Grainyhead-like genes and mammalian neural tube defects

Lead Research Organisation: University College London
Department Name: Institute of Child Health

Abstract

During early pregnancy, a crucial event in the developing embryo is the formation of the neural tube, which will later develop into the brain and spinal cord. Failure of the neural tube to form correctly leads to a group of birth defects called neural tube defects (NTDs), in which the brain and/or spinal cord of the fetus become irreversibly damaged, resulting in death before or shortly after birth, or handicap in surviving babies. Overall, NTDs occur in around 1 per 1,000 pregnancies although the rate varies and is significantly higher in some regions (e.g. Northern Ireland and Scotland). Worldwide, approximately 130,000 cases occur every year. The risk of NTDs depends on both inherited genetic factors and non-genetic factors such as diet, but the exact causes are not well understood. We are studying mouse strains that are predisposed to develop NTDs that resemble the corresponding human birth defects, with the aim of understanding why the defects develop and finding ways to prevent them. In one of these strains, curly tail, we found that reduced expression of a gene called grainyhead-like-3, causes NTDs. We now have evidence to suggest that increased expression of the same gene or a related gene, grainyhead-like-2, can also cause NTDs, and we propose to use genetic approaches to test this idea. We will then investigate how the altered expression of the grainyhead-like genes changes cellular behaviours and thereby alters the mechanical properties of the developing embryo such that the neural tube fails to close. Finally, we will test whether dysregulation of grainyhead-like-2 and -3 cause NTDs through similar or differing effects on the expression of other ?downstream? genes. Identification of the genes that cause NTDs in mice may indicate genes that may be causative in humans. Knowledge of the genes responsible for NTDs in humans may then allow more accurate counselling for affected families who are considering a further pregnancy, and may allow development of novel therapeutic strategies.

Technical Summary

Neural tube defects (NTDs), such as spina bifida and anencephaly, are severe congenital malformations caused by failure of closure of the embryonic neural tube. Folic acid supplementation can prevent some NTDs but a significant proportion (at least 30%) are unresponsive, and these defects remain a major health issue in the UK and worldwide. The causes of human NTDs are still largely unknown, but appear to involve multiple genetic and environmental factors. Therefore, our long term aim is to use detailed knowledge of the molecular and developmental basis of the pathogenesis of NTDs in mouse models, to assist identification of genetic risk factors for the corresponding human birth defects and development of primary preventive strategies. Several lines of evidence suggest that spinal neurulation depends on precise regulation of expression of members of the grainyhead-like (Grhl) gene family, and that their dysregulation confers susceptibility to NTDs. We previously found that reduced expression of Grhl3 causes spinal NTDs in curly tail mice, a model for folate-resistant and inositol-responsive NTDs. We now have preliminary data to suggest that over-expression of Grhl3 can also cause spinal NTDs, and that this may involve a distinct mechanism from that in the curly tail model. Furthermore, mapping and expression studies in another partially penetrant model for spinal NTDs, the Axial defects (Axd) mouse, suggest that the causative genetic defect involves a regulatory mutation resulting in over-expression of another Grhl member, Grhl2. We will build on these findings by, (1) testing the hypothesis that Grhl2 over-expression is the direct cause of NTDs in Axd. Suppression of Grhl2 function in Axd mice is predicted to rescue neurulation defects, whereas, transgenic over-expression of Grhl2 in wild-type embryos is predicted to cause NTDs. Next, (2) a series of studies will investigate the cellular and developmental basis of NTDs in Grhl2 and Grhl3 over-expression models, to determine whether the mechanism is shared with, or distinct from, that which we previously found to operate in the curly tail model. We will test whether NTDs caused by over-expression of Grhl3 involve a re-opening defect, which would represent a novel mechanism for development of spinal NTDs. Finally, (3) we will test the idea that over-expression of Grhl2 results in suppression of Grhl3 function, through disruption of transcription factor dimerisation, such that NTDs in the Axd (Grhl2 over-expression) and curly tail (Grhl3 under-expression) models, share a similar molecular basis in terms of aberrant expression of downstream genes.

