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Independent and coordinate trafficking of single Drosophila germ plasm mRNAs

A Corrigendum to this article was published on 25 February 2016

This article has been updated

Abstract

Messenger RNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. Whereas different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single-molecule imaging, we analysed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells. We find that the germ-cell-destined transcripts nanos, cyclin B and polar granule component travel within the oocyte as ribonucleoprotein particles containing single mRNA molecules but co-assemble into multi-copy heterogeneous granules selectively at the posterior of the oocyte. The stoichiometry and dynamics of assembly indicate a defined stepwise sequence. Our data suggest that co-packaging of these transcripts ensures their effective segregation to germ cells. In contrast, compartmentalization of the germline determinant oskar mRNA into different granules limits its entry into germ cells. This exclusion is required for proper germline development.

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Figure 1: Measuring absolute mRNA content of nos granules.
Figure 2: nos granule assembly occurs exclusively at the posterior oocyte cortex.
Figure 3: Co-localization of polar granule components begins during late oogenesis.
Figure 4: mRNA content of posterior granules is heterogeneous.
Figure 5: osk mRNA forms multimeric complexes on entry into the oocyte.
Figure 6: Dynamics of osk granule assembly.
Figure 7: osk mRNA is continuously segregated from germ plasm components.
Figure 8: osk localization to germ plasm impairs germline development.

Change history

  • 01 February 2016

    In the version of this Article originally published, the sentence ‘13% of GFP–Vas granules contain nos mRNAs and 11% contain cycB’ in the caption of Fig. 4h–j was incorrect; it should have read ‘69% of GFP–Vas granules contain nos and 51% contain cycB’. This has been corrected in all online versions of the Article.

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Acknowledgements

We thank W. Eagle (Princeton University, USA), P. Lasko (McGill University, Canada), P. Macdonald (University of Texas, Austin, USA) and D. St Johnston (Gurdon Institute, UK) for fly stocks and reagents, S. Chatterjee and S. Kyin for technical assistance, and E. Abbaszadeh, S. Blythe and B. He for comments on the manuscript. This work was financially supported by National Institute of Health grant R01GM067758 (E.R.G.) and the Howard Hughes Medical Institute (E.F.W.).

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S.C.L., K.S.S. and E.R.G. designed the experiments. S.C.L., K.S.S., J.J.L. and E.R.G. performed the experiments S.C.L. and E.R.G. analysed the data. S.C.L., E.R.G. and E.F.W. wrote the manuscript.

Corresponding author

Correspondence to Elizabeth R. Gavis.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Unlocalized nos mRNAs are found as individual molecules, whereas localized granules contain many mRNAs.

(a) Imaged particles are diffraction-limited objects. Normalized intensities of 50 nm fluorescent beads (black dashed line), unlocalized nos (green), and polar granules (blue) are plotted. Red: fitted Gaussian distribution. (b) nos particle density decreases upon localization. Blue: normalized fluorescence signal density at n.c. 3. Vertical line: border of the localization domain where fluorescence exceeds that found in the anterior. Red: particles/μm3. (c) Histogram of intensities (arbitrary units, log scale) of all particles found in the left half, posterior third of an embryo. Vertical line separates dim and bright peaks. (d) Fraction of objects >164 A.U. at given distance from the posterior pole. Vertical line demarcates the localization domain, where the majority of bright objects are found. (e) Absolute RT-qPCR yields 2.5 +/− 0.8106 nos molecules per embryo. Blue: dilution series of plasmid DNA template. Red: dilution series of in vitro transcribed mRNA. Inset: dilution of single embryos (n = 6 embryos). (f) Poisson model of random co-localization: probability that imaging volumes contain n = (0,1,2… ) mRNAs when particles occupy 0.35 μm3. Inset: probabilities of particles containing n ≥ 1 mRNA. (g) Non-uniform distribution of nos throughout interior of embryo. Pixels lacking signal (red) cover 52% of the image. (h) In two colour labelling, rotation of one image stack by 90o yields a 10% co-localization frequency. (i) Poisson model as in (f) with 0.2 μm3 imaging volumes and particles occupying 60% of available volume. (j) Pair-correlation functions showing density of nos (blue) or pgc (red) particles as a function of distance from 594 reference nos particles. Error bars: standard deviations (SD). Mean densities and SD were calculated within bins of 2 pixel width at intervals of 2 pixels. Inset: P-values from paired sample t-test comparing density in first bin to densities at all other distances. Densities are not significantly different (dashed line: P-value of 0.1). (k) Histogram of intensities of cortical objects after normalization to the most commonly observed intensity. Granules have >4 mRNAs. Inset: in dual-colour labelling, normalized data fall on a line of slope = 1, indicating 4% measurement error. (l) Cumulative probability distribution of nos mRNA in granules containing >4 nos mRNAs as a function of distance from the posterior pole. Inset: number of localized granules as a function of distance. (m) Histogram of nos mRNA content in oskA87/+ oocytes (stage 10b). (n) Histogram of nos mRNA content in vas PD/vas D1 oocytes (stage 11–12). (o) Mean nos mRNA content of granules with >4 mRNAs as a function of distance from the posterior pole. Upper inset: variance divided by mean (Fano factor). For a process of random arrival of mRNAs at assembly sites, predicted Fano factor is 1 (red line). Lower inset: fractional SD of mRNA content (SD/mean) as a function of distance.

