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Curr Biol. Author manuscript; available in PMC Jul 22, 2009.
Published in final edited form as:
PMCID: PMC2581475
NIHMSID: NIHMS60420
Changes in bicoid mRNA anchoring highlight conserved mechanisms during the oocyte-to-embryo transition
Timothy T. Weil,1 Richard Parton,2,3 Ilan Davis,2,3 and Elizabeth R. Gavis1*
1Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
2Department of Biochemistry, The University of Oxford, South Parks Road, Oxford OX1 3QU, UK
3Former address: Wellcome Trust Centre for Cell Biology, The University of Edinburgh, The Kings Buildings, Edinburgh EH9 3JR, UK
*Author for correspondence: lgavis/at/princeton.edu; Phone: (609) 258-3857; FAX: (609) 258-1343
Intracellular mRNA localization directs protein synthesis to particular subcellular domains to establish embryonic polarity in a wide range of vertebrates and invertebrates. In Drosophila, bicoid (bcd) mRNA is pre-localized at the anterior of the oocyte. After fertilization, this RNA is translated to produce a Bcd protein gradient that determines anterior cell fates [1, 2]. Recent analysis of bcd mRNA during late stages of oogenesis led to a model for steady-state localization of bcd by continual active transport [3]. Here, we elucidate the path and mechanism of sustained bcd mRNA transport by direct observation of bcd RNA particle translocation in living oocytes. However, this mechanism cannot explain maintenance of bcd localization throughout the end of oogenesis, when microtubules disassemble in preparation for embryogenesis [4, 5] or retention of bcd at the anterior in mature oocytes, which can remain dormant, but developmentally competent, for weeks prior to fertilization [6]. Through temporal analysis of bcd RNA particle dynamics, we show that bcd mRNA shifts from continuous active transport to stable actin-dependent anchoring at the end of oogenesis, ensuring the developmental integrity of the oocyte during dormancy. Egg activation triggers release of bcd from the anterior cortex for proper deployment in the fertilized egg, probably through reorganization of the actin cytoskeleton. These findings uncover a surprising parallel between flies and frogs, as cortically tethered Xenopus Vg1mRNA undergoes a similar redistribution during oocyte maturation [7]. Our results highlight a conserved mechanism used by invertebrates and vertebrates to regulate mRNA anchoring and redeployment during the oocyte-to-embryo transition.
Using live cell imaging of mRNA labeled in vivo with GFP or RFP, we previously determined that bcd mRNA accumulates at the anterior cortex primarily late in oogenesis, as the ovarian nurse cells extrude their cytoplasm into the oocyte, a process known as nurse cell dumping [3]. This localization requires a special population of microtubules that are anchored to the anterior cortical actin cytoskeleton. Concurrently, nanos (nos) mRNA accumulates at the oocyte posterior, but unlike bcd, nos localization is microtubule-independent [8]. Fluorescence recovery after photobleaching (FRAP) analysis showed that, at steady state, the population of bcd mRNA at the anterior cortex remains in flux. In contrast, the population of nanos (nos) mRNA localized at the posterior pole of the oocyte is static [3]. To elucidate the mechanistic basis for this difference between bcd and nos, we visualized RNA particles in oocytes expressing GFP-labeled bcd or nos mRNA (bcd*GFP or nos*GFP) [3, 8] using widefield imaging methods that allow increased sensitivity and temporal resolution.
Drosophila oogenesis progresses through 14 morphologically defined stages. Dumping, which transfers bcd and nos to the oocyte, initiates at stage 10b, and is accompanied by vigorous movement of the oocyte cytoplasm (ooplasmic streaming) that mixes the oocyte and incoming nurse cell cytoplasm. Particles of bcd*GFP are highly dynamic during stages 10b-13, even after dumping and ooplasmic streaming have ceased (Figure 1A, 1B, 1Ba–Bf, 1E, 1F; Movie S1; data not shown). In contrast, little movement was detected among nos*GFP particles visualized at the oocyte posterior (Fig 1C, D; Movie S1).
Figure 1
Figure 1
bcd and nos mRNA particle dynamics in late oocytes
To establish whether bcd mRNA particles move in a directed manner, we monitored all particles within representative regions of the anterior or posterior oocyte cortex. Particles were scored as static (no significant detectable movement), jiggling (movement within one particle length in any direction), or directed (sustained runs of more than 1 µm in a single direction) (Fig 1E, F). Among 11 stage 10b-13 oocytes, we observed movement by 85% (n = 283/332) of bcd*GFP particles (Table 1). Nearly 25% of these show directed runs, but this value is likely an underestimate since we were unable to track particles that moved rapidly in or out of the plane of focus. While the majority of particles were detected within 5 µm of the cortex, bcd*GFP particles undergoing directed movement could be detected as far as 20 µm from the anterior.
