Role for dynamic protrusions in epithelial patterning

Lead Research Organisation: University College London
Department Name: MRC Laboratory of Molecular Cell Biology

Abstract

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.

Technical Summary

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.

Planned Impact

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 health and 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.
 
Description We have discovered a way that cell division is coupled to tissue patterning. This helps to ensure that patterning happens at a discrete time and goes to fruition.

We have also shown how mechanical pulling on a signalling molecule can helps cells to signal to one another during patterning.

We have showed that Myosin dependent tissue tension affects cell-cell signalling via long range protrusions
Exploitation Route Now the work is published we expect it to inspire research in other systems.
Ginger Hunter no plans to pursue this as a group leader at Clarkson Uni
Sectors Healthcare

 
Description Spoke about tissue patterning on Radio 4 as part of a series on Kiplings stories to an audience of several million heard by millions on prime time Radio 4: http://www.bbc.co.uk/programmes/b01pthk9
First Year Of Impact 2010
Sector Education
Impact Types Cultural

 
Description AMS careers team - team science working group
Geographic Reach National 
Policy Influence Type Membership of a guidance committee
URL http://www.acmedsci.ac.uk/policy/policy-projects/team-science/
 
Title Drosophila lines 
Description For genetic perturbations in imaging backgrounds for investigating protrusion mediated patterning in the Drosophila notum 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2014 
Provided To Others? Yes  
Impact Sharing within London community to advance research 
 
Description Collaboration with image analysis team studying order 
Organisation University College London
Department Department of Computer Science
Country United Kingdom 
Sector Academic/University 
PI Contribution It has been a longterm collaboration to study biological order. We have 2 joint students working on this
Collaborator Contribution They have been doing the image analysis and developing the mathematical theory
Impact a paper
Start Year 2011
 
Description Collaboration with theorist - Nick Monk 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution Theoretical framework for systems approach
Collaborator Contribution Help with defining the trajectory of signalling during patterning
Impact in progress
Start Year 2014
 
Description Ginger - Giniger 
Organisation National Institutes of Health (NIH)
Country United States 
Sector Public 
PI Contribution NIH Bethesda
Collaborator Contribution The Ginnier lab is hosting Ginger Hunter while she completes this work
Impact Paper in progress
Start Year 2015
 
Description IPLS 
Organisation University College London
Department Institute for the Physics of Living Systems
Country United Kingdom 
Sector Academic/University 
PI Contribution I have helped to set up IPLS. We have hired to Theorists who have joined UCL to work with researchers.
Collaborator Contribution I sit on the committee and helped to set it up - building upon UCL Systems Biology
Impact none yet
Start Year 2014
 
Description MDCK tissue stretch 
Organisation University College London
Department London Centre for Nanotechnology
Country United Kingdom 
Sector Academic/University 
PI Contribution Stretching tissue with Guillaume Charras
Collaborator Contribution We are working together to test the impact of stretch on tissue mechanics and cell division
Impact several publications and devices
Start Year 2013
 
Description Mammalian epithelial monolayers - Guillaume Charras 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Understanding of actin dynamics in epithelia
Collaborator Contribution Help with research direction
Impact in progress
Start Year 2011
 
Description Perrimon lab 
Organisation Harvard University
Department Harvard Medical School
Country United States 
Sector Academic/University 
PI Contribution We have combined work to study patterning in the fly notum.
Collaborator Contribution Li He in the Perrimon lab has contributed a fly line to study signalling in real time, and a cell culture assay to show that tension affects Notch-Delta signalling
Impact a paper
Start Year 2014
 
Description Tissue stretching 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Stretching to determine how physical force impacts cell behaviour in the fly Yanlan Mao's lab
Collaborator Contribution She has been looking at the impact of tissue stretch on N signalling and cell division
Impact none
Start Year 2016
 
Description CRICK Lates 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I gave a CRICK late talk about "patterning" as part of the "pattern" exhibition at the FCI.
Year(s) Of Engagement Activity 2018
 
Description EMBO workshop on morphogenesis in Chile 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Spoke on evolution of morphogenesis
Year(s) Of Engagement Activity 2019
 
Description Imperial college - maths and biology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Gave a talk to mathematicians and physicists.
Year(s) Of Engagement Activity 2016
 
Description Just so science 
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 Just so science was a radio series about animals.
I spoke on - how the leopard got its spots to an audience of ~4 million.
Year(s) Of Engagement Activity 2014
URL http://www.bbc.co.uk/programmes/b01pthk9
 
Description Lecture to Phd students at Pasteur as part of international phd programme 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Gave seminar on evolution
Year(s) Of Engagement Activity 2020
 
Description Meeting on cytonemes 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I sent post-doc Ginger Hunter to speak at this meeting in my place since I couldnt attend.
This was a field we helped to establish, hence the importance.
Year(s) Of Engagement Activity 2015
 
Description PhD student seminar at IGC portugal on morphogenesis 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact taught students
Year(s) Of Engagement Activity 2020
 
Description Plenary talk at US fly meeting San Diego 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I gave a plenary talk on cell division in flies
Year(s) Of Engagement Activity 2018
 
Description Profile in JCB 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact lots of nice feedback

Post-doc applicants
Year(s) Of Engagement Activity 2014
 
Description helped run workshop in India (by remote) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Organised and spoke by remote (3 lectures) to international students on programme
Year(s) Of Engagement Activity 2019
URL http://indico.ictp.it/event/8736/