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The regular organization of the ommatidial lattice in the Drosophila eye originates in the precise regulation of the proneural gene atonal (ato), which is responsible for the specification of the ommatidial founder cells R8. Here we show that Rough eye (Roi), a dominant mutation manifested by severe roughening of the adult eye surface, causes defects in ommatidial assembly and ommatidial spacing. The ommatidial spacing defect can be ascribed to the irregular distribution of R8 cells caused by a disruption of the patterning of ato expression. Disruptions in the recruitment of other photoreceptors and excess Hedgehog production in differentiating cells may further contribute to the defects in ommatidial assembly. Our molecular characterization of the Roi locus demonstrates that it is a gain-of-function mutation of the bHLH gene amos that results from a chromosomal inversion. We show that Roi can rescue the retinal developmental defect of ato1 mutants and speculate that amos substitutes for some of ato's function in the eye or activates a residual function of the ato1 allele.

SIX5 belongs to a family of highly conserved homeodomain transcription factors implicated in development and disease [1–3]. The mammalian SIX5/SIX4 gene pair is likely to be involved in the development of mesodermal structures [4–6]. Moreover, a variety of data have implicated human SIX5 dysfunction as a contributor to myotonic dystrophy type 1 (DM1), a condition characterized by a number of pathologies including muscle defects and testicular atrophy [7–9]. However, this link remains controversial. Here, we investigate the Drosophila gene, D-Six4 , which is the closest homolog to SIX5 of the three Drosophila Six family members [10]. We show by mutant analysis that D-Six4 is required for the normal development of muscle and the mesodermal component of the gonad. Moreover, adult males with defective D-Six4 genes exhibit testicular reduction. We propose that D-Six4 directly or indirectly regulates genes involved in the cell recognition events required for myoblast fusion and the germline:soma interaction. While the exact phenotypic relationship between D-Six4 and SIX4/5 remains to be elucidated, the defects in D-Six4 mutant flies suggest that human SIX5 should be more strongly considered as being responsible for the muscle wasting and testicular atrophy phenotypes in DM1.

In Drosophila neurogenesis, proneural genes encode bHLH proteins that are required for neural precursor selection. But many vertebrate homologues are expressed later and are postulated to have multiple roles during neurogenesis. We have isolated a new Drosophila gene, cato, which encodes a protein with a bHLH domain that is closely related to that of the proneural protein Atonal. cato expression is restricted to the developing PNS, where it is expressed in between the stages of precursor selection and terminal differentiation (and therefore later than the proneural genes). We present evidence from loss-of-function and misexpression experiments that cato is involved in sensory neurone morphology. Moreover, in prospero mutants, in which axon and dendrite outgrowth is defective, cato is strongly derepressed in the developing CNS.

In a variety of organisms, early neurogenesis requires the function of basic-helix-loop-helix (bHLH) transcription factors. For the Drosophila PNS, such transcription factors are encoded by the proneural genes (atonal and the achaete–scute complex, AS-C). We have identified a proneural gene, amos, that has strong similarity with atonal in its bHLH domain. We present evidence that amos is required for olfactory sensilla and is regulated by the prepattern gene lozenge. Between them, amos, atonal, and the AS-C can potentially account for the origin of the entire PNS.

The proneural genes (atonal and the genes of the achaete–scute complex (AS-C)) are required for the selection of sense organ precursors. They also endow these precursors with sense organ subtype information. In most of the ectoderm, atonal is required for precursors of chordotonal sense organs, whereas AS-C are required for those of most external sense organs, such as bristles. To address the question of how proneural genes influence subtype identity, we have made use of the Gal4/UAS system of misexpression. Unlike previous misexpression experiments, we found that under specific conditions of misexpression, atonal shows high subtype specificity of ectopic sense organ formation. Moreover, atonal can even transform wild-type external sense organs to chordotonal organs, although scute cannot perform the reciprocal transformation. Our evidence demonstrates that atonal's subtype determining role is not to activate directly chordotonal fate, but to repress the activation of cut, a gene that is necessary for external sense organ fate, thereby freeing its precursors to follow the alternative chordotonal organ fate.

