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. 2012 Dec;14(12):1305-13.
doi: 10.1038/ncb2627. Epub 2012 Nov 25.

Drosophila patterning is established by differential association of mRNAs with P bodies

Timothy T Weil  1 , Richard M Parton, Bram Herpers, Jan Soetaert, Tineke Veenendaal, Despina Xanthakis, Ian M Dobbie, James M Halstead, Rippei Hayashi, Catherine Rabouille, Ilan Davis
Affiliations

Drosophila patterning is established by differential association of mRNAs with P bodies

Timothy T Weil et al. Nat Cell Biol. 2012 Dec.

Abstract

The primary embryonic axes in flies, frogs and fish are formed through translational regulation of localized transcripts before fertilization. In Drosophila melanogaster, the axes are established through the transport and translational regulation of gurken (grk) and bicoid (bcd) messenger RNA in the oocyte and embryo. Both transcripts are translationally silent while being localized within the oocyte along microtubules by cytoplasmic dynein. Once localized, grk is translated at the dorsoanterior of the oocyte to send a TGF-α signal to the overlying somatic cells. In contrast, bcd is translationally repressed in the oocyte until its activation in early embryos when it forms an anteroposterior morphogenetic gradient. How this differential translational regulation is achieved is not fully understood. Here, we address this question using ultrastructural analysis, super-resolution microscopy and live-cell imaging. We show that grk and bcd ribonucleoprotein (RNP) complexes associate with electron-dense bodies that lack ribosomes and contain translational repressors. These properties are characteristic of processing bodies (P bodies), which are considered to be regions of cytoplasm where decisions are made on the translation and degradation of mRNA. Endogenous grk mRNA forms dynamic RNP particles that become docked and translated at the periphery of P bodies, where we show that the translational activator Oo18 RNA-binding protein (Orb, a homologue of CEPB) and the anchoring factor Squid (Sqd) are also enriched. In contrast, an excess of grk mRNA becomes localized inside the P bodies, where endogenous bcd mRNA is localized and translationally repressed. Interestingly, bcd mRNA dissociates from P bodies in embryos following egg activation, when it is known to become translationally active. We propose a general principle of translational regulation during axis specification involving remodelling of transport RNPs and dynamic partitioning of different transcripts between the translationally active edge of P bodies and their silent core.

