Quantification of gene expression has become a central tool for understanding genetic networks. In many systems the only viable way to measure protein levels is by immunofluorescence, which is notorious for its limited accuracy. Using the early Drosophila embryo as an example, we show that careful identification and control of experimental error allows for highly accurate gene expression measurements. We generated antibodies in different host species, allowing for simultaneous staining of four Drosophila gap genes in individual embryos. Careful error analysis of hundreds of expression profiles reveals that less than ∼20% of the observed embryo-to-embryo fluctuations stem from experimental error. These measurements make it possible to extract not only very accurate mean gene expression profiles but also their naturally occurring fluctuations of biological origin and corresponding cross-correlations. We use this analysis to extract gap gene profile dynamics with ∼1 min accuracy. The combination of these new measurements and analysis techniques reveals a two-fold increase in profile reproducibility due to a collective network dynamics that relays positional accuracy from the maternal gradients to the pair-rule genes.
Dubuis et al. - Data_Fig_2B
Source data for Figure 2B: Invagination of the membrane for 8 embryos (delta x), mean (mean delta) and adjusted mean in fixed tissues (mean delta fixed) as function of time during n.c. 14. NaN indicates that furrow canal could not be detected at that particular time.
Dubuis et al. - Raw_Profiles
Each file contains the source data for raw gap gene (Hb, Kr, Gt, Kni) dorsal intensity profiles measured using immunofluorescence techniques in 163 Drosophila embryos during n.c. 14. Column 1 contains the embryo number referring to its position on the slide (see Images.zip). Column 2 contains an information about the azimuthal orientation of the embryo on the slide ('1' if the confocal plane is closer to midsagittal plane, '2' if it is closer to the coronal plane). Column 3 contains the furrow canal depth (delta_FC) measured in micrometers. Column 4 contains the corresponding estimated age in minutes. Columns 5 to 1004 contain a 1x1000 vector representing the dorsal intensity profile of the gap gene (Hb, Kr, Gt, Kni) in the embryo. The 1000 points are equally spaced along the AP axis. Thus, Intensity345 represents the intensity at 34.5%EL. NaN means that we couldn't reliably detect the profile intensity at that position (usually near the edges). Columns 1-4 are identical in the four files.
Dubuis et al. - Processed_Profiles
Each file contains the source data for gap gene (Hb, Kr, Gt, Kni) processed dorsal gene expression levels measured using immunofluorescence techniques in 23 Drosophila embryos with FC depth comprised between 10 and 20 microns. We only selected embryos that were imaged close to their midsagittal plane (Orientation '1'). Column 1 contains the embryo number referring to its position on the slide (see Images.zip). Column 2 contains an information about the azimuthal orientation of the embryo on the slide ('1' if the confocal plane is closer to midsagittal plane, '2' if it is closer to the coronal plane). Column 3 contains the furrow canal depth (delta_FC) measured in micrometers. Column 4 contains the corresponding estimated age in minutes. Columns 5 to 1004 contain a 1x1000 vector representing the dorsal time-corrected gene expression level of the gap gene (Hb, Kr, Gt, Kni) in the embryo. The 1000 points are equally spaced along the AP axis. Thus, g345 represents the gene expression level at 34.5%EL. NaN means that we coudn't reliably detect the profile intensity at that position (usually near the edges). Columns 1-4 are identical in the four files.
Dubuis et al. - Source_Data_Files
Complete source data for Dubuis et al. contains two folders: 1. Folder "FC_Calibration" contains the source data used for calibration of the time-dependent furrow canal depth. It displays the chi_2 minimized furrow canal depth (delta_FC) for the 8 live imaged Drosophila embryos as function of time during n.c. 14 (see Materials and Methods). It also shows the mean furrow canal depth averaged over the 8 embryos (mean_delta_FC) and adjusted mean in fixed tissues (mean_delta_FC_fixed) that we used to estimate the age of the 163 fixed embryos. Column 1 contains the time in minutes after the onset of n.c. 14. Columns 2 to 9 contain the chi_2 minimized furrow canal depths (delta_FC_x, x being the embryo number) in for the 8 live imaged embryos. NaN means that we couldn't detect the furrow canal at that particular time. Column 10 contains the mean furrow canal depth averaged over the 8 embryos (mean_delta_FC) measured in micrometers. Column 11 contains the the furrow canal depth, measured in micrometers, which we used to estimate the age of fixed embryos. It was obtained by a 5% shrinkage of the previous column (see Materials and Methods). 2. Folder "Images" contains the original images of 201 Drosophila embryos at blastoderm stage immunostained against the four main gap genes (Kni, Kr, Gt, Hb). For each embryo with provide 5 12-bit images, each corresponding to a different optical channel (see Materials and methods). Channel 0 -- rat @ Kni (488nm); Channel 1 -- bright field (delta_FC); Channel 2 -- guinea pig @ Gt (568nm); Channel 3 -- rabbit @ Kr (594nm); Channel 4 -- mouse @ Hb (647nm). Images were taken with a Leica 20x HC PL APO NA 0.7 oil immersion objective, and with sequential excitation wavelengths of 488, 546, 594 and 633 nm. The bandwidth of the detection filters were set up as shown in Figure 1A to minimize fluorophore cross-talk while still allowing good detection in each optical channel. For each embryo, three high-resolution images (1024x1024 pixels, with 12 bits and at 100 Hz) were taken along the anterior-posterior axis (focused at the midsagittal plane) at 1.7 magnified zoom and averaged together. With these settings, the linear pixel dimension corresponds to 0.44 um.
