Molecular Genetics of Mammalian Cilia in Development and Disease

Lead Research Organisation: University of Edinburgh

Abstract

Patterning of the embryo is a tightly controlled process which requires the correct integration and interpretation of key developmental signals. A cell makes a decision of whether to differentiate, divide or migrate in response to these environmental cues. Small microtubule-based projections, or cilia, found on almost every mammalian cell play a critical role in signal transduction during development and disease. This is because cilia have a unique composition that is enriched in many receptors and signalling components, analogous to an antenna for focusing signalling responses. Dysfunction of the cilium underlies a group of human genetic disorders, termed ciliopathies. The inability to maintain a cilium results in a spectrum of health problems ranging from skeletal defects and cystic kidney disease, to cognitive and behavioural defects, as well as obesity. Despite a growing list of genes implicated in cilia biology, we have very little idea what the encoded proteins actually do in cells, or how mutations cause the disease. Consequently most of these cilia-related diseases are currently untreatable. Using mouse as a genetic model of human disease, we aim to understand how cilia are maintained as a specialized signalling compartments. We are also searching for cell-type specific differences how traffic to the cilia, necessary for environmental sensing, is regulated. Identifying novel ciliary components and determining their functions will lead to a better understanding for the diagnosis and clinical management within ciliopathy spectrum.

Technical Summary

Cilia are small, microtubule-based projections from the surface of most mammalian cells. Cilia regulate the responsiveness of the cell to key developmental signals, including Hedgehog (Hh) and Wnts. Cilia are distinct sub-compartments of the cell with unique membrane and cytosolic compositions, enriched in many receptors and signalling components. While recent studies have shown that mutations in genes required for cilia structure in both mice and humans result in a broad spectrum of developmental disorders, termed ciliopathies, little is known about how this compartmentalization of the cilia itself is maintained.

The aim of our work is to investigate the genetic and molecular mechanisms regulating mammalian cilia compartmentalization necessary for development and tissue homeostasis. Using a combination of mouse models and primary cell culture, we hope to identify novel mechanisms of regulating traffic into the ciliary compartment. These studies will lead to a better understanding for the diagnosis and clinical management within ciliopathy spectrum.
The objectives for this project are:
1) To investigate sorting mechanisms regulating traffic into the ciliary compartment.
2) To determine cell-specific physiological consequences of loss of ciliary trafficking.
3) To identify post-translational modifications which ”tag” cargo for dynamic and specific trafficking into and out of the cilia.

Our overall aim is to generate new mouse models of human disease candidates, to correlate genotype, cellular phenotype, and overall health. We examine the effects of these mutations integrating microscopy, transcriptomics and proteomics. Taken together, we hope this work will help us evaluate how a genetic mutation leads to specific cellular and physiological consequences that will help to develop evidence-based strategies for clinical management of ciliopathies.

Publications

10 25 50
 
Description House of Commons Science & Technology Committee Inquiry on Genomics and Genome Editing in the NHS - primary ciliary dyskinesia
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Gave evidence to a government review
 
Description BBSRC Project Grant
Amount £9,344 (GBP)
Funding ID BB/P00122X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2016 
End 11/2019
 
Description Carnegie Collaborative Research Grant
Amount £49,882 (GBP)
Funding ID 035424/1 
Organisation Carnegie Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2015 
End 07/2016
 
Description Clinical Starter Grant: Molecular mechanisms of RPGR in photoreceptor degeneration in mouse models of Retinitis Pigmentosa, Awardee: Roly Megaw (Mill Lab)
Amount £18,814 (GBP)
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2017 
End 10/2018
 
Description Institutional Strategic Support Award 2 (ISSF2): Development of small molecule HDR agonists for In Vivo Therapeutic Gene Editing
Amount £27,482 (GBP)
Organisation University of Edinburgh 
Department Institutional Strategic Support Fund
Sector Charity/Non Profit
Country United Kingdom
Start 08/2016 
End 02/2017
 
Description Institutional Strategic Support Fund (ISSF2): In vivo correction of human retinitis pigmentosa mutations in mouse model
Amount £35,892 (GBP)
Organisation University of Edinburgh 
Department Institutional Strategic Support Fund
Sector Charity/Non Profit
Country United Kingdom
Start 05/2016 
End 05/2017
 
