Repurposing the lineage-determining transcription factor Atoh1 without redistributing its genomic binding sites.

Although the lineage-determining ability of transcription factors is often modulated according to cellular context, the mechanisms by which such switching occurs are not well known. Using a transcriptional programming model, we found that Atoh1 is repurposed from a neuronal to an inner ear hair cell (HC) determinant by the combined activities of Gfi1 and Pou4f3. In this process, Atoh1 maintains its regulation of neuronal genes but gains ability to regulate HC genes. Pou4f3 enables Atoh1 access to genomic locations controlling the expression of sensory (including HC) genes, but Atoh1 + Pou4f3 are not sufficient for HC differentiation. Gfi1 is key to the Atoh1-induced lineage switch, but surprisingly does not alter Atoh1's binding profile. Gfi1 acts in two divergent ways. It represses the induction by Atoh1 of genes that antagonise HC differentiation, a function in keeping with its well-known repressor role in haematopoiesis. Remarkably, we find that Gfi1 also acts as a co-activator: it binds directly to Atoh1 at existing target genes to enhance its activity. These findings highlight the diversity of mechanisms by which one TF can redirect the activity of another to enable combinatorial control of cell identity.

The dynamics of protein localisation to restricted zones within Drosophila mechanosensory cilia.

The Drosophila chordotonal neuron cilium is the site of mechanosensory transduction. The cilium has a 9 + 0 axoneme structure and is highly sub-compartmentalised, with proximal and distal zones harbouring different TRP channels and the proximal zone axoneme also being decorated with axonemal dynein motor complexes. The activity of the dynein complexes is essential for mechanotransduction. We investigate the localisation of TRP channels and dynein motor complexes during ciliogenesis. Differences in timing of TRP channel localisation correlate with order of construction of the two ciliary zones. Dynein motor complexes are initially not confined to their target proximal zone, but ectopic complexes beyond the proximal zone are later cleared, perhaps by retrograde transport. Differences in transient distal localisation of outer and inner dynein arm complexes (ODAs and IDAs) are consistent with previous suggestions from unicellular eukaryotes of differences in processivity during intraflagellar transport. Stable localisation depends on the targeting of their docking proteins in the proximal zone. For ODA, we characterise an ODA docking complex (ODA-DC) that is targeted directly to the proximal zone. Interestingly, the subunit composition of the ODA-DC in chordotonal neuron cilia appears to be different from the predicted ODA-DC in Drosophila sperm.

Strongly Truncated Dnaaf4 Plays a Conserved Role in Drosophila Ciliary Dynein Assembly as Part of an R2TP-Like Co-Chaperone Complex With Dnaaf6.

Axonemal dynein motors are large multi-subunit complexes that drive ciliary movement. Cytoplasmic assembly of these motor complexes involves several co-chaperones, some of which are related to the R2TP co-chaperone complex. Mutations of these genes in humans cause the motile ciliopathy, Primary Ciliary Dyskinesia (PCD), but their different roles are not completely known. Two such dynein (axonemal) assembly factors (DNAAFs) that are thought to function together in an R2TP-like complex are DNAAF4 (DYX1C1) and DNAAF6 (PIH1D3). Here we investigate the Drosophila homologues, CG14921/Dnaaf4 and CG5048/Dnaaf6. Surprisingly, Drosophila Dnaaf4 is truncated such that it completely lacks a TPR domain, which in human DNAAF4 is likely required to recruit HSP90. Despite this, we provide evidence that Drosophila Dnaaf4 and Dnaaf6 proteins can associate in an R2TP-like complex that has a conserved role in dynein assembly. Both are specifically expressed and required during the development of the two Drosophila cell types with motile cilia: mechanosensory chordotonal neurons and sperm. Flies that lack Dnaaf4 or Dnaaf6 genes are viable but with impaired chordotonal neuron function and lack motile sperm. We provide molecular evidence that Dnaaf4 and Dnaaf6 are required for assembly of outer dynein arms (ODAs) and a subset of inner dynein arms (IDAs).

The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly.

Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans, the impairment of these motors through mutation results in the disease primary ciliary dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. Here we report the characterisation of the Drosophila orthologue of the less-known assembly factor DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and the cells that generate spermatozoa, The only two Drosophila cell types bearing cilia/flagella containing dynein motors. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly.

Homeostatic maintenance and age-related functional decline in the Drosophila ear.

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.

Social creatures: Model animal systems for studying the neuroendocrine mechanisms of social behaviour.

