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Role for dynamic protrusions in epithelial patterning

Reference	BB/J008532/1
Principal Investigator / Supervisor	Professor Buzz Baum
Co-Investigators / Co-Supervisors	Dr Alexandre Benedetto, Dr Guillaume Charras
Institution	University College London
Department	MRC Laboratory of Molecular Cell Biology
Funding type	Research
Value (£)	346,706
Status	Completed
Type	Research Grant
Start date	07/01/2013
End date	06/01/2016
Duration	36 months

Abstract

According to the prevailing view of lateral inhibition, the binding of Delta to a cell expressing Notch results in the cleavage of the intracellular portion of Notch followed by translocation to the nucleus, where it down-regulates pro-neural gene expression and Delta transcription. In the notum, this is known to contribute to the emergence of evenly-spaced bristle precursors (pI cells). Importantly, computational models of the process suggest that this simple view of patterning cannot quantitatively explain the spacing of pI cells or the gradual process of pattern refinement observed in vivo. Using the notum as a model system, we recently identified a previously unappreciated role for dynamic filopodia in this process, as pI cell spacing and pattern order were affected when filopodial protrusion and dynamics were inhibited. In addition, a theoretical analysis revealed that the intermittent cell-cell signalling events resulting from protrusion-mediated contacts are likely to play a critical role in robust lateral inhibition-mediated patterning. However, this work lacked a detailed molecular and cellular framework. Here we therefore propose studying the molecular mechanisms that generate basal filopodial dynamics, the changes in filopodial dynamics that follow a change in cell-cell signalling and the role of these filopodia in determining overall tissue pattern order in the notum. More specifically, we will carry out a detailed quantitative analysis of filopodial morphology and dynamics to ask what parameters are good predictors of cell fate. Next, we will identify regulators of filopodial dynamics using an RNAi screen concentrating on actin-associated proteins and downstream effectors of Notch and Delta. Finally, we will examine the robustness of patterning when challenged with global perturbations but also with perturbations applied only to one half of the notum. We expect the result to give a detailed quantitative understanding of the patterning process.

Summary

The back of an adult fly carries a beautiful array of bristles. This has long been used as a simple model to understand pattern formation in biology. When imaged live fly embryos it is clear that this pattern of bristles develops over time through a process of self-organisation, which from messy beginnings yields a well-ordered final state. Remarkably, such patterns can be achieved even when challenged with wide range of perturbations. The possibility to manipulate fly genetics and to image fly development live make the fly thorax (notum) an ideal model with which to explore general principles in this process of biological pattern formation. Fundamental discoveries in this area are likely to have implications for our understanding of processes as diverse as the generation of the organisation in the human inner ear or the gut. In a recent study of this process we discovered that the cells that make up the notum (a flat tissue) have numerous fingerlike protrusions (filopodia) that span several cell diameters. These formed a dense web of cell-cell contacts underneath the tissue that enabled non-neighbouring cells to contact one another and, importantly, to exchange information at a distance. Like most cellular protrusions, these are dynamic, with an average lifetime of less than 10 minutes. Because of this, cells contact different neighbours over time as the pattern of bristles is being established, enabling each cell to ensure that it is correctly positioned with respect to other cells in the vicinity before deciding on its fate. Inhibition of protrusion formation resulted in patterning that was severely disrupted and in which the average separation between bristles was decreased. These data suggest that filopodial protrusions play an important role in sending the signals that regulate bristle patterning. This generated interest across the scientific community because many researchers suspect protrusions may mediate long range signalling in their own systems of study. Here, in order to build on this novel finding, we aim to identify the molecular mechanisms that underpin protrusion formation, dynamics, and protrusion mediated signalling in flies. We will use approaches taken from nanotechnology together with genetic techniques to determine the role of the dynamic filopodia in the generation of a well-ordered and patterned epithelium. First, we will image individual cells in developing flies using high power light microscopy, paying particular attention to the dynamics and morphology of their protrusions. By using markers of signalling together with markers of filopodia, we will be able to see what differentiates cells that become bristles from those that do not by measuring parameters related to filopodial length and duration of contact with other cells. Next, we will search for proteins that regulate the dynamics of filopodial protrusions. To do this, we will reduce the number of candidate proteins and ask if their depletion affects filopodial form and/or dynamics. In this way we expect to identify proteins that are physical components of the filopodia and proteins that regulate their assembly acting downstream of cell-cell signalling events. To find out what these protrusions do and to understand the link between individual filopodia and whole tissue patterning we will disrupt filopodia dynamics in specific tissue regions and ask how bristle spacing changes across the perturbed region of tissue, comparing results with simulations in a computational model of the process. Finally, we will ask if filopodial dynamics contribute to the robustness of this patterning process following mutation or environmental shock. We believe this work will provide us with a better understanding of this new type of patterning that relies on dynamic changes in cell-cell contacts. We anticipate this having broad implications for other patterning processes during development, homeostasis and disease, and for tissue engineering.

