Abstract
BackgroundSophisticated tools for manipulation of gene expression in select neurons, including neurons that regulate sexually dimorphic behaviors, are increasingly available for analysis of genetic model organisms. However, we lack comparable genetic tools for analysis of non-model organisms, including Aedes aegypti, a vector mosquito which displays sexually dimorphic behaviors that contribute to pathogen transmission. Formaldehyde-assisted isolation of regulatory elements followed by sequencing (FAIRE-seq) recently facilitated genome-wide discovery of putative A. aegypti cis-regulatory elements (CREs), many of which could be used to manipulate gene expression in mosquito neurons and other tissues. The goal of this investigation was to identify FAIRE DNA elements that promote gene expression in the olfactory system, a tissue of vector importance.ResultsEight A. aegypti CREs that promote gene expression in antennal olfactory receptor neurons (ORNs) were identified in a Drosophila melanogaster transgenic reporter screen. Four CREs identified in the screen were cloned upstream of GAL4 in a transgenic construct that is compatible with transformation of a variety of insect species. These constructs, which contained FAIRE DNA elements associated with the A. aegypti odorant coreceptor (orco), odorant receptor 1 (Or1), odorant receptor 8 (Or8) and fruitless (fru) genes, were used for transformation of A. aegypti. Six A. aegypti strains, including strains displaying transgene expression in all ORNs, subsets of these neurons, or in a sex-specific fashion, were isolated. The CREs drove transgene expression in A. aegypti that corresponded to endogenous gene expression patterns of the orco, Or1, Or8 and fru genes in the mosquito antenna. CRE activity in A. aegypti was found to be comparable to that observed in D. melanogaster reporter assays.ConclusionsThese results provide further evidence that FAIRE-seq, which can be paired with D. melanogaster reporter screening to test FAIRE DNA element activity in select tissues, is a useful method for identification of mosquito cis-regulatory elements. These findings expand the genetic toolkit available for the study of Aedes neurobiology. Moreover, given that the CREs drive comparable olfactory neural expression in both A. aegypti and D. melanogaster, it is likely that they may function similarly in multiple dipteran insects, including other disease vector mosquito species.
Highlights
Sophisticated tools for manipulation of gene expression in select neurons, including neurons that regulate sexually dimorphic behaviors, are increasingly available for analysis of genetic model organisms
It was hypothesized that Formaldehyde-assisted isolation of regulatory elements (FAIRE) DNA elements flanking these genes, which are known to be expressed in Aedes antennal olfactory receptor neurons (ORNs) [26], would regulate gene expression in these neurons
Identification of A. aegypti Cis-regulatory element (CRE) that function in the ORNs of divergently-related dipteran insects The results of this investigation indicated that the A. aegypti odorant receptor 1 (Or1), odorant receptor 8 (Or8), orco and fru CREs identified through FAIRE-seq open chromatin profiling [17] can promote transgene expression patterns in the A. aegypti antenna that are comparable to the endogenous Or1, Or8, orco and fru gene transcript expression patterns in A. aegypti larval (Fig. 2) and adult (Fig. 3) antennae
Summary
Sophisticated tools for manipulation of gene expression in select neurons, including neurons that regulate sexually dimorphic behaviors, are increasingly available for analysis of genetic model organisms. Formaldehyde-assisted isolation of regulatory elements followed by sequencing (FAIRE-seq) recently facilitated genome-wide discovery of putative A. aegypti cis-regulatory elements (CREs), many of which could be used to manipulate gene expression in mosquito neurons and other tissues. The goal of this investigation was to identify FAIRE DNA elements that promote gene expression in the olfactory system, a tissue of vector importance. It is critical that we identify new tools to both study mosquito biology and combat these insect vectors of human disease-causing pathogens Mosquito behaviors such as blood meal acquisition, courtship and oviposition have attracted the attention of biologists for years. While hundreds of neuron-specific GAL4 driver lines enable sophisticated perturbation of the fruit fly nervous system [3, 4], we lack comparable genetic tools for analysis of the neurophysiological basis of behaviors in most insects, including mosquitoes
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