Precise developmental gene expression arises from globally stochastic transcriptional activity

Abstract

Early embryonic patterning events are strikingly precise, a fact that appears incompatible with the stochastic gene expression observed across phyla. Using single-molecule mRNA quantification in Drosophila embryos, we determine the magnitude of fluctuations in the expression of four critical patterning genes. The accumulation of mRNAs is identical across genes and fluctuates by only ∼8% between neighboring nuclei, generating precise protein distributions. In contrast, transcribing loci exhibit an intrinsic noise of ∼45% independent of specific promoter-enhancer architecture or fluctuating inputs. Precise transcript distribution in the syncytium is recovered via straightforward spatiotemporal averaging, i.e., accumulation and diffusion of transcripts during nuclear cycles, without regulatory feedback. Common expression characteristics shared between genes suggest that fluctuations in mRNA production are context independent and are a fundamental property of transcription. The findings shed light on how the apparent paradox between stochastic transcription and developmental precision is resolved.

Figure 1. Counting of absolute transcript number in Drosophila embryos

A: Confocal section through the nuclear layer of a WT embryo during interphase 13 labeled with 114 fluorescent oligonucleotide probes against hunchback, oriented anterior to the left. Scale bar: 25 μm. Inset: Low magnification image identifying the region shown in A. B,C: Magnified views of anterior (B) and posterior (C) boxed regions in (A). Scale bars: 5 μm. D: Particle intensity histogram showing thresholds separating transcripts from noise (red line) and from the long tail of bright transcription sites (green line). E: hb transcript distribution in axial cross-section through a nucleus centered at x=0. z=0 represents apical surface. Color indicates mean particle density in relative units (red=high, blue=low). Dashed box: cylindrical summation volume. F: Intensity scatter plot in two channels using probes of alternating colors. Data point density given by color. Inset: Cross-sections of scatter plot in (F) along the correlated (red) and anti-correlated direction (blue) shows Gaussian distributions with σ=20% (red) and σ=12% (blue) after normalization to mean cytoplasmic particle intensity (1 “cyto unit”). See also Figures S1 and S2.

Figure 2. Precision and reproducibility of cytoplasmic hb profiles

A: Absolute cytoplasmic hb mRNA counts per standardized volume as a function of AP position. Data for four embryos at nuclear cycle 12 (blue), 13 (green), early 14 (red), and late 14 (magenta). Position is shown as distance from inflection point x_transition (see also Figure S3C). Inset: fractional SD σ^max/N^max in the spatial domain of highest mRNA accumulation as a function of the mean count (N^max) for 101 embryos. Dashed line at 8%. B: Cytoplasmic hb mRNA counts (N^max) as a function of time. Ages estimated by visual inspection of DAPI staining; relative width of mitoses (gray shading) and interphases according to Alberts and Foe (1983). Reproducibility of counts in 12^th and 13^th mitoses is 8% and 11%, respectively. Inset: estimated reproducibility $\widehat{\sigma }$ as a function of time. Data points: running averages of root-mean-square displacement from smoothed timeline over 15 consecutive data points normalized to mean. Dashed line: average $\widehat{\sigma }$ (17%). See also Figure S3

Figure 3. Variability of transcriptional activity at nascent transcription sites

A: Scatter plot of total nascent hb mRNA per nucleus using probes of alternating colors for an embryo in nuclear cycle 13 after normalization to the mean cytoplasmic particle intensity (C.U.). Intensities follow a direct proportionality relation with slope 0.90±0.09 (n=5 embryos). Inset: root mean square normalized deviation from linear fit; scatter = 5% (arrows). B: Two-color scatter plot of nascent mRNA content in which probes bearing the same fluorophore are clustered on the 5’ (green channel) and 3’ (red channel) portions of the transcript. Cyan: measurements using 57 green and 57 red-labeled probes; observed slope: 1.3. Yellow: results with 78 green and 36 red-labeled probes; observed slope: 1.6. Green line in A is plotted for comparison. C: Transcriptional activity per nucleus as a function of position along the AP axis for four embryos in nuclear cycle 12 (blue), 13 (green), 14 early (red), and 14 late (magenta) in binned averages of 10, 20, 40 and 40 nuclei, respectively. Error bars: SDs within bins. Position is shown as distance from inflection point x_transition. D: Transcriptional activity per nucleus as a function of absolute AP position for the embryo in interphase 13 in C. E: Transcription noise for 10 embryos in nuclear cycle 13 plotted as fractional SD across nuclei as a function of cytoplasmic hb counts within the spatial domain of highest accumulation. Transcription activity noise remains constant throughout interphase at 22±3%. See also Figure S4.

