Ciliary tip actin dynamics regulate photoreceptor outer segment integrity.

As signalling organelles, cilia regulate their G protein-coupled receptor content by ectocytosis, a process requiring localised actin dynamics to alter membrane shape. Photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip of highly-specialised connecting cilia, into which photosensitive GPCRs are concentrated. Discs are shed and remade daily. Defects in this process, due to mutations, cause retinitis pigmentosa (RP). Whilst fundamental for vision, the mechanism of photoreceptor disc generation is poorly understood. Here, we show membrane deformation required for disc genesis is driven by dynamic actin changes in a process akin to ectocytosis. We show RPGR, a leading RP gene, regulates actin-binding protein activity central to this process. Actin dynamics, required for disc formation, are perturbed in Rpgr mouse models, leading to aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. Actin manipulation partially rescues this, suggesting the pathway could be targeted therapeutically. These findings help define how actin-mediated dynamics control outer segment turnover.

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.

Mechanisms of Photoreceptor Death in Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is the most common cause of inherited blindness and is characterised by the progressive loss of retinal photoreceptors. However, RP is a highly heterogeneous disease and, while much progress has been made in developing gene replacement and gene editing treatments for RP, it is also necessary to develop treatments that are applicable to all causative mutations. Further understanding of the mechanisms leading to photoreceptor death is essential for the development of these treatments. Recent work has therefore focused on the role of apoptotic and non-apoptotic cell death pathways in RP and the various mechanisms that trigger these pathways in degenerating photoreceptors. In particular, several recent studies have begun to elucidate the role of microglia and innate immune response in the progression of RP. Here, we discuss some of the recent progress in understanding mechanisms of rod and cone photoreceptor death in RP and summarise recent clinical trials targeting these pathways.

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.

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.

Coordinate post-transcriptional repression of Dpp-dependent transcription factors attenuates signal range during development.

Precise control of the range of signalling molecule action is crucial for correct cell fate patterning during development. For example, Drosophila ovarian germline stem cells (GSCs) are maintained by exquisitely short-range BMP signalling from the niche. In the absence of BMP signalling, one GSC daughter differentiates into a cystoblast (CB) and this fate is stabilised by Brain tumour (Brat) and Pumilio (Pum)-mediated post-transcriptional repression of mRNAs, including that encoding the Dpp transducer, Mad. However, the identity of other repressed mRNAs and the mechanism of post-transcriptional repression are currently unknown. Here, we identify the Medea and schnurri mRNAs, which encode transcriptional regulators required for activation and/or repression of Dpp target genes, as additional Pum-Brat targets, suggesting that tripartite repression of the transducers is deployed to desensitise the CB to Dpp. In addition, we show that repression by Pum-Brat requires recruitment of the CCR4 and Pop2 deadenylases, with knockdown of deadenylases in vivo giving rise to ectopic GSCs. Consistent with this, Pum-Brat repression leads to poly(A) tail shortening and mRNA degradation in tissue culture cells, and we detect a reduced number of Mad and shn transcripts in the CB relative to the GSC based on single molecule mRNA quantitation. Finally, we show generality of the mechanism by demonstrating that Brat also attenuates pMad and Dpp signalling range in the early embryo. Together our data serve as a platform for understanding how post-transcriptional repression restricts interpretation of BMPs and other cell signals in order to allow robust cell fate patterning during development.

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 gene regulatory cascade linking proneural specification with differentiation in Drosophila sensory neurons.

In neurogenesis, neural cell fate specification is generally triggered by proneural transcription factors. Whilst the role of proneural factors in fate specification is well studied, the link between neural specification and the cellular pathways that ultimately must be activated to construct specialised neurons is usually obscure. High-resolution temporal profiling of gene expression reveals the events downstream of atonal proneural gene function during the development of Drosophila chordotonal (mechanosensory) neurons. Among other findings, this reveals the onset of expression of genes required for construction of the ciliary dendrite, a key specialisation of mechanosensory neurons. We determine that atonal activates this cellular differentiation pathway in several ways. Firstly, atonal directly regulates Rfx, a well-known highly conserved ciliogenesis transcriptional regulator. Unexpectedly, differences in Rfx regulation by proneural factors may underlie variations in ciliary dendrite specialisation in different sensory neuronal lineages. In contrast, fd3F encodes a novel forkhead family transcription factor that is exclusively expressed in differentiating chordotonal neurons. fd3F regulates genes required for specialized aspects of chordotonal dendrite physiology. In addition to these intermediate transcriptional regulators, we show that atonal directly regulates a novel gene, dilatory, that is directly associated with ciliogenesis during neuronal differentiation. Our analysis demonstrates how early cell fate specification factors can regulate structural and physiological differentiation of neuronal cell types. It also suggests a model for how subtype differentiation in different neuronal lineages may be regulated by different proneural factors. In addition, it provides a paradigm for how transcriptional regulation may modulate the ciliogenesis pathway to give rise to structurally and functionally specialised ciliary dendrites.

Post-meiotic transcription in Drosophila testes.

Post-meiotic transcription was accepted to be essentially absent from Drosophila spermatogenesis. We identify 24 Drosophila genes whose mRNAs are most abundant in elongating spermatids. By single-cyst quantitative RT-PCR, we demonstrate post-meiotic transcription of these genes. We conclude that transcription stops in Drosophila late primary spermatocytes, then is reactivated by two pathways for a few loci just before histone-to-transition protein-to-protamine chromatin remodelling in spermiogenesis. These mRNAs localise to a small region at the distal elongating end of the spermatid bundles, thus they represent a new class of sub-cellularly localised mRNAs. Mutants for a post-meiotically transcribed gene (scotti), are male sterile, and show spermatid individualisation defects, indicating a function in late spermiogenesis.