Description MRC Confidence in Concept
Amount £124,791 (GBP)
Funding ID MRC/CIC3/021 
Organisation University of Edinburgh 
Sector Academic/University
Country United Kingdom
Start 06/2015 
End 07/2015
 
Description Starter Grant for Clinical Lecturers: In vivo correction of a human mutation in a mouse model of retinitis pigmentosa. Awardee: Roly Megaw, Mill lab
Amount £29,923 (GBP)
Organisation Academy of Medical Sciences (AMS) 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2016 
End 01/2018
 
Title In vivo gene editing reporter 
Description We have generated a mouse model for rapid and scaleable monitoring gene editing events in situ, allowing tracking of potentially damaging non-homologous end-joining (NHEJ) versus therapeutically desirable homology-driven repair (HDR) events in a whole organism, tissue or field of primary cells. This model will be essential for optimizing gene editing reagents and vehicles to establish robust protocol for therapeutic gene editing in target tissues of interest. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact MRC Confidence in Concept award. 
 
Title PCD Mouse models 
Description We have generated several mouse models of PCD disease genes with a range of human disease-like mutations (indels, duplications, insertions and inversions) which we will use for testing therapeutic benefit of somatic gene editing. In parallel, these models will also be used in collaboration with the UK CF Gene Therapy Consortium looking at repurposing reagents for gene replacement therapies for PCD. These animals are excellent models of human PCD disease, from pathology to molecular mechanism. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Carnegie Collaborative Research Grant 
 
Description COST Action BEAT-PCD 
Organisation Beat-PCD
Country Belgium 
Sector Private 
PI Contribution As member of this European-based consortium, we are part of the Basic Science work group, that is intended to encourage communication between basic and clinical scientists working on cilia and PCD. In particular, we will be providing models of PCD (animal and cellular) with the aim of developing them to evaluate pre-clinical studies for novel therapies for PCD.
Collaborator Contribution COST Actions provide funds for cooperation, collaboration and the development of ideas through meetings, workshops, and short term lab visits. It aims to help seek more direct forms of funding down the way.
Impact Funding for meetings, workshops and travel. I presented a podium talk at the Gordon Research Conference (February 2017, Galveston Texas) Cilia, Mucus and Mucociliary Interactions meeting.
Start Year 2015
 
Description Defining Interaction Networks of Human Ciliopathy Proteins 
Organisation University College Dublin
Department Systems Biology Ireland
Country Ireland 
Sector Academic/University 
PI Contribution Provision of affinity captured samples for label-free proteomics analysis in human and fly samples. Validation of potential hits by IP-WB, IF and PLA. Characterization of interaction networks in null cells/animals.
Collaborator Contribution Preparation and analysis of mass spec proteomics.
Impact Publications: Diggle CP, Moore DJ, Mali G, zur Lage P, Ait-Lounis A, Schmidts M, Shoemark A, Garcia Munoz A, Halachev MR, Gautier P, Yeyati PL, Bonthron DT, Carr IM, Hayward B, Markham AF, Hope JE, von Kriegsheim A, Mitchison HM, Jackson IJ, Durand B, Reith W, Sheridan E, Jarman AP, Mill P. HEATR2 plays a conserved role in assembly of the ciliary motile apparatus.PLoS Genet. 2014 Sep 18;10(9):e1004577. doi: 10.1371/journal.pgen.1004577. eCollection 2014 Sep. Yeyati, PL, Schiller, R, Mali, G, Kasioulis, I, Kawamura, A, Adams, IR, Playfoot, C, Gilbert, N, Heyningen, VH, Wills, JH, Kriegsheim, AV, Finch, A, Sakai, J, Schofield, CJ, Jackson, IJ, Mill, P. KDM3A coordinates actin dynamics with intraflagellar transport to regulate cilia stability. J Cell Biol Feb 2017, jcb.201607032; DOI: 10.1083/jcb.201607032 Hall, EA, Nahorski, MS, Murray, LM, Shaheen, R, Perkins, E, Dissanayake, KN, Kristaryanto, Y, Jones, RA, Vogt, J, Rivagorda, M, Handley, MT, Mali, GR, Quidwai, T, Soares, DC, Keighren, MA, McKie, L, Mort, RL, Gammoh, N, Garcia-Munoz, A, Davey, T, Vermeren, M, Walsh, D, Budd, R, Aligianis, IA, Faqei, E, Quigley, AJ, Jackson, IJ, Kulathu, Y, Jackson, M, Ribchester, R, Kriegsheim, AV, Alkuraya, FS, Woods, CG, Maher, ER, Mill, P. Mutations in PLAA cause a lethal infantile epileptic encephalopathy by disrupting ubiquitin-mediated endolysosomal degradation of synaptic proteins. American Journal of Human Genetics (revised version submitted).
Start Year 2014
 