The interaction of animals with conspecifics, termed social behaviour, has a major impact on the survival of many vertebrate species. Neuropeptide hormones modulate the underlying physiology that governs social interactions, and many findings concerning the neuroendocrine mechanisms of social behaviours have been extrapolated from animal models to humans. Neurones expressing neuropeptides show similar distribution patterns within the hypothalamic nucleus, even when evolutionarily distant species are compared. During evolution, hypothalamic neuropeptides and releasing hormones have retained not only their structures, but also their biological functions, including their effects on behaviour. Here, we review the current understanding of the mechanisms of social behaviours in several classes of animals, such as worms, insects and fish, as well as laboratory, wild and domesticated mammals.

Survey of the Ciliary Motility Machinery of Drosophila Sperm and Ciliated Mechanosensory Neurons Reveals Unexpected Cell-Type Specific Variations: A Model for Motile Ciliopathies.

The motile cilium/flagellum is an ancient eukaryotic organelle. The molecular machinery of ciliary motility comprises a variety of cilium-specific dynein motor complexes along with other complexes that regulate their activity. Assembling the motors requires the function of dedicated "assembly factors" and transport processes. In humans, mutation of any one of at least 40 different genes encoding components of the motility apparatus causes Primary Ciliary Dyskinesia (PCD), a disease of defective ciliary motility. Recently, Drosophila has emerged as a model for motile cilia biology and motile ciliopathies. This is somewhat surprising as most Drosophila cells lack cilia, and motile cilia are confined to just two specialized cell types: the sperm flagellum with a 9+2 axoneme and the ciliated dendrite of auditory/proprioceptive (chordotonal, Ch) neurons with a 9+0 axoneme. To determine the utility of Drosophila as a model for motile cilia, we survey the Drosophila genome for ciliary motility gene homologs, and assess their expression and function. We find that the molecules of cilium motility are well conserved in Drosophila. Most are readily characterized by their restricted cell-type specific expression patterns and phenotypes. There are also striking differences between the two motile ciliated cell types. Notably, sperm and Ch neuron cilia express and require entirely different outer dynein arm variants-the first time this has been clearly established in any organism. These differences might reflect the specialized functions for motility in the two cilium types. Moreover, the Ch neuron cilia lack the critical two-headed inner arm dynein (I1/f) but surprisingly retain key regulatory proteins previously associated with it. This may have implications for other motile 9+0 cilia, including vertebrate embryonic nodal cilia required for left-right axis asymmetry. We discuss the possibility that cell-type specificity in ciliary motility machinery might occur in humans, and therefore underlie some of the phenotypic variation observed in PCD caused by different gene mutations. Our work lays the foundation for the increasing use of Drosophila as an excellent model for new motile ciliary gene discovery and validation, for understanding motile cilium function and assembly, as well as understanding the nature of genetic defects underlying human motile ciliopathies.

SUMOs Mediate the Nuclear Transfer of p38 and p-p38 during Helicobacter Pylori Infection.

The p38 mitogen activated protein kinase (MAPK) signaling pathway has been suggested to play a significant role in the gastric mucosal inflammatory response to chronic Helicobacter pylori (H. pylori) infection. Nuclear translocation is thought to be important for p38 function, but no nuclear translocation signals have been found in the protein and no nuclear carrier proteins have been identified for p38. We have investigated the role of small ubiquitin-related modifier (SUMO) in the nuclear transfer of p38 in response to H. pylori infection. Exposure of human AGS cells to H. pylori induced the activation of p38 and the expression of SUMOs, especially SUMO-2. SUMO knockdown counteracted the effect of H. pylori infection by decreasing the resulting p38 mediated cellular apoptosis through a reduction in the nuclear fraction of phosphorylated p38. We identified a non-covalent interaction between SUMOs and p38 via SUMO interaction motifs (SIMs), and showed that SUMO-dependent nuclear transfer of p38 was decreased upon mutation of its SIMs. This study has identified a new pathway of p38 nuclear translocation, in response to H. pylori infection. We conclude that in the presence of H. pylori SUMO-2 has a major role in regulating nuclear levels of p38, through non-covalent SUMO-p38 interactions, independent of the p38 phosphorylation state.

ZMYND10 functions in a chaperone relay during axonemal dynein assembly.

Molecular chaperones promote the folding and macromolecular assembly of a diverse set of 'client' proteins. How ubiquitous chaperone machineries direct their activities towards specific sets of substrates is unclear. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone that confers specificity for the FKBP8-HSP90 chaperone complex towards axonemal dynein clients required for cilia motility. Loss of ZMYND10 perturbs the chaperoning of axonemal dynein heavy chains, triggering broader degradation of dynein motor subunits. We show that pharmacological inhibition of FKBP8 phenocopies dynein motor instability associated with the loss of ZMYND10 in airway cells and that human disease-causing variants of ZMYND10 disrupt its ability to act as an FKBP8-HSP90 co-chaperone. Our study indicates that primary ciliary dyskinesia (PCD), caused by mutations in dynein assembly factors disrupting cytoplasmic pre-assembly of axonemal dynein motors, should be considered a cell-type specific protein-misfolding disease.

