Genetic interactions regulate hypoxia tolerance conferred by activating Notch in excitatory amino acid transporter 1-positive glial cells in Drosophila melanogaster

Abstract

Hypoxia is a critical pathological element in many human diseases, including ischemic stroke, myocardial infarction, and solid tumors. Of particular significance and interest of ours are the cellular and molecular mechanisms that underlie susceptibility or tolerance to low O2. Previous studies have demonstrated that Notch signaling pathway regulates hypoxia tolerance in both Drosophila melanogaster and humans. However, the mechanisms mediating Notch-conferred hypoxia tolerance are largely unknown. In this study, we delineate the evolutionarily conserved mechanisms underlying this hypoxia tolerant phenotype. We determined the role of a group of conserved genes that were obtained from a comparative genomic analysis of hypoxia-tolerant D.melanogaster populations and human highlanders living at the high-altitude regions of the world (Tibetans, Ethiopians, and Andeans). We developed a novel dual-UAS/Gal4 system that allows us to activate Notch signaling in the Eaat1-positive glial cells, which remarkably enhances hypoxia tolerance in D.melanogaster, and, simultaneously, knock down a candidate gene in the same set of glial cells. Using this system, we discovered that the interactions between Notch signaling and bnl (fibroblast growth factor), croc (forkhead transcription factor C), or Mkk4 (mitogen-activated protein kinase kinase 4) are important for hypoxia tolerance, at least in part, through regulating neuronal development and survival under hypoxic conditions. Becausethese genetic mechanisms are evolutionarily conserved, this group of genes may serve as novel targets for developing therapeutic strategies and have a strong potential to be translated to humans to treat/prevent hypoxia-related diseases.

Keywords: Drosophila melanogaster; Hypoxia; Notch signaling; eaat1-posive glia; genetic interactions.

Figure 1

Distribution of Eaat1-positive glial cells during development. The Eaat1-positive glial cells were labeled by GFP in the progeny of [Eaat1-GAL4]×[UAS-GFP]crosses. The expression of glial marker (repo) and neuronal marker (elav) were labeled by immunostaining. (A–D): the distribution of Eaat1-positive glial cells at first (A), second (B), and third (C) instar larval as well as adult (D) brain. Colocalization of GFP and Repo within the same cell was highlighted in (C) (insert, image represents a Z-projection of 5–12 slices). GFP (green), Repo (red), and Elav (blue). Scale bar = 50 µm.

Figure 2

Eaat1-positive glial cells are important for the development and survival of D.melanogaster. Eaat1-Gal4 virgin female was crossed with male UAS-rpr with transgene inserted on 2nd or X chromosome (Chr2 or ChrX) to deplete the Eaat1-positive glial cells by rpr-induced cell death. The crosses of Eaat1-Gal4 with UAS-rpr (m, Chr2) were lethal in both males and females, none of the embryos reached adult stage. In the cross between female Eaat1-Gal4 and male UAS-rpr (m, ChrX), only male flies were obtained, demonstrating that the Eaat1-positive glial cells are essential for the development and survival of D.melanogaster.

Figure 3

Characterization of the dual-UAS/Eaat1-Gal4 system. The EN line was crossed with UAS-LacZ or the background D.melanogaster strains (w¹¹¹⁸ or yw) for the transgenic lines to test the efficiency of this system. No alterations on survival were observed in the crosses and controls under room air condition. A significant enhancement of hypoxia survival was detected in the [EN] × [w1118], [EN] × [yw], and [EN] × [UAS-LacZ] as compared to the controls (w1118, yw, and UAS-LacZ) under 5% O₂, demonstrating that double the dosage of UAS-transgenes Eaat1-Gal4 > UAS-NICD/UAS-LacZ (the progeny of [EN] × [UAS-LacZ]) did not significantly affect the hypoxia tolerant phenotype showing in Eaat1-Gal4 > UAS-NICD (the progeny of [EN] × [w1118] or [EN] × [yw]). Bars represent mean ± SD (n = 3 vials) for each group/treatment. Ordinary one-way ANOVA [F_{(13, 28)}= 108.3, P < 0.0001]with Turkey’s multiple comparison tests (*** P < 0.001; ns: not significant).

Figure 4

Genetic interactions regulating hypoxia tolerance conferred by activating Notch in Eaat1-positive glial cells. Specific UAS-RNAi line targeting bnl, croc, Egfr, grn, h, inv or Mkk4 was crossed with the EN line to knock down the expression of the targeted genes individually on the background of Notch activation in the Eaat1-positive glial cells. The eclosion rate of the progeny under hypoxic condition (5% O₂) were measured. A significant reduction of Notch activation-conferred hypoxia survival was observed in the crosses with (A) bnl knock down [Ordinary one-way ANOVA; F_(4,10) = 18.25, P = 0.0001], (B) croc knock down [Ordinary one-way ANOVA; F_(4,10) = 11.09, P = 0.0011], (C) Mkk4 knock down [Ordinary one-way ANOVA; F_(4,10) = 13.44, P = 0.0005], or (G) h knock down [F_(4,10) = 44.24, P < 0.0001]. In contrast, knock down (D) Egfr [Ordinary one-way ANOVA; F_(4,10) = 37.23, P < 0.0001), (E) grn [Ordinary one-way ANOVA; F_(4,10) = 23.66, P < 0.0001]or (F) inv [Ordinary one-way ANOVA; F_(4,10) = 56.93, P < 0.0001]did not significantly diminish the hypoxia survival rate to control levels. Bars represent mean ± SD (n = 3 to 6 vials) for each group (*P < 0.05, ** P < 0.01, ***P < 0.001; Turkey’s multiple comparison tests).

Figure 5

Effect of ubiquitous knocking down of candidate genes on hypoxia tolerance. Specific UAS-RNAi line targeting bnl, croc, Egfr, grn, h, inv, or Mkk4 was crossed with the da-Gal4 driver to ubiquitously knock down the expression of the targeted genes individually in D.melanogaster. The eclosion rate of the progeny under 5% O₂ hypoxic condition was measured. A significant enhancement of hypoxia survival was detected in the flies with specific knocking down of grn, inv, and Mkk4. In contrast, the crosses with croc and h knocking down showed a similar survival rate with the controls. And ubiquitous knocking down of bnl or Egfr was lethal. Bars represent means ± SD (n = 3 to 6 vials) for each cross. Ordinary one-way ANOVA [F_(8,48) = 31.93, P < 0.0001]with Turkey’s multiple comparison tests (**P < 0.01).

Figure 6

Schematic illustration of hypothetical functional output of Notch/bnl, Notch/croc and Notch/Mkk4 interactions under hypoxia. Hypoxia tolerance conferred by Notch activation in the Eaat1-positive glial cells requires functional synchronization with bnl, croc and Mkk4 to regulate cell survival and proliferation, such functional synchronization is essential for organismal survival under prolonged severe hypoxic conditions.