Regulation of cell shape by the cytoskeleton during tissue morphogenesis

Lead Research Organisation: MRC Laboratory of Molecular Biology

Abstract

Epithelial sheets of cells are one of the major tissue types in all animals. They also form the basic building blocks of all tubular organs, such as lungs, kidney, vasculature, in both vertebrates such as us humans, but also in invertebrates such as fruit flies. Many severe developmental defects are the result of a failure of tubes forming correctly, such as neural tube closure defects, and furthermore 80% of cancers originate from epithelial tissues. We want to dissect the basic mechanisms of how simple epithelial sheets deform in a highly coordinated manner to turn into complex tissue shapes. We use a process of tube formation in the fruit fly embryo as our main model: the formation of the tubes of the salivary glands. The macroscopic process of this tube formation is conserved and very similar to for instance the early formation of tubes in the development of lungs in mouse or humans. In contrast to vertebrates, fruit flies have the advantage of excellent genetic tools and allow easy imaging and manipulation. We use a combination of genetic methods, live imaging and computational tools to understand how healthy organs form, and his will allow us in the future to better understand the causes of diseases or malformation of tubular epithelia.

Technical Summary

At the start of organ development during embryogenesis most animals consist of a simple polarised epithelial sheet that transforms into complex 3D shapes over time. Correct organ shape is critical for proper organ function. But what determines organ shape? It is the shape and arrangement of cells within an organ that ultimately determines its shape. And cell shape itself is determined by the cytoskeleton, in particular by the actomyosin cortex and microtubule filaments. My lab’s core interest is how cell shape is determined by the cytoskeleton and how neighbouring cells are coordinated with one another to determine organ shape.
Many of the important organ systems in mammals and in invertebrate model systems retain their epithelial nature, and are often tubular in structure. This is true for the intestinal tract, kidney, liver, lung, and vasculature. With defects in tube formation and failure in tube homeostasis leading to severe disease phenotypes including Spina Bifida or Polycystic Kidney Disease, it is clear that understanding the mechanisms that drive faithful organ morphogenesis is key to a better understanding, and eventually treatment, of such defects.
Tubular organs can form through a number of mechanisms, including folding and wrapping of as well as budding from epithelial sheets. My lab focuses on two models of tube morphogenesis: the budding of the salivary glands in the Drosophila embryo and recently we have also begun to use human renal organoids grown from iPSCs as a second model. These represent simple processes of tube formation that are highly amenable to approaches such as live imaging, genetic perturbation and single cell analyses. Using these systems, we address fundamental questions at different scales. Achieving a deep knowledge of tube formation will inform understanding of more complex processes of tissue morphogenesis.
At the molecular level, we analyse the control of cytoskeletal dynamics and architecture, e.g. proteins that control activation or accumulation of for instance actomyosin. The functions of actomyosin and microtubules are coordinated, and thus one research focus is on a particular class of cytoskeletal crosslinkers that can interact with both.
At the cellular level, cell-cell adhesion receptors play an essential role in coordinating cell shape changes between neighbouring cells. Intriguingly, this coordination through adhesion is intimately linked to effects on the cytoskeleton near to cell-cell junctions. Several components of cell-cell junctions are mechano-sensitive and will therefore modulate junctional responses depending on tensile stresses. We analyse different classes of homophilic and heterophilic adhesion receptors and their effects on coordinating cytoskeletal behaviour. We are also using single cell RNAseq approaches to understand how cells are primed for specific morphogenetic routes.
The molecular and cellular mechanisms culminate at the tissue level in exquisitely coordinated behaviours of cells within a tissue. Some key behaviours at the cell and tissue level have been identified, such as apical constriction or cell wedging leading to tissue bending. We use quantitative morphometric methods to identify and quantify cell behaviours across our model tissue, both in 2D within the apical domain of the epithelial cells as well as in 3D, where we developed novel methods to approximate 3D cell shape changes quantitatively.

Publications

10 25 50
 
Description Modelling of 3D tube formation in silico 
Organisation University of British Columbia
Country Canada 
Sector Academic/University 
PI Contribution Prof. James Feng's group at UBC Dept. of Chemical and Biological Engineering and Dept. of Mathematics are interested in using 3D vertex models to model a tube formation process in silico. As and example they have chosen the formation of the salivary glands in the Drosophila embryo as a simple model example. My lab has studied this process fro many years and we have thus teamed up with Prof. Feng to provide the biological data (quantitative morphometrics) to use as input and boundary conditions in their modelling.
Collaborator Contribution Prof. Feng's team undertakes the in silico modelling based on 3D vertex model of epithelial cells.
Impact no outputs yet
Start Year 2019
 
Description Modulation of Rok membrane dissociation rate by Crb/aPKC triggers Rok planar polarisation during morphogenesis 
Organisation University of Toronto
Country Canada 
Sector Academic/University 
PI Contribution My postdoc Dr Clara Sidor has been investigating the molecular mechanism driving planar polarisation of Rok activity during epithelial morphogenesis, using a combination of fly genetics, imaging of live and fixed samples, biochemistry and modelling (in collaboration with Dr Tim Stevens within our division of Cell Biology at the LMB).
Collaborator Contribution Drs Tony Harris and Matt Bailey at the University of Toronto in conjunction with Dr Kenneth E. Prehoda at the University of Oregon have performed in vitro kinase assays using recombinant Rok to illustrate its ability to be phosphorylated by aPKC, and Drs Tony Harris and Matt Bailey have also generated transgenic fly lines allowing expression of non-phosphorylatable versions of Rok in the fly that we are using.
Impact manuscript in preparation
Start Year 2017
 