The Drosophila ear, Johnston’s organ (JO), is housed in its antenna. At the heart of the JO are intrinsically mechanosensitive ciliated sensory neurons called chordotonal neurons. Development of the JO requires formation and patterning of the antennal primordium in the larva, the specification of the precursor cells of the mechanosensory units, the differentiation of the chordotonal neurons, and the targeting of their axons to the antennal mechanosensory and motor center (AMMC) of the brain. JO chordotonal neurons combine the functions of vertebrate hair cells and auditory neurons. Although they differ in structure, clear developmental, genetic, and physiological parallels between the Drosophila and vertebrate auditory systems suggest that chordotonal neurons and hair cells are orthologous cell types and that the auditory systems evolved from the same ancestral mechanoreceptive organ.

Neurogenesis is the process by which neurons are born during the development of an animal. The nervous system entails great cellular diversity and is the most complex of any organ system. A developing animal needs to harness sophisticated cellular and genetic mechanisms to ensure the orderly generation of large numbers of neurons in the right places and times, and in the right numbers. A long history of molecular genetic studies in the fruit fly, Drosophila melanogaster, has provided many insights into how this is achieved. Neurogenesis involves the initial patterning of the ectoderm, the formation of neural precursor cells from the ectoderm and the formation of neurons from these precursors through fixed lineages involving asymmetric cell division. Many of the concepts uncovered apply to neurogenesis in other animals. Indeed, many of the molecular mechanisms are highly conserved. Key Concepts: The fruit fly has relatively few neurons in its nervous system, making it tractable to detailed developmental analysis. The arrangement of these neurons is, as far as is known, very precisely determined by the mechanisms controlling neurogenesis during development. Neurons are the progeny of neural precursor cells that derive from the ectoderm. The precursor cells are specified by the action of proneural transcription factors, which are opposed by Notch signalling in the process of lateral inhibition. At their formation, the precursors are already specified to contribute specific neuronal progeny via fixed lineages of cell division. For the PNS, neural precursor identity is endowed by proneural factors, their cofactors. For the CNS, neuroblasts have unique identities according to their location, due to their inheriting different combinations of ectodermal patterning factors. Within the fixed division lineages, cellular diversity arises through asymmetric cell division, which is driven largely by asymmetrically localised and inherited cell fate determinants. Keywords: cell type specification; neurogenesis; neuroblasts; sensory neurons; asymmetric cell division

In contrast to the progress in understanding ciliogenesis and cilium function, we know less about the transcriptional regulation of ciliogenesis genes and how this regulatory program is modulated to generate diverse cilia. Drosophila sensory neurons have ciliary dendrites that are structurally and functionally specialised for receiving different sensory modalities. Time-course gene expression profiling of differentiating chordotonal (Ch) mechanosensory neurons allowed us to determine how Atonal, a proneural bHLH factor, regulates events leading to mechanosensory cilium formation and specialisation. Atonal regulates ciliogenesis via activation of two downstream transcription factors: the well-known cilia gene regulator, Rfx, and a novel factor of the Forkhead family (Fd3F). Rfx regulates a variety of ciliogenesis genes in all ciliated sensory neurons. In contrast, Fd3F is unique to Ch neurons, where it regulates a cohort of genes required for ciliary motility – a unique specialisation of Ch cilia in Drosophila and an essential part of the hearing mechanism. Among the targets of Fd3F are genes with human homologues linked to primary ciliary dyskinesia, a congenital condition resulting from defective ciliary motility. We provide evidence that Fd3F is a previously unrecognised orthologue of Foxj1, the so-called master regulator of motile ciliated cells in vertebrates. Interestingly, Fd3F and Rfx cooperate to regulate motility target genes directly via adjacent DNA binding sites, thus providing a mechanism for how Rfx can regulate genes for cilium specialisation in addition to ‘core’ ciliogenesis genes. Our study suggests how Foxj1 and Rfx factors may interact in vertebrates to generate ciliary diversity. http://www.ed.ac.uk/schools-departments/integrative-physiology/staff-profiles/andrew-jarman

