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Axons are the up-to-meter long cellular processes of neurons that form the biological cables wiring nervous systems. Axons must be sustained for an organism's lifetime which makes them key lesion sites during healthy ageing, in injury and neurodegenerative diseases. Axon degeneration is considered as the cause rather than consequence for neuron decay in the context of various neurodegenerative diseases. Parallel bundles of microtubules (MTs) are the structural backbones of axons which provide the highways for life-sustaining long-distance transport between cell bodies and the distal growth cones or synaptic endings. MTs are polymers made of alpha/beta-tubulin heterodimers. Little is known about how tubulin heterodimers are made available in axons, i.e., large distances away from the transcriptional machinery in the cell body. The aim of my Ph.D. project was to explore the possibility that tubulin can be provided in axons through local biogenesis, i.e., local translation and chaperone-mediated folding. Using single molecule in situ hybridisation approaches in Drosophila primary neurons, I found alpha/beta-tubulin mRNA is present in axons, which were depleted upon deficiency of tubulin genes and significantly reduced upon loss of kinesin-1 and/or -3 function, suggesting involvement of active mRNA transport. I found axonal localisation of ribosomal proteins, P-body components and detected active axonal translation using puromycin assays. Furthermore, I could show that at least two of the necessary chaperone machineries (prefoldin, CCT, TBC) are present in axons. Of these, TBC is the most specific for tubulin processing, presenting the last step in the folding process. Using systematic knockdown of TBCA-E, I tested how each TBC subunit impacts axon length, MT polymerisation (assessed via Eb1), the presence of mRNA in axons and protein levels of alpha/beta-tubulin (via Western blots). The results revealed differential effects when knocking down individual subunits. This suggests that the complex does not act in an all-or-nothing fashion, but that there are either different maturation stages of tubulins that cause different phenotypic outcomes, or that individual subunits have additional roles outside the complex.

A hexanucleotide repeat expansion in a non-coding region of the C9orf72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases with considerable clinical and pathological overlap. FTD and ALS are invariably fatal and there is no cure. An understanding of the mechanisms through which the C9orf72 mutation leads to FTD/ALS will be crucial for development of effective therapeutics. There is evidence to support three possible pathogenic mechanisms underlying C9orf72 toxicity: (1) haploinsufficiency of C9orf72; (2) repetitive RNA transcripts arising from the repeat are toxic; (3) repeat-associated non-AUG (RAN) translation produces five different toxic dipeptide repeat proteins (DPRs) - AP, GP, GA, PR, and GR - from repeat RNA. The majority of evidence points to a gain of function mechanism and DPRs as the main toxic species, but does not rule out possible contributions from RNA toxicity and haploinsufficiency via synergistic mechanisms. Despite many studies into DPR-mediated toxicity in vitro and in vivo, the precise mechanisms through which DPR expression leads to neurodegeneration are still poorly understood. Drosophila models of DPR toxicity have been instrumental in the discovery that DPRs are the main drivers of toxicity. However, there remains a lack of consistent and physiologically relevant DPR models. Most express up to only 100 repeat units, whereas in patients, the expansion is several hundreds to thousands of repeats in length. Furthermore, despite the average age of onset of C9orf72-related FTD/ALS being 57 years of age, there are few studies investigating the effect of increasing age on DPR toxicity. The aims of this study were to address the lack of Drosophila models expressing DPRs of a physiological repeat length, fully characterise the effect of DPR expression on age-related neurodegenerative phenotypes, and subsequently use these models to unravel the mechanisms underpinning DPR toxicity. Firstly, we showed that 1000 repeat DPRs are stable in the Drosophila genome for over 3 years (~100 generations), and flies pan-neuronally expressing DPRs are viable with a reasonable lifespan. Each DPR was associated with a distinct age-related phenotypic profile. In support of previous studies, GR1000 showed the greatest age-related toxicity. However, AP1000, generally considered the least toxic of the DPRs, showed neurodegeneration, reduced climbing speed, and electrophysiological defects. Given that all DPRs have the capacity to be present in the same cell in patients, we next looked to see if co-expressing DPRs can alter their phenotypic profiles. Co-expression revealed a novel bang-sensitive seizure susceptibility not previously observed in DPR fly models. Subsequently, a dominant modifier screen was designed to identify mutations that could exacerbate seizure susceptibility in DPR flies. The screen implicated genes involved in mitochondrial function, oxidative stress, microtubule dynamics, and synaptic inhibition in DPR-mediated neurotoxicity. In summary, novel Drosophila models expressing physiologically relevant repeat length DPRs have been generated. Characterisation revealed age- and DPR- specific phenotypic profile, and highlights the importance of co-expression. We anticipate that this model will be useful for future studies elucidating the mechanisms underpinning DPR toxicity in C9orf72-related FTD/ALS.

