Investigation of the role of the primary cilia and centrosome during signalling events in development using the chicken mutant talpid3

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

It is thought that every cell in our bodies has a small antenna poking out of it, called a primary cilia. For many years biologist thought that the primary cilia was no more than a vestigial organ, something left behind from when we were single-celled organisms swimming through the oceans using a flagella (the type of tail sperm and other motile cells use to move). However now it seems that the primary cilia is more than just a stumpy tail, it is in fact essential to normal development of the embryo. Clues as to the importance of cilia, first came from investigation into human diseases in which motile cilia were absent or impaired. Not surprisingly, these people often suffer from respiratory and reproductive problems, as they don't have cilia to move mucus, eggs or sperm. In addition it was found that many of these people also have situs inversus, a condition when the internal organs such as the heart are found on the wrong side of the body. This suggested that cilia somehow play an important role in the development of the embryo, somehow controlling which side organs should form on. To understanding how cilia could control such important events, recent research has aimed to determine what cilia consist of. Using many different techniques including studying mouse and human mutations which result tell-tale cilia syndromes such as the development of extra fingers and kidney problems, scientists have begun to understand which genes are needed to form normal cilia. By studying precisely what goes wrong in animals which lack cilia, we now understand that cilia are required to control important intracellular signalling events, particularly in pathways important in development and cancers known as the Hedgehog and Wnt pathways. Using high powered imaging techniques, researchers have been able to look inside cilia and have found that some important proteins become highly concentrated in cilia, using cilia as a special compartment in which vital protein interactions can take place. But we have little idea how this occurs. We study a chicken flock in which some of the embryos fail to hatch. It was discovered that these embryos die in the egg from serious developmental disorders. By examining the anatomy of these embryos known as talpid3, we discovered that important signalling events in the Hedgehog signalling pathway, go awry, leaving embryos with central nervous system, limb, head, blood vessel and bone defects. Excitingly when the defects in talpid3 embryos are compared to those in humans and mice in which cilia are defective there are many areas of similarity and it now seems talpid3 embryos also have defective cilia. Furthermore the gene which causes talpid3, localises in the centrosome, a structure essential for normal cilia development. Talpid3 embryos offer a unique approach to studying cilia function which is difficult to undertake in mice. Because embryos develop outside the mother (in the egg) it is possible to manipulate embryos while they are still developing. We plan to use talpid3 embryos to examine which genes are important in cilia growth, by over expressing or reducing expression of genes known to be important in cilia formation or Hedgehog signalling in the embryos and then examining cilia formation, or patterning of the nervous system controlled by Hedgehog signalling. We can also use both these embryo manipulations and cell culture techniques to see where proteins localise in the cell. Talpid3 embryos also have one other interesting defect, in that they don't form normal veins. Although this could be because of the abnormal Hedgehog signalling in talpid3 we would also like to investigate if this is due to another function of cilia, which can sense mechanical stresses such as flow. We think it is possible that cilia not only act as a focal point for signalling in the cells but may act to coordinate the many varied signalling events that determine how we develop.

Technical Summary

Primary non-motile cilia (9+0) and their associated centrosomal components, the centriole are essential components of the Wnt, PDGF and Hedgehog (Hh) signalling pathways. Cilia are also important mechanosensory organelles. Defects in genes associated with cilia formation result in a wide range of human developmental disorders, characterised by phenotypes such as central nervous system disorders, kidney failure, blindness and polydactyly. It is known that components of signalling pathways are preferentially concentrated in the cilia but it is unclear how this protein sorting is achieved. Talpid3 is a chicken mutant with a defect in KIAA0586, a gene associated with the centrosome. Talpid3 embryos lack cilia, have developmental defects which resemble phenotypes seen in the human ciliopathies and have a well characterized Hh signalling defect. As developing chicken embryos are readily available and easily to experimentally manipulate, we will use this ciliary mutant in order to understand the mechanisms involved in cilia based intracellular signalling. Using in vitro and in ovo complementation experiments and co-localisation studies, we will observe the effect on cilia and Hh signalling, by manipulating both known components of the Hh pathway and genes involved in ciliogenesis. We will also use this approach to test candidate genes identified as potentially involved in Hh signalling/ciliogenesis in a previous analysis. The mechanosensory role of cilia in the kidney is clear, but the contribution of mechanical stimulation as a driver of morphogenesis is less well understood, although it is known that blood flow contributes to the patterning of vasculature. Talpid3 embryos have defective vascular patterning. We will investigate how the loss of cilia contributes to this phenotype. This work may elucidate the way in which cilia co-ordinate these diverse signalling events to direct morphogenesis.