Supplementary Figure 2 Localization results from the formation of granules that maintain nos content during rapid transit.

(a) Upper: Normalized nos fluorescence density as a function of position during late oogenesis. Vertical lines indicate position at which density exceeds that found in the oocyte anterior. Lower: Plot showing fraction of all objects containing >4 mRNAs as a function of position. Nearly all bright granules are found within the localization domain. (bd) Calibrated mRNA content (red) of four selected highly motile nos particles during rapid displacement (blue). Selected frames for each particle from Supplementary Video 2 are shown. Red arrowheads indicate motile particles.

Supplementary Figure 3 Co-localization of nos, cycB, and pgc.

(ad) nos, cycB, and pgc do not co-localize in nurse cells at stage 8. 2D histograms show fluorescence intensities of cycB channel at positions where nos particles are found (a), nos at cycB positions (b), pgc at nos positions (c), and nos at pgc positions (d). Vertical green and horizontal red lines indicate thresholds separating signal from imaging noise. (e) Time course of cycB and nos localization. Scale bars: 5 μm (upper, middle); 10 μm (lower). (f) 2D histograms of nos and cycB content at stages 10a, 12, and 13. cycB begins assembling into large granules slightly earlier than nos, likely because of high levels of cycB expression at earlier times compared to nos. In contrast to the cycB mRNA that accumulates at late stages of oogenesis, this initial population of localized cycB mRNA shows only weak co-localization with nos. Color indicates relative density of data points, with red showing highest density. Red and green lines indicate thresholds separating localized and unlocalized particles. For all particles surpassing the localized threshold in one channel, the fraction surpassing the localization threshold in the second channel is shown in the upper right quadrant (green: cycB, red: nos). The fraction of localized particles containing 0 mRNAs in the second channel is shown in the upper left (for localized nos) or lower right (for localized cycB) quadrants. Very little nos surpasses the localized threshold at early time points.

Supplementary Figure 4 Assembly by random selection does not explain co-localization.

(a) Data fit to a model where nos and cycB mRNAs compete for integration into granules. Plot shows the fraction of localized granules containing 0 nos or cycB as a function of the sum of nos and cycB mRNA content. Total mRNA content for each localized granule was determined by summing nos and cycB. For granules with a given total content, the fraction containing 0 nos (blue) or 0 cycB (red) was calculated and plotted as a function of the total. Due to measurement uncertainty, granules can possess apparent non-integer mRNA numbers. Therefore, x-axis values were generated in increments of 0.25 mRNA, using bins of total content spanning ± 0.5 mRNAs. Lines indicate best fit curves (1 − p) n where n is total mRNA content (that is, fitting observations to binomial distribution). Fitting yields probabilities of P = 0.17 for incorporation of nos mRNA and P = 0.08 for incorporation of cycB mRNA into a forming granule. Actual total granule content is likely larger, given the enrichment of many genes in the germ plasm. Thus, these probabilities represent upper bounds. (be) Probabilities obtained in (a) were used to predict the distribution of nos and cycB content as a function of total content (b,d). Predictions were compared to measurements (c,e). Heat maps indicate relative densities. Magenta arrows: discrepancy between model and observation, namely, where data points predicted by model are absent in observations despite the presence of many granules completely lacking one of the two mRNAs. Because the actual total content is likely larger, the apparent relationship between observed content and total content (that is, the slope of a line fitted to the data clouds in c and e) is likely to be lower. However, the change in slope does not account for the absence of data points (arrows) in the observations. Data falling on the line of slope = 1 are those granules that contain only nos (c) or only cycB (e); in actuality these granules co-localize with other mRNAs (for example, pgc), and therefore have a higher total content than suggested by these plots.