Neural precursor cells in Drosophila arise from the ectoderm in the embryo and from imaginal disc epithelia in the larva. In both cases, this process requires daugh - terless and the proneural genes achaete, scute and lethal-of-scute of the achaete-scute complex. These genes encode basic helix-loop-helix proteins, which are nuclear transcription factors, as does the asense gene of the achaete-scute complex. Our studies suggest that asense is a neural precursor gene, rather than a proneural gene. Unlike the proneural achaete-scute gene products, the asense RNA and protein are found in the neural precursor during its formation, but not in the proneural cluster of cells that gives rise to the neural precursor cell. Also, asense expression persists longer during neural precursor development than the proneural gene products; it is still expressed after the first division of the neural precursor. Moreover, asense is likely to be down-stream of the proneural genes, because (1) asense expression is affected in proneural and neurogenic mutant backgrounds, (2) ectopic expression of asense protein with an intact DNA-binding domain bypasses the requirement for achaete and scute in the formation of imaginal sense organs. We further note that asense ectopic expression is capable of initiating the sense organ fate in cells that do not normally require the action of asense. Our studies therefore serve as a cautionary note for the inference of normal gene function based on the gain-of-function phenotype after ectopic expression.

Background bHLH transcription factors play many roles in neural development. cousin of atonal (cato) encodes one such factor that is expressed widely in the developing sensory nervous system of Drosophila. However, nothing definitive was known of its function owing to the lack of specific mutations. Results We characterised the expression pattern of cato in detail using newly raised antibodies and GFP reporter gene constructs. Expression is predominantly in sensory lineages that depend on the atonal and amos proneural genes. In lineages that depend on the scute proneural gene, cato is expressed later and seems to be particularly associated with the type II neurons. Consistent with this, we find evidence that cato is a direct target gene of Atonal and Amos, but not of Scute. We generated two specific mutations of cato. Mutant embryos show several defects in chordotonal sensory lineages, most notably the duplication of the sensory neuron, which appears to be caused by an extra cell division. In addition, we show that cato is required to form the single chordotonal organ that persists in atonal mutant embryos. Conclusions We conclude that although widely expressed in the developing PNS, cato is expressed and regulated very differently in different sensory lineages. Mutant phenotypes correlate with cato's major expression in the chordotonal sensory lineage. In these cells, we propose that it plays roles in sense organ precursor maintenance and/or identity, and in controlling the number of cell divisions in the neuronal branch of the lineage arising from these precursors.

The results of a number of recent studies indicate that eye development in insects and vertebrates may have more features in common than hitherto suspected. The results support the possibility that insect and vertebrate eyes evolved from a complex ancestral organ.

The major positive regulatory activity of the human alpha-globin gene complex has been localized to an element associated with a strong erythroid-specific DNase I hypersensitive site (HS -40) located 40 kb upstream of the zeta 2-globin mRNA cap site. Footprint and gel shift analyses of the element have demonstrated the presence of four binding sites for the nuclear factor GATA-1 and two sites corresponding to the AP-1 consensus binding sequence. This region resembles one of the major elements of the beta-globin locus control region in its constitution and characteristics; this together with evidence from expression studies suggests that HS -40 is a primary element controlling alpha-globin gene expression.

A key point in neural development is the commitment of progenitor cells to a specific neural fate. In all animals studied, proneural proteins - transcription factors of the basic helix-loop-helix (bHLH) family - are central to this process. The function of these factors is strongly influenced by the spatial and temporal context in which they are expressed. It is important to understand the molecular mechanisms by which developmental context interacts with and modifies the intrinsic functions and properties of the proneural proteins. Recent insights have been obtained in Drosophila and vertebrates from analysis of how bHLH proteins interact with other transcription factors to regulate target genes.

