Controlling gene expression in time and space

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An inducible expression system that allows temporal and dosage control of genes can be used to study how gene expression gradients influence embryonic development.

The importance of gene dosage is well established, especially in developmental processes such as body patterning and limb growth, which rely on the exquisite, coordinated control of the activity of multiple genes. Many such genes act along gradients in which gene activity peaks in cells from certain embryonic regions and then tapers off in more distally situated cells as mRNAs and proteins diffuse outward. A cell's position within a gradient is often reflected in the extent of the cell's response to the gradient factor, but this isn't always the case. For example, in Drosophila larvae, the cells responsible for wing formation—the wing imaginal disc—grow in response to the morphogen Decapentaplegic (DPP), but even though DPP levels form a sharp gradient, cell growth is even throughout the disc, seemingly independent of DPP levels.

Until recently, nobody really knew why. "There were various different explanations that people suggested, and there wasn't really very good support for any of them," explains Kenneth Irvine of Rutgers University. "It was sort of a dirty little secret in the field—we'd all just say that DPP patterns influence growth, but we sort of glossed over the fact that we really didn't know how this worked!" Hoping to solve that riddle, Irvine and graduate student Dragana Ragulja developed a gene control system that would allow them to finely manipulate the timing and extent of DPP response at a level that was not previously possible.

They generated transgenes for a constitutively activated form of the DPP receptor with an upstream activating element that responds to the binding of a transactivator fused to a progesterone receptor domain, expressed from a second transgene. In the presence of an activator compound—the progesterone analog RU486—the activated receptor is rapidly produced, simulating DPP activation. According to Irvine, timing is everything in developmental studies. "Most signaling pathways have some kind of feedback," he says, "and so responses are dampened over time. And technically, because of the way these genetic experiments are done, people have only looked at long-term responses, where long-term is like two days after you turn on the pathway." This system, on the other hand, allows rapid and dosage-dependent gene activation, allowing studies with far shorter time scales. Working with chimeric animals—with genetically modified cells positioned at different locations within the wing disc—Irvine and Ragulja were able to manipulate DPP-response gradients and immediately observe the impact on cell growth in different regions. The data suggested that the essential factor for cell growth is not the concentration of DPP, but rather the slope of the gradient, with growth resulting where neighboring cells show markedly different levels of DPP activity.

Irvine's group is now applying the same system to other gradient-dependent molecules, but Irvine adds that there has also been considerable interest from other groups looking to achieve equally sensitive modulation for their own genes of interest. "There's a number of different systems where it's nice to add this element of temporal control," he says, "and this is a pretty simple way to do it."

Michael Eisenstein

References

Rogulja, D. & Irvine, K.D. Regulation of cell proliferation by a morphogen gradient. Cell 123, 449–461 (2005). | Article | PubMed |