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.

31Environmental factors such as the availability of oxygen are instructive cues to regulate 32 stem cell maintenance and differentiation. We used a genetically encoded biosensor to 33 monitor the hypoxic state of neural cells in the larval brain of Drosophila. The biosensor 34 reveals brain compartment and cell type specific levels of hypoxia. The values correlate 35 with differential tracheolation that is observed throughout development between the 36 central brain and the optic lobe. Neural stem cells in both compartments show the 37 strongest hypoxia response while intermediate progenitors, neurons and glial cells 38 reveal weaker responses. We demonstrate that the distance between a cell and the 39 next closest tracheole is a good predictor of the hypoxic state for that cell. Our model 40 concludes that oxygen availability is the major factor controlling the hypoxia response 41 in the developing Drosophila brain but cell intrinsic and cell-type specific factors 42 contribute to modulate the response in an unexpected manner. 43

Environmental factors such as the availability of oxygen are instructive cues that regulate stem cell maintenance and differentiation. We used a genetically encoded biosensor to monitor the hypoxic state of neural cells in the larval brain of Drosophila. The biosensor reveals brain compartment and cell-type specific levels of hypoxia. The values correlate with differential tracheolation that is observed throughout development between the central brain and the optic lobe. Neural stem cells in both compartments show the strongest hypoxia response while intermediate progenitors, neurons and glial cells reveal weaker responses. We demonstrate that the distance between a cell and the next closest tracheole is a good predictor of the hypoxic state of that cell. Our study indicates that oxygen availability appears to be the major factor controlling the hypoxia response in the developing Drosophila brain and that cell intrinsic and cell-type specific factors contribute to modulate the response in an unexpected manner.This article has an associated First Person interview with the first author of the paper.

Synaptic proteins and synaptic transmission are under homeostatic control, but the relationship between these two processes remains enigmatic. Here, we systematically investigated the role of E3 ubiquitin ligases, key regulators of protein degradation-mediated proteostasis, in presynaptic homeostatic plasticity (PHP). An electrophysiology-based genetic screen of 157 E3 ligase-encoding genes at the Drosophila neuromuscular junction identified thin, an ortholog of human tripartite motif-containing 32 (TRIM32), a gene implicated in several neurological disorders, including autism spectrum disorder and schizophrenia. We demonstrate that thin functions presynaptically during rapid and sustained PHP. Presynaptic thin negatively regulates neurotransmitter release under baseline conditions by limiting the number of release-ready vesicles, largely independent of gross morphological defects. We provide genetic evidence that thin controls release through dysbindin, a schizophrenia-susceptibility gene required for PHP. Thin and Dysbindin localize in proximity within presynaptic boutons, and Thin degrades Dysbindin in vitro. Thus, the E3 ligase Thin links protein degradation-dependent proteostasis of Dysbindin to homeostatic regulation of neurotransmitter release.