Regulation and function of genes involved in drosophila ciliogenesis
Proneural proteins are transcription factors of the bHLH family and have a conserved role in directing neurogenesis from invertebrate to mammals. In Drosophila, proneural proteins are required for early developmental specification of precursor cells of sense organs (SOPs). Despite considerable progress having been made in this field, it remains unknown how proneural proteins organise the well-orchestrated process that facilitates each type of SOP to acquire both generic neuronal properties and individual neuronal subtype identity during the progression from specification to differentiation. To approach this question, we investigate the gene regulatory network by proneural protein Ato by means of the microarray analysis. Ato directs the formation of the Drosophila chordotonal organs (Ch), important proprioceptive sense organs (Jarman et al., 1993b). The microarray study generated a list of candidate Ato target genes (Cachero et al., 2011). My PhD project entails the characterisation of two potential Ato target genes arising from this screen: Rfx and dila. To determine their positions in the gene regulatory network, I analysed the regulation and function of these genes. First, I demonstrated that both Rfx and dila are activated during Ch neurogenesis as direct targets of Ato. This was established by characterising their expression patterns, cis-regulation analyses and identifying the potential Ato binding sites by site-directed mutagenesis. RFX is a well-known ciliogenic regulator (Dubruille et al., 2002; El Zein et al., 2009; Swoboda et al., 2000), and its activation by Ato is consistent with Ch neurons having ciliated dendrites. However, the role of dila was completely unknown, but its sequence suggested that it may be involved in neuronal differentiation rather than gene regulation. I generated several dila mutant alleles and demonstrated that dila mutants exhibit severe uncoordination, due to a series of defects in ciliated neurons. These defects were linked to a disruption in the ciliogenesis machinery, particularly in the process known as intraflagellar transport (IFT). dila mutants also display reduced male fertility because of aberrant basal body function, which leads to a disorder in sperm individualisation. Thus DILA is required for the differentiation of all ciliated cells in Drosophila. Visualisation of tagged protein localised DILA to the basal body and transition zone of the sensory cilia. Further analysis revealed the genetic interaction between DILA and UNC (another basal body protein) during ciliogenesis. Taken together I propose that DILA regulates IFT at the base of the cilia in collaboration with UNC. Given that dila is an evolutionarily conserved gene, dila homologues could be candidate genes for human ciliopathies. Rfx is essential for ciliogenesis in both Ch and the external sense (ES) organs, which have distinctive cilia. Despite of this common role of RFX, I discovered that Rfx is expressed differently in Ch and ES lineages, which led me to hypothesise that the difference in Rfx expression modulates ciliogenesis in these two lineages. I obtained preliminary data that support this hypothesis. Overall, my study demonstrates important links between Ato and the regulation of ciliogenesis, which is an important process in Ch neuron differentiation. The data support a model in which Ato controls ciliogenesis both directly (e.g. via activating a ciliary genes like dila) and indirectly (e.g. via regulating the transcriptional factors essential for ciliogenesis, like RFX).
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