Andrés Dekanty

Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting.

Drosophila tracheal terminal branches are plastic and have the capacity to sprout out projections toward oxygen-starved areas, in a process analogous to mammalian angiogenesis. This response involves the upregulation of FGF/Branchless in hypoxic tissues, which binds its receptor Breathless on tracheal cells. Here, we show that extra sprouting depends on the Hypoxia-Inducible Factor (HIF)-alpha homolog Sima and on the HIF-prolyl hydroxylase Fatiga that operates as an oxygen sensor. In mild hypoxia, Sima accumulates in tracheal cells, where it induces breathless, and this induction is sufficient to provoke tracheal extra sprouting. In nontracheal cells, Sima contributes to branchless induction, whereas overexpression of Sima fails to attract terminal branch outgrowth, suggesting that HIF-independent components are also required for full induction of the ligand. We propose that the autonomous response to hypoxia that occurs in tracheal cells enhances tracheal sensitivity to increasing Branchless levels, and that this mechanism is a cardinal step in hypoxia-dependent tracheal sprouting.

The insulin-PI3K/TOR pathway induces a HIF-dependent transcriptional response in Drosophila by promoting nuclear localization of HIF-α/Sima

The hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of a constitutively expressed HIF-β subunit and an oxygen-regulated HIF-α subunit. We have previously defined a hypoxia-inducible transcriptional response in Drosophila melanogaster that is homologous to the mammalian HIF-dependent response. In Drosophila, the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) are the functional homologues of the mammalian HIF-α and HIF-β subunits, respectively. HIF-α/Sima is regulated by oxygen at several different levels that include protein stability and subcellular localization. We show here for the first time that insulin can activate HIF-dependent transcription, both in Drosophila S2 cells and in living Drosophila embryos. Using a pharmacological approach as well as RNA interference, we determined that the effect of insulin on HIF-dependent transcriptional induction is mediated by PI3K-AKT and TOR pathways. We demonstrate that stimulation of the transcriptional response involves upregulation of Sima protein but not sima mRNA. Finally, we have analyzed in vivo the effect of the activation of the PI3K-AKT pathway on the subcellular localization of Sima protein. Overexpression of dAKT and dPDK1 in normoxic embryos provoked a major increase in Sima nuclear localization, mimicking the effect of a hypoxic treatment. A similar increase in Sima nuclear localization was observed in dPTEN homozygous mutant embryos, confirming that activation of the PI3K-AKT pathway promotes nuclear accumulation of Sima protein. We conclude that regulation of HIF-α/Sima by the PI3K-AKT-TOR pathway is a major conserved mode of regulation of the HIF-dependent transcriptional response in Drosophila.

Drosophila genome-wide RNAi screen identifies multiple regulators of HIF-dependent transcription in hypoxia.

Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF–dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF–related pathologies, including heart attack, cancer, and stroke.

Cellular and developmental adaptations to hypoxia: a Drosophila perspective.

The fruit fly Drosophila melanogaster, a widely utilized genetic model, is highly resistant to oxygen starvation and is beginning to be used for studying physiological, developmental, and cellular adaptations to hypoxia. The Drosophila respiratory (tracheal) system has features in common with the mammalian circulatory system so that an angiogenesis-like response occurs upon exposure of Drosophila larvae to hypoxia. A hypoxia-responsive system homologous to mammalian hypoxia-inducible factor (HIF) has been described in the fruit fly, where Fatiga is a Drosophila oxygen-dependent HIF prolyl hydroxylase, and the basic helix-loop-helix Per/ARNT/Sim (bHLH-PAS) proteins Sima and Tango are, respectively, the Drosophila homologues of mammalian HIF-alpha (alpha) and HIF-beta (beta). Tango is constitutively expressed regardless of oxygen tension and, like in mammalian cells, Sima is controlled at the level of protein degradation and subcellular localization. Sima is critically required for development in hypoxia, but, unlike mammalian model systems, it is dispensable for development in normoxia. In contrast, fatiga mutant alleles are all lethal; however, strikingly, viability to adulthood is restored in fatiga sima double mutants, although these double mutants are not entirely normal, suggesting that Fatiga has Sima-independent functions in fly development. Studies in cell culture and in vivo have revealed that Sima is activated by the insulin receptor (InR) and target-of-rapamycin (TOR) pathways. Paradoxically, Sima is a negative regulator of growth. This suggests that Sima is engaged in a negative feedback loop that limits growth upon stimulation of InR/TOR pathways.

Oxygen Sensing in Drosophila: Multiple Isoforms of the Prolyl Hydroxylase Fatiga Have Different Capacity to Regulate HIFα/Sima

Background The Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen. Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit, promoting its proteasomal degradation in normoxia. Three HIF-prolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals. PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear. PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent. The physiologic significance of the diversity of the PHD oxygen sensors is intriguing. Methodology and Principal Findings We have analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing. FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3. Through a combination of genetic experiments in vivo and molecular analyses in cell culture, we show that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA. The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter. Conclusions and Significance Diversity of PHD isoforms is a conserved feature in evolution. As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain. Our results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain.

Central Role of the Oxygen-dependent Degradation Domain of Drosophila HIFα/Sima in Oxygen-dependent Nuclear Export

The Drosophila HIFalpha homologue, Sima, is localized mainly in the cytoplasm in normoxia and accumulates in the nucleus upon hypoxic exposure. We have characterized the mechanism governing Sima oxygen-dependent subcellular localization and found that Sima shuttles continuously between the nucleus and the cytoplasm. We have previously shown that nuclear import depends on an atypical bipartite nuclear localization signal mapping next to the C-terminus of the protein. We show here that nuclear export is mediated in part by a CRM1-dependent nuclear export signal localized in the oxygen-dependent degradation domain (ODDD). CRM1-dependent nuclear export requires both oxygen-dependent hydroxylation of a specific prolyl residue (Pro850) in the ODDD, and the activity of the von Hippel Lindau tumor suppressor factor. At high oxygen tension rapid nuclear export of Sima occurs, whereas in hypoxia, Sima nuclear export is largely inhibited. HIFalpha/Sima nucleo-cytoplasmic localization is the result of a dynamic equilibrium between nuclear import and nuclear export, and nuclear export is modulated by oxygen tension.

S12-02 Oxygen-dependent plasticity of the Drosophila tracheal system

Drosophila tracheal terminal branches are plastic and have the capacity to sprout-out projections towards oxygen-starved areas, in a process analogous to mammalian angiogenesis. It was previously shown that this sprouting response involves the upregulation of the FGF homolog Branchless in hypoxic tissues, which binds its receptor Breathless on tracheal cells, thereby attracting the outgrowth of terminal cells. We have found that tracheal extra-sprouting depends on the Hypoxia Inducible Factor alphasubunit Sima, as well as on the HIF prolyl hydroxylase Fatiga that operates as an oxygen sensor. In mild hypoxia, Sima accumulates mainly in tracheal terminal cells, where it promotes transcriptional upregulation of the receptor breathless. Strikingly, this induction is sufficient to provoke extra-sprouting of tracheal terminal branches. In non-tracheal cells, Sima contributes to induction of the ligand branchless, whilst over-expression of Sima fails on itself to attract terminal branch outgrowth, suggesting that HIFindependent components are also required for full induction of the ligand. We propose that the autonomous response to hypoxia that occurs in tracheal cells enhances tracheal sensitivity to increasing levels of the ligand Branchless, and that this mechanism is a cardinal step in hypoxia-dependent tracheal sprouting.