Table 1
Table 1
Temporal analysis of RNA particle dynamics
Tracking of individual particles showed that 70% travel toward or laterally along the anterior cortex and 30% travel away from the anterior, with average instantaneous velocities of 0.36–2.15 µm/sec (Fig 1E, F). Occasionally, bcd*GFP particles exhibit pauses during runs and, more rarely, direction reversals (Movie S2, S3). In addition, complex trajectories were observed that suggest translocation of particles between microtubules (Movie S4). In contrast to bcd, only 12% (n = 21/174) of nos*GFP particles at the posterior cortex exhibit any detectable movement and all of this movement is due to jiggling (Table 1). We also detected flow of nos*GFP particles in concert with yolk granules during ooplasmic streaming, consistent with passive movement (Fig 1D; Movie S1).
The directed runs of bcd particles we observed suggest translocation on microtubules. We tested this hypothesis by covisualizing microtubules labeled with tau-GFP together with bcd*GFP in stage 12 oocytes. Our results show individual bcd*GFP particles moving along microtubule tracks, providing direct in vivo evidence for microtubule-based RNA transport (Figure 2A–2C; Movie S5). Previous work is consistent with transport of bcd mRNA in late oocytes being Dynein-dependent [3]. To test this hypothesis directly, we analyzed the effect of hypomorphic mutations in Dynein heavy chain (Dhc), the force generating motor subunit, on bcd particle movement. We found that all movement of bcd*GFP particles is completely eliminated in Dhc mutant oocytes, including both unidirectional and bidirectional motility (Movie S6). These observations are consistent with recent evidence that Dynein can mediate both minus- and plus-end directed transport [911]. The minimal amount of bcd detected at the anterior cortex could be due to residual Dynein function of the hypomorphic alleles used, which is sufficient at least to permit oogenesis (Figure 2D–2F). Alternatively, this RNA may have been localized during mid-oogenesis, when anchoring is microtubule-independent [3], a notion that is supported by the fact that in contrast to bcd*GFP particles at the anterior of wild-type oocytes, residual bcd*GFP particles in Dhc mutant oocytes are stationary. Taken together, these results provide direct visual evidence that bcd is transported to the anterior on microtubules by dynein, and that sustained transport achieves steady-state anterior localization.
Figure 2
Figure 2
bcd RNA particles are transported on microtubules by dynein
Following dumping, oogenesis proceeds very rapidly to produce a mature oocyte in approximately 8 hours. In contrast, mature (stage 14) oocytes are long lived and can be stored for weeks by the female prior to fertilization. These stored oocytes appear to be physiologically stable as they give rise to viable progeny[6]. It remained unclear whether bcd mRNA transport is sustained during stage 14 for long periods by continuous active transport or, alternatively, whether bcd mRNA transitions to a stably anchored state to ensure maintenance over long time periods. We distinguished between these two possibilities by analyzing the dynamics of bcd RNA particles at successive stages of oogenesis after the onset of dumping. We found a marked decrease in the frequency of directed runs as oogenesis progresses in laying females (Table 1). By stage 14, all mobility declines as particles coalesce to form large, stationary foci at the anterior cortex (Figure 3A–3E, Movie S7). These foci remain associated with the anterior cortex through stage 14 and appear to be composed of many individual bcd particles (Figure 3F; Movie S8). Consistent with their ability to support normal development, bcd remains localized in stage 14 oocytes held for several days by unfertilized females (data not shown).bcd RNA particles contain Staufen (Stau), a double-stranded RNA binding protein required for bcd localization from stage 10b onward [3, 12], and this association is maintained when particles coalesce into foci (Figure 3F–3H).nos particles, which are anchored to the actin cytoskeleton, do not change their distribution at the posterior cortex during this time (data not shown). These results are consistent with the acquisition of a stable cortical anchoring mechanism for bcd toward the end of oogenesis.
