Timothy Thomas Weil

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

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.

Distinguishing direct from indirect roles for bicoid mRNA localization factors.

Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential for patterning the anteroposterior body axis in the early embryo. bicoid mRNA localizes in a complex multistep process involving transacting factors, molecular motors and cytoskeletal components that remodel extensively during the lifetime of the mRNA. Genetic requirements for several localization factors, including Swallow and Staufen, are well established, but the precise roles of these factors and their relationship to bicoid mRNA transport particles remains unresolved. Here we use live cell imaging, super-resolution microscopy in fixed cells and immunoelectron microscopy on ultrathin frozen sections to study the distribution of Swallow, Staufen, actin and dynein relative to bicoid mRNA during late oogenesis. We show that Swallow and bicoid mRNA are transported independently and are not colocalized at their final destination. Furthermore, Swallow is not required for bicoid transport. Instead, Swallow localizes to the oocyte plasma membrane, in close proximity to actin filaments, and we present evidence that Swallow functions during the late phase of bicoid localization by regulating the actin cytoskeleton. In contrast, Staufen, dynein and bicoid mRNA form nonmembranous, electron dense particles at the oocyte anterior. Our results exclude a role for Swallow in linking bicoid mRNA to the dynein motor. Instead we propose a model for bicoid mRNA localization in which Swallow is transported independently by dynein and contributes indirectly to bicoid mRNA localization by organizing the cytoskeleton, whereas Staufen plays a direct role in dynein-dependent bicoid mRNA transport.

mRNA localization in the Drosophila germline

Localization and the associated translational control of mRNA is a well established mechanism for segregating cellular protein expression. Drosophila has been instrumental in deciphering the prevailing mechanisms of mRNA localization and regulation. This review will discuss the diverse roles of mRNA localization in the Drosophila germline, the cis-elements and cellular components regulating localization and the superimposition of translational regulatory mechanisms. Despite a history of discovery, there are still many fundamental questions regarding mRNA localization that remain unanswered. Take home messages, outstanding questions and future approaches that will likely lead to resolving these unknowns in the future are summarized at the end.

Subcellular mRNA localisation at a glance

ABSTRACT mRNA localisation coupled to translational regulation provides an important means of dictating when and where proteins function in a variety of model systems. This mechanism is particularly relevant in polarised or migrating cells. Although many of the models for how this is achieved were first proposed over 20 years ago, some of the molecular details are still poorly understood. Nevertheless, advanced imaging, biochemical and computational approaches have started to shed light on the cis-acting localisation signals and trans-acting factors that dictate the final destination of localised transcripts. In this Cell Science at a Glance article and accompanying poster, we provide an overview of mRNA localisation, from transcription to degradation, focusing on the microtubule-dependent active transport and anchoring mechanism, which we will use to explain the general paradigm. However, it is clear that there are diverse ways in which mRNAs become localised and target protein expression, and we highlight some of the similarities and differences between these mechanisms.

A single and rapid calcium wave at egg activation in Drosophila

Activation is an essential process that accompanies fertilisation in all animals and heralds major cellular changes, most notably, resumption of the cell cycle. While activation involves wave-like oscillations in intracellular Ca2+ concentration in mammals, ascidians and polychaete worms and a single Ca2+ peak in fish and frogs, in insects, such as Drosophila, to date, it has not been shown what changes in intracellular Ca2+ levels occur. Here, we utilise ratiometric imaging of Ca2+ indicator dyes and genetically encoded Ca2+ indicator proteins to identify and characterise a single, rapid, transient wave of Ca2+ in the Drosophila egg at activation. Using genetic tools, physical manipulation and pharmacological treatments we demonstrate that the propagation of the Ca2+ wave requires an intact actin cytoskeleton and an increase in intracellular Ca2+ can be uncoupled from egg swelling, but not from progression of the cell cycle. We further show that mechanical pressure alone is not sufficient to initiate a Ca2+ wave. We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca2+ transient. Based on this data we propose the following model for egg activation in Drosophila: exposure to lateral oviduct fluid initiates an increase in intracellular Ca2+ at the egg posterior via osmotic swelling, possibly through mechano-sensitive Ca2+ channels; a single Ca2+ wave then propagates in an actin dependent manner; this Ca2+ wave co-ordinates key developmental events including resumption of the cell cycle and initiation of translation of mRNAs such as bicoid.

