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Figures
- Fig. 1.
Construction of an oxygen-sensitive fluorescent protein-based biosensor. (A) Schematic representations of full-length Sima protein (dHIF1-alpha; top), with its O2-dependent degradation domain (ODD; aa 673-895), of the GFP-ODD fusion protein (middle), and of the GFP-ODD construct with the P850 residue mutated into alanine [GFP-ODD(P850A); bottom]. (B) Embryos showing the sensitivity of GFP-ODD to O2 tension. GFP-ODD signals (top panels) were reduced in embryos incubated at 21% O2 compared to embryos incubated at 5% O2. The P850A mutation renders the GFP-ODD construct largely insensitive to changing O2 levels (bottom panels). Scale bar: 100 µm. (C) Schematic representation of the ratiometric sensor design. The red fluorescence from a control protein (mRFP-nls) remains relatively constant under changing O2 concentrations, whereas GFP-ODD is sensitive to O2 levels, providing a measure of the cellular hypoxia response state.
- Fig. 2.
GFP-ODD responds to changes in ambient O2 concentrations. Embryos incubated at three different O2 concentrations show decreasing levels of GFP-ODD fluorescence with the highest levels under hypoxia (5% O2; A), lower levels under normoxia (21% O2; B) and further reduced levels under hyperoxia (60% O2; C). (A′,B′,C′) Conversely, fluorescence intensity of mRFP-nls shows comparatively little changes in the three O2 conditions. (A″,B″,C″) The merge of the two channels, indicating the GFP-ODD (green)/mRFP-nls (magenta) ratio, as basis for the ratiometric analysis. (D) Analysis of the hypoxic state of individual cells. GFP-ODD and mRFP-nls intensities were analysed in nuclei of embryonic tracheal cells. The scatter plot shows normalised intensities (a.u., arbitrary units) of each channel, representing changes in fluorescence signals. Each point corresponds to a single nucleus. The number of embryos (n) analysed for each condition is indicated. (E) Changes in individual fluorescence channels and GFP-ODD/mRFP-nls ratios for each O2 condition. Each box plot represents data from a single embryo in which fluorescence was measured in at least 35 cells. Scale bar: 100 µm. ***P≤0.001; Mann-Whitney U-test.
- Fig. 3.
GFP-ODD responds to modulation of HIF pathway components. Levels of GFP-ODD fluorescence (green) were analysed in wing imaginal discs of third-instar larvae, in which regulators of the hypoxia response were either depleted by RNAi or overexpressed in the posterior compartment using the en-Gal4 driver. Cells in the posterior compartment (marked ‘P’ in panel A″) are labelled by the en-Gal4-driven expression of UAS-mCherry-nls (A, magenta in A″). The anterior compartment (marked ‘A’ in panel A″) serves as an internal control. In control larvae (lacZ-RNAi; A-A″), GFP-ODD levels in the anterior and posterior compartment are indistinguishable. RNAi-mediated knockdown of dVHL (B-B″) or fatiga (C-C″) in the posterior compartment results in accumulation of GFP-ODD. Conversely, overexpression of Fatiga-A leads to lower levels of GFP-ODD (D-D″). (E-E″) Depletion of PTEN in the posterior compartment results in accumulation of GFP-ODD. (F-F″) Inhibition of the mitochondrial respiratory chain through COXVb RNAi causes accumulation of GFP-ODD. Scale bar: 100 µm. (G) Bar graph showing ratios between mean GFP-ODD intensities in the posterior (GFP[P]) and anterior compartment (GFP[A]). Ratios were normalised to the values of the RNAi control (en>lacZ RNAi). Bars indicate mean values, error bars represent the standard deviation. The number (n) of imaginal discs analysed for each genotype is indicated.
- Fig. 4.
Different tracheolation densities within the brain correlate with distinct cellular hypoxic states. (A) 3D reconstruction of the tracheal system in a brain hemisphere of a larva (96 hALH). Maximum intensity projection of anti-Dlg antibody staining (blue) shows outline of the brain. Tracheoles are coloured red in the central brain (CB) and yellow in the optic lobe (OL). The brain's midline is to the left and anterior (a) and posterior (p) are indicated. See also Movie 1. (B) Box plot showing quantification of total tracheal surface within the CB (blue) and OL (orange) (CB=27720.1 µm2, OL=4647.8 µm2; n=8 brains; Mann–Whitney U-test, P=0.00015). The box plot shows maximum and minimum observation, upper and lower quartile, and median. (C) Single frontal confocal section of a brain hemisphere of a larva (96 hALH) expressing ubi-GFP-ODD and ubi-mRFP-nls. The colour code (upper right) indicates average GFP-ODD/mRFP-nls ratios for each nucleus. (C′) Maximum intensity projection of the brain's tracheal system (white). (C″) Superposition of the images shown in (C) and (C′) illustrates the topographical correlation between the differential tracheolation of CB and OL regions and their different hypoxic states. Low ratios correlate with dense tracheolation in the CB. Higher ratios correlate with the sparsely tracheolated OL. Note that cells adjacent to the OL lateral tracheoles (arrows) exhibit lower ratios, consistent with O2 diffusion across few cell diameters. (D) Single frontal confocal section of a brain hemisphere of a larva (96 hALH) expressing ubi-GFP-ODD(P850A) and ubi-mRFP-nls. The colour code (upper right) indicates average GFP-ODD(P850A)/mRFP-nls ratios for each nucleus. (E) Histogram representing the frequency distribution of GFP-ODD/mRFP-nls ratios for the CB and the OL, showing a clear separation, n=7 brain hemispheres. (F) Histogram representing the frequency distribution of GFP-ODD(P850A)/mRFP-nls ratios for CB and OL, which show a large overlap, n=7 brain hemispheres. Data in E and F is presented as box plots. (G) Box plots showing the average GFP-ODD/mRFP-nls ratios, calculated from the data shown in E. Note the significantly higher average ratios in the OL compared to the CB (1.11 in OL vs 0.83 in CB; student's t-test, P=1.256e-06). (H) Box plots showing the average ODD(P850A)/mRFP-nls ratios for CB and OL, calculated from the data shown in F. Note the overlap in average ratios between the non-tracheolated OL region and the CB (1.02 in OL vs 0.97 in CB; student's t-test, P=0.03). Box plots in E,F,G and H show maximum and minimum observation, upper and lower quartile, and median. Scale bars: 40 µm.
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