David Holloway
Fruit flies are models for understanding the genetic regulation involved in specifying the complex body plans of higher animals. The head-to-tail (anterior-posterior) axis of the fly (Drosophila) is established in the first hours of development. Maternally supplied factors form concentration gradients which direct embryonic (zygotic) genes where to be activated to express proteins. These protein patterns specify the positions and cell types of the body's tissues. Recent research has shown, comparing between embryos, that the zygotic gene products are much more precisely positioned than the maternal gradients, indicating an embryonic error reduction mechanism. Within embryos, there is the additional aspect that DNA and mRNA operate at very low copy number, and the associated high relative noise has the potential to strongly affect protein expression patterns. In recent work, we have focused on the noise aspects of positional specification within individual embryos, and what molecular mechanisms confer robustness to the process.
We simulate activation of hunchback (hb), a primary target of the maternal Bicoid (Bcd) protein gradient, which forms an expression pattern dividing the embryo into anterior and posterior halves. We use a master equation approach to simulate the stochastic dynamics of hb regulation, including the binding/unbinding of Bcd molecules at the hb promoter (the portion of DNA regulating hb transcription), hb transcription, subsequent translation to Hb protein, binding/unbinding of Hb at the promoter (self-regulation), and diffusion of the Bcd and Hb proteins. Model parameters were set by deterministically matching large scale pattern features for a series of experimental expression patterns: wild-type (WT) embryos; hb mutants lacking self-regulation; and constructs in which portions of the hb promoter were used to express a reporter gene (lacZ). The model was then solved stochastically to predict the noise output in these different experiments. In subsequent noise measurements we experimentally corroborated several predictions: including that mRNA is noisier than protein and that Hb self-regulation reduces noise.
Results indicate that WT (self-regulatory) Hb output noise is predominantly dependent on the transcription and translation dynamics of its own expression, and is uncorrelated with Bcd fluctuations. In the constructs and mutant, which lack self-regulation, we find that increasing the number and strength of Bcd binding sites (there are 6 in the core hb promoter) provides a rudimentary level of noise reduction. We have recently incorporated a known inhibitor of hb, Kr�ppel (Kr), into the model � preliminary indications are that the Hb-Kr activator-inhibitor dynamics increase precision at the mid-embryo boundary.
The analysis of hb shows common modes of gene regulation (e.g. multiple regulatory sites, self-regulation) that are involved in noise reduction, which can be applied generally to reproducibility and determinacy of spatial patterning in other developmental phenomena.