Axons are the long, cable-like processes of neurons which form the nerves that wire our bodies. The maintenance of these delicate structures requires continuous parallel bundles of filamentous polymers called microtubules, which run all along the axonal core. In ageing and neurodegeneration, axonal microtubules often become curled and disorganised, causing detrimental accumulations of organelles and vesicles. The hypothesis of Local Axon Homeostasis suggests that the disorganisation of axon bundles is caused by the constant mechanical stress from the activity of motor proteins performing life-sustaining axonal cargo transport. In healthy axons, microtubule-regulating proteins prevent this from happening. If such proteins are dysfunctional, microtubules have a heightened probability of becoming disorganised. In this scenario, we would expect that microtubule phenotypes should appear similar, regardless of which microtubule regulatory proteins are absent; their images seem to suggest this. Therefore, my task was to develop objective parametric analyses to describe and compare microtubule phenotypes of different genetic conditions. First, I tested a number of existing software packages (Imaris, CellProfiler, NeuronJ, FiberApp), none of which proved suitable for a number of reasons. Especially curvature descriptions of the curled microtubules were impossible with any of these software packages. I therefore investigated different algorithms that would allow such analyses, and chose the Smoothing Spline Fitting method, which I turned into computational code in MATLAB. Tests of these algorithms using spirals and ellipses of defined parameters revealed a fairly stable reproduction of their curvatures. In addition, I used Hough Transforms to establish tools to identify and analyse straight lines. In my validation tests using sample images, these performed better than classic metrics. In the next step, I designed ALFRED (Advanced Labelling, Fitting, Recognition and Enhancement of Data) as user-friendly software in which the above algorithms can be applied. The development of ALFRED included strategies for image processing (from reduction of background noise to skeletonisation), ROI selection (with the help of MATLAB user interface tools) and path recognition (skeletons transformed into graphs and the shortest path algorithm is applied). When using ALFRED to analyse biological images of neurons, length and area measurements were matching closely with manual analyses. Curvature and straightness analyses of neurons of different genotype revealed a clear distinction between normal and mutant neurons, and different mutant conditions revealed similar valued, as is consistent with the hypothesis. The software is now available for wider application. As a complementary approach, I attempted to compare different phenotypes using machine learning. Data clean-up of neuron images was performed with Ilastik, but no accurate outcomes were achieved in the time available. Two pre-trained classification networks (VGG16 and ResNet18) were used to classify different genotypes, but results were inconclusive, likely also due to the insufficient data clean-up of images.