Figure 3
Figure 3
Accumulation and actin-dependent anchoring of bcd foci at stage 14
Maintenance of bcd at the anterior cortex during stages 10b-13 is both microtubule and actin-dependent. The actin-dependence at these stages is indirect, however, through a requirement for actin in anchoring microtubules to the anterior cortex [3]. To test the role of the cytoskeleton in stable bcd mRNA anchoring at stage 14, we studied the effects of actin and microtubule depolymerizing drugs on bcd mRNA localization at the final stage of oogenesis. Treatment of stage 14 oocytes with the microtubule depolymerizing drug colcemid, either in culture or through ingestion by females, did not affect maintenance of bcd*GFP foci at the anterior (data not shown). In contrast, treatment of cultured stage 14 oocytes with cytochalasin D (cyto D) to disrupt the actin cytoskeleton resulted in dissociation of bcd*GFP foci from the anterior and their dispersion into individual particles (Figure 3I, 3J). We conclude that maintenance of bcd by continual transport is replaced by an actin-based anchoring mechanism by the end of oogenesis, ensuring the developmental integrity of the mature oocyte.
Embryogenesis marks a new phase, in which pre-localized bcd mRNA is translated for the first time to produce the morphogenetic Bcd gradient. Since Bcd protein concentration is linearly related to the amount of bcd mRNA [13], cortically anchored bcd foci could provide concentrated point sources in the fertilized embryo to generate the peak Bcd concentration. However, through visualization of bcd*GFP, we found that bcd undergoes a final transition from its localization in cortical foci at stage 14, becoming dispersed as fine particles throughout the anterior region of the newly fertilized embryo (Figure 4A–4C). These particles retain Stau, suggesting that the RNA particle composition is not grossly altered during this reorganization (Figure 3K–3M). Time lapse imaging showed that bcd*GFP particles do not exhibit directed movement in the embryo but remain fixed in place as though tethered (data not shown).
Figure 4
Figure 4
Redistribution of bcd and cortical actin during the transition from oogenesis to embryogenesis
The transition from oogenesis to embryogenesis requires calcium dependent events, collectively termed "egg activation", that occur inside the female to allow the newly fertilized egg to complete meiosis and initiate embryonic development [1417]. We tested whether egg activation triggers the redistribution of bcd RNA, using stage 14 oocytes activated in vitro. We found that egg activation eliminates or severely reduces foci of bcd*GFP; instead, bcd*GFP particles become dispersed within the anterior region, resembling their distribution in the embryo (Figure 4D–4F). Time lapse imaging showed that foci begin to dissipate within 3–5 minutes of activation (Figure S1A–S1C). The requirement for actin in bcd anchoring at stage 14 prompted us to investigate whether egg activation might affect bcd localization by regulating the actin cytoskeleton. The actin cytoskeleton was monitored using a GFP fusion to the F-actin binding tail of moesin (GFP-moe), which decorates cortical actin without disrupting actin function [18]. When visualized at stage 14, the actin cytoskeleton appears dense and tightly apposed to the oocyte cortex (Figure 4G, 4Ga, 4Gb). In vitro egg activation causes a rapid alteration in actin organization, producing a diffuse actin network that extends into the interior of the egg (Figure 4H, 4Ha, 4Hb). A similar distribution of actin is observed in the newly fertilized embryo (Figure 4I). Moreover, this rearrangement of the actin cytoskeleton can be detected within 3 minutes of activation, corresponding to the onset of bcd dispersal (Figure S1D–S1F).
To confirm that the change in bcd localization is indeed a physiological consequence of egg activation, we examined the effect of mutations that disrupt egg activation on bcd localization in the early embryo. sarah(sra) encodes calcipressin, an inhibitor of the calcium dependent phosphatase calcineurin, and acts early in the egg activation pathway [16, 17]. In contrast to wild-type embryos, bcd*GFP remains cortically associated in sra mutant embryos and forms foci characteristic of stage 14 oocytes (Figure 5A–5C). However, mutations in two other genes required during egg activation, cortex (cort), which encodes a Cdc20 family member [5, 19] and grauzone (grau), which activates cort expression [20], do not prevent redistribution of bcd*GFP (Figure 5D–5F). The timing of bcd dispersal and its dependence on sra, but not cort or grau, lead us to conclude that an early, sarah-dependent event during egg activation triggers redistribution of bcd RNA for embryogenesis. Given Sra's function as a calcium regulator [16, 17], we propose that this event is likely to involve calcium signalling.
