Journal:

Article Title: Control of the Hypoxic Response in Drosophila melanogaster by the Basic Helix-Loop-Helix PAS Protein Similar

doi: 10.1128/MCB.22.19.6842-6853.2002

Figure Lengend Snippet: A hypoxia-inducible transcriptional response in Drosophila. Physiological characterization of the hypoxic response in vivo. (A) Schematic representation of the LDH-LacZ transcriptional reporter: A 233-bp fragment of the murine LDH-A enhancer from bp −186 to +47 (relative to the transcription initiation site) controls β-Gal expression. The sequences correspond to HRE and CRE elements present in the enhancer. Bases in bold lettering mark the HRE consensus, and the numbers above indicate the position relative to the transcription initiation site. (B) The LDH-LacZ reporter is induced in embryos maintained at 5% O2 for 8 h (•) compared to normoxia (⧫). The x axis represents time course of the β-Gal reaction. (C and D) Stage 17 transgenic embryos carrying the LDH-LacZ reporter maintained in normoxia (C) or hypoxia (8 h at 5% O2) (D) were stained with X-Gal in a reaction developed overnight. Whereas in embryos exposed to hypoxia the X-Gal staining was ubiquitous, in normoxic individuals the expression of the reporter was restricted to a small anterior domain. (E) Modulation of the hypoxic response during development. Individuals bearing the LDH-LacZ reporter were synchronized at different stages of development and were subjected to hypoxia (5% O2) for 4 h. The hypoxia/normoxia β-Gal activity ratio is shown. Hypoxic induction of the reporter is maximal at late embryogenesis, although it is also high at larval stages 1 and 2. P, pupa; A, adult. (F) Effect of oxygen concentration. Stage 16 to 17 embryos bearing the LDH-LacZ reporter were exposed to different oxygen concentrations for 4 h, the β-Gal activity was compared with that of normoxic controls, and the ratios of the activities were calculated. Triplicate determinations of the activity were performed. Induction of the reporter was maximal between 3 and 5% oxygen concentration.

Article Snippet: Rabbit anti-β-Gal antibodies (Cappel), rat anti-Sima antibody ( 1 ), rat anti-Trh antibody ( 51 ), and mouse monoclonal 2A12 antitracheal lumen antibody (Developmental Studies Hybridoma Bank, Iowa University) were used.

Techniques: In Vivo, Expressing, Transgenic Assay, Staining, Activity Assay, Concentration Assay

Journal:

Article Title: Control of the Hypoxic Response in Drosophila melanogaster by the Basic Helix-Loop-Helix PAS Protein Similar

doi: 10.1128/MCB.22.19.6842-6853.2002

Figure Lengend Snippet: Spatially restricted induction of the transcriptional response to hypoxia. The expression pattern of LDH-LacZ (A to C) and LDH-Gal4 (D to L) reporters was examined in transgenic embryos or larvae exposed to hypoxia. (A) X-Gal staining of a stage 15 LDH-LacZ embryo subjected to 5% O2 for 4 h. Some branches of the tracheal system (arrows) are stained. (B) Double immunostaining showing the tracheal lumen in brown (arrows) and β-Gal expression in blue (arrowheads). The reporter is expressed at some branches of the tracheal system. (C) In normoxia, no expression of the reporter is seen. (D to F) An unsynchronized population of embryos was exposed to 5% oxygen for 4 h and induction of the reporter was analyzed. Double immunofluorescent confocal image shows that the β-Gal reporter (green) colocalizes with the tracheal marker Trachealess (red). Note that some extratracheal cells express the reporter as well. Expression of the reporter cannot be detected at embryonic stage 11 (see panel D). By stage 13 (panel E), scattered tracheal cells (arrowheads) begin to express the reporter, with further cells responding to hypoxia at stage 14 (arrowheads) (F). (G) By the end of embryogenesis and throughout larval stages, the LDH-Gal4/UAS-TAU.GFP reporter is expressed in the whole tracheal system upon hypoxia (arrowheads, lower panel), but in normoxia no expression is seen in the tracheae (upper panel), although expression of the reporter can be detected in salivary glands (arrows). (H and I) In breathlessMZ13 homozygous embryos tracheal cells (red) fail to migrate (arrowhead), but the hypoxic reporter (UAS-nGFP.LacZ) is induced normally (arrows) in hypoxia. (I) Higher magnification of panel H. (J and K) First-instar larvae subjected to 4% O2 for 16 h express the UAS-nGFP reporter in nontracheal tissues, as well as in the esophagus (e), gut (g), ectoderm (ec), fat body (fb), and tracheae (t). Expression in the salivary glands (sg) is constitutive. (L) Hypoxic induction of the UAS-nGFP reporter is seen in the legs of adult flies maintained at 5% O2 for 24 h (lower panel) compared with normoxia (upper panel). (M) Schematic representation of the LDH-Gal4 transcriptional reporter. A dimerized 51-bp fragment of the murine LDH-A enhancer including the HREs and CRE was constructed as a dimer controlling expression of Gal4.

