In vivo functional characterization of nicotinic acetylcholine receptors in Drosophila melanogaster
AuthorLuong, Hang Ngoc Bao
Document TypePhD thesis
Access StatusOpen Access
© 2018 Dr. Hang Ngoc Bao Luong
Nicotinic acetylcholine receptors (nAChRs) are responsible for fast excitatory synaptic transmission in insect central nervous system. Their role as targets for commercial insecticides have resulted in extensive studies on their structure and pharmacological properties. However, many other aspects of their fundamental biology remain less understood. For example, what behaviours are underpinned by the activity of nicotinic acetylcholine receptors? Here, we used reverse genetics to address this question. The precise genome editing power of CRISPR/Cas9 technology was used to generate a collection of Drosophila melanogaster lines harbouring precise genomic deletions of the genes of interest, including the subunits for the nicotinic acetylcholine receptors as well as a couple of their accessory proteins. The overall strategy was to remove as much as of the genomic locus as possible by having two sgRNAs directing Cas9 to cut at the 5’ and 3’ ends of the gene’s coding sequence and relying on non-homologous end joining repair to ligate the termini together creating a deletion. In total, nine knockout strains were generated for four genes, successfully removing genomic sequences ranging from 4 to 83kb in length. For three genes, Dα4, Dα6 and DmRIC3, the same allele was recapitulated for three backgrounds. The role of nAChRs in regulating sleep behaviour in vinegar flies was investigated using null alleles of the receptor subunits. For seven of the ten subunits, flies harbouring null alleles were viable as adults for behavioural assays. All mutants showed changes in total sleep amount compared to their controls, which most strongly correlated with changes in sleep episode duration. Additionally, genotypes carrying partial deletions or point mutations displayed different sleep changes, suggesting that allelic variation within subunits can yield different phenotypes. These data confirmed a role in sleep regulation for most nAChR subunits. Furthermore, the role of the nAchR accessory proteins were considered. Lines with a deletion of the nAChR-specific chaperone DmRIC3 responded to two commercial insecticides in similar manner to loss of the subunit Dα1. Those lines also phenocopied sleep behaviour of flies lacking receptor subunits. This is the first in vivo evidence of the functional significance of DmRIC3 to nAChRs in D. melanogaster. Altogether, these results show that significant behavioural changes might be considerable fitness costs beyond viability for resistant alleles of genes with important functions in the central nervous system such as nAChRs. However, resistance could still arise from disruption to other proteins interacting and regulating nAChRs with less severe costs.
KeywordsDrosophila; nachrs; sleep; insecticide; CRISPR
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