Figure 5
Figure 5
Effect of mutations that disrupt egg activation on bcd localization in the early embryo
Together, our studies show that as oogenesis proceeds toward formation of a developmentally competent egg, bcd mRNA transitions from being continuously transported to a stably anchored state. By providing a mechanism to ensure bcd localization for long periods of time in dormant stage 14 oocytes, stable anchoring may play an important role in buffering reproduction against environmental challenges. The delay in stable anchoring of bcd mRNA at the anterior may be caused by the rapid influx of nurse cell cytoplasm occurring at the oocyte anterior during dumping. Alternatively, deployment of this anchoring mechanism may require factors provided to the oocyte during dumping. In either case, continual transport provides a mechanism to retain bcd at the anterior until other developmental processes are complete. Previous work has shown that cortically associated microtubules are replaced by short, randomly oriented cytoplasmic microtubules as the oocyte matures during stages 13–14 [4]. These cytoplasmic microtubules disappear by fertilization [5], when microtubules are nucleated by centrosomes for nuclear divisions and migration [21]. Although we have not been able to determine if and when the specific anterior population of microtubules that transport bcd is disassembled, it is likely that disassembly of oocyte microtubules in preparation for embryogenesis is coordinated with, or required for bcd mRNA anchoring.
A surprising finding is the release of bcd from its anterior cortical tether during egg activation. In the early embryo, bcd mRNA particles contain Stau, indicating release from the anterior cortex does not result from disassembly of bcd/Stau complexes (Figure 3K–M). Rather, our results suggest a mechanism for release of bcd from the cortex by an activation-dependent re-structuring of the actin cytoskeleton. Interestingly, a similar mechanism may be used in Xenopus oocytes for the disassociation of localized Vg1 mRNA from the vegetal cortex. Like bcd, release of Vg1 mRNA is triggered by egg activation and can be mimicked by pharmacological disruption of the actin cytoskeleton [7]. Dispersal of Vg1 mRNA prior to cleavage divisions presumably promotes its inheritance by vegetal cells, where production of Vg1 protein is required for mesoderm induction and embryonic patterning [22]. We propose that, in addition to cell cycle progression, a conserved feature of egg activation is the redeployment of mRNAs localized during oogenesis for their embryonic functions.
Conclusion
Although the molecular determinants of embryonic patterning may differ between invertebrates and vertebrates, mRNA localization plays a fundamental role in the deployment of key patterning molecules in both Drosophila and Xenopus. It is well recognized that while RNA cargos may not be conserved, the machinery for delivering these cargos to the correct destinations is [23, 24]. Our findings suggest that these parallels go even deeper to include mRNA anchoring and redeployment mechanisms that act during the transition from oogenesis to embryogenesis to ensure proper patterning.
Fly strains
The following mutants and transgenic lines were used: Dhc6-6, Dhc6-12(ref [25]); sraA108, sra687 (ref [16]); cortQW55, cortRH65, grauQQ36, grauRG1 (refs [5, 26]); hsp83-MCP-GFP, hsp83-MCP-RFP, bcd-(ms2)6(ref [3]); nos-(ms2)18(ref [27]), tau-GFP(ref [28]); GFP-Stau(ref [29]); GFP-moe (sGMCA; ref [18]). The bcd-(ms2)6 and nos-(ms2)18 transgenes completely rescue the bcd and nos mutant patterning defects, respectively [3, 27].
Manipulation and confocal imaging of oocytes and embryos
Oocytes were cultured and treated with cytochalasin D (Sigma; 10 µg/ml final concentration) or colcemid (Sigma; final concentration 50 µg/ml) as previously described [3]. For colcemid feeding, fresh females were starved for 2–3 days, then for 4 hours with fed yeast paste supplemented to 50 µg/ml colcemid. In vitro activation of stage 14 oocytes was carried out according to Page and Orr-Weaver [30] with modification: individual stage 14 oocytes from well fed females were dissected in Schneider’s insect culture medium (GIBCO-BRL) and individual egg chambers were transferred to uncoated #1.5 glass bottom culture dishes (MatTek) and covered with a 1 mm2 coverslip cut from a #1.5 glass coverslip (Corning). Schneider’s medium was then removed and replaced by activation buffer [30]. Embryos were collected for 0–2 hour periods on yeasted apple juice agar plates, dechorionated for 1 minute in 50% bleach, then mounted on No. 1 coverslips and covered with Series 95 halocarbon oil. Time-lapse and Z-series imaging were carried out using a Leica TCS SP5 confocal microscope, 63x/1.3 NA objective.