Supplementary Figure 5 Quantification of osk mRNA in localized granules.

(a,b) Confocal section taken near the cortical surface (a) or near the mid-sagittal plane (b) of an early (n.c. 3) embryo (anterior left, dorsal up) labeled with osk probes. Green boxed region in inset of (a) shows area displayed in main panel. Middle and right panels show magnified views of red (upper panels) and yellow (lower panel) boxes. Two contrast levels are used to display the extreme brightness of localized granules compared to unlocalized mRNAs. Scale bars: 20 μm (left panels); 2 μm (magnified views). (c) Absolute RT-qPCR for osk using DNA standard (blue) or in vitro transcribed mRNA standard (red) compared to a dilution series of single embryos (inset). (d) In embryos, unlocalized particles display a heavy bias toward 2-4 mRNA, similar to that seen in oocytes. (eh) Estimates of osk mRNA content in four motile granules. (e′–h′) Selected frames from time-lapse movies of granules analysed in (eh). Red arrowheads indicate granule traced and displayed in eh. In h′, three granules (arrowheads) converge to the same point, resulting in (h) in apparent increased mRNA content at around 30 s (when particles denoted by green and red arrowheads merge) and 50 s (merging of particle denoted by yellow arrowhead). (i) osk mRNAs are often found on track-like structures along with nos in nurse cells during mid-oogenesis. (j) Log normal distribution of localized granule content. (k) osk content of localized granules in an early embryo. (l) Estimated total accumulated osk in the posterior localization domain shows a marked increase during late oogenesis. Time points are estimated from ref. 1 and correspond to stages 7, 8, 9, 10b, 12, and 13.

Supplementary Figure 6 Localization of gfp-nos3’UTR mRNA to polar granules does not affect pole cell formation.

(a,b) FISH analysis of gfp-nos3’UTR (GN) embryos with nos and gfp probes. Z-series projections of the posterior cortex at n.c. 3 (a) and n.c. 11 (b) show colocalization of GN and nos. Individual channels are shown at right. Scale bars: 10 μm (a); 5 μm (b). (c) Anti-Vas immunofluorescence in wild-type (upper) and GN (lower) embryos. (d) Box plot showing mean (red line), 25th and 75th quartiles (blue boxes), and range (black lines) of Vas-positive pole cell number (WT, n = 29; GN, n = 30). Supplementary Video 1 : Subsection of a confocal stack of an early (n.c. 3) embryo posterior labeled with nos probes. First section is adjacent to the lateral cortex. Stack is shown twice, followed by magnified views of unlocalized and localized regions. To facilitate visualization of unlocalized punctae, the stack is displayed at high contrast such that localized granules appear saturated. However, no saturated pixels are present in raw images, as illustrated with a single cortical image slice displayed at alternating high and low contrast settings. Z-sections are separated by 340 nm. Supplementary Video 2 : Time lapse movie of germ plasm RNP particles containing nosGFP at the oocyte posterior. Maximum projection of 5 z-sections spanning a total of 6 μm and a total time of 5 min. Supplementary Table 1: Oligonucleotide sequences used to design probes for this study.

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Subsection of a confocal stack of an early (n.c. 3) embryo posterior labeled with nos probes.

First section is adjacent to the lateral cortex. Stack is shown twice, followed by magnified views of unlocalized and localized regions. To facilitate visualization of unlocalized punctae, the stack is displayed at high contrast such that localized granules appear saturated. However, no saturated pixels are present in raw images, as illustrated with a single cortical image slice displayed at alternating high and low contrast settings. Z-sections are separated by 340 nm. (AVI 26019 kb)

Time lapse movie of germ plasm RNP particles containing nos*GFP at the oocyte posterior.

Maximum projection of 5 z-sections spanning a total of 6 μm and a total time of 5 min. (AVI 797 kb)

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Little, S., Sinsimer, K., Lee, J. et al. Independent and coordinate trafficking of single Drosophila germ plasm mRNAs. Nat Cell Biol 17, 558–568 (2015). https://doi.org/10.1038/ncb3143

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