2 is expressed late in the developing third antennal segment, an anatomical structure associated with smell. In addition, we find that DmDyrk2 is expressed in the morphogenetic furrow of the developing eye, that loss of DmDyrk2 in the eye produced a subtle but measurable defect, and that ectopic DmDyrk2 expression in the eye produced a strong rough eye phenotype characterized by increased secondary, tertiary and bristle interommatidial cells. This phenotype was dependent on DmDyrk2 kinase activity and was only manifest when expressed in post-mitotic non-neuronal progenitors. Together, these data indicate that DmDyrk2 is expressed in developing sensory systems, that it is required for the development of the visual system, and that the eye is a good model to identify DmDyrk2 targets.

The role of N‐Linked glycosylation during Drosophila development

N‐linked glycosylation is a key post‐translational modification for many secretory pathway targeted proteins. Endoplasmic reticulum glycosyltranferases construct a 14‐sugar precursor on a dolichol carrier, which is transferred to protein consensus sites. alg9 and alg10 N‐glycosylation mutants were used to examine the role of this modification during Drosophila development. alg10 encodes the enzyme catalyzing terminal glucose addition to the sugar‐precursor prior to transfer, while alg9 encodes an enzyme acting five steps earlier. In embryos, loss of alg9 and alg10 caused severe and pleotrophic defects. Central nervous system (CNS) neurons were specified in both alg9 and alg10 embryos. alg10 loss disrupted axon pathfinding, while alg9 embryos lacked mature neurons. Drosophila eye development in the absence of alg9 and alg10 yielded small rough adult eyes, but alg9 eyes were more severe. Examination of molecular markers in alg9 and alg10 late 3rd instar eye imaginal discs suggested adult small rough adult eyes might be due to neuronal apoptosis. These eye discs also showed defects in axon pathfinding, as shown by the loss of Bolwig's nerve and disrupted axon tracks. These results suggest loss of alg10 may disrupt the maturation of a subset of N‐glycoproteins, since alg9, which acts earlier, has more severe developmental defects. Funding sources: UD Undergrad Research Program, NSF and HHMI

DNF-YB plays dual roles in cell death and cell differentiation during Drosophila eye development

Nuclear transcription factor Y (NF-Y) is well characterized in eukaryotes. It consists of three different subunits, NF-YA, NF-YB and NF-YC, all of which are required for formation of the NF-Y complex and DNA-binding. There is a high homology in NF-YB among Drosophila species with 75% identity and 95% similarity overall, especially in the histone-fold motif (HFM) (95% identity and 100% similarity). In the present study, specific knockdown of Drosophila NF-YB (dNF-YB) in eye imaginal discs induced a rough eye phenotype in adults and this phenotype was the result of induction of caspase-dependent apoptosis followed by apoptosis-induced proliferation. Furthermore, knockdown specifically inhibited R7 photoreceptor cell differentiation, independent of the apoptotic function. dNF-YB and dNF-YA indeed form complexes in vivo where they impair R7 photoreceptor cell differentiation by down regulating the mitogen-activated protein kinase (MAPK) pathway. Expression of the sev gene, or the D-raf gene, a downstream component of the MAPK cascade, could rescue the rough eye phenotype and the loss of R7 signals in dNF-YB knockdown flies. The death executioner Bcl-2 (debcl) is the homolog of Bcl-2 in Drosophila melanogaster and its promoter contains four dNF-Y-binding consensus sequences which play positive roles in promoter activity. In chromatin immunoprecipitation assays with anti-dNF-YB antibody and S2 cells, the debcl gene promoter region containing the NF-Y consensus was effectively amplified in immunoprecipitates by polymerase chain reaction. Taken together, these results indicate that dNF-Y regulates debcl gene expression.

