Evolution, Expression, and Function of Nonneuronal Ligand-Gated Chloride Channels in Drosophila melanogaster

Abstract

Ligand-gated chloride channels have established roles in inhibitory neurotransmission in the nervous systems of vertebrates and invertebrates. Paradoxically, expression databases in Drosophila melanogaster have revealed that three uncharacterized ligand-gated chloride channel subunits, CG7589, CG6927, and CG11340, are highly expressed in nonneuronal tissues. Furthermore, subunit copy number varies between insects, with some orders containing one ortholog, whereas other lineages exhibit copy number increases. Here, we show that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. We used promoter-GFP expression analysis, RNA-sequencing, and in situ hybridization to examine cell type and tissue-specific localization of the three D. melanogaster subunits. CG6927 is expressed in the nurse cells of the ovaries. CG7589 is expressed in multiple tissues including the salivary gland, ejaculatory duct, malpighian tubules, and early midgut. CG11340 is found in malpighian tubules and the copper cell region of the midgut. Overexpression of CG11340 increased sensitivity to dietary copper, and RNAi and ends-out knockout of CG11340 resulted in copper tolerance, providing evidence for a specific nonneuronal role for this subunit in D. melanogaster Ligand-gated chloride channels are important insecticide targets and here we highlight copy number and functional divergence in insect lineages, raising the potential that order-specific receptors could be isolated within an effective class of insecticide targets.

Keywords: copper tolerance; copy number variation; gene duplication; ligand-gated chloride channel; nonneuronal expression.

Figure 1

Maximum likelihood phylogenetic tree of Insect Group I LGCCs. Node labels indicate branch support as a percentage over 1000 bootstrap replicates. The scale bar denotes the expected number of substitutions per site along branches in the tree. * indicates manually annotated or corrected.

Figure 2

Expression pattern of CG7589 in third instar larvae (A–C) and adults (D–F). Larvae: (A) GFP expression in the larval salivary gland, in secretory cells (sc) and in the imaginal rings (imr), (B) anterior portion of the midgut, and (C) malpighian tubules. Adults: (D) GFP expression in the salivary glands (sg) directly following the salivary duct cells, and the early midgut (mg), (E) the stellate cells of the malpighian tubules, and the (F) male ejaculatory duct. GFP, green fluorescent protein.

Figure 3

Expression pattern of CG6927. (A and B) Adult female germline. GFP is localized to the nuclei of the nurse cells of oocytes from early on in oocyte development. (C and D) In situ hybridization of CG6927 in female reproductive tissue of adult D. melanogaster. (C) Negative control using sense probe for CG6927. (D) Positive staining observed in nurse cell cytoplasm using antisense probe for CG6927. GFP, green fluorescent protein.

Figure 4

Expression pattern of CG11340. (A and B) The third instar larval midgut, (am- anterior midgut; mm- middle midgut). GFP expression is localized primarily to the middle midgut, divided into cc (copper cells), LFC (large flat cells), and fe (iron cells). (C and D) Malpighian tubule expression in third instar larvae (C) and adults (D). (E and F) The adult midgut. GFP expression is localized to regions R1, R2a, and R3 (50). Also shows malpighian tubules (mt). GFP, green fluorescent protein.

Figure 5

RNA-seq expression levels in third instar larval midgut. (A) CG7589 signal is highest in the M1 region, reducing over the M2–5. (B) CG11340 signal begins in the M3–5 portion and reaches its peak in M6, reducing in M7–8. (C) Summary of gene expression patterns for CG11340 (purple), CG7589 (green), and CG6927 (red), combining promotor-GFP, in situ hybridization, and RNA-seq results. Light to dark colored shading represents moderate to high expression levels. GFP, green fluorescent protein. Line drawings adapted from Miller (1950) and Murakami et al, (1994).

Figure 6

(Top) Overexpression and RNAi knockdown of CG11340 and CG7589 in the midgut, malpighian tubules, and fat body (5′HR driver). Survival on reduced available copper was tested with the addition of 0.5 M of the copper chelator BCS to the media, and increased copper by the addition of 1–2 mM CuSO₄. Overexpression of CG11340 showed reduced survival on 1 mM CuSO₄ compared to w¹¹¹⁸ control and other lines tested, whereas CG11340 knockdown provided increased survival on 1 mM CuSO₄. (Bottom) Larval to adult mortality of CG11340 knockout and w¹¹¹⁸ control on 6 doses of CuSO₄ (1.75–5 mM). Mortality of the CG11340 knockout line is significantly reduced compared to controls (95% C.I. shown). BCS, bathocuproine disulphonate; CuSO₄, copper (II) sulfate.