A gradient of bicoid protein in Drosophila embryos

The maternal gene bicoid (bcd) organizes anterior development in Drosophila. Its mRNA is localized at the anterior tip of the oocyte and early embryo. Antibodies raised against bcd fusion proteins recognize a 55-57 kd doublet band in Western blots of extracts of 0-4 hr old embryos. This protein is absent or reduced in embryonic extracts of nine of the 11 bcd alleles. The protein is concentrated in the nuclei of cleavage stage embryos. It cannot be detected in oocytes, indicating temporal control of bcd mRNA translation. The bcd protein is distributed in an exponential concentration gradient with a maximum at the anterior tip, reaching background levels in the posterior third of the embryo. The gradient is probably generated by diffusion from the local mRNA source and dispersed degradation.     

Spatial regulation of the gap gene giant during Drosophila development

We describe the regulated expression of the segmentation gene giant (gt) during early embryogenesis. The gt protein is expressed in two broad gradients in precellular embryos, one in anterior regions and the other in posterior regions. Double immunolocalization studies show that the gt patterns overlap with protein gradients specified by the gap genes hunchback (hb) and knirps (kni). Analysis of all known gap mutants, as well as mutations that disrupt each of the maternal organizing centers, indicate that maternal factors are responsible for initiating gt expression, while gap genes participate in the subsequent refinement of the pattern. The maternal morphogen bicoid (bcd) initiates the anterior gt pattern, while nanos (nos) plays a role in the posterior pattern. Gene dosage studies indicate that different thresholds of the bcd gradient might trigger hb and gt expression, resulting in overlapping but noncoincident patterns of expression. We also present evidence that different concentrations of hb protein are instructive in defining the limits of kni and gt expression within the presumptive abdomen. These results suggest that gt is a bona fide gap gene, which acts with hb, Krüppel and kni to initiate striped patterns of gene expression in the early embryo.     

The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner

The bicoid (bcd) protein in a Drosophila embryo is derived from an anteriorly localized mRNA and comes to be distributed in an exponential concentration gradient along the anteroposterior axis. To determine whether the levels of bcd protein are directly related to certain cell fates, we manipulated the density and distribution of bcd mRNA by genetic means, measured the resultant alterations in height and shape of the bcd protein gradient, and correlated the gradient with the fate map of the respective embryos. Increases or decreases in bcd protein levels in a given region of the embryo cause a corresponding posterior or anterior shift of anterior anlagen in the embryo. The bcd protein thus has the properties of a morphogen that autonomously determines positions in the anterior half of the embryo.     

Bicoid - morphogen function revisited

Bicoid (Bcd) functions as a morphogen during Drosophila development. Accordingly, bcd mRNA is maternally localized to the anterior pole of the embryo, and Bcd forms an anterior/posterior gradient, which functions in a concentration dependent fashion. Thus, nuclei receiving identical amounts of Bcd should express the same target genes. However, we found that ectopic, uniform expression of Bcd causes anterior gene expression in the posterior with mirror image polarity, indicating that one or several additional factors must provide positional information. Recently, we have shown that one of these factors is Capicua (Cic), a ubiquitous maternal repressor that is down-regulated at the embryonic termini by maternal Torso, a key component of the maternal terminal system. Cic acts on Bcd dependent enhancer elements by repression and thereby controls the posterior limit of Bcd target gene expression. Based on these new findings, we propose that spatial control of gene expression in the anterior region of the embryo is not solely the result of Bcd morphogen action. Rather, it relies on a "morphogenic network" that integrates the terminal system and Bcd activities, providing both polarity and spatial information to the prospective head region of the developing embryo.     

Gamma-tubulin37C and gamma-tubulin ring complex protein 75 are essential for bicoid RNA localization during drosophila oogenesis

bicoid (bcd) RNA localization requires the activity of exuperantia and swallow at sequential steps of oogenesis and is microtubule dependent. In a genetic screen, we identified two novel genes essential for bcd RNA localization. They encode maternal gamma-Tubulin37C (gammaTub37C) and gamma-tubulin ring complex protein 75 (Dgrip75), both of which are gamma-tubulin ring complex components. Mutations in these genes specifically affect bcd RNA localization, whereas other microtubule-dependent processes during oogenesis are not impaired. This provides direct evidence that a subset of microtubules organized by the gamma-tubulin ring complex is essential for localization of bcd RNA. At stage 10b, we find gammaTub37C and Dgrip75 anteriorly concentrated and propose the formation of a microtubule-organizing center at the anterior pole of the oocyte.     

Changes in bicoid mRNA anchoring highlight conserved mechanisms during the oocyte-to-embryo transition

Intracellular mRNA localization directs protein synthesis to particular subcellular domains to establish embryonic polarity in a variety of organisms. In Drosophila, bicoid (bcd) mRNA is prelocalized at the oocyte anterior. After fertilization, translation of this RNA produces a Bcd protein gradient that determines anterior cell fates [1] and [2]. Analysis of bcd mRNA during late stages of oogenesis suggested a model for steady-state bcd localization by continual active transport [3]. However, this mechanism cannot explain maintenance of bcd localization throughout the end of oogenesis, when microtubules disassemble in preparation for embryogenesis [4] and [5], or retention of bcd at the anterior in mature oocytes, which can remain dormant for weeks before fertilization [6]. Here, we elucidate the path and mechanism of sustained bcd mRNA transport by direct observation of bcd RNA particle translocation in living oocytes. We show that bcd mRNA shifts from continuous active transport to stable actin-dependent anchoring at the end of oogenesis. Egg activation triggers bcd release from the anterior cortex for proper deployment in the embryo, probably through reorganization of the actin cytoskeleton. These findings uncover a surprising parallel between flies and frogs, as cortically tethered Xenopus Vg1 mRNA undergoes a similar redistribution during oocyte maturation [7]. Our results thus highlight a conserved mechanism for regulating mRNA anchoring and redeployment during the oocyte-to-embryo transition.     

