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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.

Background Protein-protein interaction (PPI) data are widely used to generate network models that aim to describe the relationships between proteins in biological systems. The fidelity and completeness of such networks is primarily limited by the paucity of protein interaction information and by the restriction of most of these data to just a few widely studied experimental organisms. In order to extend the utility of existing PPIs, computational methods can be used that exploit functional conservation between orthologous proteins across taxa to predict putative PPIs or 'interologs'. To date most interolog prediction efforts have been restricted to specific biological domains with fixed underlying data sources and there are no software tools available that provide a generalised framework for 'on-the-fly' interolog prediction. Results We introduce Bio::Homology::InterologWalk, a Perl module to retrieve, prioritise and visualise putative protein-protein interactions through an orthology-walk method. The module uses orthology and experimental interaction data to generate putative PPIs and optionally collates meta-data into an Interaction Prioritisation Index that can be used to help prioritise interologs for further analysis. We show the application of our interolog prediction method to the genomic interactome of the fruit fly, Drosophila melanogaster. We analyse the resulting interaction networks and show that the method proposes new interactome members and interactions that are candidates for future experimental investigation. Conclusions Our interolog prediction tool employs the Ensembl Perl API and PSICQUIC enabled protein interaction data sources to generate up to date interologs 'on-the-fly'. This represents a significant advance on previous methods for interolog prediction as it allows the use of the latest orthology and protein interaction data for all of the genomes in Ensembl. The module outputs simple text files, making it easy to customise the results by post-processing, allowing the putative PPI datasets to be easily integrated into existing analysis workflows. The Bio::Homology::InterologWalk module, sample scripts and full documentation are freely available from the Comprehensive Perl Archive Network (CPAN) under the GNU Public license.