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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

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.

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.