The formation of the Bicoid morphogen gradient requires protein movement from anteriorly localized mRNA

The Bicoid morphogen gradient directs the patterning of cell fates along the anterior-posterior axis of the syncytial Drosophila embryo and serves as a paradigm of morphogen-mediated patterning. The simplest models of gradient formation rely on constant protein synthesis and diffusion from anteriorly localized source mRNA, coupled with uniform protein degradation. However, currently such models cannot account for all known gradient characteristics. Recent work has proposed that bicoid mRNA spatial distribution is sufficient to produce the observed protein gradient, minimizing the role of protein transport. Here, we adapt a novel method of fluorescent in situ hybridization to quantify the global spatio-temporal dynamics of bicoid mRNA particles. We determine that >90% of all bicoid mRNA is continuously present within the anterior 20% of the embryo. bicoid mRNA distribution along the body axis remains nearly unchanged despite dynamic mRNA translocation from the embryo core to the cortex. To evaluate the impact of mRNA distribution on protein gradient dynamics, we provide detailed quantitative measurements of nuclear Bicoid levels during the formation of the protein gradient. We find that gradient establishment begins 45 minutes after fertilization and that the gradient requires about 50 minutes to reach peak levels. In numerical simulations of gradient formation, we find that incorporating the actual bicoid mRNA distribution yields a closer prediction of the observed protein dynamics compared to modeling protein production from a point source at the anterior pole. We conclude that the spatial distribution of bicoid mRNA contributes to, but cannot account for, protein gradient formation, and therefore that protein movement, either active or passive, is required for gradient formation.     

Localization of bicoid mRNA in late oocytes is maintained by continual active transport

Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential to produce the Bicoid protein gradient that patterns the anterior-posterior axis of the embryo. Previous studies have characterized a microtubule-dependent pathway for bicoid mRNA localization during midoogenesis, when bicoid first accumulates at the anterior. We show that the majority of bicoid is actually localized later in oogenesis, when the only known mechanism for mRNA localization is based on passive trapping. Through live imaging of fluorescently tagged endogenous bicoid mRNA, we identify a temporally distinct pathway for bicoid localization in late oocytes that utilizes a specialized subpopulation of anterior microtubules and dynein. The directional movement of bicoid RNA particles within the oocyte observed here is consistent with dynein-mediated transport. Furthermore, our results indicate that association of bicoid with the anterior oocyte cortex is dynamic and support a model for maintenance of bicoid localization by continual active transport on microtubules.     

Bicoid Signal Extraction with a Selection of Parametric and Nonparametric Signal Processing Techniques

The maternal segmentation coordinate gene bicoid plays a significant role during Drosophila embryogenesis. The gradient of Bicoid, the protein encoded by this gene, determines most aspects of head and thorax development. This paper seeks to explore the applicability of a variety of signal processing techniques at extracting bicoid expression signal, and whether these methods can outperform the current model. We evaluate the use of six different powerful and widely-used models representing both parametric and nonparametric signal processing techniques to determine the most efficient method for signal extraction in bicoid. The results are evaluated using both real and simulated data. Our findings show that the Singular Spectrum Analysis technique proposed in this paper outperforms the synthesis diffusion degradation model for filtering the noisy protein profile of bicoid whilst the exponential smoothing technique was found to be the next best alternative followed by the autoregressive integrated moving average.     

In vivo analysis of Drosophila bicoid mRNA localization reveals a novel microtubule-dependent axis specification pathway.

Drosophila bicoid mRNA is synthesized in the nurse cells and transported to the oocyte where microtubules and Exuperantia protein mediate localization to the anterior pole. Fluorescent bicoid mRNA injected into the oocyte displays nonpolar microtubule-dependent transport to the closest cortical surface, and the oocyte microtubule cytoskeleton lacks clear axial asymmetry. Nonetheless, bicoid mRNA injected into the nurse cell cytoplasm, withdrawn, and injected into a second oocyte shows microtubule-dependent transport to the anterior cortex. Nurse cells require microtubules and Exuperantia to support anterior transport of bicoid mRNA, and microtubules are required for bicoid mRNA-Exuperantia particle coassembly. We propose that microtubule-dependent Exuperantia-bicoid mRNA complex formation in the nurse cell cytoplasm allows anterior-specific transport on a grossly nonpolar oocyte microtubule network.     