Description Morphometric analysis of tube formation in 3D 
Organisation University of Cambridge
Department Department of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution We perform the in vivo imaging analysis, genetic analysis and conceptual ideas.
Collaborator Contribution Dr Guy Blanchard has written software allowing segmentation and tracking of cells in time-lapse movies, followed by morphometric analysis to determine strain rates as proxies of forces acting in the tissue. We are using his software and collaborating with him on expanding the analysis software and interpretation of the data.
Impact We are currently writing up a manuscript describing our joint analysis.
Start Year 2014
 
Description Quantitative Analysis of Cell Behaviours during tube formation 
Organisation University of Cambridge
Department Department of Physiology, Development and Neuroscience
Country United Kingdom 
Sector Academic/University 
PI Contribution All lab based research was performed at the LMB, our collaborator has over many years established quantitate computational methods to analyse in vivo data, and for this project we have collaboratively established new aspects of this technology.
Collaborator Contribution software development and implementation
Impact Paper published in 2018: Radially patterned cell behaviours during tube budding from an epithelium. Sanchez-Corrales YE, Blanchard GB, Röper K. Elife. 2018 Jul 17;7. pii: e35717. doi: 10.7554/eLife.35717. PMID: 30015616
Start Year 2016
 
Description The Gas2 family protein Pigs is a microtubule +TIP that affects cytoskeleton organisation 
Organisation University of North Carolina at Chapel Hill
Country United States 
Sector Academic/University 
PI Contribution In depth analysis of the localisation and function of the cytolinker protein Pigs. Dr Gemma Girdler in the lab performed this analysis both in vivo in Drosophila tissues as well as in vitro in true culture.
Collaborator Contribution Dr Stephen Rogers and his postdoc Derek Applewhite had data complimentary to our analysis, in particular mutations of SxIP motifs in the protein, and so we decided to combine our finding for publication and share and exchange expression constructs made.
Impact Girdler, G.C., Applewhite, D.A., Perry, W.M.G., Rogers, S.L., and Röper, K. (2016). The Gas2 family protein Pigs is a microtubule +TIP that affects cytoskeleton organisation. J. Cell Sci. 129: 121-134 (featured as cover picture)
Start Year 2015
 
Description 'Big Biology Day' in Cambridge 
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 Cambridge Big Biology Day is a one-day science festival, hosted by Hills Road Sixth Form College, which is open to all and celebrates the life sciences through hands-on activities, crafts and displays, bringing together a number of biological organisations under one roof.
Year(s) Of Engagement Activity 2019
 
Description Artists in Residence Lunch Event (LMB, Cambridge , UK) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Artists in Residence Lunch: A postdoc from the lab participated and the participants talked about the influence of temperature on diverse aspects of every day lab work in relation to the kitchen and cooking preparation at home.
Year(s) Of Engagement Activity 2016
URL https://www.youtube.com/watch?v=kcsmf1X0n5Y&feature=youtu.be
 
Description Cambridge Science Festival (Cambridge, UK) 
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 Cambridge Science Festival: a postdoc helped running a Microbit session for children interested in programming.
Year(s) Of Engagement Activity 2016
URL http://www.microbit.co.uk
 
Description Hosting of Artist (MRC-LMB, Cambridge, UK) 
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 A local artists, Kate Winter, visited the LMB to sketch scenes of daily lab life. Our lab hosted her for a large part of this.
The sketches are now available to view within he LMB but will be accessible to a wider audience soon.
Year(s) Of Engagement Activity 2016
 
Description LMB Open Day 2017 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This was the second Open day held at the new LMB in Cambridge. As last time, all divisions opened their doors and led tours throughout the day to showcase their research, and inanition many interactive stations were set up in the atrium, hands-on activities for the whole family. My lab ran part of the Division of Cell Biology Tour, showcasing how the fruit fly Drosophila can inform our understanding of how tissues are formed during development, and how this knowledge is crucial to be able to understand what goes wrong is disease phenotypes.
Year(s) Of Engagement Activity 2017
 
Description Lifelab Events Cambridge 
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 Interactive Science stalls in Peterborough Shopping Centre to get general public interested in basic biology
Year(s) Of Engagement Activity 2018
URL https://camlifelab.co.uk/cambridge
 
Description London International Youth Science Forum 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact science talks to student audience over a 2-week residential course in London
Year(s) Of Engagement Activity 2018
URL https://www.liysf.org.uk
 
Description School Visit (Microscopes for Schools, Cambridge, UK) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Four researchers combined from the MRC-LMB in Cambridge and the Gurdon Institute in Cambridge took a set of microscopes into a Year 2 group at Morley Memorial Primary School, a local Primary School. With one microscope between 3 children and a variety of pre-prepared samples to look at and study the children learned about the small in comparison to the big, learned about life cycle and growing. This was combined with a presentation explaining the basics of microscopy and magnification/resolution.
Year(s) Of Engagement Activity 2016
 
Description Sci-Bar science talk (Cambridge, UK) 
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 a postdoc gave a lay talk about her work as part of 'Sci-Bar' (part of the Cambridge Science Festival)
Year(s) Of Engagement Activity 2016
URL http://www.sciencefestival.cam.ac.uk/events/scibar-cambridge
 
Description Science Talk to Wolfson College Fellows and Student body 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Science talk of Postdoctoral fellow who is involve din Wolfson college to explain their particular research interest in the lab to the wider college body (fellows and students)
Year(s) Of Engagement Activity 2018