Background Movement of cells, either as amoeboid individuals or in organised groups, is a key feature of organ formation. Both modes of migration occur during Drosophila embryonic gonad development, which therefore provides a paradigm for understanding the contribution of these processes to organ morphogenesis. Gonads of Drosophila are formed from three distinct cell types: primordial germ cells (PGCs), somatic gonadal precursors (SGPs), and in males, male-specific somatic gonadal precursors (msSGPs). These originate in distinct locations and migrate to associate in two intermingled clusters which then compact to form the spherical primitive gonads. PGC movements are well studied, but much less is known of the migratory events and other interactions undergone by their somatic partners. These appear to move in organised groups like, for example, lateral line cells in zebra fish or Drosophila ovarian border cells. Results We have used time-lapse fluorescence imaging to characterise gonadal cell behaviour in wild type and mutant embryos. We show that the homeodomain transcription factor Six4 is required for the migration of the PGCs and the msSGPs towards the SGPs. We have identified a likely cause of this in the case of PGCs as we have found that Six4 is required for expression of Hmgcr which codes for HMGCoA reductase and is necessary for attraction of PGCs by SGPs. Six4 affects msSGP migration by a different pathway as these move normally in Hmgcr mutant embryos. Additionally, embryos lacking fully functional Six4 show a novel phenotype in which the SGPs, which originate in distinct clusters, fail to coalesce to form unified gonads. Conclusion Our work establishes the Drosophila gonad as a model system for the analysis of coordinated cell migrations and morphogenesis using live imaging and demonstrates that Six4 is a key regulator of somatic cell function during gonadogenesis. Our data suggest that the initial association of SGP clusters is under distinct control from the movements that drive gonad compaction.

Background The regulation of proneural gene expression is an important aspect of neurogenesis. In the study of the Drosophila proneural genes, scute and atonal, several themes have emerged that contribute to our understanding of the mechanism of neurogenesis. First, spatial complexity in proneural expression results from regulation by arrays of enhancer elements. Secondly, regulation of proneural gene expression occurs in distinct temporal phases, which tend to be under the control of separate enhancers. Thirdly, the later phase of proneural expression often relies on positive autoregulation. The control of these phases and the transition between them appear to be central to the mechanism of neurogenesis. We present the first investigation of the regulation of the proneural gene, amos. Results Amos protein expression has a complex pattern and shows temporally distinct phases, in common with previously characterised proneural genes. GFP reporter gene constructs were used to demonstrate that amos has an array of enhancer elements up- and downstream of the gene, which are required for different locations of amos expression. However, unlike other proneural genes, there is no evidence for separable enhancers for the different temporal phases of amos expression. Using mutant analysis and site-directed mutagenesis of potential Amos binding sites, we find no evidence for positive autoregulation as an important part of amos control during neurogenesis. Conclusion For amos, as for other proneural genes, a complex expression pattern results from the sum of a number of simpler sub-patterns driven by specific enhancers. There is, however, no apparent separation of enhancers for distinct temporal phases of expression, and this correlates with a lack of positive autoregulation. For scute and atonal, both these features are thought to be important in the mechanism of neurogenesis. Despite similarities in function and expression between the Drosophila proneural genes, amos is regulated in a fundamentally different way from scute and atonal.

The Drosophila peripheral nervous system comprises four major types of sensory element: external sense organs (such as mechano-sensory bristles), chordotonal organs (internal stretch receptors), multiple dendritic neurons, and photoreceptors. During development, the selection of neural precursors for external sense organs requires the proneural genes of the achaete-scute complex, which encode basic-helix-loop-helix transcription factors. These genes do not, however, control precursor selection for chordotonal organs or photoreceptors, raising the question of whether other proneural genes exist or a different mechanism of neurogenesis operates. Here we show that atonal (ato), originally isolated as a proneural gene for chordotonal organs, is also the proneural gene for photoreceptors. Pattern formation in the Drosophila eye involves a succession of cell fate specifications. Of the eight photoreceptors within each ommatidium of the compound eye, the photoreceptor R8 is the first to appear in the eye imaginal disc, right behind the morphogenetic furrow. The appearance of other photoreceptors (R1-7) follows in a defined sequence that is thought to arise by induction from R8 (refs 8, 9, 11, 12). We find that photoreceptor formation requires the function of atonal at the morphogenetic furrow and that atonal is specifically required for R8 selection. Formation of other photoreceptors does not directly require atonal function, but does depend on R8 selection by atonal. Thus, photoreceptors are selected by two mechanisms: R8 by a proneural mechanism, and R1-7 by local recruitment.