Overexpression of Drosophila Rfx3 Induces Apoptosis and Interferes with Differentiation of Photoreceptor Cells

Regulatory Factor X (RFX) is a protein containing a characteristic DNA binding domain that is called RFX domain. Members of the RFX family are known to be present in human, mouse, Drosophila, Caenorhabditis elegans, Schizosaccharomyces pombe and Saccharomyces cerevisiae. Two RFX proteins, Drosophila RFX (dRFX) and dRFX2 have been identified in Drosophila so far. It is known that dRFX is involved in the differentiation of central and peripheral nerves, while dRFX2 is essential to the cell cycle progression and it may be involved in regulation of apoptosis. A novel protein, dRFX3 carrying RFX domain was identified by a Drosophila genome database search. dRFX3 is a likely homolog to the human RFX5, because of a significant sequence similarity in RFX domains between these two proteins. Two lines of transgenic fly carrying the HA-dRFX3 cDNA were established. Adult flies expressing dRFX3 specifically in the eye and the wing imaginal discs exhibited severe rough eye and atrophied wing phenotypes, respectively. 5-Bromo-2’-deoxyuridine incorporation assays and the immunological detection using anti-Cyclin B antibody indicated that the overexpression of dRFX3 in the eye imaginal discs exerted no effect on the cell cycle progressions. On the other hand, overexpression of dRFX3 in the eye imaginal discs interfered with the differentiation of R2/R5 photoreceptor cells and also induced apoptosis. These data suggest that the dRFX3 plays a negative role for the differentiation of R2/R5 photoreceptor cells and plays a positive role in the regulation of apoptosis.

Genetic Link between Heme Oxygenase and the Signaling Pathway of DNA Damage in<i> Drosophila</i> <i>Melanogaster</i>

Heme oxygenase (HO) is a rate-limiting step of heme degradation, which catalyzes the conversion of heme into biliverdin, iron, and CO. HO has been characterized in microorganisms, insects, plants, and mammals. The mammalian enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli. The present study reports that eye imaginal disc-specific knockdown of the Drosophila HO homologue (dHO) conferred serious abnormal eye morphology in adults, resulting in the generation of reactive oxygen species and apoptosis in third-instar larvae. Oxidative stress frequently induces DNA lesions that are recognized by damage sensors, including ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and rad3-related (ATR) proteins. The knockdown of dHO took place in G0/G1-arrested cells posterior to the morphogenetic furrow and thus prevented these cells from entering S-phase, with an increase in the level of histone H2A.V, a DNA damage marker. Moreover, the knockdown of dHO resulted in the enhancement of the rough eye phenotype in ATM-deficient flies or was lethal in ATR-deficient flies. These results indicate that dHO functions in control of the signal pathway of DNA damage. On the other hand, genetic crosses with a collection of Drosophila deficiency stocks allowed us to identify eight genomic regions, each deletion of which caused suppression of the rough eye phenotype induced by dHO knockdown. This information should facilitate the identification of HO regulators in Drosophila and clarification of the roles of HO in eye development.

Parvin Overexpression Uncovers Tissue-Specific Genetic Pathways and Disrupts F-Actin to Induce Apoptosis in the Developing Epithelia in Drosophila

Parvin is a putative F-actin binding protein important for integrin-mediated cell adhesion. Here we used overexpression of Drosophila Parvin to uncover its functions in different tissues in vivo. Parvin overexpression caused major defects reminiscent of metastatic cancer cells in developing epithelia, including apoptosis, alterations in cell shape, basal extrusion and invasion. These defects were closely correlated with abnormalities in the organization of F-actin at the basal epithelial surface and of integrin-matrix adhesion sites. In wing epithelium, overexpressed Parvin triggered increased Rho1 protein levels, predominantly at the basal side, whereas in the developing eye it caused a rough eye phenotype and severely disrupted F-actin filaments at the retina floor of pigment cells. We identified genes that suppressed these Parvin-induced dominant effects, depending on the cell type. Co-expression of both ILK and the apoptosis inhibitor DIAP1 blocked Parvin-induced lethality and apoptosis and partially ameliorated cell delamination in epithelia, but did not rescue the elevated Rho1 levels, the abnormal organization of F-actin in the wing and the assembly of integrin-matrix adhesion sites. The rough eye phenotype was suppressed by coexpression of either PTEN or Wech, or by knock-down of Xrp1. Two main conclusions can be drawn from our studies: (1), high levels of cytoplasmic Parvin are toxic in epithelial cells; (2) Parvin in a dose dependent manner affects the organization of actin cytoskeleton in both wing and eye epithelia, independently of its role as a structural component of the ILK-PINCH-Parvin complex that mediates the integrin-actin link. Thus, distinct genetic interactions of Parvin occur in different cell types and second site modifier screens are required to uncover such genetic circuits.