Publications

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Bangs F (2009) 16-P002 The developmental mutant talpid3 lacks primary cilia in Mechanisms of Development

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Dunn IC (2011) The chicken polydactyly (Po) locus causes allelic imbalance and ectopic expression of Shh during limb development. in Developmental dynamics : an official publication of the American Association of Anatomists

 
Title Chicken Wearing SOX 
Description Image submitted to RPS International Images for Science 2015 
Type Of Art Image 
Year Produced 2016 
Impact Public engagement 
URL http://www.rps.org
 
Description TALPID3 protein is a centrosomal protein

It is postulated through analysis of TALPID3 homologues that TALPID3 has a Eumetazoan origin

TALPID3 has 2 essential coiled coiled domains

TALPID3 has 1 essential region of intrisic disorder

TALPID3 is essential for motile and non-motile cilia development

TALPID3 is essential for migration and orientation of centrosomes during ciliogenesis

TALPID3 is not essential for centriole length

TALPID3 is not essential for centriol duplication

TALPID3 is not essential for axoneme formation

Telencephalic choroid plexus forms in in talpid3-/- chicken embryos

Lung development is abnormal in talpid3-/- and phenocopies human ciliopathy syndromes

Liver development is abnormal in talpid3-/- and phenocopies human ciliopathy syndromes

Inner ear development is abnormal in talpid3-/- and phenocopies human ciliopathy syndromes

talpid3-/- embryos have polycystic kidneys

talpid3-/- embryos have planar cell polarity type anatomical defects

talpid3-/- embryos/cells have intracellular mislocalisation of core planar cell polarity proteins

TALPID3 is not epistatic to KIF7

TALPID3 is not epistatic to SMO

TALPID3 mutations in human cause Joubert syndrome
Exploitation Route TALPID3 may be a target to regulation aberrant Hedgehog signalling (i.e. a significant cause of pancreatic and other cancers)

TALPID3 mutations may be used to diagnose Joubert syndrome
Sectors Healthcare

 
Description TALPID3 mutations have been found to cause human Joubert Syndrome. Preimplantation diagnosis has been used to slelect unaffected embryos in families with Joubert Syndrome.
First Year Of Impact 2015
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Policy & public services

 
Description British Society for Developmental Biology Travel Award
Amount £400 (GBP)
Organisation British Society for Developmental Biology 
Sector Learned Society
Country United Kingdom
Start 06/2013 
End 06/2014
 
Description CoB Small Meeting
Amount £2,000 (GBP)
Organisation Company of Biologists 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2013 
End 05/2013
 
Description Nordic Centrosome and Cilia Network 
Organisation Nordic Cilia and Centrosome Network
Country Global 
Sector Charity/Non Profit 
PI Contribution Training partnerships, conferences
Collaborator Contribution Training partnerships, conferences
Impact Training[ proteomics
Start Year 2012
 
Title Preimplantion diagnosis for Joubert Syndrome 
Description Families with Joubert syndrome have now selected unaffected embryos through pre-implantation diagnosis used whole exome sequencing to discover mutations in TALPID3 
Type Diagnostic Tool - Non-Imaging
Current Stage Of Development Initial development
Year Development Stage Completed 2016
Development Status Under active development/distribution
Impact Detection of TALPID3 mutations in children with Jeune's syndrome 
 
Description Doors Open Day Roslin 2013 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Sparked interest and questions

School students sought out information on STEM careers
Year(s) Of Engagement Activity 2013
 
Description Media Interest (hand development) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Interview with radio Scotland, coverage article in The Times, The Metro

Many people have contacted me regarding interest in becoming a scientist, and learning more about the use of animals in research
Year(s) Of Engagement Activity 2014
 
Description Midlothian Science Festival 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact increased awareness of use of animals in research, sparked interest in becoming scientist

Students said they were open to career in STEM
Year(s) Of Engagement Activity 2014
 
Description National Science and Engineering Competition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact The participant from the lab went on to take STEM subject at university

The student went on to STEM subjects at university
Year(s) Of Engagement Activity 2013
 
Description Nuffield Research Placement 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact Encouraged STEM careers

Students went on to take STEM subjects at university
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013
 
Description School visit (Edinburgh) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk, practical, discussion

Participants were more open to animal research
Year(s) Of Engagement Activity 2013
 
Description Soapbox Science Edinburgh 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Soapbox Science is an informal forum in which female scientists present their work in public using a 'soapbox' format in a public area. The passing public are encouraged to stop and listen and ask questions of the scientists. Social media was used extensively before and after the event.
Year(s) Of Engagement Activity 2016
URL https://youtu.be/WEvzU_soOJ4
 
Description TEDX Talk-"This is a Golden Age of Science- Grab it with Both hands 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact This was an invited TEDX talk which has been view online more than 700 times and was the most highly viewed talk of the TEDX event. The talk covered the impacts of genome sequencing technology and open access publication. Subsequently Dr Davey was invited to attend other public engagement events.
Year(s) Of Engagement Activity 2016
URL https://youtu.be/vw7sTgQiltI?list=PL1c4W6FJ_82YOOiJVbZUZlfj2ysg_4aYX