Axons are slender, cable-like projections of neurons which electrically wire our nervous system and bodies. They are the longest cellular processes and this unique architecture has to be maintained for an organism's lifetime. Notably, axons contain dense bundles of parallel microtubules (MTs) which form the structural backbones as well as the tracks for long-distance transport between cell bodies and synaptic endings. We lose about 50% of axons towards high age, and this is often accompanied by disorganisation of MT bundles in axon swellings. My host group proposes the model of 'local homeostasis' to explain how MT bundles are maintained. The model proposes that (a) MT bundles are highly dynamic with constant turnover to prevent senescence caused by wear and tear; (b) in the axonal environment the dynamic MTs tend to go off-track into disorganisation; (c) different mechanisms mediated by MT regulators 'tame' MTs into axonal bundles. The aim of my project was to identify further molecular mechanisms contributing to MT bundle formation and maintenance, focussing on candidate MT regulators which are all linked to neurodegeneration. These proteins were (1) Futsch/MAP1B (expected to act as a MT cross-linker, potentially in conjunction with the spectraplakin Short stop/Shot), (2) Spastin (expected to sustain MT turnover through its MT severing activity), and (3) the motor protein kinesin-1/Kinesin heavy chain (Khc) (expected to be involved in the even distribution of newly forming MTs through its MT sliding activity). For my studies, I capitalised on the versatile Drosophila primary neurons. I used combinatorial genetics and pharmacological approaches, combined with informative subcellular readouts including MT organisation, axon length, mitochondrial shape and distribution, presynaptic proteins distribution and endoplasmic reticulum. Detailed studies of all three regulators failed to confirm the above proposed working hypotheses, with loss of Futsch, of Spastin or of the Khc-driven MT sliding function failing to generate any MT disorganisation even in long-term studies or when challenged in different ways. Nevertheless, my work on all three candidates produced important new data and concepts. First, my results shifted the focus from Futsch to Shot, in particular its plakin-repeat region (PRR; specific to one isoform abundant in neurons). My work provided first analysis on a new CRISPR-mediated shotPRR allele lacking the PRR, which causes MT disorganisation, providing indication of the importance of PRR in MT bundling. Second, my results shifted the focus from Spastin to Katanin 60, another MT severer which I found to cause strong MT disorganisation when functionally depleted; future work will show whether it performs the roles I proposed for Spastin. Third, my main focus lay on Khc which causes strong MT disorganisation when depleted. However, this was not caused by absence of Khc-mediated MT sliding, but linked to its roles in cargo/vesicle transport (absence of the Kinesin light chain/Klc linker) and mitochondrial dynamics (absence of Miro or Milton linkers). My experiments aiming to explain how these sub-functions of Khc relate to MT bundle organisation led into new ways of thinking about kinesin-1-linked neurodegeneration. So far, the best supported model involves oxidative stress downstream of mitochondrial dysfunction. The second model involves force imbalance caused by loss of specific motor proteins from specific MT bundle subtracts; it is experimentally less well supported, but consistent with my experimental results so far. Although my experiments failed to support my original hypotheses for roles of Futsch, Spastin and Khc within the model of local axon homeostasis, the work I present opens up new concepts for thinking about axonal cell biology and provides new working hypotheses, candidate molecules and molecular tools readily available for future studies.