Impact Summary

Impact will be ensured through a range of activities (see plan for details). Training: AB will receive continual cross-disciplinary training and mentoring, which will aid his progression towards an independent group leader position. In addition, AB will benefit from generic skills gained on training courses at UCL, through co-authoring papers, grants and reviews and by presenting this work at international meetings. Both BB and GC are actively involved in interdisciplinary training activities at UCL. BB is head of Systems Biology and co-runs the MRes-PhD training programme. GC runs the Systems Biology "quantitative biology" module, and BB runs the introduction to biological complexity module of the CoMPLEX and Systems Biology MRes courses. The project described here will be ideal for introducing students from different backgrounds to interdisciplinary research in the life sciences. Moreover, a large number of students will benefit from involvement in this interdisciplinary systems level research through rotation projects in the 2 labs and through MRes and tutorial activities associated with these programmes. This will include exposure to modelling via the collaboration with the Zaikin lab. Similarly, undergraduates will be exposed to this work through internships and short projects. Finally, by publicising this work, we expect to allow the UK to attract better and brighter students and researchers. Research and technology: Notch signalling and actin dynamics are implicated in neurogenesis and cancer development. Since there are usually studied separately, our approach may yield original insights, which could have far-reaching implications for our ability to understand and treat cancer and neurological defects that result from injury or age-related diseases, where Notch signaling plays roles in neural stem cell proliferation, survival, self-renewal and differentiation. In the long-term, this research is therefore likely to have an impact on lifelong human healthand well being. UK Plc will directly benefit from this high profile research as technological developments will be commercialised through the LCN and will be made available to UK companies working in tissue engineering. Outreach: Previously members of the team have been involved in interactions with the wider community through media appearances, public discussions and through school visits. Through this type of outreach we expect this work to reach a wide audience; giving the public a better understanding of processes that underlie health and disease and an appreciation of the remarkable natural world in which we live. Moreover, through our involvement in HFSP, the EMBO YIP forum, EU and Weizmann-UK networks, we expect this work to reach the global scientific community. To ensure impact we will: i) present the work at high profile conferences that cover the different aspects of the work: tissue engineering development actin and cell biology drosophila genetics ii) expect to publish the main biological findings in 1 or 2 papers in high impact journals. We expect interest from a wide audience based upon our work in Developmental Cell, which was reviewed in Current Biology (Sept 2010), in Dev (Feb 2011), in Science (April 2011), and as a highlight in Nat. Reviews Mol. Cell Biology (Oct 2010). iii) ensure that any novel methods and tools developed during the course of this study are made widely available, subject to patent filing. Published fly stocks developed during the course of this work will be given to Bloomington to distribute. Management Ensuring the smooth running of this project will be the primary responsibility of BB, who will act as line-manager and primary mentor to AB, and the LMCB will serve as AB's primary home. However, to ensure the success of this interdisciplinary collaboration, GC will attend all project discussions and AB will attend all GC and BB lab meetings, and joint cytoskeletal group meetings.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	Synthetic Biology
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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