Reporter Strains.

<p>a—Copy number was determined via qPCR, using <i>ama-1</i> as a control, detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s011" target="_blank">S1 Text</a>, Section 5.</p><p>Reporter Strains.</p

Interindividual variation in expression of <i>P</i>_{<i>hsp-16</i>.<i>2</i>}-<i>GFP</i> reporter strains by flow and by image cytometry.

<p>a—For flow measurements, numbers come from about 500 animals measured for each strain, repeated in ten runs on ten different days. CV is the Coefficient of Variation, or relative standard deviation (CV = Standard Deviation/Mean).</p><p>b—For microscopic measurements numbers come from the summed values of all intestine cells measured with image cytometry, which is a total of three measured CV values for each reporter.</p><p>c—Mean expression measured in flow. We could not compare mean expression data acquired by microscopy because we needed to increase the PMT detector gain for the single copy reporter.</p><p>d—Strain TJ3001.</p><p>e—Strain TJ375.</p><p>Interindividual variation in expression of <i>P</i>_{<i>hsp-16</i>.<i>2</i>}-<i>GFP</i> reporter strains by flow and by image cytometry.</p

Quantification of single-cell expression by nuclear fluorescence signal.

<p><b>A)</b> For each cell, from a confocal stack, we chose the z slice with the largest nuclear perimeter, then, using ImageJ, drew the nuclear boundary using the ellipse tool, and then used the selection brush tool to correct the boundary (approximated here by the red dashed line). We took the average pixel intensity inside these boundaries. Additional detail is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s007" target="_blank">S7 Fig</a>, which shows the proper identification of the nuclear equatorial plane in actual confocal micrographs. <b>B)</b> Relationship between cytoplasmic and nuclear concentration of fluorescent proteins in intestine cells. GFP or mCherry expression was driven by the <i>daf-21</i> promoter in strains RBW2661 (<i>P_daf-21-GFP</i>) and RBW2642 (<i>P_daf-21-mCherry</i>), respectively. Cytoplasmic and nuclear concentrations of fluorescent protein were measured and plotted as x,y coordinates. Red dots are from mCherry expressing cells; green dots are from GFP expressing cells. We measured the nucleus as in <b>A.</b> To sample the cytoplasm, we measured a nucleus sized area of the cytoplasm in the same z slice as the equatorial plane of the nucleus, with care to avoid autofluorescent granules in the cytoplasm (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s003" target="_blank">S3 Fig</a>). Three randomly selected intestine cells were measured in each of four different animals (12 cells total), two expressing GFP and two expressing mCherry. <b>C)</b> The relationship between cytoplasmic and nuclear concentration of fluorescent proteins in intestine cells of heat shocked animals is shown. GFP or mCherry expression was driven by the <i>hsp-16</i>.<i>2</i> promoter in TJ3001 (<i>P</i>_{<i>hsp-16</i>.<i>2</i>}-<i>GFP</i>) or TJ3002 (<i>P</i>_{<i>hsp-16</i>.<i>2</i>}-<i>mCherry</i>) animals, respectively. Measurements were made as in <b>B</b>, but cytoplasmic measures were difficult because there was more autofluorescence in heat shocked animals’ cells, and we had to sample a few more cells (15 cells total) to see a decent correlation due to this “noise” in the cytoplasmic measurements. <i>P</i> < 10^–7 for the correlations in <b>B</b> and <b>C</b>.</p

Layout of cells comprising the adult <i>C</i>. <i>elegans</i> intestine.

<p>Top panel shows the location of the left-handed helical half twist. Bottom panel shows the intestine within an animal. Anteriormost ring is comprised of four cells, remaining rings are made up of two cells, all arranged around a hollow core with the half twist between ring V and ring VI. Cells are identified by proper intestine cell name (e.g., int5L), progenitor cell (lineage, e.g., Ealp), and number (e.g., #12). Cells in L lineage are even numbers and cells in R lineage are odd numbers. We refer to these cells by their numbers in the x axis of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.g004" target="_blank">Fig 4</a>. A more anatomically detailed cartoon with corresponding microscopic images that are also anatomically detailed is available as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s001" target="_blank">S1 Fig</a>; identification of the nuclei in the cells in the twist in microscopic images is also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s006" target="_blank">S6 Fig</a>.</p

Expression of two differently colored <i>P</i>_{<i>hsp-16</i>.<i>2</i>} reporters in the same animal using multicopy and single copy reporters.