Re-examining the role of Drosophila Sas-4 in centrosome assembly using two-colour-3D-SIM FRAP

Centrosomes have many important functions and comprise a ‘mother’ and ‘daughter’ centriole surrounded by pericentriolar material (PCM). The mother centriole recruits and organises the PCM and templates the formation of the daughter centriole. It has been reported that several important Drosophila PCM-organising proteins are recruited to centrioles from the cytosol as part of large cytoplasmic ‘S-CAP’ complexes that contain the centriole protein Sas-4. In a previous paper (Conduit et al., 2014b) we showed that one of these proteins, Cnn, and another key PCM-organising protein, Spd-2, are recruited around the mother centriole before spreading outwards to form a scaffold that supports mitotic PCM assembly; the recruitment of Cnn and Spd-2 is dependent on another S-CAP protein, Asl. We show here, however, that Cnn, Spd-2 and Asl are not recruited to the mother centriole as part of a complex with Sas-4. Thus, PCM recruitment in fly embryos does not appear to require cytosolic S-CAP complexes. DOI: http://dx.doi.org/10.7554/eLife.08483.001

Localized Translation of gurken/TGF-α mRNA during Axis Specification Is Controlled by Access to Orb/CPEB on Processing Bodies

Summary In Drosophila oocytes, gurken/TGF-α mRNA is essential for establishing the future embryonic axes. gurken remains translationally silent during transport from its point of synthesis in nurse cells to its final destination in the oocyte, where it associates with the edge of processing bodies. Here we show that, in nurse cells, gurken is kept translationally silent by the lack of sufficient Orb/CPEB, its translational activator. Processing bodies in nurse cells have a similar protein complement and ultrastructure to those in the oocyte, but they markedly less Orb and do not associate with gurken mRNA. Ectopic expression of Orb in nurse cells at levels similar to the wild-type oocyte dorso-anterior corner at mid-oogenesis is sufficient to cause gurken mRNA to associate with processing bodies and translate prematurely. We propose that controlling the spatial distribution of translational activators is a fundamental mechanism for regulating localized translation.

Post-transcriptional regulation of early embryogenesis.

Gene expression is controlled by diverse mechanisms before, during, and after transcription. Chromatin modification factors as well as transcriptional repressors, silencers, and enhancers all feed into how eukaryotes transcribe RNA in the nucleus. However, there is increasing evidence that post-transcriptional regulation of gene expression is as widespread as transcriptional control if not more so. Studies of specific transcripts in oocytes and embryos are at the core of our mechanistic understanding of many post-transcriptional events. Coupled with genome-wide and large-scale experimental approaches, research is bringing to light how these regulatory events function independently and in concert to regulate protein expression.

Translational control of gurken mRNA in Drosophila development

ABSTRACT Localized mRNA translation is a widespread mechanism for targeting protein synthesis, important for cell fate, motility and pathogenesis. In Drosophila, the spatiotemporal control of gurken/TGF-α mRNA translation is required for establishing the embryonic body axes. A number of recent studies have highlighted key aspects of the mechanism of gurken mRNA translational control at the dorsoanterior corner of the mid-stage oocyte. Orb/CPEB and Wispy/GLD-2 are required for polyadenylation of gurken mRNA, but unlocalized gurken mRNA in the oocyte is not fully polyadenylated.1 At the dorsoanterior corner, Orb and gurken mRNA have been shown to be enriched at the edge of Processing bodies, where translation occurs.2 Over-expression of Orb in the adjacent nurse cells, where gurken mRNA is transcribed, is sufficient to cause mis-expression of Gurken protein.3 In orb mutant egg chambers, reducing the activity of CK2, a Serine/Threonine protein kinase, enhances the ventralized phenotype, consistent with perturbation of gurken translation.4 Here we show that sites phosphorylated by CK2 overlap with active Orb and with Gurken protein expression. Together with our new findings we consolidate the literature into a working model for gurken mRNA translational control and review the role of kinases, cell cycle factors and polyadenylation machinery highlighting a multitude of conserved factors and mechanisms in the Drosophila egg chamber.