Article Snippet: Rabbit anti-β-Gal antibodies (Cappel), rat anti-Sima antibody ( 1 ), rat anti-Trh antibody ( 51 ), and mouse monoclonal 2A12 antitracheal lumen antibody (Developmental Studies Hybridoma Bank, Iowa University) were used.

Techniques: Expressing, Transgenic Assay, Staining, Double Immunostaining, Marker, Construct

Journal:

Article Title: Control of the Hypoxic Response in Drosophila melanogaster by the Basic Helix-Loop-Helix PAS Protein Similar

doi: 10.1128/MCB.22.19.6842-6853.2002

Figure Lengend Snippet: Role of bHLH-PAS proteins in the transcriptional response to hypoxia. (A) Wild-type expression of the β-Gal reporter in the tracheal system of LDH-Gal4/UAS-nGFP.LacZ transgenic embryos subjected to hypoxia (arrowheads). (B) Absence of reporter induction in tango5 homozygous embryos subjected to hypoxia. Only constitutive reporter expression is seen in the salivary glands (sg) and cells of the gut (arrow). Typical tracheal defects are observed in tango mutants (B, arrowheads). (C and D) Sima cannot be detected in normoxic wild-type embryos (C) but is induced upon hypoxia (5% O2 for 14 h) in a pattern that follows the tracheal tree (D). (E) Western blot analysis of lysates of embryos bearing the LDH-Gal4/UAS-GFP.LacZ reporter subjected to hypoxia (5% O2 for 14 h) or maintained in normoxia showing hypoxic induction of both Sima and LDH-Gal4/UAS-GFP.LacZ. (F) Slot blot analysis of RNA extracted from hypoxic or normoxic embryos treated as in panel E. β-Gal mRNA exhibited strong hypoxic induction (9.7- to 10.4-fold), whereas Sima mRNA was induced only slightly (1.3- to 1.5-fold).

Article Snippet: Rabbit anti-β-Gal antibodies (Cappel), rat anti-Sima antibody ( 1 ), rat anti-Trh antibody ( 51 ), and mouse monoclonal 2A12 antitracheal lumen antibody (Developmental Studies Hybridoma Bank, Iowa University) were used.

Techniques: Expressing, Transgenic Assay, Western Blot, Dot Blot

Journal:

Article Title: Control of the Hypoxic Response in Drosophila melanogaster by the Basic Helix-Loop-Helix PAS Protein Similar

doi: 10.1128/MCB.22.19.6842-6853.2002

Figure Lengend Snippet: Regulation of Sima subcellular localization by hypoxia. Sima (A and E) or SimaΔ692-863 (I and M) were expressed ectopically through an en-Gal4 driver, and subcellular localization was detected with an anti-Sima antibody. (B, F, J, and N) The embryos also contained a UAS-nGFP.LacZ reporter (bearing a nuclear localization signal) detected with an anti-β-Gal antibody that was used to mark the nuclei. In normoxia, Sima was localized exclusively in the cytoplasm (A to C) and became nuclear in hypoxia (5% O2 for 8 h) (E to G). SimaΔ692-863 was constitutively detected in the nuclei irrespective of oxygen levels (I to K and M to O). (D, H, L, and P) Sima or SimaΔ692-863 ectopic expression driven by en-Gal4 was also performed in embryos bearing the direct LDH-LacZ hypoxic reporter and lacking the UAS-nGFP.LacZ element. Upon ectopic expression of Sima, the LDH-LacZ reporter was strongly induced in hypoxia (H) but only weakly in normoxia (D), a finding consistent with the subcellular localization of Sima. Expression of SimaΔ692-863 produced strong expression of the reporter irrespective of oxygen levels (L and P).

Article Snippet: Rabbit anti-β-Gal antibodies (Cappel), rat anti-Sima antibody ( 1 ), rat anti-Trh antibody ( 51 ), and mouse monoclonal 2A12 antitracheal lumen antibody (Developmental Studies Hybridoma Bank, Iowa University) were used.

Techniques: Expressing, Produced