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.
Highlights
Spatial patterning during embryonic development is orchestrated through concentration gradients of regulatory molecules known as morphogens (Turing 1952; Wolpert 1969)
To gain some preliminary insight into what to expect when imaging with the Lattice light-sheet microscopy (LLSM), we assumed an isotropic distribution of molecules and a 400nm-thick excitation sheet and estimated a range on the order of 103–101 BCD molecules per imaging plane in a single nucleus
Our initial observation of the low-affinity nature of BCD binding to chromatin is partially congruent with the classical view of BCD as a concentration-dependent morphogen
Summary
Spatial patterning during embryonic development is orchestrated through concentration gradients of regulatory molecules known as morphogens (Turing 1952; Wolpert 1969). The ability of BCD and other morphogens to activate different target genes at locations along concentration gradients is classically thought to arise from variations in the number and strength of cognate DNA-binding sites within different enhancers (Burz et al 1998; Lebrecht et al 2005; Xu et al 2015), with sharp expression domain boundaries set through cooperative binding (Ephrussi and St Johnston 2004; Lebrecht et al 2005) Under this model, enhancers with lower-affinity binding sites would be activated only at high concentrations, while enhancers with higher-affinity sites would be activated at lower concentrations. Since the question of how BCD molecules can find their targets in these short times requires dynamic measurements, genomic assays and biochemical approaches that provide static snapshots have proven inadequate to resolve outstanding mechanistic questions about morphogen activity
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