Role of S5b/PSMD5 in Proteasome Inhibition Caused by TNF-α/NFκB in Higher Eukaryotes

The ubiquitin-proteasome system is essential for maintaining protein homeostasis. However, proteasome dysregulation in chronic diseases is poorly understood. Through genome-wide cell-based screening using 5,500 cDNAs, a signaling pathway leading to NFκB activation was selected as an inhibitor of 26S proteasome. TNF-α increased S5b (HGNC symbol PSMD5; hereafter S5b/PSMD5) expression via NFκB, and the surplus S5b/PSMD5 directly inhibited 26S proteasome assembly and activity. Downregulation of S5b/PSMD5 abolished TNF-α-induced proteasome inhibition. TNF-α enhanced the interaction of S5b/PSMD5 with S7/PSMC2 in nonproteasome complexes, and interference of this interaction rescued TNF-α-induced proteasome inhibition. Transgenic mice expressing S5b/PSMD5 exhibited a reduced life span and premature onset of aging-related phenotypes, including reduced proteasome activity in their tissues. Conversely, S5b/PSMD5 deficiency in Drosophila melanogaster ameliorated the tau rough eye phenotype, enhanced proteasome activity, and extended the life span of tau flies. These results reveal the critical role of S5b/PSMD5 in negative regulation of proteasome by TNF-α/NFκB and provide insights into proteasome inhibition in human disease.

Overexpression of ubiquitin carboxyl terminal hydrolase impairs multiple pathways during eye development in Drosophila melanogaster

UCH-L1 (ubiquitin carboxyl terminal hydrolase L1) is well known as an enzyme that hydrolyzes polyubiquitin at its C-terminal to release ubiquitin monomers. Although the overexpression of UCH-L1 inhibits proteasome activity in cultured cells, its biological significance in living organisms has not been clarified in detail. Here, we utilized Drosophila as a model system to examine the effects of the overexpression of dUCH, a Drosophila homologue of UCH-L1, on development. Overexpression in the eye imaginal discs induced a rough eye phenotype in the adult, at least partly resulting from the induction of caspase-dependent apoptosis followed by compensatory proliferation. Genetic crosses with enhancer trap lines marking the photoreceptor cells also revealed that the overexpression of dUCH specifically impaired R7 photoreceptor cell differentiation with a reduction in activated extracellular-signal-regulated kinase signals. Furthermore, the dUCH-induced rough eye phenotype was rescued by co-expression of the sevenless gene or the Draf gene, a downstream component of the mitogen-activated protein kinase (MAPK) cascade. These results indicate that the overexpression of dUCH impairs R7 photoreceptor cell differentiation by down-regulating the MAPK pathway. Interestingly, this process appears to be independent of its pro-apoptotic function.

Neuropathogenic role of adenylate kinase-1 in Aβ-mediated tau phosphorylation via AMPK and GSK3β

Abnormally hyperphosphorylated tau is often caused by tau kinases, such as GSK3β and Cdk5. Such occurrence leads to neurofibrillary tangle formation and neuronal degeneration in tauopathy, including Alzheimer's disease (AD). However, little is known about the signaling cascade underlying the pathologic phosphorylation of tau by Aβ(42). In this study, we show that adenylate kinase 1 (AK1) is a novel regulator of abnormal tau phosphorylation. AK1 expression is markedly increased in the brains of AD patients and AD model mice and is significantly induced by Aβ(42) in the primary neurons. Ectopic expression of AK1 alone augments the pathologic phosphorylation of tau at PHF1, CP13 and AT180 epitopes and enhances the formation of tau aggregates. Inversely, downregulation of AK1 alleviates Aβ(42)-induced hyperphosphorylation of tau. AK1 plays a role in Aβ(42)-induced impairment of AMPK activity and GSK3β activation in the primary neurons. Pharmacologic studies show that treatment with an AMPK inhibitor activates GSK3β, and a GSK3β inhibitor attenuates AK1-mediated tau phosphorylation. In a Drosophila model of human tauopathy, the retinal expression of human AK1 severely exacerbates rough eye phenotype and increases abnormal tau phosphorylation. Further, neural expression of AK1 reduces the lifespan of tau transgenic files. Taken together, these observations indicate that the neuronal expression of AK1 is induced by Aβ(42) to increase abnormal tau phosphorylation via AMPK-GSK3β and contributes to tau-mediated neurodegeneration, providing a new upstream modulator of GSK3β in the pathologic phosphorylation of tau.