Alzheimer's Disease (AD) is the most prevalent form of dementia among elderly people. This disease is a major world health issue as the numbers of patients with this type of dementia will significantly increase in the next few decades. The aetiology of AD is not completely fully understood. The amyloid cascade hypothesis that identifies Beta-amyloid peptide (AB) as the initiator of the AD pathology leads the drug development efforts for AD. Currently only five drugs are approved by the FDA for AD but these drugs just ameliorate the symptoms of the disease for a limited time and do not target the underlaying pathology. Unfortunately, none of the drugs targeting the underlying disease has fully succeeded in clinical trials. In this project, we screened a drug library of 174 compounds that have been previously approved and/or undergone clinical trials and five drugs that have been previously identified to have a potential effect as AD therapies. We used SH-SY5Y cells acutely treated with AB42, and SH-SY5Y695 cells, as models to mimic the neurotoxic effects of AB in AD. First, we evaluated the feasibility and optimised different in vitro assays to be used as primary and secondary screening assays. MTT was selected as one of the primary screening methods as cells treated with AB42 decreased MTT reduction compared to non-treated cells. Mesoscale Discovery (MSD) immunoassays were also selected as another primary screening assay using SH-SY5Y695 cells. This immunoassay allowed us to simultaneously measure the concentration of the following proteins involved in APP processing: sAPP-alpha, sAPPB, AB38, AB40 and AB42. The primary screening using MTT identified SEN304, EGCG and castalagin as hits. MSD immunoassays identified compounds 4 (Acetyl-beta-methylcholine), 8 (arecoline), 133 (Tranylprimine), 169 (tropisetron), castalagin and SEN304 as possible hits. The first part of secondary screening focused on assessing the potency of the hits identified from the MTT primary screening and evaluating the possible advantage of a two-drug combination approach. EGCG was the most potent compound at inhibiting AB toxicity measured by MTT, followed by SEN304, castalagin and vescalagin. The two-drug combination approach did not show any synergetic effect. The second part of the secondary screening focused in assessing whether the hits from both MT and MSD immunoassays could hinder the increase of ROS generated in SH-SY5Y cells treated with AB42. EGCG was the only drug that decreased the ROS caused by AB42. A Drosophila melanogaster model overexpressing AB42 decreased the longevity of fruit flies compared to a control fly model not expressing AB We performed survival assays to evaluate whether EGCG or SEN304 increased the lifespan of the AB42 flies. None of the drugs evaluated increased the life span of flies overexpressing AB42. Overall the assays presented in this thesis allowed us to identify possible hits for the treatment of AD. The results suggest that SEN304, EGCG, vescalagin an castalagin have multiple mechanism of action to inhibit AB toxicity. Mesoscale Discovery immunoassays are a promising drug screening platform as it is possible to evaluate various targets simultaneously.

In fibroblasts integrin receptors are the primary adhesion receptors and act as the "sensory organs" of cells to sense external signals. My aim was to understand how integrins, together with the adhesion-associated plaque proteins Talin and Vinculin, regulate sensing of extracellular biochemical and mechanical stimuli in neurons. A first set of experiments served to characterise the sensing behaviour of CNS-derived primary cells and cell lines on different extracellular matrix (ECM). Integrin antibodies helped to characterise their adhesion receptor profiles and showed that specific integrin family members were involved in adhesion and axon/neurite outgrowth when plated on ECM proteins of laminin (LN), fibronectin, and collagen. Data showed that integrin-ECM interactions affected axon/neurite outgrowth and pathfinding. Presentation of LN promoted axon/neurite outgrowth and furthermore, presenting it in stripes dramatically enhanced outgrowth speeds showing that not only ECM but how it is presented directs growth. In a further set of experiments I established procedures that enabled me to determine the impact of mechanosensing in axon/neurite outgrowth. The established methodology helped to define point contact areas at the base of filopodia as sites of traction force exertion. Growth cones exerted dynamic stresses of around 10-30 Pa. To examine whether and how growth cones sense mechanical differences in their surrounding environment axon/neurite outgrowth was tracked when cells were plated on polyacrylamide. Data showed that axon/neurite outgrowth lengths and speeds were significantly longer and faster, respectively, on soft (0.4 kPa) than on stiff (8 kPa) substrates. Interestingly, the depletion of vinculin in neurons blocked the ability of growth cones to determine between soft and stiff substrates. Altogether, the interdisciplinary approach in this study helped to gain new insight into how neurons sense biochemical and mechanical properties of the ECM.

