1. Rapid Transcription Fosters Coordinate snail Expression in the Drosophila Embryo 
Alistair Nicol Boettiger, Michael Levine 
Cell Reports 
Volume 3, Issue 1, Pages 8-15 (January 2013)
DOI: /j.celrep
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2. Cell Reports 2013 3, 8-15DOI: (10.1016/j.celrep.2012.12.015)
Copyright © 2013 The Authors Terms and Conditions

3. Figure 1 Single-Molecule mRNA Counting
(A) Representative individual confocal section of several adjacent nuclei, showing bright, diffraction limited spots.
(B) Gaussian filtered version of the image in (A).
(C) Plot of the number of separate objects identified versus intensity threshold applied. The script selects the intensity threshold, theta, which maximizes the number of separate objects.
(D) Resulting image after threshold determined in (C) is applied. Note under dense conditions, several spots remain fused (white arrows indicate examples).
(E) Segmented image after a watershed algorithm is applied to unlink spots joined by the threshold in (D) (see white arrows).
(F) White crosses mark the centroids of all the mRNA molecules identified in the image, which are assigned to parent nuclei using the computed nucleoid region map, indicated by gray partitions (see Experimental Procedures).
(G) Three-dimensional-projection of “stacked disks” (red/yellow) identified in each image plane in the previous steps. Projection of volume reconstructions of nuclei are shown in blue.
(H) These disks are clustered along z to identify which dots correspond to different focus planes from the same molecule. White ovals indicate some examples.
(I) Three-dimensional reassembly of individual snail mRNA transcripts (denoted by small red spheres), yellow mRNA driven from a single-copy snail BAC transgene (green spheres), and nuclei (Draq5 labeled DNA, blue). Approximate cellular boundaries have been outlined.
See also Figure S1.
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4. Figure 2 Homogenous Expression of snail in Mesodermal Cells
(A) Comparison of the coefficient of variation (CoV) for genes expression in D. melanogaster (blue) M. mus. (cyan) (Itzkovitz et al., 2011), S. cerevisiae (magenta) (Zenklusen et al., 2008; To and Maheshri, 2010) and mammalian cultured CHO cells (red) (Raj et al., 2006). “Snail early” refers to CoV measured at the onset of cycle 14 (telophase of cycle 13). “Snail steady state” is the mid cycle 14 stable levels. lacZ-1 and lacZ-2 are from ubiquitous induction of two different UAS-lacZ lines with different maternal drivers. Scr measurements from Paré et al. (2009). Error bars indicate SD in CoV between embryos (for Drosophila) or by bootstrapping the population of single-cell measurements.
(B) Spatial distribution of mRNA counts per cell for ubiquitously induced lacZ expression during cycle 14. The colored tiles outline the “nucleoid region” around each nucleus. The color of the tile indicates the number of mRNA molecules counted within (see color bar next to C).
(C) Spatial distribution of snail mRNA counts per cell during cycle 14.
(D) Fano factor comparison for lacZ, snail and all previously published data shown in (A). Error bars indicate SD in Fano-factor between embryos (for Drosophila data) or by bootstrapping the population of single-cell measurements (for cultured cell data).
(E) Comparison of median mRNA counts per cell over all cycle 14 embryos for snail, lacZ, and previously published data. Error bars indicate SD in median counts between embryos (for Drosophila data) or the SD among cells in the population (cultured cell data).
See also Figure S2.
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5. Figure 3 Dynamics of mRNA Expression at the Single-Cell Level
(A) Heatmap representation of the number of mRNAs in each cell for progressively older embryos (i–iv) in cycle 14. The colored tiles outlines the “nucleoid region” around each nucleus. The color of the tile indicates the number of mRNA molecules counted within (see color bar after iv). A single confocal slice from the box in (iv) is shown at right.
(B) mRNA counting results. Each column represents a single embryo, each dot a single cell, and the y position indicates the number of mRNA found in that cell. Embryos are sorted approximately by age. The color of the dot indicates the age class as determined by nuclear density and nuclear morphology. Representative nuclei images for each class are shown in the insets below.