During Drosophila eye development, the proneural gene atonal specifies founding R8 photoreceptors of individual ommatidia, evenly spaced relative to one another in a pattern that prefigures ommatidial organisation in the mature compound eye. Beyond providing neural competence, however, it has remained unclear to what extent atonal controls specific R8 properties. We show here that reduced Atonal function gives rise to R8 photoreceptors that are functionally compromised: both recruitment and axon pathfinding defects are evident. Conversely, prolonged Atonal expression in R8 photoreceptors induces defects in inductive recruitment as a consequence of hyperactive EGFR signalling. Surprisingly, such prolonged expression also results in R8 pattern formation defects in a process associated with both Hedgehog and Receptor Tyrosine Kinase signalling. Our results strongly suggest that Atonal regulates signalling and other properties of R8 precursors.

The selection of Drosophila melanogaster sense organ precursors (SOPs) for sensory bristles is a progressive process: each neural equivalence group is transiently defined by the expression of proneural genes (proneural cluster), and neural fate is refined to single cells by Notch- Delta lateral inhibitory signalling between the cells. Unlike sensory bristles, SOPs of chordotonal (stretch receptor) sense organs are tightly clustered. Here we show that for one large adult chordotonal SOP array, clustering results from the progressive accumulation of a large number of SOPs from a persistent proneural cluster. This is achieved by a novel interplay of inductive epidermal growth factor-receptor (EGFR) and competitive Notch signals. EGFR acts in opposition to Notch signalling in two ways: it promotes continuous SOP recruitment despite lateral inhibition, and it attenuates the effect of lateral inhibition on the proneural cluster equivalence group, thus maintaining the persistent proneural cluster. SOP recruitment is reiterative because the inductive signal comes from previously recruited SOPs.

The Drosophila gene atonal encodes a basic helix-loop-helix protein similar to those encoded by the proneural genes of the achaete–scute complex (AS-C). The AS-C are required in the Drosophila PNS for the selection of neural precursors of external sense organs. We have isolated mutants of atonal, which reveal that this gene encodes the proneural gene for chordotonal organs and photoreceptors. In atonal mutants, all observable adult chordotonal organs, and almost all embryonic chordotonal organs fail to form; all adult photoreceptors are missing. For both types of sense organ, this defect is already apparent at the level of precursor formation. Therefore it is a failure in the epidermal-neural decision process i.e. a proneural defect. The failure to form photoreceptors results in atrophy of the atonal mutant imaginal disc, due to apoptosis and lack of stimulation of division. Lack of photoreceptors should also eliminate signalling that arises from differentiating photoreceptors and is required for morphogenetic furrow movement in the wild-type eye disc. Nevertheless, a remnant morphogenetic furrow is still observed in the atonal mutant disc. This presumably reflects the process of furrow initiation, which would not depend on signals from developing photoreceptors.

Important questions concerning early neurogenesis include: how do certain ectodermal cells decide to become neural precursors; and how do these cells choose to differentiate as one of many possible neural subtypes? Owing to its relative simplicity, and our detailed knowledge of its anatomy and ontogeny, the Drosophila peripheral nervous system (PNS) has been a good model system for identifying and characterizing genes involved in these fate decisions.

Although the control of cell proliferation has been studied intensively at the level of the single cell, less is known about how cell numbers are controlled in developing populations and organs. Often, proliferation provides a pool of cells for organ construction, but the rate of this proliferation must be coordinated with patterning to avoid imbalances in cell numbers. Recent research on the development of the Drosophila eye and the proliferation signals (mitogens) can act to coordinate cell numbers.

