Trick switch

Recent work by the group, which has been submitted for publication, addressed how the muscle-differentiation binary switch actually works by identifying targets of the Prdm1 transcription factor.

Initially, they took a candidate-gene approach - an educated guess - looking for expression of downstream, fibre type-specific elements.

They showed that the slow forms of the genes tested are not expressed in the absence of Prdm1, and the fast ones are expressed inappropriately with no Prdm1 expressed. Then, most recently, they used ChIP assays to do a genome-wide analysis.

This approach was made possible by a new promoter chip for zebrafish, generated by Fiona Wardle and Jim Smith at the Gurdon Institute at the University of Cambridge, in collaboration with Hazel Smith at the Whitehead Institute in Boston.

"This approach has proved really powerful and quite revealing, and the bottom line is that Prdm1 works in two ways," Ingham says. First, Prdm1, which is a known transcriptional repressor, promotes slow-fibre differentiation by repressing some other transcriptional repressor. So, the slow-specific genes are being switched on indirectly.

Secondly, and at the same time, Prdm1 is directly repressing all of the fast-specific genes. "So, it has to actively repress that alternative differentiation pathway to have its effect. We thought a binary switch would be simple to work out - you either turn on A or B, but it is seemingly not the case - it is far more complex and interesting than that."

The ultimate goal of Ingham's current project is to understand the molecular complexities of these muscle development pathways. "We want to generate a whole muscle transcriptome - a regulatory transcriptional network that underlies the generation of these different muscle fibre types right from the initial multipotent progenitor cells through differentiation.

"We work with a very nice system for doing this, because literally you go from an uncommitted myoblast to committed cell in really what is just one step -exposing specific cells to Hh switches on Prdm1 and then everything else happens."

Ingham's main motivation remains to understand how the Hedgehog signalling system directs multipotent cells down specific pathways of differentiation: the zebrafish muscle system is a very good model for this. And despite the leaps the field has made in recent years, there is clearly much more to know, even just about muscle fibre types in zebrafish.

"We are also actively pursuing how different levels of Hh can induce these different cell types. We want to understand more about the position of fibre-specific progenitor cells in the embryo, their origin, and why those particular cells are responding to high levels of Hh.

"Is it the position in which they lie on the embryo or is it something about the time over which they remain exposed to the signal?" This issue of morphogen concentration compared to position in the embryo remains a key question in developmental cell biology.