Axons are up-to-a-meter-long cable-like cellular processes of neurons. The proper function of nervous systems requires that axons grow and wire up correctly during development or regeneration. The uniquely challenging architecture of axons has to be sustained for an organism's lifetime, and renders them key lesion sites during healthy ageing, in injury and neurodegenerative diseases. Notably, axon degeneration is considered as the cause rather than consequence for neuron decay in the context of various neurodegenerative diseases. The structural backbones of axons are formed by parallel bundles of microtubules (MTs) which also provide the highways for life-sustaining long-distance transport between cell bodies and the growth cones or synaptic endings. To better understand axon development, regeneration, maintenance and degeneration during ageing, my PhD project has focused on mechanisms underpinning the regulation of MT bundles in axons. For this, I have capitalised on fast and genetically and experimentally amenable research possible in Drosophila neurons, both in primary culture and in vivo. I have used systematic combinatorial genetics and pharmacological approaches to unravel mechanisms and roles of actin as well as the cortical collapse factor Efa6 in MT regulation during axon formation and maintenance. I was able to gain a number of novel mechanisms contributing to the de novo alignment and maintenance of ordered MT bundles. First, it has been proposed that Spectraplakins (large actin-microtubule linkers) guide the extension of polymerising MTs along cortical F-actin, thus directly laying axonal MTs out into parallel bundles. Here, I have used manipulations of actin networks as well as hybrid constructs of Shot where the actin binding domain was replaced by actin associating domains of other molecules. My data strongly suggest that Shot's ABD domain has unique properties that can sense specific properties of F-actin networks, and this is important for its ability to appropriately regulate MT behaviours. Second, using combinations of actin and Shot manipulations, I found that Shot displays not only these actin-dependent guidance functions, but it displays novel actin-independent function in MT bundle maintenance for which I present a working hypothesis. Third, I found a novel and Shot-independent role of axonal actin in maintaining MTs and promoting axon growth, and my results suggest that these functions involve promotion of MT polymerisation. MT maintenance is therefore mediated through two complementary mechanisms involving Shot on the one hand and actin on the other, and simultaneous removal of Shot and actin leads to entire loss of axons. Finally, I have unravelled novel axonal functions of the cortical collapse factor Efa6 which serves as a check point in MT bundle maintenance by eliminating "off track" MTs that have escaped the axonal bundle organisation. In the absence of this factor, a gradual increase of disorganised, criss-crossed MTs occurs as a matter of days. These new mechanisms strongly suggest that different MT-regulatory mechanisms act in parallel in axons and complement each other in one common mechanism of MT bundle formation and maintenance. I propose here a local homeostasis model of axonal MT bundle maintenance which provides new ways to think about problems of ageing as well as a range of different neurodegenerative diseases.

During development of the nervous system axons must extend to their specific target cells,such as other neurons or muscles, in order to connect into the functional networks that formthe basis of brain function. Due to the high precision required, this process is susceptible toperturbations which cause a host of neurodevelopmental disorders including forms of mentalretardation. Furthermore, many of the same mechanisms which drive axon growth arereiterated during axon regeneration following injury, stroke or degeneration. Axon extension is performed by growth cones (GCs) at the tip of the extending axons,guided to their targets through extracellular signals. Intracellularly, the cytoskeletal machinerydrives the morphogenetic processes implementing GC behaviour. Whereas F-actinpredominantly regulates the directionality of axon growth, microtubules (MTs) essentiallyimplement axon extension, and sustain the structural integrity of axons and the transportbetween soma and GCs. Therefore, an important aim of axon growth research is to understandhow MT networks are regulated. This is the overarching objective of my project. MT-binding proteins (MTBPs) interact with MTs, with one another and with F-actin,thus forming complex regulatory networks. This complexity can be tackled with efficientgenetic approaches possible in the fruit fly Drosophila, using its well established axon growthreadouts in vivo complementary to the high resolution readouts for cytoskeleton in culturedprimary neurons. I focussed my work on a specific class of MTBPs, so called +TIP, whichbind to the polymerising plus ends of MTs to regulate their dynamics, a process thatessentially contributes to MT network formation and organisation. To be able to carry out my project, I first optimised the use of the culture system. Iestablished approaches to assess MT network organisation, and MT dynamics. My initialstudies showed that fundamental properties of MT networks are highly conserved betweenvertebrates and Drosophila. I used these new approaches to contribute to ongoing work onfunctions of the actin-MT linker Shot during axon growth. I demonstrated novel roles of Shotin regulating MT polymerisation dynamics and guidance which simultaneously validated thenewly established imaging strategies. I then carried out a systematic analysis of thelocalisation patterns of different +TIPs (CLASP, CLIP, APC1, APC2, p150glued and Lis1)which I found to be highly conserved with patterns of their vertebrate homologues describedin the literature. I then used loss of function analyses identified promising axonal growthphenotypes to be used in my further study. I selected CLIP-190 as a promising candidate because of its prominent localisation inGCs and its strong mutant axon overgrowth phenotype. Well into my studies, further testsunfortunately revealed the axon growth phenotype to be due to loss of the neighbouring gene,Lrch, an actin-binding protein for which my experiments revealed a putative role in MTregulation downstream of F-actin networks. Meticulous loss of CLIP-190 analyses, includingdouble- and triple mutant combinations with all of the above +TIP members, failed to revealany neuronal phenotypes in primary neurons. Neither did CLIP-190 loss show axon growth orNMJ phenotypes in vivo. Accordingly, in neurons CLIP-190 is tethered in large patches in theGCs, via its central coiled-coil domain, and a link to myosin VI, but shows little affinity forMT plus ends, via its EB1-binding domain. Parallel studies with the close mammalianhomologue CLIP-170 showed the same localisation patterns in neurons, suggesting that CLIP-170 doesn’t play a significant role in the nervous system. Overall, my studies in Drosophilaneurons have firmly established this system for the systematic analysis of MT regulation,likely to produce mechanistic understanding that can be applied also to mammals.

Epithelia act as protective barriers and it is therefore essential that wounded epithelia are rapidly repaired to maintain barrier function. Cells surrounding epithelial wounds become motile following wounding, which involves generating dynamic actin structures that drive closure of the wound. These actin structures include filopodia which are important in the final stage of epithelial closure in which the opposing epithelial edges are joined together. The molecular mechanisms that trigger wound edge cells to become motile are not well understood. Using Drosophila wound healing and the morphogenetic process dorsal closure as models, we find that phosphatidylinositol 3,4,5-triphosphate (PIP3) regulates epithelial closure by promoting the formation of filopodia at epithelial edges. PIP3 accumulates at epithelial edges and genetically depleting PIP3 results in reduced filopodia and defects in epithelial closure. We demonstrate that the GTPase Rac and guanine nucleotide exchange factor Myoblast City function downstream of PIP3 to promote filopodia formation. We also demonstrated that the scaffolding protein Par3/Bazooka and the lipid phosphatase PTEN are responsible for restricting the localisation of PIP3 and consequently the downstream signals to the epithelial leading edge, so acting to determine the location of filopodia formation. This project reveals a novel mechanism by which actin protrusions, required for epithelial closure, are formed in response to epithelial damage. Additionally, we have identified an additional role for PIP3 in regulating the extrusion of cells from epithelial sheets in the Drosophila embryo. This finding implicates PIP3 in the regulation of tissue homoeostasis, and could contribute to our understanding of tumour initiation as unregulated tissue growth can result in the formation of tumours.

