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. 2018 Jun 18;28(12):1872-1881.e3.
doi: 10.1016/j.cub.2018.04.037. Epub 2018 May 31.

Stochastic Seeding Coupled with mRNA Self-Recruitment Generates Heterogeneous Drosophila Germ Granules

Matthew G Niepielko  1 , Whitby V I Eagle  1 , Elizabeth R Gavis  2
Affiliations

Stochastic Seeding Coupled with mRNA Self-Recruitment Generates Heterogeneous Drosophila Germ Granules

Matthew G Niepielko et al. Curr Biol. .

Abstract

The formation of ribonucleoprotein assemblies called germ granules is a conserved feature of germline development. In Drosophila, germ granules form at the posterior of the oocyte in a specialized cytoplasm called the germ plasm, which specifies germline fate during embryogenesis. mRNAs, including nanos (nos) and polar granule component (pgc), that function in germline development are localized to the germ plasm through their incorporation into germ granules, which deliver them to the primordial germ cells. Germ granules are nucleated by Oskar (Osk) protein and contain varying combinations and quantities of their constituent mRNAs, which are organized as spatially distinct, multi-copy homotypic clusters. The process that gives rise to such heterogeneous yet organized granules remains unknown. Here, we show that individual nos and pgc transcripts can populate the same nascent granule, and these first transcripts then act as seeds, recruiting additional like transcripts to form homotypic clusters. Within a granule, homotypic clusters grow independently of each other but depend on the simultaneous acquisition of additional Osk. Although granules can contain multiple clusters of a particular mRNA, granule mRNA content is dominated by cluster size. These results suggest that the accumulation of mRNAs in the germ plasm is controlled by the mRNAs themselves through their ability to form homotypic clusters; thus, RNA self-association drives germ granule mRNA localization. We propose that a stochastic seeding and self-recruitment mechanism enables granules to simultaneously incorporate many different mRNAs while ensuring that each becomes enriched to a functional threshold.

