Quantitative morphogen analysis of periodic ruga patterning

Lead Research Organisation: King's College London
Department Name: Craniofacial Dev Orthodon and Microbiol

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Title 'An astonishingly intricate architecture': Visual Music of the Brain and Mind 
Description Terry Trickett, inspired by a series of discussions with me, the award holder, about the research done under the award, created a digital video and musical score and performed and spoke at a number of international venues, on the role, nature and elaboration of spatial patterns. 
Type Of Art Artwork 
Year Produced 2017 
Impact Performances at a number of new media events, e.g. "Conciousness Reframed!" in China. 
 
Description We have developed ways of analysing patterns of gene expression to uncover and understand the way in which cells release and respond to chemicals during embryonic development to organise themselves into the correct structures in the body. We have used a combination of mathematics and experiments on living pieces of tissue to establish the "logic circuits" that predict how the organisation responds to changes in chemical levels. This knowledge will help understand how birth defects happen and provide a foundation for regenerating organised structures for repairing defects, injuries or diseased tissues in the future. We have also advanced the technical mathematical tools for this type of work.
Exploitation Route The methods add to our quantitative understanding of how systems of morphogens work together to create structure in developing (and potentially regenerating) tissues in the body
Sectors Education

Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Description This project served to train the postdoc employed on it and other members of the research group in new areas of image analysis, systems biology and mathematics. It has also led to a number of public outreach events on the understanding of pattern formation in biology and the legacy of Alan Turing. It has also inspired professional artist and performer, Terry Trickett, to a number of new works and a panel discussion at the 2018 Electronic Visual Arts conference in London. It was the basis of consultation for an educational YouTube video on patterning (https://www.youtube.com/watch?v=alH3yc6tX98) which has been watched over 500,000 times and for a small section of a BBC Brian Cox programme on this topic.
First Year Of Impact 2017
Sector Education,Culture, Heritage, Museums and Collections
Impact Types Cultural

Societal

 
Description Project Grant - Epithelial bending
Amount £435,467 (GBP)
Funding ID BB/L002965/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2014 
End 02/2017
 
Description Jaeger Network Inference collaboration 
Organisation Centre for Genomic Regulation (CRG)
Country Spain 
Sector Academic/University 
PI Contribution We identified the mouse palate epithelium as a Turing morphogen patterning system with iterative gene expression regulation and proposed a project to infer the underlying gene network using the partner's methodology.
Collaborator Contribution Dr. Johannes Jaeger developed a method of inferring transcriptional network structure from dense gene expression analysis by in situ measurement. He helped construct a proposal to apply this method for the first time to a mammalian tissue, namely the oral palate epithelium that we had studied
Impact Grant proposal to the BBSRC funded from June 2012 for three years. Has led to significant coverage in the media on our research on animal patterning
Start Year 2011
 
Description Article in Daily Telegraph print edition 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I was asked to comment on a recently published article on the origin of the stripes on tabby cats in relation to so-called Turing Reaction-Diffusion patterning (the "Turing" being Alan Turing who published only one but a very influential paper on biological patterning and self-organisation). The journalist published a short article in the paper edition of the Daily Telegraph quoting me by name.
Year(s) Of Engagement Activity 2021
 
Description Career presentation for Inspiring Futures (careers teacher organisation) hosted at Wellcome Trust 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact I made a presentation and participated in workshop discussions with a group of secondary school teachers involved in giving career advice
Year(s) Of Engagement Activity 2015
 
Description Consult with Brian Cox & BBC producers for "Human Universe" series 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Brian Cox presented a section on how Turing mechanisms self-organise shapes and patterns in biology. I spoke extensively with one of the producers and also corresponded directly with Prof. Cox himself, including on the specific wording of the script for that segment.
Year(s) Of Engagement Activity 2014
 
Description Helped design YouTube video on how animals get their spots 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Consulted extensively and provided illustrative material for YouTube series MinuteEarth
Year(s) Of Engagement Activity 2016
URL https://www.youtube.com/watch?v=alH3yc6tX98
 
Description Presentation at Electronic Visualisation and the Arts 2018 conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Some 50 attendees were present in a plenary session of the Electronic Visualisation and the Arts 2018 (EVA2018) conference, a group of artists and computer scientists using computers to generate 2D and 3D fine art and members of the general public interested in this type of art. The session "Turing's Genius: Defining an Apt Microcosm" consisted of a talk by me on the intellectual background of Alan Turing's interest in spatial pattern and commentaries by three visual artists in this area.
Year(s) Of Engagement Activity 2018
URL https://www.scienceopen.com/document?vid=6bca6e6f-9363-465e-8e2b-6f92138f3d50
 
Description Quanta Magazine (Jennifer Ouillette) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Interview was written up covering our work on Turing patterning mechanisms and published on the Quanta magazine website. I do not have access to web traffic information.
Year(s) Of Engagement Activity 2013
URL https://www.quantamagazine.org/20130325-biologists-home-in-on-turing-patterns/
 
Description School visits (Careers), London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Visits to several different secondary and primary schools in classes and smaller and larger groups describing the science we do and the career pathways in science

influenced several school students to pursue science-based A-levels and degree options
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2017
 
Description Science Live/European Researchers Night at Natural History Museum 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact More than 3,000 members of the public attended the event as a whole, an open evening at the Natural History Museum, with about 800 scientists including NHM staff and invited others, including myself. I was at the "Science Bar" available to members of the public wanting to meet or question a real scientist about any issue relating to science. Each year of this activity, I spoke to between 10 and 20 members of the public in detail over the course of the evening.

Talked at length with a range of members of the public many of whom were thirsty for science knowledge having somehow missed out at school. One individual said that she had learned more talking to me that evening than in the whole of her school career!
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description Web Report (Kat Arney, mosaicscience.com) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Interview was written up in web article on this site, sponsored by the Wellcome Trust. I don't have data on reads.
Year(s) Of Engagement Activity 2014
URL http://mosaicscience.com/story/how-zebra-got-its-stripes-alan-turing