Description Functional Characterization of Candidates in Human Skeletal Dysplasias 
Organisation Complutense University of Madrid
Department Grupo de Genética Humana y Patología Molecular, Instituto de Investigaciones Biomédicas
Country Spain 
Sector Academic/University 
PI Contribution Functional characterization of skeletal dysplasia candidates in mammalian cell and mouse models.
Collaborator Contribution Identification of novel skeletal dysplasia candidates.
Impact A manuscript on the role of WDR35 in EVC is in preparation for submission the end of this year. Mouse models of putative ciliopathy by CRISPR are being generated.
Start Year 2013
 
Description Functional Conservation in Ciliary Trafficking 
Organisation University of Edinburgh
Department Center for Intergative Physiology
Country United Kingdom 
Sector Academic/University 
PI Contribution Characterization of function of ciliary candidate genes in mammalian cell culture and mouse mutant models.
Collaborator Contribution Identification and characterization of putative ciliary candidate genes in Drosophila cilia.
Impact PAPERS: Hall,E.A., Keighren, M., Ford, M., Davey, T., Jarman, A.P., Smith, L.B.S, Jackson I.J. and Mill, P. (2013). Acute Versus Chronic Loss of Mammalian Azi1/Cep131 Results in Distinct Ciliary Phenotypes. PLoS Genetics. 9(12): e1003928. doi:10.1371/journal.pgen.1003928. Diggle CP, Moore DJ, Mali G, zur Lage P, Ait-Lounis A, Schmidts M, Shoemark A, Garcia Munoz A, Halachev MR, Gautier P, Yeyati PL, Bonthron DT, Carr IM, Hayward B, Markham AF, Hope JE, von Kriegsheim A, Mitchison HM, Jackson IJ, Durand B, Reith W, Sheridan E, Jarman AP, Mill P. HEATR2 plays a conserved role in assembly of the ciliary motile apparatus. PLoS Genet. 2014 Sep 18;10(9):e1004577. doi: 10.1371/journal.pgen.1004577. eCollection 2014 Sep. PhD candidate: Girish Mail
Start Year 2007
 
Description Genome editing of HEATR2 with CRISPR technology 
Organisation University of Tsukuba
Department Department of Anatomy and Embryology
Country Japan 
Sector Academic/University 
PI Contribution Generation and characterization of CRISPR edited mouse mutant alleles.
Collaborator Contribution Construction and testing of plasmids for injections.
Impact I was invited to a talk in Tsukuba Global Science Week 2014. My student was also invited to give a talk where he won a "Young Scientist Award".
Start Year 2013
 
Description Role of Centriolar Satellites in Mammalian Development 
Organisation Cancer Research UK Cambridge Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution In collaboration with Fanni Gegely, we have generated null alleles as well as SNAP-tagged alleles of the key centriolar satellite gene PCM-1 in mouse by CRISPR gene editing. If PCM1 is essential for centriolar satellites in vivo as it is in vitro, we would expect mutant embryos to be embryonic lethal, with profound defects in ciliogenesis, centriole duplication and DNA damage response. Key human disease genes implicated in centrosomes, cilia and DNA repair dynamically localize to centriolar satelittes where it is proposed these structures play key roles is regulating access between cellular compartments. We will test this during embryogenesis as well as adult homeostasis.
Collaborator Contribution Fanni Gegely has a unique set of PCM1 interacting proteins, many of which are novel and are potential human disease genes for ciliopathies.
Impact This collaboration is in its early stages, we have generated the mice but no further outcomes yet.
Start Year 2016
 