(C) Average mRNA per cell for embryos in each age class as a function of cell distance from the snail boundary. Dashed lines represent ± SD. Color codes are shown as in (B).
(D) Distribution of average mRNA counts per mesodermal cell, for embryos in each age class. The mesodermal boundary is defined as the area where the mRNA count, averaged across a line perpendicular to the boundary, drops below half its maximal value. The box spans from the lower to upper quartile. The median is indicated by the black dot. Whiskers extend to greatest and smallest data point.
(E) Box plots of coefficient of variation for embryos in each age class.
See also Figure S3.
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6. Figure 4 Dosage Compensation by Weak Negative Feedback
(A) Counting results from 98 cycle 14 embryos. Genotypes are snail-BAC (cyan) wild-type snail locus, homozygous for a snail-BAC (pink) wild-type snail locus homozygous for a snail-BAC (pink) and heterozygotes for a snail deletion (red).
(B) Average spatial profiles of mRNA expression from embryos at mitosis of cycle 13: endogenous snail (blue), BAC transgene (cyan), or both (pink). 1× snail embryos cannot be identified at mitotic stages due to the absence of nascent transcripts, see (D). Dashed lines bound a region the width of one SD.
(C) Average spatial profiles as in (B) but for cycle 14.
(D) Identification of 1× snail embryos by counting nascent transcripts. Note the median number of detectable nascent transcripts per cell provides a reliable indication of the copy number for snail.
(E) Box-and-whisker plot summarizing effect of copy number on mRNA levels. Whiskers mark the positions of the highest and lowest count in sample. Boxes indicate interquartile range and median.
See also Figure S4.
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7. Figure S1
Efficient Detection of Single Transcripts, Related to Figure 1
(A) Schematic of approach for measuring labeling efficiency: transcripts are labeled with a collection of short red and green probes. If labeling efficiency is low, a substantial number of transcripts will be detected in only one channel. The fraction of total transcripts detected should be approximately the square root of the fraction of double labeled transcripts if both green and red probes hybridize equally well.
(B) Zoom in on 3D reconstruction of a region of mRNAs double labeled with red and green probes against snail transcripts.
(C) 2D max projection of red and green channels for an arbitrary region expressing the target transcript. Crosses (red channel) or circles (green channel) mark the spots detected in both colors, yellow ∗ mark spots which were detected only in the other channel.
(D) Quantification of the fraction of yellow transcripts relative to all red or all green transcripts. The number of detected transcripts is given above the graph.
(E) Schematic to determine if dots contain multiple transcripts. Individual probes labeled in red or green compete for the same target sequence. If a single spot contains multiple transcripts, than most spots should be yellow. If each spot is an individual transcript, spots should be either red or green.
(F) Comparison of the mRNA counts per cell measured in red and green, to an embryo of the same age labeled with our standard snail probes. The average red dots per active cell is 36 (standard deviation = 9) and average green counts is 33 (standard deviation = 8). Embryo at this stage (onset of cc14) have an average of 80 to 110 transcripts per cell (yellow distribution). This suggests an additional 10%–35% increase in missed detection for our single large probes, which enter cells less easily and provide weaker signal than ∼1.5 kb tiled by short probes. (G) Zoom in on 3D reconstruction of a region of mRNAs double labeled with competitive red and green probes against the 3′ end of the snail transcript.
(H) 2D max projection of red and green channels for an arbitrary region expressing the target transcript. Crosses (red channel) or circles (green channel) mark the spots detected in each channel, yellow ∗ mark the location of spots detected only in the other channel.