Localization of nanos RNA controls embryonic polarity.

Anterior-posterior polarity of the Drosophila embryo is initiated during oogenesis through differential maternal RNA localization. The RNA of the anterior morphogen bicoid is localized to the anterior pole of the embryo, where bicoid protein controls head and thorax development. The RNA of the posterior morphogen nanos is localized to the posterior pole, where nanos protein is required for abdomen formation. Here we show that the nanos 3' untranslated region, like that of the bicoid RNA, is sufficient for RNA localization. We have used the bicoid RNA localization signal to mislocalize nanos, producing embryos with two sources of nanos protein. Such embryos form two abdomens with mirror image symmetry. Embryos with nanos RNA localized only to the anterior have greater nanos gene activity than embryos with nanos RNA localized posteriorly. We propose a role for RNA localization in regulating nanos activity.     

Sequence of swallow, a gene required for the localization of bicoid message in Drosophila eggs.

We report the sequence of the Drosophila maternal effect gene swallow, one of the genes whose product is required for the localization of bicoid message during Drosophila oogenesis. The inferred swallow protein contains a domain that is predicted to be an amphipathic alpha-helix similar to those implicated in protein:protein associations in other systems. Another part of the predicted protein appears to be a diverged RNA-binding motif. We discuss these structural features in light of the function of the swallow protein in the bicoid message localization process.     

Swallowing dynein: a missing link in RNA localization?

Localization of bicoid messenger RNA to the anterior cortex of the developing oocyte is essential for correct anterior-posterior patterning of the Drosophila embryo. It now seems that the Swallow protein functions as an adaptor, bridging bicoid mRNA to dynein, a molecular motor that would transport the complex anteriorly along microtubules.     

Extreme divergence of a homeotic gene: the bicoid case

Evolutionary developmental genetics (evo-devo) reveals that the plasticity of development is so important that every developmental biology project should carefully take this point into consideration. The example of bicoid, the first discovered morphogen, illustrates how an essential gene can change its function during evolution. The search for bicoid homologues showed that this gene is surprisingly specific to flies (cyclorraphan diptera) and absent in other insects. In fact, recent studies demonstrate that bicoid is a very derived Hox3 homeotic gene. During insect evolution, the ancestral Hox3 gene lost its homeotic function and acquired new roles in oocytes and embryonic annexes. Then, in the lineage leading to modern flies, a duplication of this new gene, followed by functional divergence, led to the formation of bicoid and zerknüllt. Both genes are located within the Drosophila Hox complex; however, they have no homeotic function. Thanks to the power of Drosophila genetics, it is possible to suggest that torso and hunchback may constitute the insect primitive anterior organizer. The bicoid evolutionary history reveals several fundamental mechanisms of the evolution of developmental genes, such as changes of gene regulation, modifications of protein sequences and gene duplication. It also shows the need for studying a wider range of model organisms before generalisations can be made from data obtained with one particular species.     

Miranda couples oskar mRNA/Staufen complexes to the bicoid mRNA localization pathway

The double-stranded RNA binding protein Staufen is required for the microtubule-dependent localization of bicoid and oskar mRNAs to opposite poles of the Drosophila oocyte and also mediates the actin-dependent localization of prospero mRNA during the asymmetric neuroblast divisions. The posterior localization of oskar mRNA requires Staufen RNA binding domain 2, whereas prospero mRNA localization mediated the binding of Miranda to RNA binding domain 5, suggesting that different Staufen domains couple mRNAs to distinct localization pathways. Here, we show that the expression of Miranda during mid-oogenesis targets Staufen/oskar mRNA complexes to the anterior of the oocyte, resulting in bicaudal embryos that develop an abdomen and pole cells instead of the head and thorax. Anterior Miranda localization requires microtubules, rather than actin, and depends on the function of Exuperantia and Swallow, indicating that Miranda links Staufen/oskar mRNA complexes to the bicoid mRNA localization pathway. Since Miranda is expressed in late oocytes and bicoid mRNA localization requires the Miranda-binding domain of Staufen, Miranda may play a redundant role in the final step of bicoid mRNA localization. Our results demonstrate that different Staufen-interacting proteins couple Staufen/mRNA complexes to distinct localization pathways and reveal that Miranda mediates both actin- and microtubule-dependent mRNA localization.     