Figure 4. Fluctuations in hb transcription are dominated by intrinsic noise

A: Transcriptional activity of loci on optically resolved sister chromatids is uncorrelated (Pearson correlation coefficient R=0.02), compared to the tight correlation (R=0.97) in a control experiment using probes of alternating colors (with 4% imaging noise). B: Transcriptional variability arises from fluctuations in inputs (extrinsic noise) and from the process of transcription itself (intrinsic noise). Two extreme scenarios are presented in cartoon form. Upper panel: a fluctuating extrinsic input leads to correlated activities of transcription sites within a given nucleus; its contribution to the fractional SD is independent of the number of transcribing loci k. Lower panel: intrinsic mechanistic noise affects all transcription sites independently; the fractional SD scales as inverse square root of available transcription sites. Left: the measured transcription noise in WT and hbΔ/+ embryos (22±3% and 33±6%, respectively) shows scaling behavior characteristic of intrinsic noise with magnitude ~45% $(\sqrt{4}\ast 22=44\%;\sqrt{2}\ast 33=47\%)$.

Figure 5. Mutations in hb and runt impair timely repression of hb expression

A,B: Cytoplasmic hb mRNA counts per standardized volume as a function of AP position of similarly staged zygotic hb mutants (A) and WT siblings (B). Smooth profiles and error bars obtained as in Figure 2A. C,D: runt mutants show delayed repression of hb transcription in nc14. C: Solid lines: runt mutants; dashed lines: WT siblings. Black: embryos of similar age (mid nc14) as judged by DAPI. Profile of an early nc14 WT embryo (red line) resembles mid-stage runt mutants, whereas a very late runt mutant (magenta line) is similar to earlier WT siblings. D: Transcriptional activity in anterior nuclei of the embryos shown in C. Boxplot depicts median, quartiles, and range of nascent transcription site activity for each embryo. runt mutants display consistently higher hb activity compared to WT siblings, leading to the inappropriately high hb transcript counts at late times as shown in C. See also Figure S5.

Figure 6. Universal properties of gap gene transcription

A: Cytoplasmic profiles of four gap genes (mRNA concentration per standard volume AP position) measured in two embryos of the same age (second half of nuclear cycle 13; indicated by dotted line in panel E) processed with hb (blue) & gt (red) and with Kr (magenta) & kni (green) probes. B-E: Gap gene expression characteristics within each gene's region of maximum expression. B: Noise in cytoplasmic counts as a function of counts per nucleus (dashed line: 8%). C: Noise in transcriptional activity as a function of activity level (dashed line: 23%). D: mRNA expression (mean mRNA count per standard volume) in embryos from cycle 12 to early 14 co-stained with FISH probes against hb and Kr mRNA. Data from WT embryos (blue) coincides with those from embryos deficient for one copy of hb (hbΔ/+; yellow) or Kr (Kr¹/+; cyan) when the concentration of the respective mRNA is rescaled by a factor of 2. Inset: Raw data (not rescaled). E: Levels of hb (blue), kni (green) and gt (red) versus Kr. Data from WT, hbΔ/+ and Kr¹/+ embryos is combined by rescaling as in D (also see Figure S5C-D). hb data as in A; kni and gt were assessed in cycles 13 and 14. Slopes of fit lines indicate ratio of absolute production rates; all are within 15% of unity.