Ciliary dynein motor preassembly is regulated by Wdr92 in association with HSP90 co-chaperone, R2TP.

The massive dynein motor complexes that drive ciliary and flagellar motility require cytoplasmic preassembly, a process requiring dedicated dynein assembly factors (DNAAFs). How DNAAFs interact with molecular chaperones to control dynein assembly is not clear. By analogy with the well-known multifunctional HSP90-associated cochaperone, R2TP, several DNAAFs have been suggested to perform novel R2TP-like functions. However, the involvement of R2TP itself (canonical R2TP) in dynein assembly remains unclear. Here we show that in Drosophila melanogaster, the R2TP-associated factor, Wdr92, is required exclusively for axonemal dynein assembly, likely in association with canonical R2TP. Proteomic analyses suggest that in addition to being a regulator of R2TP chaperoning activity, Wdr92 works with the DNAAF Spag1 at a distinct stage in dynein preassembly. Wdr92/R2TP function is likely distinct from that of the DNAAFs proposed to form dynein-specific R2TP-like complexes. Our findings thus establish a connection between dynein assembly and a core multifunctional cochaperone.

Atoh1 in sensory hair cell development: constraints and cofactors.

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.

The Drosophila homologue of Rootletin is required for mechanosensory function and ciliary rootlet formation in chordotonal sensory neurons.

BACKGROUND: In vertebrates, rootletin is the major structural component of the ciliary rootlet and is also part of the tether linking the centrioles of the centrosome. Various functions have been ascribed to the rootlet, including maintenance of ciliary integrity through anchoring and facilitation of transport to the cilium or at the base of the cilium. In Drosophila, Rootletin function has not been explored. RESULTS: In the Drosophila embryo, Rootletin is expressed exclusively in cell lineages of type I sensory neurons, the only somatic cells bearing a cilium. Expression is strongest in mechanosensory chordotonal neurons. Knock-down of Rootletin results in loss of ciliary rootlet in these neurons and severe disruption of their sensory function. However, the sensory cilium appears largely normal in structure and in localisation of proteins suggesting no strong defect in ciliogenesis. No evidence was found for a defect in cell division. CONCLUSIONS: The role of Rootletin as a component of the ciliary rootlet is conserved in Drosophila. In contrast, lack of a general role in cell division is consistent with lack of centriole tethering during the centrosome cycle in Drosophila. Although our evidence is consistent with an anchoring role for the rootlet, severe loss of mechanosensory function of chordotonal (Ch) neurons upon Rootletin knock-down may suggest a direct role for the rootlet in the mechanotransduction mechanism itself.

Piezo Is Essential for Amiloride-Sensitive Stretch-Activated Mechanotransduction in Larval Drosophila Dorsal Bipolar Dendritic Sensory Neurons.

Stretch-activated afferent neurons, such as those of mammalian muscle spindles, are essential for proprioception and motor co-ordination, but the underlying mechanisms of mechanotransduction are poorly understood. The dorsal bipolar dendritic (dbd) sensory neurons are putative stretch receptors in the Drosophila larval body wall. We have developed an in vivo protocol to obtain receptor potential recordings from intact dbd neurons in response to stretch. Receptor potential changes in dbd neurons in response to stretch showed a complex, dynamic profile with similar characteristics to those previously observed for mammalian muscle spindles. These profiles were reproduced by a general in silico model of stretch-activated neurons. This in silico model predicts an essential role for a mechanosensory cation channel (MSC) in all aspects of receptor potential generation. Using pharmacological and genetic techniques, we identified the mechanosensory channel, DmPiezo, in this functional role in dbd neurons, with TRPA1 playing a subsidiary role. We also show that rat muscle spindles exhibit a ruthenium red-sensitive current, but found no expression evidence to suggest that this corresponds to Piezo activity. In summary, we show that the dbd neuron is a stretch receptor and demonstrate that this neuron is a tractable model for investigating mechanisms of mechanotransduction.

Stretching the imagination beyond muscle spindles - stretch-sensitive mechanisms in arthropods.

Much attention has been given to mammalian muscle spindles and their role in stretch-mediated muscle proprioception. Recent studies, particularly, have sought to determine the molecular mediators of stretch-evoked mechanotransduction, which these endings rely upon for functionality. Nonetheless, much about these endings remains unknown. Opportunities may be presented from consideration of extensive parallel research in stretch receptor mechanisms in arthropods. Such systems may provide a useful source of additional data and powerful tools for dissecting the complex systems of stretch transduction apparatus. At the least, such systems provide tractable exemplars of how organisms solve the problem of converting stretch stimuli to electrical output. Potentially, they may even provide molecular mechanisms and candidate molecular mediators of direct relevance to mammalian muscle spindles. Here we provide a brief overview of research on arthropod stretch receptors.

