Hedgehog and the need for speed

In a New York Times article in 1994, Professor Philip Ingham was quoted as saying he would "resign from science" if landmark findings about the role of Hedgehog genes in vertebrate model organisms did not turn out to be equally important for human development.

Fortunately he is still very much in science and will share his latest findings on this key family of signalling molecules at the Hunter Cell Biology meeting starting today.

In 1993, Ingham's group at the Molecular Embryology Laboratory of the Imperial Cancer Research Fund in Oxford pooled efforts with two groups at Harvard University, resulting in the key Hedgehog findings and three papers in the same issue of Cell.

The work heralded the critical role of Hedgehog genes in vertebrate morphogenesis in three animal models - mouse, fish, and chicken. The findings opened up a whole new era of developmental biology by uncovering one of the key pieces of a developmental puzzle that had been worked on for over 25 years prior - how an organism sets up its body pattern.

Developing embryos have to accomplish just two tasks - they have to grow and they have to set up and orchestrate a plan for their mature body. Sounds easy enough, but to accomplish this, embryonic cells must proliferate extensively to generate sufficient progenitor cells, and they have to instruct these cells where to go and when to differentiate into the ordered arrays of specific cell types characteristic of different organs.

A central challenge in developmental biology, according to Ingham, now deputy director of the Institute of Molecular and Cell Biology (IMCB) in Singapore, is working out exactly how this happens at the cellular and molecular level.

For instance, how does an embryo know where and when to grow a limb or specific digit and how long it should be, or exactly how many neurons are needed to establish complex neural network and where they should be sent?

"Remarkably, all of these processes, even across species, are orchestrated by a limited repertoire of signalling molecules from just a few protein families," Ingham says.

It is this phenomenon that has interested many researchers, including Ingham, for a long time. "Many, many years ago, we started looking for what regulates body patterning during Drosophila melanogaster development," he says. This interest led him to the Hedgehog family of secreted proteins (Hh), which turned out to be one of only a few protein families responsible for all vertebrate and invertebrate embryonic development.

Ingham pioneered genetic analysis of the Hh signalling pathway in Drosophila, identifying its receptor, Patched, and transmembrane transducer, Smoothened. He was also one of the first to use Danio rerio (zebrafish) as a model system for developmental genetics in the late 1980s.

The Hh proteins are the morphogens (literally meaning "structure makers") that many people were looking for at the time. Morphogens are secreted molecules that direct body patterning by setting up a temporally and spatially controlled signal gradient. The Hh family and its role in the developing embryo has been the core of Ingham's research ever since.

Hedgehog in the fast or slow lane

The control of skeletal muscle differentiation, growth and regeneration in the zebrafish embryo is a major focus of Ingham's group's work nowadays. This research is important, core discovery science as well as being relevant to the understanding of human muscle disease. His lab uses a variety of cell and molecular biology approaches, including transgenesis, in vivo imaging, chromatin immunoprecipitation (ChIP) and proteomic analysis.

All of this work is done in the zebrafish vertebrate model, not only because it is a fabulous model for development biology and very pretty, but also because of its high proportion of skeletal muscle and the similarity between fish and mammalian genetic pathways.

"What we found quite early on was that zebrafish embryos develop slow- and fast-twitch muscle fibres pretty rapidly during embryogenesi," he says. "We also showed that fibre type is specified in response to Hh signalling."

Ingham says that at some level there is a binary choice between the two different lineages of slow or fast. "In simplistic terms, the presence of Hh means you get slow fibres and if you don't have it, you get fast muscle. However, different cell populations are singled out within both lineages of fibre type in response to different levels of Hh. In other words, first Hh sets up the lineages, but then it sets up distinctions within each lineage.

"This temporal element of a changing competence in the cells to respond to Hh is something that we are very interested in and currently investigating, although it is proving rather difficult to crack."

What his team has made much more progress with is taking that binary switch - the initial decision between slow and fast - and asking how it is interpreted by the cells. "So, what is the mechanism that allows the muscle precursor cells to respond to Hh and what are the downstream consequences?"

In a Nature Genetics paper in 2004, Ingham reported a gene critical for this fibre switch in response to Hh signalling. Called Blimp1 or Prdm1, it encodes a transcription factor that was in fact already well characterised in the haematopoietic system where it is required for B cell maturation into plasma cells.

This work shed new light on the origin of different muscle lineages in response to positional cues in the vertebrate embryo.

Ingham realised then that Prdm1 seemed to be the key to all the downstream effects of Hh. "Essentially what happens is that Hh activates transcription of the Prdm1 gene in the progenitors of the slow muscle cells and this activation immediately drives all other aspects of the muscle differentiation process."

---PB--- 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.