Molecular and biophysical investigation of epithelial cell sheet invagination

Lead Research Organisation: University College London
Department Name: London Centre for Nanotechnology

Abstract

Early morphogenetic movements transform the new embryo from an amorphous ball of cells into a complex compartimentalised structure in which the main organs are laid out. Examples of such movements are gastrulation which creates the primitive gut and neurulation which creates the spinal chord. Invagination is a morphogenetic movement during which a cell sheet buckles (along a line in the case of neurulation and along a circle in the case of gastrulation). Abnormalities in invagination lead to conditions such as spina bifida that affect approximately 1 in 1000 live births. Cell sheets also undergo invagination during the creation of branched organs (lungs, kidneys, blood vessels) or during cancer when the tumour causes new blood vessels to grow to provide it with nutrients. During invagination, one group of cells has to express different genes than the surrounding cells. Invagination has been extensively studied in embryos and several theories have been proposed to explain the mechanism through which a cell sheet invaginates. However, since all cells in embryos undergo a defined sequence of gene expression, it is difficult to know what is responsible for invagination and what is just a side effect. All of the proposed theories for invagination require that one subset of the cell population express a different set of genes than the surrounding cells. Modern molecular biology techniques enable us to force cells to express a gene of choice. Microprinting techniques and microfluidic techniques enable us to selectively treat cells with micrometer precision. By combining both sets of techniques, I will devise an experimental system that will allow me to force a subset of cells within a monolayer to express a gene of my choice. In addition, the cells will be cultured on a soft substrate that they can deform. This system will enable me to test each of the different theories proposed for invagination directly. For each proposed theory, I will replicate the proposed movement by forcing a stripe of cells within the monolayer to undergo the type of movement that the theory hypothesizes is the cause of invagination. This will be done either through forced gene expression or through chemical treatment. Then, I will simply observe the cell sheet over a period of 24 hours and see if it invaginates. Once I have found which treatments give rise to invaginations, I will examine the mechanical forces at play. This project will enable us to better understand the mechanisms of invagination and identify proteins that can be targeted to inhibit it during cancer progression.

Technical Summary

Epithelial cell sheet invagination is a ubiquitous phenomenon in embryogenesis and organogenesis. During embryonic development, cells of the neural plate invaginate to form the neural tube and spinal chord. Many models of invagination have been proposed and these include contraction of an apical band of actin, cell proliferation, cell migration, or differential adhesion. Though invagination is of great importance, to date there exist no in vitro culture systems to study it. I propose to devise such a system and utilize it to study the molecular biology and biophysics of epithelial cell sheet invagination. All of the proposed models for invagination involve localized gene expression or cell behaviour. I will devise a culture system to locally transfect a stripe of cells within a sheet with the relevant gene products under the control of inducible promoters using either microfluidic channels or micropatterning. The gene product will be expressed only in the central region or only in the peripheral regions. This will enable me to replicate each of the proposed models of invagination in vitro. To test the role of apical contraction in invagination, I will perfuse the cells with contraction inducing drugs or retroviral constructs containing constitutively active forms of the small GTPase Rho or the neural crest protein shroom. To test the role of differential adhesion, cells in the central zone will be transfected with different types of cadherins. To test the role of cell proliferation, we will perfuse the cells in the central regions with promitogenic drugs. The role of cell migration will be tested by transfecting the cells in the peripheral regions with dominant forms of the small GTPase Rac. To directly assess the appearance of an invagination and investigate the forces involved, I will culture the cells on deformable acrylamide substrates and directly observe whether invaginations result from a specific pattern of gene expression.

Publications

10 25 50
 
Description Accurate spatiotemporal regulation of genetic expression and cell microenvironment are both essential to epithelial morphogenesis during development, wound healing and cancer. In vivo, this is achieved through the interplay between intrinsic cellular properties and extrinsic signals. Amongst these, morphogen gradients induce specific concentration- and time-dependent gene expression changes that influence a target cell's fate. As systems biology attempts to understand the complex mechanisms underlying morphogenesis, the lack of experimental setup to recapitulate morphogen-induced patterning in vitro has become limiting. For that reason, we developed a versatile microfluidic-based platform to control the spatiotemporal delivery of chemical gradients onto tissues grown in Petri-dishes. Using this setup combined with a synthetic inducible gene expression system, we were able to restrict a target gene's expression within a confluent epithelium to bands of cells as narrow as four cell diameters with a one cell diameter accuracy. Applied to the targeted delivery of growth factor gradients onto a confluent epithelium, this method further enabled the localized induction of epithelial-mesenchymal transitions and associated morphogenetic changes. Our approach paves the way for replicating in vitro the morphogen gradients observed in vivo to determine the relative contributions of known intrinsic and extrinsic factors in differential tissue patterning during development and cancer. It could also be readily used to spatiotemporally control cell differentiation in ES/iPS cell cultures or explants for re-engineering of complex tissues. Finally, the reversibility of the microfluidic chip assembly allows for pre- and post-treatment sample manipulations that extend the range of patternable samples to animal explants.
Exploitation Route -this method could be applied to tissue engineering to create complex tissues from stem cells. -an article is currently in revision for publication.

-our new method for controlling gene expression can be applied to any system where a morphogen gradient directs differentiation of progenitor cells in a tissue.
Sectors Healthcare

 
Description BBSRC Responsive mode
Amount £600,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2018 
End 01/2021
 
Description EMBO long term fellowship
Amount € 60,000 (EUR)
Organisation European Molecular Biology Organisation 
Sector Charity/Non Profit
Country Germany
Start 01/2015 
End 12/2015
 
Description Marie Curie Fellowship
Amount £128,418 (GBP)
Funding ID H2020-MSCA-IF-2014-658536 
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 01/2016 
End 12/2017
 
Description Role for dynamic protrusions in epithelial patterning
Amount £346 (GBP)
Funding ID BB/J008532/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2013 
End 12/2015
 
Description Patterning the notochord 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution We have begun a collaborative research project to expose mouse iPSCs to gradient of Sonic hedgehog morphogen using the device designed in this study
Start Year 2011
 
Description Interview with TV 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I participated in an interview for NHK, a major TV network in Japan. I described how charities in the UK fund research.
Year(s) Of Engagement Activity 2017
 
Description Interview with newspaper 
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 provided opinions on the risk of Brexit to Science research in the UK.
Year(s) Of Engagement Activity 2018