Identification and Characterisation of Genes Required For DIF-1 Signaling and Patterning in Dictyostelium

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

Developmental biologists are dedicated to understanding how our different body parts are arranged with respect to one another. Considering the organizational complexity of a newborn child, this is a staggering problem. In most cases this is nevertheless successfully accomplished. However, a significant number of children (1-5%) in the UK are born with major developmental defects. If we can understand what happens during normal development, we might be able to understand, prevent, treat or even cure these disorders. For example, why do we have feet rather than hands at the end of our legs? Furthermore, the leg itself is made up of many constituent parts (cells) that have different functions (e.g. skin cells for protection and muscles to power movement). How do cells choose which part (cell type) to become when there are more than a hundred choices in the human embryo? Finally, to function properly the different cell types must be organized or positioned correctly with respect to one another. These are the key problems that developmental biologists hope to solve: cell type choice and patterning.

Over the years, developmental biologists have sought to address these issues by identifying molecules that orchestrate development. One approach is to disrupt the system in a defined way and ask what happens. However, it is clearly impossible to do this with human embryos. In this light, developmental biology has been revolutionised by the finding that, not only must animals as diverse as slime moulds and humans solve the same problems, but also they often use the same molecules to orchestrate the process. Consequently, a number of organisms have become the models of choice for laboratory studies of developmental processes. The slime mould, Dictyostelium discoideum is one such model as it contains only a small number of cell types arranged into tissues along its axis and is easily maintained and manipulated in the laboratory. Furthermore, Dictyostelium illustrates a patterning mechanism that is poorly understood in any system. In this, the different cell types are initially disorganized and scattered amongst each other, but then sort out into coherent tissues.

Using Dictyostelium as a model, we aim to understand this patterning mechanism, by identifying factors required for this process. By studying how their activity is regulated and how they interact with one another, we will begin to learn how patterning is controlled. These studies should provide insights into the principles of patterning and disease.

Technical Summary

A key question in developmental biology concerns the mechanisms by which body pattern is established. Although positional information conveyed by morphogen gradients and lateral inhibition are widely accepted ways of forming pattern, an alternative method is conceivable, based on the intermingled differentiation of cells with different fates, followed by their sorting into discrete pattern elements. It has been proposed that Dictyostelium prestalk and prespore cells may behave in this way. Furthermore, there is evidence that this pattern-forming process operates in higher organisms. Through the proposed project, we aim to understand the signaling events required for this patterning process in Dictyostelium.

The developing Dictyostelium slug has a clear anterior-posterior pattern. Prestalk cells (pstA and pstO) occupy the anterior quarter, and the posterior three quarters comprise the prespore zone. Marker gene studies reveal, however, that pstO cells initially differentiate scattered amongst the prespore cells before sorting out. The diffusible signaling molecule DIF-1 is required for normal pstO cell differentiation and patterning. (1) Mutants defective in DIF biosynthesis (dmtA-) and DIF-1 responses (dimA-) have been identified. dimA encodes a bZIP transcription factor and dmtA a small molecule methyltransferase. The only characterised defect in cell type differentiation in both mutants is a failure to express a subset of pstO markers and increase in the prespore expression domain. (2) The STATc transcription factor is tyrosine phosphorylated and translocates to the nucleus of pstO cells in response to DIF-1.

These observations suggest pstO/prespore choice is driven by a process akin to lateral inhibition and that although all cells experience similar concentrations of DIF-1, initial intrinsic differences between them distinguish responding and non-responding populations. Finally, once distinct populations of cells arise, there is good evidence that subsequent tissue patterning could occur by sorting out due to differential adhesion and/or cell motility.

Using forward and reverse genetics, in addition to the powerful molecular genetic, biochemical and post-genomic tools available in Dictyostelium, this project aims to further understand DIF-1 regulated patterning and signal transduction pathway by:

1. Defining how the activity of the DIF-1 dependent transcription factor DimA is regulated
2. Identifying and characterising genes required for DIF-1 signal transduction and regulation

Through these studies, we will identify genes required for DIF-1 signal transduction, begin to define how this signaling pathway becomes activated in a subset of cells in the face of a seemingly uniform signaling environment and provide insights into the principles of this patterning mechanism.