Staufen, a gene required to localize maternal RNAs in the Drosophila egg.

The posterior group gene staufen is required both for the localization of maternal determinants to the posterior pole of the Drosophila egg and for bicoid RNA to localize correctly to the anterior pole. We report the cloning and sequencing of staufen and show that staufen protein is one of the first molecules to localize to the posterior pole of the oocyte, perhaps in association with oskar RNA. Once localized, staufen is found in the polar granules and is required to hold other polar granule components at the posterior pole. By the time the egg is laid, staufen protein is also concentrated at the anterior pole, in the same region as bicoid RNA.     

Par-1 regulates bicoid mRNA localisation by phosphorylating Exuperantia

The Ser/Thr kinase Par-1 is required for cell polarisation in diverse organisms such as yeast, worms, flies and mammals. During Drosophila oogenesis, Par-1 is required for several polarisation events, including localisation of the anterior determinant bicoid. To elucidate the molecular pathways triggered by Par-1, we have performed a genome-wide, high-throughput screen for Par-1 targets. Among the targets identified in this screen was Exuperantia (Exu), a mediator of bicoid mRNA localisation. We show that Exu is a phosphoprotein whose phosphorylation is dependent on Par-1 in vitro and in vivo. We identify two motifs in Exu that are phosphorylated by Par-1, and show that their mutation abolishes bicoid mRNA localisation during mid-oogenesis. Interestingly, exu mutants in which Exu phosphorylation is specifically affected can to some extent recover from these bicoid mRNA localisation defects during late oogenesis. These results demonstrate that Par-1 establishes polarity in the oocyte by activating a mediator of bicoid mRNA localisation. Furthermore, our analysis reveals two phases of Exu-dependent bicoid mRNA localisation: an early phase that is strictly dependent on Exu phosphorylation and a late phase that is less phosphorylation dependent.     

水稻bicoid反义基因转基因植株胚胎发育的形态变化

Bicoid是调控果蝇胚胎极性及以后分节发生过程的重要基因,这一基因编码的蛋白是一种转录调控因子,对一组早期胚胎发育过程中的多齿基因的表达具有调控作用,并被称为形态发生剂。在以前的工作中,从水稻cDNA文库中分离了一个同果蝇bicoid有相当同源性的克隆-Rb24基因（EMBL登录号：AJ2771380）。为进一步了解它在水稻中功能,将Rb的反义基因片段通过农杆菌的介导转入水稻中。通过Gus活性检测和PCR鉴定所获的抗性植株为转基因植株,转基因植株一个很明显的变化就是大约有50%的种子是败育的。通过石蜡切片观察了转基因水稻幼胚的发育情况,结果表明细胞的组织的分化在胚的发育过程中被阻断了。因此Rb基因同控制水稻胚的发育有关。     

Persistence of Hunchback in the terminal region of the Drosophila blastoderm embryo impairs anterior development

Anterior terminal development is controlled by several zygotic genes that are positively regulated at the anterior pole of Drosophila blastoderm embryos by the anterior (bicoid) and the terminal (torso) maternal determinants. Most Bicoid target genes, however, are first expressed at syncitial blastoderm as anterior caps, which retract from the anterior pole upon activation of Torso. To better understand the interaction between Bicoid and Torso, a derivative of the Gal4/UAS system was used to selectively express the best characterised Bicoid target gene, hunchback, at the anterior pole when its expression should be repressed by Torso. Persistence of hunchback at the pole mimics most of the torso phenotype and leads to repression at early stages of a labral (cap'n'collar) and two foregut (wingless and hedgehog) determinants that are positively controlled by bicoid and torso. These results uncovered an antagonism between hunchback and bicoid at the anterior pole, whereas the two genes are known to act in concert for most anterior segmented development. They suggest that the repression of hunchback by torso is required to prevent this antagonism and to promote anterior terminal development, depending mostly on bicoid activity.