HEATR2 plays a conserved role in assembly of the ciliary motile apparatus.

Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.

Mutations in ZMYND10, a gene essential for proper axonemal assembly of inner and outer dynein arms in humans and flies, cause primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a ciliopathy characterized by airway disease, infertility, and laterality defects, often caused by dual loss of the inner dynein arms (IDAs) and outer dynein arms (ODAs), which power cilia and flagella beating. Using whole-exome and candidate-gene Sanger resequencing in PCD-affected families afflicted with combined IDA and ODA defects, we found that 6/38 (16%) carried biallelic mutations in the conserved zinc-finger gene BLU (ZMYND10). ZMYND10 mutations conferred dynein-arm loss seen at the ultrastructural and immunofluorescence level and complete cilia immotility, except in hypomorphic p.Val16Gly (c.47T>G) homozygote individuals, whose cilia retained a stiff and slowed beat. In mice, Zmynd10 mRNA is restricted to regions containing motile cilia. In a Drosophila model of PCD, Zmynd10 is exclusively expressed in cells with motile cilia: chordotonal sensory neurons and sperm. In these cells, P-element-mediated gene silencing caused IDA and ODA defects, proprioception deficits, and sterility due to immotile sperm. Drosophila Zmynd10 with an equivalent c.47T>G (p.Val16Gly) missense change rescued mutant male sterility less than the wild-type did. Tagged Drosophila ZMYND10 is localized primarily to the cytoplasm, and human ZMYND10 interacts with LRRC6, another cytoplasmically localized protein altered in PCD. Using a fly model of PCD, we conclude that ZMYND10 is a cytoplasmic protein required for IDA and ODA assembly and that its variants cause ciliary dysmotility and PCD with laterality defects.

The role of Atonal transcription factors in the development of mechanosensitive cells.

Mechanosensation is an evolutionarily ancient sensory modality seen in all main animal groups. Mechanosensation can be mediated by sensory neurons or by dedicated receptor cells that form synapses with sensory neurons. Evidence over the last 15-20 years suggests that both classes of mechanosensory cells can be specified by the atonal class of basic helix-loop-helix transcription factors. In this review we discuss recent work addressing how atonal factors specify mechanosensitive cells in vertebrates and invertebrates, and how the redeployment of these factors underlies the regeneration of mechanosensitive cells in some vertebrate groups.

Acute versus chronic loss of mammalian Azi1/Cep131 results in distinct ciliary phenotypes.

Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1(Gt/Gt)). Surprisingly, Azi1(Gt/Gt) MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease.

Forkhead transcription factor Fd3F cooperates with Rfx to regulate a gene expression program for mechanosensory cilia specialization.

Cilia have evolved hugely diverse structures and functions to participate in a wide variety of developmental and physiological processes. Ciliary specialization requires differences in gene expression, but few transcription factors are known to regulate this, and their molecular function is unclear. Here, we show that the Drosophila Forkhead box (Fox) gene, fd3F, is required for specialization of the mechanosensory cilium of chordotonal (Ch) neurons. fd3F regulates genes for Ch-specific axonemal dyneins and TRPV ion channels, which are required for sensory transduction, and retrograde transport genes, which are required to differentiate their distinct motile and sensory ciliary zones. fd3F is reminiscent of vertebrate Foxj1, a motile cilia regulator, but fd3F regulates motility genes as part of a broader sensory regulation program. Fd3F cooperates with the pan-ciliary transcription factor, Rfx, to regulate its targets directly. This illuminates pathways involved in ciliary specialization and the molecular mechanism of transcription factors that regulate them.

The SUMO pathway promotes basic helix-loop-helix proneural factor activity via a direct effect on the Zn finger protein senseless.

During development, proneural transcription factors of the basic helix-loop-helix (bHLH) family are required to commit cells to a neural fate. In Drosophila neurogenesis, a key mechanism promoting sense organ precursor (SOP) fate is the synergy between proneural factors and their coactivator Senseless in transcriptional activation of target genes. Here we present evidence that posttranslational modification by SUMO enhances this synergy via an effect on Senseless protein. We show that Senseless is a direct target for SUMO modification and that mutagenesis of a predicted SUMOylation motif in Senseless reduces Senseless/proneural synergy both in vivo and in cell culture. We propose that SUMOylation of Senseless via lysine 509 promotes its synergy with proneural proteins during transcriptional activation and hence regulates an important step in neurogenesis leading to the formation and maturation of the SOPs.