A genetically encoded biosensor for visualizing hypoxia responsesin vivo

Abstract

Cells experience different oxygen concentrations depending on location, organismal developmental stage, and physiological or pathological conditions. Responses to reduced oxygen levels (hypoxia) rely on the conserved hypoxia-inducible factor 1 (HIF-1). Understanding the developmental and tissue-specific responses to changing oxygen levels has been limited by the lack of adequate tools for monitoring HIF-1 in vivo. To visualise and analyse HIF-1 dynamics in Drosophila, we used a hypoxia biosensor consisting of GFP fused to the oxygen-dependent degradation domain (ODD) of the HIF-1 homologue Sima. GFP-ODD responds to changing oxygen levels and to genetic manipulations of the hypoxia pathway, reflecting oxygen-dependent regulation of HIF-1 at the single-cell level. Ratiometric imaging of GFP-ODD and a red-fluorescent reference protein reveals tissue-specific differences in the cellular hypoxic status at ambient normoxia. Strikingly, cells in the larval brain show distinct hypoxic states that correlate with the distribution and relative densities of respiratory tubes. We present a set of genetic and image analysis tools that enable new approaches to map hypoxic microenvironments, to probe effects of perturbations on hypoxic signalling, and to identify new regulators of the hypoxia response.

Glial response to hypoxia in trachealess mutants induces synapse remodeling

“…To understand whether trh 1 /trh 2 mutant cells are under hypoxia, we used the hypoxia biosensor GFP-ODD, in which the GFP is fused to the oxygen-dependent degradation (ODD) domain of Sima, under the control of the ubiquitin-69E ( ubi ) promoter [36]. We first confirmed that GFP-ODD signal was low under normoxia (21% O 2 ) and enhanced under hypoxia (5% O 2 , Fig 2A) in wild-type late-stage embryos when tracheal tubules are already formed and functioning [36]. Indeed, enhanced GFP signal was ubiquitous under hypoxia in wild-type embryos, with some pronounced focal GFP signals (Fig 2A, upper row, and Fig S2C).…”

“…Indeed, enhanced GFP signal was ubiquitous under hypoxia in wild-type embryos, with some pronounced focal GFP signals (Fig 2A, upper row, and Fig S2C). The signal of mRFP-nls, also under the control of ubi promoter as an internal control, remained constant under hypoxia (Fig 2A, bottom row, and Fig S2D) [36]. Quantification of the GFP/RFP ratio revealed a significant difference between normoxia and hypoxia conditions (Fig 2B).…”

A fat-tissue sensor couples growth to oxygen availability by remotely controlling insulin secretion

“…6A). To confirm the hypoxic state of the fat tissue, we made use of a genetic hypoxia reporter based on a GFP fusion with the oxygendependent degradation (ODD) domain of HIF-1a 58 . Under normoxic conditions, GFP::ODD is ubiquitinylated through the activity of Hph, targeting it for degradation.…”

“…ΔGbp1,2 14 was kindly given by T. Koyama. Hph Δ9 (Fatiga Δ9 , Fga Δ9 ) was a kind gift of P. Wappner, ubi-ODD::GFP hypoxia sensor 58…”

“…The following primary antibodies were used: rat anti-DILP2 57 58 . This reporter protein is degraded at a rate proportional to tissue oxygen levels.…”