Effect ofαB-Crystallin on Protein Aggregation inDrosophila

Disorganisation and aggregation of proteins containing expanded polyglutamine (polyQ) repeats, or ectopic expression of α-synuclein, underlie neurodegenerative diseases including Alzheimer's, Parkinson, Huntington, Creutzfeldt diseases. Small heat-shock proteins, such as αB-crystallin, act as chaperones to prevent protein aggregation and play a key role in the prevention of such protein disorganisation diseases. In this study, we have explored the potential for chaperone activity of αB-crystallin to suppress the formation of protein aggregates. We tested the ability of αB-crystallin to suppress the aggregation of a polyQ protein and α-synuclein in Drosophila. We found that αB-crystallin suppresses both the compound eye degeneration induced by polyQ and the α-synuclein-induced rough eye phenotype. Furthermore, by using histochemical staining we have determined that αB-crystallin inhibits the aggregation of polyQ in vivo. These data provide a clue for the development of therapeutics for neurodegenerative diseases.

Eye Detection Based-on Color and Shape Features

This paper presents an eye detection technique based-on color and shape features. The approach consists of three steps: a rough eye localization using projection technique, a white color thresholding to extract white sclera, and an ellipse fitting to fit the ellipse shape of eye. The proposed white color thresholding utilizes the normalized RGB chromaticty diagram, where white color objects are bounded by a small circle on the diagram. Experimental result shows that the proposed technique achieves a high eye detection rate of 91%.

Building an ommatidium one cell at a time

Since the discovery of a single white-eyed male in a population of red eyed flies over 100 years ago (Morgan, 1910), the compound eye of the fruit fly, Drosophila melanogaster, has been a favorite experimental system for identifying genes that regulate various aspects of development. For example, a fair amount of what we know today about enzymatic pathways and vesicular transport is due to the discovery and subsequent characterization of eye color mutants such as white. Likewise, our present day understanding of organogenesis has been aided considerably by studies of mutations, such as eyeless, that either reduce or eliminate the compound eyes. But by far the phenotype that has provided levers into the greatest number of experimental fields has been the humble "rough" eye. The fly eye is composed of several hundred unit-eyes that are also called ommatidia. These unit eyes are packed into a hexagonal array of remarkable precision. The structure of the eye is so precise that it has been compared with that of a crystal (Ready et al., 1976). Even the slightest perturbations to the structure of the ommatidium can be visually detected by light or electron microscopy. The cause for this is two-fold: (1) any defect that affects the hexagonal geometry of a single ommatidium can and will disrupt the positioning of surrounding unit eyes thereby propagating structural flaws and (2) disruptions in genes that govern the development of even a single cell within an ommatidium will affect all unit eyes. In both cases, the effect is the visual magnification of even the smallest imperfection. Studies of rough eye mutants have provided key insights into the areas of cell fate specification, lateral inhibition, signal transduction, transcription factor networks, planar cell polarity, cell proliferation, and programmed cell death just to name a few. This review will attempt to summarize the key steps that are required to assemble each ommatidium.

Drosophila Ribosomal Protein Mutants Control Tissue Growth Non-Autonomously via Effects on the Prothoracic Gland and Ecdysone

The ribosome is critical for all aspects of cell growth due to its essential role in protein synthesis. Paradoxically, many Ribosomal proteins (Rps) act as tumour suppressors in Drosophila and vertebrates. To examine how reductions in Rps could lead to tissue overgrowth, we took advantage of the observation that an RpS6 mutant dominantly suppresses the small rough eye phenotype in a cyclin E hypomorphic mutant (cycEJP). We demonstrated that the suppression of cycEJP by the RpS6 mutant is not a consequence of restoring CycE protein levels or activity in the eye imaginal tissue. Rather, the use of UAS-RpS6 RNAi transgenics revealed that the suppression of cycEJP is exerted via a mechanism extrinsic to the eye, whereby reduced Rp levels in the prothoracic gland decreases the activity of ecdysone, the steroid hormone, delaying developmental timing and hence allowing time for tissue and organ overgrowth. These data provide for the first time a rationale to explain the counter-intuitive organ overgrowth phenotypes observed for certain members of the Minute class of Drosophila Rp mutants. They also demonstrate how Rp mutants can affect growth and development cell non-autonomously.