It has become apparent in recent years that unexpectedly large numbers of minisatellites exist within the eukaryotic genome. Their use in genetics is well known, but as with any new class of sequence, there is also much speculation about their involvement in a range of biological processes. How much is known of their biology?

The proneural gene, Atoh1, is necessary and in some contexts sufficient for early inner ear hair cell development. Its function is the subject of intensive research, not least because of the possibility that it could be used in therapeutic strategies to reverse hair cell loss in deafness. However, it is clear that Atoh1's function is highly context dependent. During inner ear development, Atoh1 is only able to promote hair cell differentiation at specific developmental stages. Outside the ear, Atoh1 is required for differentiation of a variety of other cell types, for example in the intestine and cerebellum. The reasons for this context dependence are poorly understood. So far, the pathways and key players that instruct Atoh1 to act as a mechanosensory cell fate determinant in the context of the inner ear are largely unknown. Here we review evidence that suggests that Atoh1 function in hair cell differentiation is modulated by interaction with other transcription factors. We particularly focus on the possible roles of Gfi1 and Pou4f3, drawing from studies in mouse, Drosophila and C. elegans.

Adult polycystic kidney disease (APCKD) is a common and often lethal multi-organ disease with an autosomal dominant pattern of inheritance; approximately 1 in 1,000 people carry the mutant gene. The major pathological abnormality is the development and progressive enlargement of cysts in several organs including the liver, pancreas and spleen as well as the kidneys. The basic biochemical defect which leads to the formation of cysts remains unknown. Cyst development, which is not retarded by any known therapy, leads to irreversible renal failure and death at a mean age of 51 unless dialysis or transplantation are used. Patients with the disease account for 9% of chronic dialysis requirement. The first symptoms tend to occur in the fourth decade, after most patients have reproduced. Presymptomatic diagnosis depends on the ultrasonographic detection of cysts, but exclusion cannot be achieved by this means; 34% of at-risk patients in the second decade and 14% in the third will go on to develop cysts after negative diagnosis. The low sensitivity of diagnostic techniques in this critical age-range imposes severe limitations on genetic counselling and the condition cannot be identified prenatally. Hence we have searched for a linkage marker for APCKD; we show here that the APCKD locus is closely linked to the alpha-globin locus on the short arm of chromosome 16 (zeta = 25.85, theta = 0.05).

In an analysis of a 90-kb region around the human beta-globin gene complex we have identified at least eight sites of attachment to the nuclear scaffold (SARs). While these have many potential functions, there appears to be a particular association with sequences important in the regulation of the complex. Two SARs are close to the known enhancer-like elements of the beta-globin gene. SARs flanking the complex co-habit with the boundaries of the putative beta-like globin gene regulatory domain. In contrast, we have detected no SARs within a 140-kb region of the human alpha-globin gene complex. If SARs play a role in the regulation of gene expression then this structural difference would imply a difference in the regulation of the two complexes.

Drosophila provide an excellent opportunity to study cilia formation and function as the sensory neurons are the only ciliated somatic cells, and so cilium mutations are recognisable by sensory behavioural defects. A reverse genetic screen was conducted to implicate candidate genes in ciliary processes. Candidates were selected from a chordotonal sensory neuron transcriptome analysis (Cachero et al., 2011) and refined by consideration of gene ontology, existence of human homologues, and participation in protein-protein interaction networks (Gallone et al., 2011). P element insertions and Gal4/UAS induced RNAi were used to disrupt gene function. Candidate genes were screened using a climbing assay to select for proprioceptive and gravitactic deficiency, indicating disrupted chordotonal neuron function. Positive hits from the screen are being investigated by observing the ciliary dendrite structure of pupal antennae and larval chordotonal neurons. Positive candidates are also tested for fertility defects due to disrupted flagellum formation during spermiogenesis. Our analysis has identified several novel conserved ciliary genes. Particular attention has been given to genes regulated by the transcription factor fd3F, which is required for the transcription of several genes involved in cilium motility.

