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

ABSTRACT: 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.

SUBMITTER: Little SC

PROVIDER: S-EPMC3046954 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe concentration gradient of Bicoid protein which determines the developmental pathways in early Drosophila embryo is the best characterized morphogen gradient at the molecular level. Because different developmental fates can be elicited by different concentrations of Bicoid, it is important to probe the limits of this specification by analyzing intrinsic fluctuations of the Bicoid gradient arising from small molecular number. Stochastic simulations can be applied to further the understanding of the dynamics of Bicoid morphogen gradient formation at the molecular number level, and determine the source of the nucleus-to-nucleus expression variation (noise) observed in the Bicoid gradient.ResultsWe compared quantitative observations of Bicoid levels in immunostained Drosophila embryos with a spatially extended Master Equation model which represents diffusion, decay, and anterior synthesis. We show that the intrinsic noise of an autonomous reaction-diffusion gradient is Poisson distributed. We demonstrate how experimental noise can be identified in the logarithm domain from single embryo analysis, and then separated from intrinsic noise in the normalized variance domain of an ensemble statistical analysis. We show how measurement sensitivity affects our observations, and how small amounts of rescaling noise can perturb the noise strength (Fano factor) observed. We demonstrate that the biological noise level in data can serve as a physical constraint for restricting the model's parameter space, and for predicting the Bicoid molecular number and variation range. An estimate based on a low variance ensemble of embryos suggests that the steady-state Bicoid molecular number in a nucleus should be larger than 300 in the middle of the embryo, and hence the gradient should extend to the posterior end of the embryo, beyond the previously assumed background limit. We exhibit the predicted molecular number gradient together with measurement effects, and make a comparison between conditions of higher and lower variance respectively.ConclusionQuantitative comparison of Master Equation simulations with immunostained data enabled us to determine narrow ranges for key biophysical parameters, which for this system can be independently validated. Intrinsic noise is clearly detectable as well, although the staining process introduces certain limits in resolution.

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

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