The role of Bicoid cooperative binding in the patterning of sharp borders in Drosophila melanogaster

In Drosophila embryonic development, the Bicoid (Bcd) protein establishes positional information of downstream developmental genes like hunchback (hb), which has a strong anterior expression and a sharp on-off boundary in the mid-embryo. The role of Bcd cooperative binding in the positioning of the Hb pattern has been previously demonstrated. However, there are discrepancies in the reported results about the role of this mechanism in the sharp Hb border. Here, we determined the Hill coefficient (n(H)) required for Bcd to generate the sharp border of Hb in wild-type (WT) embryos. We found that an n(H) of approximately 6.3 (s.d. 1.4) and 10.8 (s.d. 4.0) is required to account for Hb sharpness at early and late cycle 14A, respectively. Additional mechanisms are possibly required because the high n(H) is likely unachievable for Bcd binding to the hb promoter. To test this idea, we determined the n(H) required to pattern the Hb profile of 15 embryos expressing an hb(14F) allele that is defective in self-activation and found n(H) to be 3.0 (s.d. 1.0). This result indicates that in WT embryos, the hb self-activation is important for Hb sharpness. Corroborating our results, we also found a progressive increase in the required value of n(H) spanning from 4.0 to 9.2 by determining this coefficient from averaged profiles of eight temporal classes at cycle 14A (T1 to T8). Our results indicate that there is a transition in the mechanisms responsible for the sharp Hb border during cycle 14A: in early stages of this cycle, Bcd cooperative binding is primarily responsible for Hb sharpness; in late cycle 14A, hb self-activation becomes the dominant mechanism.     

Model for the robust establishment of precise proportions in the early Drosophila embryo

During embryonic development, a spatial pattern is formed in which proportions are established precisely. As an early pattern formation step in Drosophila embryos, an anterior-posterior gradient of Bicoid (Bcd) induces hunchback (hb) expression (Nature 337 (1989) 138; Nature 332 (1988) 281). In contrast to the Bcd gradient, the Hb profile includes information about the scale of the embryo. Furthermore, the resulting hb expression pattern shows a much lower embryo-to-embryo variability than the Bcd gradient (Nature 415 (2002) 798). An additional graded posterior repressing activity could theoretically account for the observed scaling. However, we show that such a model cannot produce the observed precision in the Hb boundary, such that a fundamentally different mechanism must be at work. We describe and simulate a model that can account for the observed precise generation of the scaled Hb profile in a highly robust manner. The proposed mechanism includes Staufen (Stau), an RNA binding protein that appears essential to precision scaling (Nature 415 (2002) 798). In the model, Stau is released from both ends of the embryo and relocalizes hb RNA by increasing its mobility. This leads to an effective transport of hb away from the respective Stau sources. The balance between these opposing effects then gives rise to scaling and precision. Considering the biological importance of robust precision scaling and the simplicity of the model, the same principle may be employed more often during development.     

Enhancer sequences influence the role of the amino-terminal domain of bicoid in transcription

Bicoid (Bcd) is a Drosophila melanogaster morphogenetic gradient that controls embryonic patterning by activating target gene expression in a concentration-dependent manner. In this study we describe experiments to determine how different enhancers respond to Bcd distinctively, focusing on two natural Bcd-responsive enhancer elements, hunchback (hb) and knirps (kni). Our results show that, on the hb enhancer element, the amino-terminal domain of Bcd (residues 1 to 91) plays primarily an inhibitory role, whereas on the kni enhancer element this same Bcd domain plays a positive role at low protein concentrations. We further demonstrate that while the amino-terminal domain is largely dispensable for cooperative binding to the hb enhancer element, it is preferentially required for cooperative binding to the kni enhancer element. Alteration of the arrangement of Bcd binding sites in the kni enhancer element reduces the role of the amino-terminal domain in cooperative DNA binding but increases the effectiveness of the self-inhibitory function. In addition, elimination of symmetric pairs of Bcd binding sites in the kni enhancer element reduces both DNA binding and activation by Bcd. We propose that the amino-terminal domain of Bcd is an enhancer-specific switch that contributes to the protein's ability to activate different target genes in distinct manners.     

Shaping a Morphogen Gradient for Positional Precision

Morphogen gradients, which provide positional information to cells in a developing tissue, could in principle adopt any nonuniform profile. To our knowledge, how the profile of a morphogen gradient affects positional precision has not been well studied experimentally. Here, we compare the positional precision provided by the Drosophila morphogenetic protein Bicoid (Bcd) in wild-type (wt) embryos with embryos lacking an interacting cofactor. The Bcd gradient in the latter case exhibits decreased positional precision around mid-embryo compared with its wt counterpart. The domain boundary of Hunchback (Hb), a target activated by Bcd, becomes more variable in mutant embryos. By considering embryo-to-embryo, internal, and measurement fluctuations, we dissect mathematically the relevant sources of fluctuations that contribute to the error in positional information. Using this approach, we show that the defect in Hb boundary positioning in mutant embryos is directly reflective of an altered Bcd gradient profile with increasing flatness toward mid-embryo. Furthermore, we find that noise in the Bcd input signal is dominated by internal fluctuations but, due to time and spatial averaging, the spatial precision of the Hb boundary is primarily affected by embryo-to-embryo variations. Our results demonstrate that the positional information provided by the wt Bcd gradient profile is highly precise and necessary for patterning precision.     