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Figures

Figure 1
Figure 1. Differential association of bcd and grk with P bodies
(a-d) mRNA detection at the dorsoanterior corner (for orientation, see Fig. 2a) by ISH-IEM on wild-type (WT) ultra-thin frozen sections of stage 9 oocytes. (a) bcd mRNA (5nm, green circles) is present both inside and at the edge of electron dense P bodies (dashed black line). Gold particles here cluster due to the use of a bridging antibody. (b) grk mRNA (15nm, red circles) is enriched at the edge of P bodies (dashed black line). (c) bcd mRNA (5nm, green circles) and grk mRNA (15nm, red circles) can associate with the same P body but bcd is enriched inside. (d) grk mRNA (15nm, red circles) docks at the edge of the P body (black dashed box magnified, inset bottom right), just outside of the Me31B dense core region (5nm gold), while grk transport particles, as described in Delanoue et. al., 2007(7), are detected in the cytoplasm at a short distance from the P body (15nm, red dashed circles). (e) mRNA density (gold/um2) in the P body sub-regions when compared to the surrounding cytoplasm. For comparison, randomized particles were analyzed in an identical way to RNAs. Error bars show SEM of gold density per scan (grk n=13, bcd n=11). (f,g) Fixed Me31B::GFP stage 8/9 expressing oocytes imaged using the OMX structured illumination super-resolution mode (3D-SIM) with double labeling of either (f) grk*mCherry, that mainly interdigitates with Me31B (green) while showing some colocalization at the edge (yellow) or (g) bcd*RFP grk mRNA (red), that shows significant colocalization (yellow) with Me31B (green). Scale bars, 200nm (a-d); 0.5μm (f, g).
Figure 2
Figure 2. Dynamics of grk, bcd and Me31B particles in live oocytes
(a) Low magnification wide-field image of the DA corner of a stage 8-9 egg chamber expressing Me31B::GFP. (b) Me31B::GFP expressing oocytes show small faint dynamic particles of Me31B (red arrowheads) moving between and fusing together with other often larger, bright and static (dashed cyan circles) Me31B bodies. (Supplemental Movie 1, 2). (c,d) grk*mCherry and Me31B::GFP expressing oocytes show grk mRNA particles (white arrowheads) moving independently of Me31B (Supplemental Movie 3). (d) Dynamic particles of grk (white arrowhead) are visualized docking and remaining on the edge of Me31B rich zones. Other small grk particles are seen in association with the Me31B throughout the time course (dashed cyan circles) (Supplemental Movie 4). (e) bcd*RFP and Me31B::GFP expressing oocytes show bcd particles (white arrowheads) moving independently of Me31B. (Supplemental Movie 5). (f) Average particle velocities for dynamic Me31B (n=30), grk (n=37) and bcd (n=31) particles in μm/second +/− SEM. P-values from student’s t-tests (two tails), P<0.001. (g) Fluorescence Recovery After Photobleaching of Me31B::GFP at the DA corner shows recovery to approximately 50% of total fluorescence with a half time of 4 minutes (n= 5). NC, Nurse Cell; N, nucleus. Scale bars, 10μm (a); 2μm (b-e); 20μm (g).
Figure 3
Figure 3. Oocyte P bodies exhibit zones differentially enriched in RNA associated proteins
(a-d,f,g) IEM localization of RNA associated proteins in stage 9 oocytes at the DA corner. Protein in the core of the electron dense P bodies indicated by green circles, at the edge of P bodies by red circles and in the cytoplasm by blue circles. (a) WT egg chamber, anti-Me31B (15nm) enriched inside P bodies. (b) Hrb27C (5nm) predominantly inside of P bodies. (c) Dcp2 (10nm) inside and at the edge of P bodies. (d) WT egg chamber, anti-Ribo 490 (10nm) shows ribosomes predominantly in the cytoplasm some at the edge but mostly excluded from inside of P bodies. The pool of cytoplasmic ribosomes corresponds to polysomes and those present on the ER membrane (Rough ER), some of which is present near the edge of a P body (black dashed box, inset: red arrowhead). Smooth ER is detected inside of the P body (inset: black arrowhead). (e) Graph showing protein concentration in gold/μm2 in the cytoplasm, edge of P bodies and inside of P bodies; proteins are organized into three distinct categories. Error bars are ± standard deviation (between n=10-15 scans in each case). (f) WT egg chamber, anti-orb (15nm) enriched at the edge of P bodies. (g) Sqd-GFP in Sqd::GFP expressing egg chamber using anti-GFP (10nm) and anti-Dhc (15nm, yellow circles). Sqd is enriched inside compared to at the edge of P bodies. Scale bars, 200nm (a-d,f,g).
Figure 4
Figure 4. RNA translation fate is regulated through association with P bodies
(a) IEM using anti-Grk (15nm, yellow circles) on a WT stage 9 egg chamber shows Grk protein highly enriched in the tER-Golgi unit (yellow dashed box) and detected on ER (black arrowhead) at the edge of a P body (black dashed line). (b) ISH-IEM detection of grk mRNA and Grk protein on a WT stage 9 egg chamber shows grk mRNA (15nm, red circles) mostly present at the edge of the P body (black dashed line) and Grk protein (5nm, yellow circles) on a tER-Golgi unit (yellow dashed box) but also present at the edge of the P body (black dashed line). Note the grk mRNA transport particle (dashed red circle) does not contain any detectable Grk protein (black arrowhead). (c) Injection of in vitro synthesized grk RNA (mixed solution of approximately one part Alexa dye labeled and 4 parts unlabeled) into a grk null egg chamber expressing Me31B::GFP. Images shown are a 4μm projection. Localization and translation was allowed for 40 minutes before the egg chamber was fixed and labeled with anti-Grk. (c,c″) Grk protein (green) and (c,c‴) grk RNA (red) colocalize, but do not colocalize with (c,c′) Me31B (blue). Instead, grk RNA and protein interdigitates with Me31B (arrowheads). (d-d‴) Low magnification of panel c. Injected grk RNA is localized to the dorsoanterior corner where it is translated. (e) IEM on a WT stage 8/9 egg chamber injected with a high concentration of grk RNA Biotin (500 ng/μl). Injected RNA (15nm, red circles) is detected at the core of and at the edge of the P body (dashed black line). (f) IEM on a stage 8/9 egg chamber over-expressing grk mRNA using the UAS-Gal4 system. grk mRNA (15nm) is enriched at the core of and at the edge of the P body (dashed black line). FC, follicle cell; TP, transport particle; ER, endoplasmic reticulum; N, nucleus. Scale bars, 200nm (a,b,e,f); 3μm (c); 10μm (d).
Figure 5
Figure 5. RNA association with P bodies in the early embryo
(a) Anterior region of a live early embryo expressing bcd*RFP and Me31B::GFP. The majority of bcd mRNA is detected outside of Me31B labeling, sometimes associating with the edge (arrowhead). (b,c) Time points from a stage 14 egg chamber expressing (b) Me31B::GFP or (c) Tral::YFP, prior to and following activation. Activation buffer added at t = 0 minutes. Immediately after activation, Me31B (or Tral) labeled P bodies disperse. (d) Stage 14 egg chamber expressing bcd*RFP and Me31B::GFP (d) Before treatment with activation buffer and (d′) 10 minutes after activation buffer is applied. Pre-activation, bcd mRNA and Me31B colocalize at the anterior (arrowhead). Immediately after activation, Me31B disperses and bcd no longer colocalizes with Me31B (arrowhead). (e) Model of how P bodies regulate differential translation of bcd and grk mRNA at the DA corner in a stage 8/9 oocyte. grk mRNA transport particles move independently of Me31B. grk associates at the edge of the P body where enrichment of Sqd is involved in anchoring, Orb activates translation and ribosomes support translation. grk is translated on rough ER at the edge of P bodies and Grk protein is trafficked through the secretory pathway (tER-Golgi unit) before being secreted. bcd mRNA transport particles move independently of Me31B and associate with the core of the P body where translation is not supported. Dynamic Me31B, Exu and Tral particles assemble and maintain the P bodies. Excess grk RNA is targeted into the core of P bodies where it is translationally repressed and likely degraded. FC, follicle cell. Scale bars, 5μm (a,d); 20μm (b,c).
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