Bilateral NSF/BIO-BBSRC: The design logic of Hedgehog-based pattern formation

A key challenge in biology is to understand how cells communicate and respond to one another. This is particularly important in developing embryos where cell communication is responsible for organizing tissues and creating the patterns of cell types that are the template for the formation of functioning organs. Over the last few decades rapid progress has been made in identifying the genes and so-called 'signaling pathways' involved in tissue development. However we have relatively little knowledge of the design logic and dynamics of these pathways, and usually limited ability to perturb or control these pathways in a predictable way. How do these pathways work, and what capabilities do they provide? And, how can we predict their response to perturbations or use them to control cellular behaviors? To achieve a more fundamental understanding of these issues requires a shift in approach from a qualitative molecular view to a quantitative systems analysis. Gaining insight is necessary in order to understand, for instance, how precision and reproducibility of developmental patterning is achieved and ultimately in order to understand how to control these processes when they go wrong or to engineer new tissues.

In this proposal we aim to understand the design principles that produce the dynamics and gene responses of the Hedgehog pathway. This pathway provides an experimentally tractable example of a developmentally important signaling pathway involved in diverse developmental processes and pathologies. However, although we know a lot about the proteins, interactions and feedback loops in the pathway we have little understanding of why the pathway has this architecture, how it behaves dynamically in an individual cell, and how its dynamics ensure proper tissue development. A key limitation has been the lack of direct readout and control of pathway activity at the level of individual cells, and the ability to link these data to tissue level assays. To address this deficiency we plan to combine the expertise of our labs and use a combination of quantitative single-cell and developmental biology approaches in well-defined cellular model systems. The Elowitz lab has expertise in developing and analyzing single cell quantitative data from a range of biological systems; whereas the Briscoe lab has experience and reagents to analyse Hedgehog signaling in neural tissue, where it is responsible for generating different neuronal subtypes. The project will develop reagents and methods that provide a new quantitative understanding of the Hedgehog pathway. Obtaining this systems level of view of the pathway will provide insight into how signals are communicated in developing tissues and reveal what features of the Shh pathway support this. These design principles are likely to be relevant for understanding not only Hedgehog but other signaling pathways involved in diverse developmental processes. More generally, introducing insights from quantitative and system biology to developmental and stem cell biology will advance the field and help underpin its future use in drug discovery, preclinical models of disease, and ultimately clinical applications.

A central challenge in biology is to understand the design logic and dynamic behaviour of cellular pathways. This issue is particularly important in development, where we have a great deal of information about components and molecular interactions, but relatively little quantitative knowledge. The Hedgehog (Shh) pathway provides an experimentally tractable example of this problem. It is involved in diverse developmental processes and pathologies. Nevertheless, we have little understanding of how the dynamics of the pathway are generated or how these control output. A key limitation has been the lack of direct, dynamic analysis and manipulation of signaling at the level of individual cells. Our proposal brings together two groups with recognized and complementary expertise in quantitative single-cell and developmental biology approaches. We will take advantage of recent advances in our groups to perform a new, multiscale, systems level analysis of the design logic of Hedgehog driven pattern formation. For this we have embraced several new technologies: quantitative time-lapse image analysis, CRISPR-mediated genome engineering and orthogonal gene expression control to manipulate the SHH pathway circuit in precise and facile ways. We have developed a "naïve' cell culture system to simplify and analyze specific cell- and system-level dynamic behaviors of the pathway. Finally, we have introduced new methods for ES cell (ESC) differentiation that enable the analysis of developmental mechanisms closely resembling those occurring in vivo in an accessible system amenable to high resolution quantitative analysis. Using these we will ask: How are dynamic Shh signaling gradients produced and transduced to form pattern? How do architectural features of the Shh pathway affect signaling dynamics and patterning behavior? What aspects of the design of a signaling pathway determine the precision and fidelity of the cellular response and pattern formation?

Our project will produce a number of key impacts:

- A broadly useful platform for quantitative analysis of signaling dynamics in individual cells. We will establish a platform and approach to design, construct and analyse pattern formation circuits in the laboratory. The development of tissues and the regeneration of damaged tissues require the long-range spatial organization of differentiated cells and our proposal will provide basic understanding for the mechanisms responsible. As such our proposal is basic research and the immediate impact will relate to scientific and knowledge advancement and the development of skills, capacity and capability. In the longer term, this research has the potential to impact in areas of wealth and health. Beneficiaries beyond academia therefore are the commercial private sector and the wider public.
- Determination of key design principles for the Shh morphogen system. By combining cutting edge cellular and genomic tools with image analysis and modeling, we will be able to move from a molecular view of pathways to a systems-level principles-based view. As a consequence, this project will enable a new approach to tissue engineering and regenerative medicine and deliver increased capacity and capability in these strategically important areas through the provision of training and the further development of methodologies and tools.
- Tools and reagents. We are developing new capabilities in quantitative time-lapse analysis of the engineered genetic circuits at the level of individual cells. These imaging and software tools will be of broad functionality and will be beneficial to a broad range of applications in the cell biology and tissue engineering fields. The reagents and methods also have a wide range of applications that will be of general interest.
- International collaboration. The project involves a close collaboration between UK and US groups and will strength scientific and technical exchange benefiting the research capacity of both organizations.
- Impact on the commercial sector. We anticipate potential benefits in biotechnology industries, who can benefit by recruiting highly skilled and experienced scientists trained through this project. The themes of this proposal - post-genomic research, developmental and stem cell biology, and quantitative biology - are economically important fields and are expected to undergo a large expansion as genomic data, stem cells and tissue engineering are exploited and commercial applications begin to rely on this knowledge. Thus, trained researchers with experience in appropriate fields of research are necessary and in increasing demand. Specialised genetic and stem cell research is expanding at very high rate and requires highly trained personnel to support this. Personnel with skills in functional genomics, stem cell biology and quantitative methods are likely to play a key role in the post-genomics era.
- Biomedical technology development and therapeutic strategies. In the longer term, the project outcomes might help progress in research areas such as development, cancer therapy and stem cell research. The pathway under consideration is involved in diverse pathologies and is a major target of drug development, despite the poor understanding we currently have of its fundamental design principles. For this reason, ultimately, the general public may thus benefit from our fundamental contribution to the understanding of this system.