Publications

10 25 50
 
Description Lister Research Prize Fellowship (Identifying and characterising genes that control cell fate choice and behaviour)
Amount £150,000 (GBP)
Organisation Lister Institute of Preventive Medicine 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2006 
End 09/2012
 
Description MRC Project Grant G0900069 P2X receptors for ATP: using the model organism Dictyostelium discoideum to understand their regulation and roles)
Amount £608,251 (GBP)
Funding ID G0900069 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 06/2010 
End 06/2013
 
Description NERC Project Grant NE/H020322/1 Genetic architecture and constraint in social evolution
Amount £446,357 (GBP)
Funding ID NE/H020322/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 01/2011 
End 09/2014
 
Description Wellcome Trust New Investigator Award
Amount £2,000,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2012 
End 03/2020
 
Title P2X research 
Description Our discovery of ATP gated ion channels, or P2X receptors, in dictyostelium was very novel. We then went on to show that these receptors regulate intracellular functions, whereas in mammalian cells they regulate responses at the plasma membrane. these studies highlight the possibility that there may be hitherto unknown intracellular roles for a pharmacologically important class of ion channel. furthermore, they show that Dictyostelium provides a model system for their study 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2007 
Provided To Others? Yes  
Impact Publication describing these findings was elected to Faculty of 1000 Awarded MRC Grant G0900069 - P2X receptors for ATP: using the model organism Dictyostelium discoideum to understand their regulation and roles 
 
Title Rap1 
Description Our studies described the effects of altering the levels of the small GTPase Rap1 during Dictyostelium development. They showed that Rap1 affects a novel cell adhesion mechanism. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2009 
Provided To Others? Yes  
Impact Other groups are now studying Rap1 during multicellular development in Dictyostelium 
 
Title Social studies 
Description We developed a new quantitative framework for the analysis of social behaviour 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2009 
Provided To Others? Yes  
Impact Paper describing this research was elected to Faculty of 1000 
 
Title Stochastic differentiation in Dictyostelium 
Description Described a new mechanism for patterning in Dictyostelium that is relevat to mammalian development and regeneration 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2013 
Provided To Others? Yes  
Impact publication and election of paper to Faculty 1000 
 
Description P2X Receptor Research 
Organisation University of Manchester
Department Faculty of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided technical expertise in Dictyostelium biology and manipulation
Collaborator Contribution New research directions and idea
Impact Publication PMID: 17625565 Dissemination: BBC Radio Manchester interview, Daily Mail article, Metro news article MRC Project Grant: G0900069 - P2X receptors for ATP: using the model organism Dictyostelium discoideum to understand their regulation and roles
Start Year 2006
 
Description Small GTPase regulation 
Organisation University of British Columbia
Country Canada 
Sector Academic/University 
PI Contribution We provided genetic, and developmental expertise in Dictyostelium pattern formation and signaling
Collaborator Contribution Provide assays of small GTPase activity and expertise in GTPase regulation
Impact PMID: 19126673
Start Year 2008
 
Description Studies of social behaviour 
Organisation University of Bath
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided Dictyostelium expertise and socio-biology expertise
Collaborator Contribution They have provided mathematical and theoretical ideas to our socio-biological research
Impact Publication PMID: 19631539 Dissemination: Guardian article, BBC Three Counties Radio interview NERC Project grant NE/H020322/1 awarded July 2010 Publication PMID: 20546090
Start Year 2006
 
Description genetics of socio biology 
Organisation Baylor College of Medicine
Country United States 
Sector Hospitals 
PI Contribution We instigated the project during my time at Baylor College of medicine. The experimental design, conclusions and manuscript preparation were all contributed to by us
Collaborator Contribution New ideas and research directions
Impact PMID: 18272966
 
Description BBC Radio 
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 Public/other audiences
Results and Impact Work has resulted in greta interest from media. Public are interested in how model organism research can help understand basic questions in biology. I have been interviewed on Radio 4 (Material World), BBC radio manchester and BBC three counties radio

Each media engagement has led to further interest in the subject
Year(s) Of Engagement Activity 2006,2007,2008,2009
 
Description Bolton School 
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 Hosted 6th form school students in my lab for 4-6 weeks lab experience

One student changed their intended degree course to Developmental and Cell Biology as a consequence of being enthused
Year(s) Of Engagement Activity 2007,2008,2009
 
Description Wellcome Trust 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Interviwe for Wellcome trust website. Description of research activities

asked to podcast research
Year(s) Of Engagement Activity 2012