DOI: 10.5061/dryad.35h8v
title: Data from: Accurate measurements of dynamics and reproducibility in small genetic networks

description: 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.
description: Dubuis et al. -
 Data_Fig_2BSource 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_ProfilesEach 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_ProfilesEach 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_FilesComplete 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.