Quantitative morphogen analysis of periodic ruga patterning

Following the Human Genome Project, we know the sum total of genes available to create and maintain the human body. What is still unknown is which genes are active where and when and how they all operate correctly to do their jobs. The integration of that kind of information into a large circuit diagram or computer programme-like description is referred to as a systems biology approach (by analogy to systems analysis in fields like chemical factory design). This is very difficult to do because huge amounts of data are involved, the places where genes operate have very complicated anatomical structures and the computational methods for putting the data together are still being developed. A particularly neglected aspect of this problem is how genes in different cells switch one another off at a distance via secreted chemicals (morphogens) so that in the embryo during development in the womb the right structures are made in the right places. So far, this has been analysed primarily in fruit flies because they are a much simpler system than mammals and their very early development is particularly simple. This project proposal is to study gene regulation in what we have discovered to be a similarly simple part of mammalian anatomy: the transverse ridges, or "rugae", in the roof of the mouth. Humans have four, mice have eight. Each ruga starts as a stripe of expression of one particular gene and each stripe is alike and parallel. Their characteristic spacing and sequential appearance constitute a one-dimensional "periodic" (i.e. repeating) patterning problem. To solve this patterning problem, we will analyze gene expression and morphogen action in the mouse palate in great detail, focusing on four morphogens that we have already identified as important in making these stripes. This involves staining for specific gene products (RNAs) and using engineered mouse strains whose cells light up fluorescently exactly when and where a particular gene is switched on. Digital imaging will allow these gene signals to be quantified accurately. When put together the data - if sufficiently detailed and quantitative - can be interpreted by so-called "reverse engineering" computational algorithms to work out which genes control which other genes and morphogens (including features such as time-lags, amplifiers and logic switches) so that we understand the circuit diagram and programme that makes development of correct anatomy possible. This simple system then serves as a paradigm for a general understanding of this kind of process. Ultimately, this provides not only rich insight into biological processes but also a route towards tweaking these circuits to enhance repair and regeneration for medical ends.

Periodic (i.e. repeated, iterative) patterns offer special advantages for quantitative analysis and a systems approach to gene regulation in intact tissue because they are readily identifiable and perturbations are obvious. They are also extremely important in biology in general and vertebrate anatomy in particular, including not only hairs, feathers and stripes but also tracheal rings, airway branches and ocular dominance columns. This project is to establish a new experimental system in which classical morphogen concepts can be combined with a more systems-based approach to address the spatial regulation of ensembles of genes. This system, the rugae of the mouse palate, has the unique merits in a mammalian experimental species, of extreme simplicity - a one-dimensional pattern - as well as periodicity (iterative elements) and progressive appearance. We have found that the stepwise time-evolution of the pattern, transverse stripes of Shh gene expression, can be observed ex vivo in the absence of growth. We have also found that FGF and Shh act as an activator-inhibitor pair in a classical Reaction-Diffusion Turing-type morphogen mechanism. To understand the underlying gene circuitry, we will quantify outputs of the FGF pathway in normal patterning and under FGF and inhibitor treatments using quantitative in situ hybridisation and an existing Shh-Gli mouse reporter line to determine cross-regulation. Regulation by the Shh pathway will then be analysed likewise, with in situ hybridisation and an FGF-Erk reporter line that we will construct. Finally, Wise/Sostdc1 mutants and canonical Wnt reporters implicate BMP and/or non-canonical Wnt signalling in rugal patterning. We will determine their respective roles using antibody assays and inhibitors in our explant system. The data gathered will be collated and used for established unbiased reverse-engineering computational algorithms for network structure inference following successful paradigms established in Drosophila.

This proposal is for fundamental basic research whose impact is, nonetheless, potentially great. The paradigm for this kind of impact is the studies done on growth factor morphogens in the late 1980s and early 1990s by the applicant, among others. Those studies helped embed the idea (by no means obvious at the time) that directed differentiation of embryonic cells was possible and specifically showed that the key pathways were those of the protein growth factors, which act like the (previously theoretical) morphogens. A direct line can be drawn from that work to adult and embryonic stem cell biotechnology that is the basis for a broad range of actual and potential cellular therapeutics of today. Most notable at present are two current clinical trials for use of embryonically derived cells: one for repair of spinal cord injury (Geron, Inc.) and the other for repair of retinal damage in Stargardt's Disease/Macular Degeneration (Advanced Cell Technology, Inc.).
The studies in this proposal aim have the potential to put directed differentiation, which is currently highly "empirical" (i.e., haphazard), on a rational basis. Potential beneficiaries therefore include
- Biotechnology industries, especially those developing cellular therapeutics who will see how a systems analysis of differentiation of tissue can lead to a predictive set of tools.
- Public health services, who may benefit from the development of rational regenerative medicine approaches (whether or not they come through a commercial route)
The general public will, of course, benefit secondarily from advances in the public and private sectors towards better cellular therapeutics and control of growth factor networks for regenerative medicine.
As part of the project we are generating a mouse line that will be useful for analysis of FGF-ERK signalling in vivo. This may well be of utility and impact for model studies in both industry and health service research.

There is a second sense in which the general public may benefit and that is from a deeper understanding and appreciation of this type of biology. In particular, as Prof. Lewis Wolpert's successful public career has demonstrated, there is a place for a highly mechanistic understanding of biology in banishing quasi-vitalistic notions about the human body and the natural world. The applicant is making strong efforts to communicate the science to the public, via talks in schools and contacts with the media to this end.