RNA-RNA interaction is required for the formation of specific bicoid mRNA 3'' UTR-STAUFEN ribonucleoprotein particles.

The formation of the anterior pattern of the Drosophila embryo is dependent on the localization of the mRNA of the morphogen Bicoid (bcd) to the anterior pole of the egg cell. Staufen protein (STAU) is required in a late step of the localization to anchor the bcd mRNA in the anterior cytoplasm. We have shown previously that endogenous STAU associates specifically with injected bcd mRNA 3'-untranslated region (UTR), resulting in the formation of characteristic RNA-protein particles that are transported along microtubules of the mitotic spindles in a directed manner. The regions recognized by STAU in this in vivo assay are predicted to form three stem-loop structures involving large double-stranded stretches. Here, we show that the STAU interaction requires a double-stranded conformation of the stems within the RNA localization signal. In addition, base pairing between two single-stranded loops plays a major role in particle formation. This loop-loop interaction is intermolecular, not intramolecular; thus dimers or multimers of the RNA localization signal must be associated with STAU in these particles. The bcd mRNA 3' UTR can also dimerize in vitro in the absence of STAU. Thus, in addition to RNA-protein interactions, RNA-RNA interaction might be involved in the formation of ribonucleoprotein particles for transport and localization.     

Graded requirement for the zygotic terminal gene, tailless, in the brain and tail region of the Drosophila embryo

We have used hypomorphic and null tailless (tll) alleles to carry out a detailed analysis of the effects of the lack of tll gene activity on anterior and posterior regions of the embryo. The arrangement of tll alleles into a continuous series clarifies the relationship between the anterior and posterior functions of the tll gene and indicates that there is a graded sensitivity of anterior and posterior structures to a decrease in tll gene activity. With the deletion of both anterior and posterior pattern domains in tll null embryos, there is a poleward expansion of the remaining pattern. Using anti-horseradish peroxidase staining, we show that the formation of the embryonic brain requires tll. A phenotypic and genetic study of other pattern mutants places the tll gene within the hierarchy of maternal and zygotic genes required for the formation of the normal body pattern. Analysis of mutants doubly deficient in tll and maternal terminal genes is consistent with the idea that these genes act together in a common pathway to establish the domains at opposite ends of the embryo. We propose that tll establishes anterior and posterior subdomains (acron and tail regions, respectively) within the larger pattern regions affected by the maternal terminal genes.     

Components Acting in Localization of Bicoid mRNA Are Conserved among Drosophila Species

Substantial insights into basic strategies for embryonic body patterning have been obtained from genetic analyses of Drosophila melanogaster. This knowledge has been used in evolutionary comparisons to ask if genes and functions are conserved. To begin to ask how highly conserved are the mechanisms of mRNA localization, a process crucial to Drosophila body patterning, we have focused on the localization of bcd mRNA to the anterior pole of the embryo. Here we consider two components involved in that process: the exuperantia (exu) gene, required for an early step in localization; and the cis-acting signal that directs bcd mRNA localization. First, we use the cloned D. melanogaster exu gene to identify the exu genes from Drosophila virilis and Drosophila pseudoobscura and to isolate them for comparisons at the structural and functional levels. Surprisingly, D. pseudoobscura has two closely related exu genes, while D. melanogaster and D. virilis have only one each. When expressed in D. melanogaster ovaries, the D. virilis exu gene and one of the D. pseudoobscura exu genes can substitute for the endogenous exu gene in supporting localization of bcd mRNA, demonstrating that function is conserved. Second, we reevaluate the ability of the D. pseudoobscura bcd mRNA localization signal to function in D. melanogaster. In contrast to a previous report, we find that function is retained. Thus, among these Drosophila species there is substantial conservation of components acting in mRNA localization, and presumably the mechanisms underlying this process.     

Scaling of the Bicoid morphogen gradient by a volume-dependent production rate

An important feature of development is the formation of patterns that are proportional to the overall size of the embryo. But how such proportionality, or scaling, is achieved mechanistically remains poorly understood. Furthermore, it is currently unclear whether organisms utilize similar or distinct mechanisms to achieve scaling within a species and between species. Here we investigate within-species scaling mechanisms for anterior-posterior (A-P) patterning in Drosophila melanogaster, focusing specifically on the properties of the Bicoid (Bcd) morphogen gradient. Using embryos from lines artificially selected for large and small egg volume, we show that large embryos have higher nuclear Bcd concentrations in the anterior than small embryos. This anterior difference leads to scaling properties of the Bcd gradient profiles: in broad regions of the large and small embryos along the A-P axis, normalizing their positions to embryo length reduces the differences in both the nuclear Bcd concentrations and Bcd-encoded positional information. We further trace the origin of Bcd gradient scaling by showing directly that large embryos have more maternally deposited bcd mRNA than small embryos. Our results suggest a simple model for how within-species Bcd gradient scaling can be achieved. In this model, the Bcd production rate, which is dependent on the total number of bcd mRNA molecules in the anterior, is scaled with embryo volume.