<p>DIC micrographs and fluorescent images show the three posterior-most intestinal cell rings (rings VII-IX). In the figure, posterior is on the left and dorsal on the top. Top row shows an animal expressing two multicopy reporters. Bottom row shows an animal expressing two single copy reporters. In about 1/3 of the animals expressing two multicopy reporters, there is a dramatic difference in the expression of the reporters in at least one cell. The strong bias toward expression of the red allele in int9R (white arrow, top row, fourth panel) occurred in slightly more than half of the animals showing differential expression (12/21). We did not observe such pronounced expression bias in the intestine cells of the animals expressing the two single copy reporters (typical image shown in bottom row).</p

Consistency of experimental measurements.

<p><b>A & B)</b> Single-cell expression profiles of different <i>P</i>_{<i>hsp-16</i>.<i>2</i>}-<i>GFP</i> reporters in the 20 cells of the intestine from three different experiments. The different intestinal cells are laid out anterior to posterior, and numbered 1 to 20. They correspond to the cell names and lineage relationships detailed in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s001" target="_blank">S1</a>. The heights of the bars show expression level normalized to the brightest cell for each reporter in each experiment. <b>C)</b> Consistency of cell-specific expression patterns within a strain. Consistency of expression profiles of <i>P</i>_{<i>hsp-16</i>.<i>2</i>}-<i>GFP</i> reporters in individual cells in TJ3001 (single copy) and TJ375 (530 copy) animals. Results come from three experiments on three different days. Plots show average expression for a given cell from one experiment as x, and average expression for the same cell measured in a different group of animals in a different experiment as y, for all combinations of two experiments. Expression profiles were between 75 and 88% correlated between experiments. <b>D)</b> Lack of similarity of cell-specific expression patterns between single copy and 530 copy strains. Average expression values for the single copy reporter were plotted as x, and the average expression value for the same cell in different animals expressing the multicopy reporter was plotted as y. Cell-specific expression from these two different <i>P</i>_{<i>hsp-16</i>.<i>2</i>} reporter strains is not correlated.</p

Variation in expression among the same cells in different animals.

<p>Plot shows variation in <i>P</i>_{<i>hsp-16</i>.<i>2</i>}-<i>GFP</i> expression for each of the twenty intestine cells by image cytometry (3 CV values per cell, with each CV derived from 8–11 measures). X axis shows cell identity (detailed in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s001" target="_blank">S1</a>). Bars show average coefficient of variation. Error bars show standard deviation. Stars above bars indicate significant differences in CV (<i>P</i> < 0.022; Two-Way Repeated Measures ANOVA followed by a Holm-Sidak comparison procedure) between the two reporters in Cell 12 (int5L) and Cell 14 (int6L).</p

Single Cell Quantification of Reporter Gene Expression in Live Adult <i>Caenorhabditis elegans</i> Reveals Reproducible Cell-Specific Expression Patterns and Underlying Biological Variation

<div><p>In multicellular organisms such as <i>Caenorhabditis elegans</i>, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. Here, we developed microscope-based methods to quantify reporter gene expression in cells of <i>Caenorhabditis elegans</i> with low measurement error. We then quantified expression in strains that carried different configurations of <i>P_hsp-16.2-fluorescent-protein</i> reporters, in whole animals, and in all 20 cells of the intestine tissue, which is responsible for most of the fluorescent signal. Some animals bore more recently developed single copy <i>P_hsp-16.2</i> reporters integrated at defined chromosomal sites, others, “classical” multicopy reporter gene arrays integrated at random sites. At the level of whole animals, variation in gene expression was similar: strains with single copy reporters showed the same amount of animal-to-animal variation as strains with multicopy reporters. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. They should allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to molecular differences. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. Understanding such variation should help reveal its contribution to differences in complex phenotypic outcomes in multicellular organisms.</p></div

Relationship between copy number and expression in <i>P_hsp-16.2</i>-XFP reporter strains.

<p><b>A)</b> X axis is reporter diploid copy number; Y axis shows expression level in PMT counts. Solid line shows a Hill function with a Hill coefficient of 0.6 (a quantitative measure of the nonlinear increase in expression with increasing copy number), fit to the data with an R² of 0.97. For each strain, the expression data is the average of at least three flow experiments that quantified about 500 animals per experiment. <b>B)</b> Average expression level of a single copy <i>P_hsp-16.2</i><i>-GFP</i> reporter in homozygotes and heterozygotes. Error bars show S.E.M. We picked over 200 F1 hermaphrodites from each cross and measured 84 F1 heterozygotes and 49 F1 homozygotes in flow; additional details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s011" target="_blank">S1 Text</a>, Section 5: Strain Construction.</p

Fluorescent proteins.

<p>A) Body plan of fluorescent proteins. DarkCitrine is fused to the N terminus of TFP and Citrine with a GSGG linker (black bar). B) Emission spectra from yeast expressing each XFP excited by a 458 nm laser and emission collected from 460–650 nm. (Double-headed arrows indicate wavelength bands used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109940#pone-0109940-g002" target="_blank">Figure 2</a>)</p