Background: Drosophila proneural genes act in the process of selecting neural precursors from undifferentiated ectoderm. The proneural gene atonal is required for the development of precursors of both chordotonal organs (stretch receptors) and photoreceptors. Although these types of sensory element are dissimilar in structure and function, they both occur as organized arrays of neurons. Previous studies have shown that clustering of photoreceptors involves local recruitment, and that signalling by the Drosophila epidermal growth factor receptor (DER) pathway is involved in the recruitment process. We present evidence that a similar mechanism is required for the clustering of embryonic chordotonal organs. Results: We have examined the expression patterns of atonal and genes of the DER pathway in wild-type and mutant backgrounds. Expression of atonal was restricted to a subset of the atonal -requiring chordotonal precursors, which we call founder precursors. The remaining precursors required DER signalling for their selection. Signalling by the founder precursors was initiated by atonal activating, directly or indirectly, rhomboid expression in these cells. Signalling by these founder precursors then provoked a response in the surrounding ectodermal cells, as shown by the activation of expression of the DER target genes pointed and argos . The signal and response then led to recruitment of some of the ectodermal cells to the chordotonal precursor cell fate. DER hyperactivation by misexpression of rhomboid resulted in excessive chordotonal precursor recruitment. Conclusions: Increased numbers of chordotonal precursors are recruited by homeogenetic induction involving signalling via DER from founder precursors to surrounding ectodermal cells. We suggest that the reason chordotonal organs and photoreceptors share a requirement for the proneural gene atonal is that this gene activates a common pathway leading to neural aggregation.

asense is a member of the achaete-scute complex (AS-C) of helix-loop-helix genes involved in Drosophila neurogenesis. Unlike the other AS-C members, which are expressed in subsets of the ectodermal areas (proneural clusters) that give rise to neural precursors, asense is one of a number of genes that are specifically expressed in the neural precursors themselves (the neural precursor genes). We have identified a mutant asense phenotype that may reflect this later expression pattern. As a step in understanding the determination of neural precursors from the proneural clusters, we have investigated the potential role of the AS-C products as direct transcriptional activators of neural precursor genes by analysing the regulation of asense. Using genomic rescues and asense-lacZ fusion genes, the neural precursor regulatory element has been identified. We show that this element contains binding sites for AS-C/daughterless heterodimers. Deletion of these sites reduces the expression from the fusion gene, but significant expression is still achieved, pointing to the existence of other regulators of asense in addition to the AS-C. asense differs from the other AS-C members in its expression pattern, regulation, mutant phenotype and some DNA-binding properties.

Age-related hearing loss (ARHL) is a threat to future human wellbeing. Multiple factors contributing to the terminal auditory decline have been identified; but a unified understanding of ARHL - or the homeostatic maintenance of hearing before its breakdown - is missing. We here present an in-depth analysis of homeostasis and ageing in the antennal ears of the fruit fly Drosophila melanogaster. We show that Drosophila, just like humans, display ARHL. By focusing on the phase of dynamic stability prior to the eventual hearing loss we discovered a set of evolutionarily conserved homeostasis genes. The transcription factors Onecut (closest human orthologues: ONECUT2, ONECUT3), Optix (SIX3, SIX6), Worniu (SNAI2) and Amos (ATOH1, ATOH7, ATOH8, NEUROD1) emerged as key regulators, acting upstream of core components of the fly’s molecular machinery for auditory transduction and amplification. Adult-specific manipulation of homeostatic regulators in the fly’s auditory neurons accelerated - or protected against - ARHL.