Modeling the precision and robustness of Hunchback border during Drosophila embryonic development

During anterior-posterior axis specification in the Drosophila embryo, the Hunchback (Hb) protein forms a sharp boundary at the mid-point of the embryo with great positional precision. While Bicoid (Bcd) is a known upstream regulator for hb expression, there is evidence to suggest that Hb effectively filters out “noisy” data received from varied Bcd gradients. We use mathematical models to explore simple regulatory networks which filter out such noise to produce a precise Hb boundary. We find that in addition to Bcd and Hb, at least one freely evolving protein is necessary. An automated search yields a number of examples of three-protein networks exhibiting the desired precision. In all such networks, Hb diffuses much slower than the third protein. In addition, the action of Hb on the third protein is the opposite of the action of the third protein on hb (i.e. if Hb activates the third protein, then the third protein inhibits hb expression, and vice versa). Most of the discovered systems satisfy the known biological properties, that Bcd activates hb, and that Hb activates its own expression. We find that all network topologies satisfying these constraints arise among the networks exhibiting the desired precision. Investigating the dynamics of these networks, we find that under a general class of non-uniform initial conditions, Bcd can be eliminated from the system and the spatiotemporal evolution of these two proteins alone is sufficient to recapture the dynamics. We hypothesize that Bcd is needed only to spatially disturb the gradient of the third protein, and then becomes unnecessary in the further evolution of the Hb border. This provides a possible explanation as to why the Hb dynamics are robust under perturbations of the Bcd gradient. Under this hypothesis, other proteins would be able to assume the role of Bcd in our simulations (possibly in the case of evolutionary divergences or a redundancy in the process), with the only constraint that they act to positively regulate hb.     

A Two-Dimensional Simulation Model of the Bicoid Gradient in Drosophila

Background

Bicoid (Bcd) is a Drosophila morphogenetic protein responsible for patterning the anterior structures in embryos. Recent experimental studies have revealed important insights into the behavior of this morphogen gradient, making it necessary to develop a model that can recapitulate the biological features of the system, including its dynamic and scaling properties.

Methodology/Principal Findings

We present a biologically realistic 2-D model of the dynamics of the Bcd gradient in Drosophila embryos. This model is based on equilibrium binding of Bcd molecules to non-specific, low affinity DNA sites throughout the Drosophila genome. It considers both the diffusion media within which the Bcd gradient is formed and the dynamic and other relevant properties of bcd mRNA from which Bcd protein is produced. Our model recapitulates key features of the Bcd protein gradient observed experimentally, including its scaling properties and the stability of its nuclear concentrations during development. Our simulation model also allows us to evaluate the effects of other biological activities on Bcd gradient formation, including the dynamic redistribution of bcd mRNA in early embryos. Our simulation results suggest that, in our model, Bcd protein diffusion is important for the formation of an exponential gradient in embryos.

Conclusions/Significance

The 2-D model described in this report is a simple and versatile simulation procedure, providing a quantitative evaluation of the Bcd gradient system. Our results suggest an important role of Bcd binding to non-specific, low-affinity DNA sites in proper formation of the Bcd gradient in our model. They demonstrate that highly complex biological systems can be effectively modeled with relatively few parameters.     

Mutual repression enhances the steepness and precision of gene expression boundaries

Embryonic development is driven by spatial patterns of gene expression that determine the fate of each cell in the embryo. While gene expression is often highly erratic, embryonic development is usually exceedingly precise. In particular, gene expression boundaries are robust not only against intra-embryonic fluctuations such as noise in gene expression and protein diffusion, but also against embryo-to-embryo variations in the morphogen gradients, which provide positional information to the differentiating cells. How development is robust against intra- and inter-embryonic variations is not understood. A common motif in the gene regulation networks that control embryonic development is mutual repression between pairs of genes. To assess the role of mutual repression in the robust formation of gene expression patterns, we have performed large-scale stochastic simulations of a minimal model of two mutually repressing gap genes in Drosophila, hunchback (hb) and knirps (kni). Our model includes not only mutual repression between hb and kni, but also the stochastic and cooperative activation of hb by the anterior morphogen Bicoid (Bcd) and of kni by the posterior morphogen Caudal (Cad), as well as the diffusion of Hb and Kni between neighboring nuclei. Our analysis reveals that mutual repression can markedly increase the steepness and precision of the gap gene expression boundaries. In contrast to other mechanisms such as spatial averaging and cooperative gene activation, mutual repression thus allows for gene-expression boundaries that are both steep and precise. Moreover, mutual repression dramatically enhances their robustness against embryo-to-embryo variations in the morphogen levels. Finally, our simulations reveal that diffusion of the gap proteins plays a critical role not only in reducing the width of the gap gene expression boundaries via the mechanism of spatial averaging, but also in repairing patterning errors that could arise because of the bistability induced by mutual repression.