A Drosophila model for Meniere's disease: Dystrobrevin is required for support cell function in hearing and proprioception.

Meniere's disease (MD) is an inner ear disorder characterised by recurrent vertigo attacks associated with sensorineural hearing loss and tinnitus. Evidence from epidemiology and Whole Exome Sequencing (WES) suggests a genetic susceptibility involving multiple genes, including α-Dystrobrevin (DTNA). Here we investigate a Drosophila model. We show that mutation, or knockdown, of the DTNA orthologue in Drosophila, Dystrobrevin (Dyb), results in defective proprioception and impaired function of Johnston's Organ (JO), the fly's equivalent of the inner ear. Dyb and another component of the dystrophin-glycoprotein complex (DGC), Dystrophin (Dys), are expressed in support cells within JO. Their specific locations suggest that they form part of support cell contacts, thereby helping to maintain the integrity of the hemolymph-neuron diffusion barrier, which is equivalent to a blood-brain barrier. These results have important implications for the human condition, and notably, we note that DTNA is expressed in equivalent cells of the mammalian inner ear.

A general mathematical model of transduction events in mechano-sensory stretch receptors.

Crayfish (Astacus astacus) muscle stretch receptors show strong homology to mammalian muscle spindles and bipolar neurons in D. melanogaster. All are typical, non-ciliated, stretch-sensitive, afferent neurons. Such receptors are observed in many species and perform an important sensory role. However, they are poorly characterised. A previous study reported a bio-mechanical and behavioural model of A. astacus stretch receptors, which used the principles of elasticity and tension in a spring to describe the adaptation of a mechano-sensory ending. This model described the changing mechano-sensory currents in the receptor when subjected to a stretch protocol. Here, we re-implement and extend this model. Notably, we introduce additional descriptions of voltage-gated channels that are suggested to contribute to stretch receptor mechano-transduction. Our model presents a more complete picture of the initiation of the mechano-receptor potential in response to a stretching stimulus. The inclusion of voltage-dependent sodium and potassium currents in addition to the initial mechano-sensitive sodium current allowed the model to account for most of the initial stretch response of the receptor. This preliminary model has potential for extension to describe fully the behaviour of non-ciliated mechano-sensors across species and predict the molecular mediators of mechano-transduction.

Drosophila homolog of the myotonic dystrophy-associated gene, SIX5, is required for muscle and gonad development.

SIX5 belongs to a family of highly conserved homeodomain transcription factors implicated in development and disease. The mammalian SIX5/SIX4 gene pair is likely to be involved in the development of mesodermal structures. 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. 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. 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.

Mitogens match cell numbers to local demand.

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.

Developmental genetics: vertebrates and insects see eye to eye.

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.

cato encodes a basic helix-loop-helix transcription factor implicated in the correct differentiation of Drosophila sense organs.

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.

Drosophila atonal controls photoreceptor R8-specific properties and modulates both receptor tyrosine kinase and Hedgehog signalling.

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.

amos, a proneural gene for Drosophila olfactory sense organs that is regulated by lozenge.

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.

Antagonism of EGFR and notch signalling in the reiterative recruitment of Drosophila adult chordotonal sense organ 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 specificity of proneural genes in determining Drosophila sense organ identity.

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.

Requirement for EGF receptor signalling in neural recruitment during formation of Drosophila chordotonal sense organ clusters.

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.

Role of the proneural gene, atonal, in formation of Drosophila chordotonal organs and photoreceptors.

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.

Atonal is the proneural gene for Drosophila photoreceptors.

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.

asense is a Drosophila neural precursor gene and is capable of initiating sense organ formation.

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 daughterless 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 downstream 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.

The regulation and function of the helix-loop-helix gene, asense, in Drosophila neural precursors.

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 (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. Delection 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 properities.

atonal is a proneural gene that directs chordotonal organ formation in the Drosophila peripheral nervous system.

In the Drosophila peripheral nervous system, proneural genes of the achaete-scute complex (ASC) are required for formation of the precursors of external sense organs but not of chordotonal organs. We report the isolation of a gene, atonal (ato), with evidence that it is a proneural gene for the formation of chordotonal organs. This gene is expressed in the proneural clusters and sense organ precursors that give rise to the embryonic and adult chordotonal, but not external sense, organs. Chordotonal organs are eliminated in embryos carrying chromosomal deficiencies that remove ato. Like the ASC products, ato protein contains a basic-helix-loop-helix region and heterodimerizes with daughterless protein to bind to E boxes. Moreover, ectopic expression of ato promotes the formation of extra sense organs. Despite similar proneural properties, we find that ectopic expression of the ASC genes promotes external sense organ formation exclusively, whereas ato promotes chordotonal organ formation preferentially. Thus, proneural genes are major determinants of neuronal identity.

Characterization of the major regulatory element upstream of the human alpha-globin gene cluster.

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 major positive regulatory region located far upstream of the human alpha-globin gene locus.

We have identified a remote, tissue-specific, positive regulatory element that is of major importance in determining the level of human alpha-globin gene expression. Stable transformants containing this DNA segment linked to the alpha gene in mouse erythroleukemia cells expressed human alpha mRNA at levels that are indistinguishable from those seen in interspecific hybrids containing the human alpha genes in their normal context on chromosome 16. Furthermore, all transgenic mice containing the alpha genes linked to this region expressed alpha-globin mRNA at high levels in erythroid tissues; and in one such mouse, readily detectable levels of human alpha-globin chains could be demonstrated in the peripheral blood. There is considerable similarity in the position, structure, and function of this region upstream of the alpha-globin complex with previously described elements within the beta-globin dominant control region (DCR). This is m marked contrast to other structural and functional differences between the two gene clusters. It seems likely that these critical, positive regulatory regions might provide target sequences through which coordinate regulation of the alpha- and beta-like globin genes is achieved.