Drosophila DEG/ENaC pickpocket genes are expressed in the tracheal system, where they may be involved in liquid clearance

Abstract

The Drosophila tracheal system and mammalian airways are branching networks of tubular epithelia that deliver oxygen to the organism. In mammals, the epithelial Na(+) channel (ENaC) helps clear liquid from airways at the time of birth and removes liquid from the airspaces in adults. We tested the hypothesis that related Drosophila degenerin (DEG)/ENaC family members might play a similar role in the fly. Among 16 Drosophila DEG/ENaC genes, called pickpocket (PPK) genes, we found 9 expressed in the tracheal system. By in situ hybridization, expression appeared in late-stage embryos after tracheal tube formation, with individual PPK genes showing distinct temporal and spatial expression patterns as development progressed. Promoters for several PPK genes drove reporter gene expression in the larval and adult tracheal systems. Adding the DEG/ENaC channel blocker amiloride to the medium inhibited liquid clearance from the trachea of first instar larvae. Moreover, when RNA interference was used to silence PPK4 and PPK11, larvae failed to clear tracheal liquid. These data suggest substantial molecular diversity of DEG/ENaC channel expression in the Drosophila tracheal system where the PPK proteins likely play a role in Na(+) absorption. Extensive similarities between Drosophila and mammalian airways offer opportunities for genetic studies that may decipher further the structure and function of DEG/ENaC proteins and development of the airways.

Figure 1

In situ hybridization of PPK11 expression at different embryonic stages. (A) Diagram of tracheal system [adopted from Manning and Krasnow (1)]. Anterior is to the left and dorsal is to the top. DT, dorsal trunk; DB, dorsal branch; LT, lateral trunk. (B–F) Antisense probe staining from stages 15–17 (S15–S17). (G and H) Negative control of sense probe staining. Scale bar indicates 100 μM for all images in Figs. 1–6.

Figure 2

PPK4 and PPK11 promoter-driven eGFP expression in the tracheal system. Shown are eGFP florescent images (Upper) and eGFP fluorescence laid over light micrographs that show tracheal branches in the same field (Lower). (A–D) PPK11-Gal4;UAS-eGFP. (E) PPK4-Gal4;UAS-eGFP. In A, the dorsal trunk (DT) and smaller branches are shown. In B, the anterior part of the larval tracheal network is shown, including the head, anterior spiracle, and multiple small branches. In C, dorsal branch (DB) epithelial cell is shown. In D and E, terminal cells are shown.

Figure 3

In situ hybridization and promoter-mediated eGFP expression for PPK10 and PPK12. (A and B) PPK10 and PPK12. (Top) In situ hybridization staining pattern in stage-17 embryos. Dorsal trunk (DT) and TC are indicated. (Middle) PPK10 and PPK12 promoters driving eGFP expression (PPK10-Gal4;UAS-eGFP and PPK12-Gal4;UAS-eGFP) in first instar larvae. (Bottom) Enlarged views from Middle.

Figure 4

PPK genes showed distinct expression patterns in the dorsal trunk (DT). (A) PPK14 in situ hybridization in stage-17 embryos. Arrows indicate the fusion point between two segmental dorsal trunks. (B) PPK13 promoter driving eGFP expression (PPK13-Gal4;UAS-eGFP). (C and D) PPK4 expression shown by in situ hybridization and promoter-driven eGFP expression (PPK4-Gal4;UAS-eGFP).

Figure 5

PPK gene expression at the spiracles and in adult Drosophila. (A) PPK4 promoter-driven expression in the adult tracheal system. Dorsal trunk (DT) is indicated. (B) PPK10 promoter-driven eGFP expression in the adult spiracles.

Figure 6

Liquid clearance in the first instar larva. (A) Wild-type larvae with air-filled trachea. (B) Amiloride-treated larvae showing liquid-filled trachea; arrows point to regions of liquid-filled trachea. (C and D) PPK4 and PPK11 RNAi-injected larvae. (E) Percentage of larvae with defective liquid clearance. n = 223–459 for each condition; *, P < 0.01.