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Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1
Figure 1. Germ granules containing single nos or pgc transcripts or single transcripts together with small homotypic clusters
(A–C, I) Maximum intensity projections of three z-slices (450 nm) from SIM imaging of stage 10 oocytes expressing Osk-GFP. nos (magenta) and pgc (green) mRNAs were detected by smFISH and Osk-GFP (blue) was detected by direct fluorescence. (B) Enlargement of the white box in (A) showing single nos and pgc mRNAs within the bulk cytoplasm (bc). (C) Enlargement of the yellow box in (A) showing mRNA particles detected within the germ plasm (gp). (D–G) Magnification of the puncta indicated by the yellow arrow (C) that contains one nos and one pgc transcript together with Osk-GFP. (H) The average fluorescence intensity distribution of single nos and pgc mRNAs in the bulk cytoplasm (broken lines) and the fluorescence intensity distribution of single nos and pgc mRNAs in the germ plasm that are shown in (D, E; solid lines). Single nos and pgc mRNAs in the germ plasm were identified by matching intensity profiles to those of average intensity profiles of single nos and pgc mRNAs in the bulk cytoplasm. Error bars = S.E.M. (J–M) Enlargement of the Osk-GFP particle indicated by the white arrow in (I) containing a single nos transcript and a small pgc homotypic cluster. (N–Q) Enlargement of the Osk-GFP puncta indicated by the yellow arrow in (I) containing a single pgc transcript and a small nos homotypic cluster. (R–U) Enlargement of the Osk-GFP particle indicated by the red arrow in (I) containing homotypic clusters of nos and pgc. Scale bar in (A) applies to (I). Scale bar in (D) applies to (D–G, J–U). See also Figure S1.
Figure 2
Figure 2. Co-population of granules by single nos transcripts with one or more pgc transcripts
(A–D) Confocal z-series projections spanning 5 μm at the posterior of stage 10 oocytes: oocyte expressing Osk-GFP (green; A, B); wild-type (wt) oocyte (C); osk− oocyte (D). nos, pgc, and lost mRNAs (magenta) were detected by smFISH and Osk-GFP by direct fluorescence. (E–G) Quantification of co-localization from experiments shown in (A–D.) Red bars: percentage of single-transcript nos particles that are co-localized with Osk-GFP (E) or pgc (F, G) as a function of distance from the posterior pole; blue bars: co-localization between a non-localizing transcript, lost, and Osk-GFP (E) or single-transcript nos particles and non-localizing gfp-tub-3′UTR transcripts (F, G). In all images, the oocyte posterior is oriented to the right. Scale bar in (A) applies to all images. Values shown are mean ± S.E.M; n≥4 oocytes. See also Figure S1 and Figure S2.
Figure 3
Figure 3. nos homotypic clusters grow while co-localized with pgc
(A, B) Census of nos and/or pgc transcripts resident in each granule, demarcated by Osk-GFP, detected at stage 10 (A) and stage 13 (B). Each data point indicates an Osk-GFP particle (i.e., germ granule) and the heatmaps indicate the fraction of all particles with each observed RNA composition. (C) 2D histogram of pgc and nos mRNA content in ~100,000 particles detected within the germ plasm from stages 10–13. Points along the y-axis and x-axis contain only pgc and only nos, respectively, and are considered as non-colocalized. Heat map indicates relative density of data points. (D) Plot of the number of pgc and nos mRNAs in the co-localized population of particles from (C) (i.e., germ granules containing at least one of each) color coded by developmental stage: stage 10 (blue), stage 11 (cyan), stage 12 (green), stage 13 (yellow), and early embryo (red). Each data point is one granule. (E) Distribution of nos particles, binned by mRNA content, that are co-localized with at least one pgc mRNA (red bars) compared to the distribution of nos particles that do not co-localize with pgc (blue bars), from stage 10 to early embryo (em). See also Figure S3 and Video S1.
Figure 4
Figure 4. Nucleation and homotypic cluster growth depend on continued Osk assimilation
(A) Quantification of Osk-GFP fluorescence in arbitrary units (a.u.) shows that both the average (x-axis) and greatest (max; y-axis) amount of Osk-GFP per granule increased significantly between stage 10 and stage 13. (B) Osk-GFP fluorescence in arbitrary units (a.u.) compared with the number of nos mRNAs/granule at stages 10 (green) and 13 (blue); for both stages combined, r = 0.65. Each data point is one granule. (C, D) Quantification of the average number of nos and pgc transcripts (C) and co-localization frequency of nos and pgc (D) in stage 13 wild-type versus 1x osk oocytes. Values shown are mean ± S.E.M; n = 4 oocytes. ***p<0.001, as assessed by Student’s t-test.
Figure 5
Figure 5. nos and pgc homotypic clusters grow independently of each other
(A–D) Confocal z-series projections spanning 5 μm at the posterior cortex of stage 13 oocytes with nos (magenta) detected in wild-type (wt; A) and 1x nos (B) oocytes and pgc (green) detected in wild-type (wt; C) and 1x pgc (D) oocytes. (A′–D′) Heatmaps representing the absolute number of transcripts per particle quantified in the yellow boxes in A–D show that the mRNA content of germ granules is reduced in 1x genotypes. (E–H) Quantification of the average numbers (x-axis) of nos and pgc mRNAs/granule and their co-localization frequencies (y-axis) in wild-type oocytes (E, G), in oocytes with decreased or increased nos mRNA (1x nos and 4x nos; F), and oocytes with decreased pgc mRNA (1x pgc oocytes; H); n = 4 oocytes each. gfp-tub3′UTR was used as a control, non-localizing transcript. Stages are color-coded as indicated. The horizontal dashed line provides a reference for the same position in each graph. (I, J) The slope of the line that represents the average ratio of nos to pgc mRNAs per granule shifts when the amount of either is decreased (1x nos and 1x pgc; I) or when nos is increased (4x nos; J); n = 3 oocytes each. In all images, the oocyte posterior is oriented to the right. Values shown are mean ± S.E.M. p<0.0005 for comparison of slopes for wild-type and each genotype in (I, J). See also Figure S4.
Figure 6
Figure 6. Granules may contain multiple nos or pgc homotypic clusters
(A, B) Maximum intensity projection of three z-slices (450 nm) from SIM imaging of nos (magenta), pgc (red), and Osk-GFP (green) in the germ plasm of early embryos. (A′, B′) Enlargements of the boxed regions in (A) and (B). Yellow arrows point to germ granules, marked by Osk-GFP, containing two homotypic clusters of nos (A″) or two homotypic clusters of pgc (B″). White asterisks denote spatially distinct homotypic clusters. Plus signs indicate the centroid of the Osk-GFP signal. (C–F) Examples of granules containing more than one nos cluster. (G) Distributions of the number of homotypic clusters detected per germ granule (marked by Osk-GFP) in the early embryo for lost, nos, and pgc; n > 4 embryos, >6,000 particles. (H, I) Quantification of the average number of nos and pgc mRNAs per germ granule (H) and the proportion of germ granules that contain more than one homotypic nos or pgc cluster (I) in stage 13 oocytes as compared to early embryos. (J) Quantification of the average number of mRNAs relative to the average number of homotypic clusters of that mRNA per granule in the early embryo. Values shown are mean ± S.E.M and p values determined by Student’s t-test. **p<0.01; ***p<0.001. See also Figure S5.
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