Transcription factor NF-Y is involved in differentiation of R7 photoreceptor cell in Drosophila

SummaryThe CCAAT motif-binding factor NF-Y consists of three different subunits, NF-YA, NF-YB and NF-YC. Knockdown of Drosophila NF-YA (dNF-YA) in eye discs with GMR-GAL4 and UAS-dNF-YAIR resulted in a rough eye phenotype and monitoring of differentiation of photoreceptor cells by LacZ expression in seven up-LacZ and deadpan-lacZ enhancer trap lines revealed associated loss of R7 photoreceptor signals. In line with differentiation of R7 being regulated by the sevenless (sev) gene and the MAPK cascade, the rough eye phenotype and loss of R7 signals in dNF-YA-knockdown flies were rescued by expression of the sev gene, or the D-raf gene, a downstream component of the MAPK cascade. The sev gene promoter contains two dNF-Y-binding consensus sequences which play positive roles in promoter activity. In chromatin immunoprecipitation assays with anti-dNF-YA antibody and S2 cells, the sev gene promoter region containing the NF-Y consensus was effectively amplified in immunoprecipitates from transgenic flies by polymerase chain reaction, indicating that dNF-Y is necessary for appropriate sev expression and involved in R7 photoreceptor cell development.

Liquid facets-Related (lqfR) Is Required for Egg Chamber Morphogenesis during Drosophila Oogenesis

Clathrin interactor 1 [CLINT1] (also called enthoprotin/EpsinR) is an Epsin N-terminal homology (ENTH) domain-containing adaptor protein that functions in anterograde and retrograde clathrin-mediated trafficking between the trans-Golgi network and the endosome. Removal of both Saccharomyces cerevisiae homologs, Ent3p and Ent5p, result in yeast that are viable, but that display a cold-sensitive growth phenotype and mistrafficking of various vacuolar proteins. Similarly, either knock-down or overexpression of vertebrate CLINT1 in cell culture causes mistrafficking of proteins. Here, we have characterized Drosophila CLINT1, liquid-facets Related (lqfR). LqfR is ubiquitously expressed throughout development and is localized to the Golgi and endosome. Strong hypomorphic mutants generated by imprecise P-element excision exhibit extra macrochaetae, rough eyes and are female sterile. Although essentially no eggs are laid, the ovaries do contain late-stage egg chambers that exhibit abnormal morphology. Germline clones reveal that LqfR expression in the somatic follicle cells is sufficient to rescue the oogenesis defects. Clones of mutant lqfR follicle cells have a decreased cell size consistent with a downregulation of Akt1. We find that while total Akt1 levels are increased there is also a significant decrease in activated phosphorylated Akt1. Taken together, these results show that LqfR function is required to regulate follicle cell size and signaling during Drosophila oogenesis.

Testing the Therapeutic Potential of Doxycycline in a Drosophila melanogaster Model of Alzheimer Disease

Therapies for Alzheimer disease that reduce the production of pathogenic amyloid β (Aβ) peptides have been associated with a range of unwanted effects. For this reason, alternative strategies that promote the clearance of the peptide by preventing its aggregation and deposition in the brain have been favored. In this context we have studied doxycycline, a member of the tetracycline family of antibiotics that has shown neuroprotective effects in a number of models of neurodegenerative disease. We investigated the neuroprotective potential of doxycycline in a Drosophila model of Aβ toxicity and sought to correlate any effects with the aggregation state of the peptide. We found that administration of doxycycline to Aβ42-expressing flies did not improve their lifespan but was able to slow the progression of their locomotor deficits. We also measured the rough eye phenotype of transgenic flies expressing the E22G variant of Aβ42 and showed that doxycycline administration partially rescued the toxicity of Aβ in the developing eye. We correlated these in vivo effects with in vitro observations using transmission electron microscopy, dynamic light scattering, and thioflavin T binding. We found that doxycycline prevents Aβ fibrillization and favors the generation of smaller, non-amyloid structures that were non-toxic as determined by the lack of caspase 3 activation in a neuroblastoma cell line. Our confirmation that doxycycline can prevent amyloid β toxicity both in vitro and in vivo supports its therapeutic potential in AD.

Preparation of Adult <em>Drosophila</em> Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes. The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3). The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.