(I) Box plot of the fraction of yellow transcripts. Approximately 90% of transcripts show signal in only single channel. The remaining 10% that appear doubly labeled probably arise from a combination of three factors. Sites of nascent transcription include multiple mRNAs and are expected to appear yellow, a percentage of each probe is probably fragmented during synthesis and/or hybridization, allowing access of both probes to the same transcript. Some distinct mRNAs are nonetheless within the chromatic aberration distance, and are thus computationally assumed to overlap. The uncertainty in overlap due to chromatic aberration and drift during acquisition is less than 500 nm, which is used as the cut-off for centroid to centroid distance at which signals are considered to be co-localized. To minimize coincidental overlap we have chosen early cc14 embryos with an expected ∼100 transcripts per nucleus rather than later cc14 embryos.
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8. Figure S2 Multiplex Detection, Related to Figure 2
(A) Cells double stained with an expressed snail transcript (red) and mRNA probes against a non exonic sequence of similar length (intron of single-minded, green), used to measure the frequency of non-specific localizations of probe.
(B) Distribution of spot counts of snail and sim mRNA.
(C) cells expressing a single copy of the BAC spanning the snail locus driving a yellow-reporter gene (green) and two copies of the endogenous snail gene (red). The single bright green and two bright right spots in each cell mark the sites of nascent transcription.
(D) Histogram of the number of mRNA counts per cell. The average mRNA count per cell for snail is twice that of the single-copy reporter and the standard deviation is root two larger, indicative of independent transcription.
(E and F) As in (C)–(D) now with 2 copies of the yellow-reporter. The reporter shows about 10% less expression than the endogenous copies due to a locus effect.
(G) Cross-correlation of mRNA expression from the snail-yellow transgene compared to endogenous. Globally the expression is well correlated, all mesodermal cells express high levels of both, non-mesodermal cells are transcriptionally silent, and a few cells at the edge still intermediate.
(H) Histogram of correlation coefficients for 41 embryos as in (e).
(I) Zoom in on mesodermal cells showing expression on a cell-to-cell basis is almost entirely uncorrelated. This is expected if the transcriptional noise dominates rather than extrinsic factors like local concentrations of particular transcription factor or transcription machinery.
(J) Histogram for ‘mesodermal’ cells as in (g). Mesoderm cells are defined as above half-maximal expression.
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9. Figure S3 Calculations of Kinetics, Related to Figure 3
(A) Mean mRNAs counts per cell for embryos in prophase and late anaphase at the end cycle 13. Dots mark population medians. Solid line is an exponential fit with non-parametric uncertainty estimates of upper and lower quartile indicated by dashed lines. Relative times were determined by comparing mitotic stage to live imaging of mitosis in RFP-histone tagged embryos (see insets of nuclear morphology).
(B) Mean mRNA counts per cell for embryos in telophase of cycle 12, interphase of cycle 13 (random sample sorted in ascending order of mRNA count), and prometaphase of cycle 13. Dots mark population medians. Solid line is a fit to a simple uniform synthesis and decay rate rate model where the number of active alleles changes from 0 to 2 to 4 as the first mRNA are completed and then the locus is later replicated. Nonparametric uncertainty estimates for upper and lower quartiles indicated by dashed lines (details in Supplementary Note 3).
(C) Prior to replication each site of nascent transcription of snail is detectable as a single bright dot. After locus replication many of these foci can be resolved into a pair of spots at high-resolution.
(D) A transgene containing a copy of the gene yellow replacing the snail coding sequence gives substantially brighter nascent transcription foci due to the inclusion of a substantial intron. In heterozygotes this construct is present in one copy per nucleus prior to replication and becomes ‘twinned’ into two tightly clustered spots following replication.
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10. Figure S4
Snail Binds Its Own Regulatory Sequences, Related to Figure 4
Whole-genome chromatin immuno-precipitation assay results for snail protein in y,w 2-4 hr embryos from Zeitlinger et al., 2007 (Zeitlinger et al., 2007b), shown for the snail locus. The snail coding sequence appears in the center. Previously identified proximal (Ip et al., 1992) and distal (Hong et al., 2008) regulatory sequences upstream (to the right in this image, highlighted in yellow), show clear binding peaks of snail protein.
Cell Reports 2013 3, 8-15DOI: ( /j.celrep )
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