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Dense Bicoid hubs accentuate binding along the morphogen gradient.

ABSTRACT: Morphogen gradients direct the spatial patterning of developing embryos; however, the mechanisms by which these gradients are interpreted remain elusive. Here we used lattice light-sheet microscopy to perform in vivo single-molecule imaging in early Drosophila melanogaster embryos of the transcription factor Bicoid that forms a gradient and initiates patterning along the anteroposterior axis. In contrast to canonical models, we observed that Bicoid binds to DNA with a rapid off rate throughout the embryo such that its average occupancy at target loci is on-rate-dependent. We further observed Bicoid forming transient "hubs" of locally high density that facilitate binding as factor levels drop, including in the posterior, where we observed Bicoid binding despite vanishingly low protein levels. We propose that localized modulation of transcription factor on rates via clustering provides a general mechanism to facilitate binding to low-affinity targets and that this may be a prevalent feature of other developmental transcription factors.

SUBMITTER: Mir M

PROVIDER: S-EPMC5666676 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Dense Bicoid hubs accentuate binding along the morphogen gradient.

Mir Mustafa M Reimer Armando A Haines Jenna E JE Li Xiao-Yong XY Stadler Michael M Garcia Hernan H Eisen Michael B MB Darzacq Xavier X

Genes & development 20170901 17

Morphogen gradients direct the spatial patterning of developing embryos; however, the mechanisms by which these gradients are interpreted remain elusive. Here we used lattice light-sheet microscopy to perform in vivo single-molecule imaging in early <i>Drosophila melanogaster</i> embryos of the transcription factor Bicoid that forms a gradient and initiates patterning along the anteroposterior axis. In contrast to canonical models, we observed that Bicoid binds to DNA with a rapid off rate throu ...[more]

PMID: 28982761

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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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Dense Bicoid hubs accentuate binding along the morphogen gradient.

Publications

Dense Bicoid hubs accentuate binding along the morphogen gradient.

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