The I-BAR proteins are a family of actin regulators which include IRSp53 and Missing-In-Metastasis (MIM). These proteins possess an N-terminal I-BAR domain which associates with both the actin cytoskeleton and membrane phospholipids and is able to induce membrane curvature. Previous cell culture and in vitro studies have implicated I-BAR proteins in the regulation of actin protrusion formation, however their roles within the organism are poorly understood. We have used Drosophila melanogaster as a model system in which to study I-BAR protein function at the cellular and organismal level. Drosophila possess two I-BAR proteins, one homologous to IRSp53 and one homologous to MIM. Using full- length and truncated splice variants generated for both dMIM and dIRSp53, we have performed structure/function analysis to determine the role of specific domains in the localisation and actin modifying abilities of the proteins in both cell culture and in vivo. We found that dMIM overexpression typically promotes a lamellipodial morphology, with dMIM localising to the edges of extending lamellipodia. dIRSp53 expression induced a more filopodial phenotype in cell culture, which was not as notable in vivo, however expression of a dIRSp53 splice variant with a WH2 domain resulted instead in a predominantly lamellipodial morphology. Similar to dMIM, dIRSp53 localises to the tip of extending protrusions, albeit more transiently. We found that multiple domains contribute to the localisation and activity of dIRSp53 and dMIM. Following overexpression analysis, complementary loss-of-function analysis was performed in vivo using Drosophila mutants lacking dMIM, dIRSp53 or both genes together. Surprisingly these mutants were viable and morphologically normal. Absence of these genes individually or together did not greatly affect cell migration or actin dynamics in haemocytes or epithelial cells undergoing dorsal closure. However, a role for I- BAR proteins in axonal filopodia formation within primary neuronal cultures was apparent, as was a notable role in neuromuscular junction morphology. We have also identified potential redundancy between Drosophila MIM and the Drosophila F-BAR protein Cip4 in actin bundle regulation within embryonic haemocytes, with an additional novel role for Cip4 alone in haemocyte lamellipodial maintenance. Our results suggest that the Drosophila I-BAR proteins contribute to actin cytoskeleton regulation in vitro and in vivo, particularly within the CNS, and with novel shared functions with other BAR domain family proteins contributing to their regulation of actin cytoskeletal organisation and function.

The past two decades have highlighted the utility in using the fruit fly Drosophila as a model organism for unravelling the molecular and functional complexities of two important fields of research: the systems that guide retinal determination (RD) and circadian rhythms (the daily body clock or oscillator). RD and clock factors are of great interest as they are: (1) highly conserved in vertebrates; (2) also essential for other physiological systems; (3) implicated in several congenital disorders and other diseases. The RD factors operate within a network in which several of their interactions have been described. Two such factors, eyes absent (eya) and sine oculis (so), are known to function as a unit to direct transcriptional regulation during photoreceptor (PR) differentiation. The regulation of eya and so by a transcriptional repressor at the heart of the clock mechanism, vrille (vri), is here investigated. Two distinct observations advocated exploration of a link between vri and eya/ so is of interest. (1) vri is a core component of the clock and interacts with RD but the RD function is unknown. (2) Recent evidence suggests that an RD factor directly upstream of eya and so, twin-of-eyeless (toy), interacts with the oscillator mechanism through direct and indirect pathways. It is possible that the indirect influences of toy on the oscillator are mediated via eya and/ or so. Interactions between eya, so and vri during RD and within oscillator cells are investigated here.Eye development function was studied using immunohistochemistry and transgenic manipulation. VRI is not expressed within the developed PRs; rather, expression of VRI is down-regulated prior to differentiation. In addition, conversely to the hypothesised role, VRI is co-expressed with EYA in some regions. Together with data from transgenic manipulation of VRI regional expression, I propose that VRI is predominantly part of a developmental pathway but can attenuate eya and so expression.The VRI binding site has been described previously and several sites were identified within eya/ so loci, some of which were tested in an in vitro binding assay. Two such sites were located adjacent to a known enhancer of so. I generated two transgenic fly lines containing: 1) an extension of the original enhancer to contain the VRI sites; and 2) a similar construct with the VRI sites ablated. Comparison of the original enhancer to those from the current study confirmed that the VRI sites attenuate expression and that intervening regions must contain binding sites for other transcription factors.In adult brains over a circadian light-dark cycle, EYA protein was expressed in three of the central brain clock neurones. Furthermore, expression of eya and so transcripts in adult heads, PRs and the brain, changed over the light/ dark cycle independently of the clock - indicating that their expression is modulated over the light-dark cycle but not by the oscillator mechanism. These data suggest interactions between eye development factors eya/ so and oscillator components, or, the light/ dark cycle exist. These interactions may be important for tissue-specific circadian physiology as well as the overall oscillator mechanism and offer an intriguing route for future investigation.