Identification and characterisation of conserved ciliary genes expressed in Drosophila sensory neurons
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Date
28/11/2014Item status
Restricted AccessEmbargo end date
31/12/2100Author
Moore, Daniel John
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Abstract
Drosophila provide an excellent model organism in which to study cilia as there are
only two ciliated cell types; the sensory neurons and sperm cells. The chordotonal
neuron is one such ciliated cell and is required for hearing, proprioception and
gravitaxis. Mechanical manipulation of the cilium that extends from the neuronal
dendrite is required for signal transduction. Chordotonal neuronal differentiation is
regulated by a transcription factor cascade. Atonal begins the cascade, which is then
continued by RFX and Fd3F for ciliary genes (Cachero et al 2011, Newton et al
2012). Genes expressed in developing chordotonal neurons are downstream of these
transcription factors and their characterisation can further elucidate how neuronal
differentiation is regulated. Ciliary genes are highly enriched in developing
chordotonal cells; uncharacterised genes enriched in these cells can therefore be
considered candidate ciliary genes (Cachero et al 2011).
A behavioural assay was conducted to identify further genes that could have a role in
ciliary formation and function. Candidate genes were identified by combining
enrichment data with previous genomic, proteomic and transcriptomic studies of
cilia. A climbing assay of RNAi mediated knock down of these genes identified a
number of candidates for future work.
One gene found to be highly enriched in developing chordotonal neurons is
CG11253. CG11253EY10866 P element insertion mutant flies show a mild
uncoordinated phenotype in a climbing assay consistent with reduced chordotonal
organ function. Male flies are also infertile due to a lack of motile sperm. CG11253
is expressed in motile ciliated cells and is conserved in organisms with motile cilia.
CG11253 expression is also regulated by RFX and Fd3F, suggesting that it is
involved in cilium motility. This was confirmed by electron microscopy, which
showed disruption of axonemal dynein arm localisation in chordotonal cilia and
sperm flagella. A CG11253::mVenus fusion protein was found to localise mainly to
the cytoplasm and to a lesser extent the cilia of chordotonal neurons. Patients with
symptoms consistent with Primary Ciliary Dyskinesia (PCD), a condition caused by
cilium immotility, have subsequently been found to have point mutations in
ZMYND10, the human homologue of CG11253.
The identification of PCD patients with ZMYND10 mutations showed that
investigating cilium motility in Drosophila chordotonal neurons could identify novel
PCD genes. It was thought that investigating previously uncharacterised targets of
Fd3F could identify novel genes involved in cilium motility and thus candidate PCD
genes. CG31320 is a gene regulated by RFX and Fd3F and conserved in organisms
with motile cilia. RNAi mediated knock down of CG31320 resulted in both a mild
uncoordinated phenotype and male infertility due to a lack of motile sperm. Electron
microscopy showed a complete loss of axonemal dynein arms in chordotonal neuron
cilia. An mVenus fusion protein of CG6971, an inner dynein arm component, was
also mislocalised from the cilia in CG3132027 deletion mutant larvae. This shows
that CG31320 is required for the appropriate localisation of the axonemal dynein
arms and thus cilium motility. This further showed that uncharacterised genes
enriched in chordotonal neurons and regulated by Fd3F could be novel ciliary genes
required for cilium motility. Our collaborators and Horani et al (2012) showed that
the human homologue of CG31320 (HEATR2) is mutated in patients with PCD,
further confirming that this method can be used to identify PCD genes.
I have identified two factors required for cilium motility. Disruption of the axonemal
dynein arms in both cases results in reduced coordination, and lack of fertility due to
sperm immotility. Mutations in the human homologues of these genes have been
found to result in PCD. This indicates that further PCD genes could be identified
from genes enriched in Drosophila chordotonal neurons that are regulated by Fd3F.