With only two types of ciliated cells, Drosophila is a useful organism in which to study conserved aspects of ciliogenesis. Cilia with motile characteristics in Drosophila are represented just by the sperm flagella and the sensory receivers of chordotonal neurons, which are proprioceptive and auditory sensory neurons. We recently used Drosophila to identify two new putative dynein arm assembly factors, CG11253 (ZMYND10 homologue) and CG31320 (HEATR2 homologue): impairment of function of either gene results in flies with immotile sperm and defective sensory transduction due to lack of dynein arms in the cilia. We have sought to extend these observations to other known or putative dynein assembly factor homologues to determine how much of this pathway is conserved and set up Drosophila as a model for exploring the function of these proteins in further detail. This analysis has used transcriptomic data of developing chordotonal neurons, gene regulatory analysis (regulation by Rfx and Fox factor, Fd3F), genetic analysis of sensation and male fertility, and protein interaction analysis. Currently, we are also carrying out mass spectroscopy.

In contrast to the progress in understanding ciliogenesis and cilium function, we know less about the pathways for generating ciliary diversity. Drosophila has a variety of sensory neurons with ciliary dendrites that are structurally and functionally specialised for receiving different sensory modalities. For instance, chordotonal (Ch) neurons have mechanosensory ciliary dendrites and are required for proprioception and hearing. Time-course gene expression profiling of differentiating Ch neurons allowed us to characterise the roles of two transcription factors for ciliogenic gene regulation: the well-known cilia gene regulator, Rfx, and a factor of the Forkhead family (Fd3F), which appears to be a diverged homologue of FOXJ1. Fd3F and Rfx cooperate to regulate a cohort of genes required for ciliary motility - in Drosophila this is a specialisation unique to Ch neuron cilia and is essential for the hearing mechanism. Analysing the target genes of Fd3F has led to the implication of new factors in the assembly of axonemal dynein complexes. Two of these are also mutated in human primary ciliary dyskinesia. Further analysis of these genes in Drosophila and mouse suggests that ZMYND10 may be linked to an emerging chaperone pathway, while HEATR2 appears to have a distinct function related to transport.

The structural and functional roles of many of the 800-1000 proteins that make up the microtubule core and specialized membranes of cilia and flagella are poorly understood. Following from our recent expression study to identify putative ciliary candidates in Drosophila sensory neurons, we focused on a subset that were targets of the transcription factor Fd3f, which regulates functional specialization of mechanosensory cilia. Bioinformatic enrichment for known ciliary domains as well as orthologous protein-protein interaction network modelling provided a list of putative ciliary genes for further functional characterization. One such candidate, CG31320, has been initially characterized in Drosophila. Little is known about this gene, except the encoded protein contains HEAT repeats – belonging to an armadillo-like fold family associated with intracellular transport. In situ analysis confirms that CG31320 mRNA is highly expressed in the ciliated chordotonal neurons. RNAi-mediated knock-down resulted in abnormal chordotonal ciliary morphology and locomotory defects, consistent with impaired mechanosensory cilium function. Currently, we are studying whether the ortholog Heatr2 is also required for mammalian cilia. Protein localization studies suggest that Heatr2 plays a role in trafficking to primary cilia. RNAi knock-down and protein interaction studies using mammalian cells are underway to functionally dissect Heatr2 roles; results will be presented. We are generating a Heatr2 conditional mouse mutant to investigate its function in different types of cilia and sperm flagella. We present a multisystem experimental pipeline for functional characterization of novel genes expressed in cilia as well as putative ciliopathy candidates.

Primary cilia are essential for mouse development, and are important for key signalling events, particularly Hedgehog (Hh) signalling. Mouse mutants for cilia genes show perturbed Hh responses. We developed a cell-based RNAi screen to identify new genes involved in cilia formation and/or function. We are screening candidate genes identified by cross-species analysis plus proteomic and transciptomic studies. The screen provides two readouts. An image-based readout identifies genes required for cilia formation, assayed by high-throughput immunofluorescence microscopy. A second, functional, readout measures Hh responsiveness, for which cilia are necessary. The screen has revealed several candidate genes, which may have a role in ciliogenesis. We have performed an in depth analysis of one such candidate gene, Azi1 for which we have generated a gene trap mouse line. Identification of novel ciliogenic genes will aid the analysis of diverse functions of primary cilia in development and help explain the varied phenotypes seen in human ciliary diseases, or ciliopathies.