Description UK GTC 
Organisation UK Cystic Fibrosis Gene Therapy Consortium
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution My lab is the named collaborator on a Wellcome Portfolio application "Respiratory Gene Therapy Portfolio (RGTP)" that is in the final stages of review with the Wellcome Trust. We will provide expertise, reagents, mouse models and primary cell culture systems of PCD disease genes for the repurposing of non-viral and viral delivery methods for gene replacement therapy studies.
Collaborator Contribution The UK CF GTC has provided access to their non-viral (GL67) as well as viral vectors for repurposing for therapeutic gene editing to airways for PCD. More direct clinical links to the PCD unit at the Brompton will also be established to speed gene discovery, functional analysis and potential therapeutic developments.
Impact We were funded for a MRC Confidence in Concept grant for therapeutic gene editing for PCD. We will be applying for the next tranche of translational funding in late 2016 to continue this work in partnership with UK CF GTC. The UK CF GTC has a Wellcome Portfolio application which is awaiting a decision regarding repurposing of their reagents for other pulmonary genetic diseases: in collaboration with Dr. Chris Boyd (Centre for Genomics and Experimental Medicine, University of Edinburgh) we will lead the PCD arm of the program.
Start Year 2015
 
Description House of Commons Science and Technology Committee Inquiry on Genomics and Genome Editing in the NHS - diagnosis of developmental disorders 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Together with Prof Jane Lucas, Southamton (Faculty of Medicine, Clinical and Experimental Sciences and Primary Ciliary Dyskinesia Centre, University Hospital Southampton) we co-wrote a response to the House of Commons Science and Technology Committee Inquiry on Genomics and Genome Editing in the NHS - diagnosis of developmental disorders entitled 'Primary ciliary dyskinesia - an exemplar of the need for NHS-integrated genomics and the potential for genome editing'. It outlined the need for improved rapid genetic diagnosis early and development of alternate therapeutic strategies for this rare highly polygenic childhood disease. As a result, I have been invited to attended a small round-table discussion with policymakers, academics and industry on genome editing March 2018.
Year(s) Of Engagement Activity 2017
 
Description PM TV interview for the PLAA story 
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 Our press release for our PLAA story entitled 'Cause of fatal childhood disorder revealed in gene study' had a large media pick-up in print media across the UK, digital media across the world and a TV interview with STV 6pm evening news. It resulted in two editorial pieces to be written by the Saudi government for a Nature-based publication (TBC).
Year(s) Of Engagement Activity 2017
 
Description PT Genome Editing at Open Doors Day 
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 The IGMM hosted an very successful Open Doors day for the general public, whereupon post-doc Peter Tennant represented our research in a lay poster he presented and helped guide groups on Institute tours. There were 232 people who attended, from a whole age spectrum, from families with young children who greatly enjoyed our activities, to teenagers and undergraduate students wondering about their next career steps, to adults and early-rising retired visitors who were fascinated by the building and the science we do here. Excellent feedback and questions on the work we do in the IGMM.
Year(s) Of Engagement Activity 2017
 
Description RM Patient Engagement Session- Eye Development and Degeneration Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact As part of the Eye Development & Degeneration conference, the IGMM hosted a patient-facing event, welcoming 40 patients suffering from vision-impairing conditions as well as their family members and guide dogs. Over half of the visitors opted in for a tour of the Institute, which showcased the Institute's state-of-the-art imaging facilities and extensive zebrafish facility.
At the main event, researchers and clinicians gave brief presentations about current research on macular degeneration and eye abnormalities such as being born with no iris (a condition called aniridia), and also talked about retinal disease therapy with the help of genome editing and stem cells.
The talks were followed by a Q and A session with a panel of leading researchers and clinicians with an expertise in eye disorders. Panel members included Dr Roly Megaw, who has just published his research on the differences in light-sensing cells (photoreceptors) between healthy people and those suffering from X-linked retinitis pigmentosa, and Prof. Alan Wright, whose contributions to the field of genetics over the past 25 years have fostered many emerging vision projects.
The gratitude and respect the visitors had for the scientists championing their cause was a humbling and rewarding experience. Such events allow scientists to take a step back and remind themselves why they do a lot of their research in the first place - in order to discover the reason for, and ultimately, the cure to, conditions that affect many people worldwide.
Year(s) Of Engagement Activity 2017