For co-visualization of bcd and Stau, ovaries expressing bcd*RFP and GFP-Stau were dissected into Schneider's medium and isolated egg chambers were imaged directly. Embryos from females expressing bcd*GFP were fixed in 1:5 4% paraformaldehyde/PBS:heptane for 20 minutes, washed with PBS, and vitelline membranes were removed manually. Anti-Stau immunostaining was performed using 1:2000 rabbit anti-Stau (gift of D. St Johnston) and 1:1000 Alexa Fluor 568 goat anti-rabbit (Invitrogen/Molecular Probes). Embryos were mounted in Aqua PolyMount (Polysciences, Inc) and imaged with a Zeiss LSM510 confocal microscope.
Live imaging and analysis of RNA particles
Ovaries from well fed females were dissected in Series 95 halocarbon oil (KMZ Chemicals) onto No. 1 coverslips (Corning) and separated from ovarioles using tungsten needles. Time lapse imaging was performed using a wide-field DeltaVision microscope (Applied Precision, Olympus IX70 microscope, Coolsnap HQ [Roper] camera) with 100x/1.35NA objective. Images were deconvolved using Sedat/Agard algorithms with Applied Precision software [31]. Particle movement was analyzed and tracked semi-manually, using Image-Pro Plus (Leeds) and Metamorph (Universal Imaging Corporation) software packages.
01: Supplemental Figure S1. Temporal correlation between bcd mRNA and cortical actin redistribution during egg activation
(A–C) Confocal Z-series projections of the anterior of oocyte expressing bcd*GFP collected prior to activation in vitro (A), 3.5 minutes after the onset of activation in vitro (B), and 7 minutes after the onset of activation in vitro. (D–F) Single confocal sections of the anterior of an oocyte expressing GFP-moe collected prior to activation (D), 4.5 minutes after the onset of activation in vitro (E), and 6.5 minutes after the onset of activation in vitro (F).
02: Movie S1: bcd and nos mRNA particle dynamics in late oocytes
Time lapse movies of bcd*GFP (left) and nos*GFP (right) in stage 12 oocytes. Images were collected at approximately 3.5 frames per second and 50 frames are displayed at 15 frames per second.
03: Movie S2: Individual bcd RNA particle run with pausing
Trail movie showing a directed movement of a single particle of bcd*GFP at the the anterior of a stage 12/13 oocyte. Images were collected at approximately 3.5 frames per second and 24 frames are displayed at 15 frames per second.
04: Movie S3: Bidirectional movement of a single bcd RNA particle
Single particle of bcd*GFP at the the anterior of a stage 12/13 oocyte showing directional reversal. Images were collected at approximately 3.5 frames per second and 24 frames are displayed at 15 frames per second.
05: Movie S4: Single bcd particle with complex trajectory
Single particle of bcd* GFP showing directed movement at the the anterior of a stage 12/13 oocyte: trail movie (left) and original collection (right). Images were collected at approximately 3.5 frames per second and 42 frames are displayed at 15 frames per second.
06: Movie S5: bcd RNA particles traveling on microtubules
Simultaneous imaging of bcd*GFP and tau-GFP near the anterior cortex of a stage 12 oocyte. Images were collected at approximately 3.5 frames per second and 36 frames are displayed at 25 frames per second.
07: Movie S6: bcd RNA particles are transported by dynein
bcd*GFP particles at the anterior cortex of a stage 13 Dhc6-6/ Dhc6–12 oocyte. Images were collected at approximately 3.5 frames per second and 120 frames are displayed at 25 frames per second.
08: Movie S7: Transition to static anchoring at the end of oogenesis
Time-lapse movies of bcd*GFP in a small region at the anterior of stage 13 (left) and stage 14 (right) oocytes. Both movies are displayed using the same parameters. Images were collected at approximately 3.5 frames per second and 50 frames are displayed at 15 frames per second.
09: Movie S8: Foci of bcd RNA are composed of many small particles
High magnification, three dimensional projection of a Z-series showing a focus of bcd*GFP at the anterior of a stage 14 egg chamber.
Acknowledgements
We thank T. Hays, D. Kiehart, A. Martin, T. Schüpbach, M. Welte, and M. Wolfner for fly stocks, D. St Johnston for fly stocks and anti-Stau antibody, J. Goodhouse for assistance with confocal microscopy, and T. Schüpbach and E. Wieschaus for comments on the manuscript. This work was supported by a grant from the National Institutes of Health (GM067758) to E.R.G. and Senior Fellowship from the Wellcome Trust to I.D.
Footnotes
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