Identification of Aedes aegypti cis-regulatory elements that promote gene expression in olfactory receptor neurons of distantly related dipteran insects

Abstract

Background: 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. 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.

Results: Eight 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.

Conclusions: These 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.

Keywords: Aedes aegypti; Antenna; Dengue; Drosophila melanogaster; Enhancer; FAIRE; Mosquito; Neuron; Sensory; Zika.

Fig. 1

A D. melanogaster screen identifies A. aegypti CREs that promote gene expression in antennal ORNs. EGFP reporter expression driven by FAIRE DNA sequences flanking the indicated genes (e93 in a, onecut in b, acj6 in c, fru in d, i, and i1, orco in e and e1, Or1 in f and f1, Or8 in g and g1, Or16 in h and h1) was assessed in a total of 40 female and 40 male adult antennae prepared from replicate experiments. EGFP expression patterns ranged from expression in all (e) or many (a- c) antennal ORNs to very specific subsets of ORNs (f-h). Sex-specific EGFP expression was detected in the adult male antenna (d; compare to female antenna in i). Embryonic expression of EGFP was detected in the Or16 reporter line (h1), but not in the orco (e1), Or1 (f1), Or8 (g1) or fru (i1) Drosophila reporter lines. Proximal is oriented upward in a-i, and anterior is oriented upward in e1-i1

Fig. 2

CRE activity in the A. aegypti larval antenna. FAIRE DNA elements associated with the orco (a1), Or1 (b1, b2), fru (c1) and Or8 (d1, d2) genes promote GAL4 reporter expression in A. aegypti larval ORNs. The orco (a1) CRE promotes transgene expression in all larval ORNs, while the Or1 (b1, b2), fru (c1) and Or8 (d1, d2) CREs drive transgene expression in subsets of ORNs. These expression patterns are comparable to the patterns of native orco (a), Or1 (b), fru (c) and Or8 (d) transcripts in the larval antenna. CRE activity is comparable in two separate Or1-GAL4 lines (a in panel b1 and b in panel b2), as well as two separate Or8-GAL4 strains (a in panel d1 and b in panel d2). Mean gray value analyses for the cell marked by the yellow arrowheads in b1 versus b2 revealed no significant differences in transgene signal intensity levels (P > 0.05). Likewise, no differences in transgene signal intensity levels were detected for the cell marked by the blue arrowheads in d1 versus d2. Proximal is oriented upward in all panels

Fig. 3

CRE activity in A. aegypti adult antennal ORNs. Expression of GAL4 transcripts driven by FAIRE DNA elements adjacent to the fru (male in a1), orco (c1), Or1 (d1, d2) and Or8 (e1, e2) genes are comparable to expression of native fru (male in a), orco (c), Or1 (d) and Or8 (e) transcripts in the adult A. aegypti antenna. No fru transcript is detected in the A. aegypti female antenna (b), and GAL4 expression is not driven by the fru CRE in the female antenna (b1). CRE activity is comparable in two separate Or8-GAL4 lines (a in e1 and b in e2), as well as two separate Or1-GAL4 strains (a in panel d1 and b in panel d2). Mean gray value analyses for the cells marked by the yellow, blue, or purple arrowheads in d1 versus d2 revealed no significant differences in transgene signal intensity levels (P > 0.05). Likewise, no significant differences were detected in the transgene signal intensity levels of the cells marked by the magenta, cyan, or green arrowheads in e1 versus e2 (P > 0.05). The fru (male in a1), Or1 (d1, d2) and Or8 (e1 and e2) CREs are active in subsets of ORNs, while the orco (c1) CRE promotes gene expression in all ORNs. With the exception of a and a1, in which male antennae are shown, female antennae oriented proximal upward are displayed in all panels