Publications

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Copp AJ (2013) Neural tube defects--disorders of neurulation and related embryonic processes. in Wiley interdisciplinary reviews. Developmental biology

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Copp AJ (2011) Regional differences in the expression of laminin isoforms during mouse neural tube development. in Matrix biology : journal of the International Society for Matrix Biology

 
Description Child Health Research Charitable Incorporated Organisation Studentship
Amount £76,334 (GBP)
Funding ID CHR CIO PhD17/18STU-2 
Organisation The Child Health Research Charitable Incorporated Organisation 
Sector Academic/University
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description Newlife Project Grant
Amount £119,000 (GBP)
Organisation Newlife 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2012 
End 03/2014
 
Description Newlife Start-Up Grant
Amount £13,259 (GBP)
Funding ID SG/15-16/04 
Organisation Newlife 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2016 
End 04/2017
 
Title Grhl2 mutant mice 
Description A gene-trap line of Grhl2 mice which carry a loss of function allele of Grhl2. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact We used Grhl2 mutant mice in a complementation experiment to demonstrate that neural tube defects in Axial Defects mutant mice result from over-expression of Grhl2. We found that loss of Grhl2 function causes neural tube defects. These findings indicate Grhl2 as a candidate gene for human NTDs. 
 
Title LC-MS/MS method - inositol 
Description We developed a novel method for quantification of inositol in biological matrices. This involved a novel HPLC step to separate inositol from other 6 carbon sugars and sugar alcohols. 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact Improvement in accuracy of inositol assays 
 
Title Grhl2 and Grhl3 RNA-Seq datasets 
Description RNA Seq data for Grhl3 and Grhl2 loss and gain of function models 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Imporved understanding of the molecular mechanisms mediated by Grhl gene function. 
 
Title Grhl3 - curly tail microarray 
Description Data from microarray analysis performed in order to identify genes whose expression is altered in the curly tail mouse model of neural tube defects 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact Genes whose altered expression may contribute to development of neural tube defects have been identified. These genes are under analysis in functional studies. 
 
Description Axial Defects mice 
Organisation University of Amsterdam
Country Netherlands 
Sector Academic/University 
PI Contribution We have carried out extensive analysis of the Axial Defects mutant mouse - a model for spina bifida. This includes gene expression analysis, measurements of cell proliferation, morphological studies and genetic experiments to identify the causative gene.
Collaborator Contribution Collaborator supplied Axial Defects mutant mouse and shared data on linkage analysis which mapped the causative mutation.
Impact We have identified the causative gene in the Axial Defects mouse model (manuscript published)
Start Year 2008
 
Description Grainyhead-like genes 
Organisation University of California, Irvine
Country United States 
Sector Academic/University 
PI Contribution We have performed genetic and cellular analysis of mice carrying conditional targeted mutation of the Grhl3 gene.
Collaborator Contribution Our collaborators provided a conditional knockout mouse strain for Grhl3. They also shared data on conditional knokout in specific tissues.
Impact Data will contribute to manuscripts currently under preparation.
Start Year 2011
 
Description Karolinska - Genetics of NTDs 
Organisation Karolinska Institute
Country Sweden 
Sector Academic/University 
PI Contribution We have been investigating the development and prevention of neural tube defects in grainyhead-like3 mutant mice. These studies have involved generation of novel transgenic mouse strains. Proteomic analysis has identified a modifier gene, which is a candidate to be involved in human neural tube defects.
Collaborator Contribution Our collaborator has contributed to design and analysis of microarray and transgenic investigations of grainyhead-like mutant mice. In addition Dr Gustavsson has collected patient DNA samples for analysis of candidate genes for neural tube defects.
Impact We showed that reduced expression of grainyhead-like3 is the cause of neural tube defects in the curly tail mouse model. We identified a polymorphism as a modifier of neural tube defects risk in curly tail mice and began analysis in human patients. Publications: 18683893 and 17720888
Start Year 2006
 
Description Lamin B1 Cell Biology 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution We identified a variant form of lamin B1 in mice that are predisposed to spina bifida. We carried otu genetic experiments that showed that the lamin B1 variant strongly influences risk of spina bifida. We generated constructs and primary cell lines for analysis by our collaborators.
Collaborator Contribution Our collaborators carried out FLIP assay and cell morphometric analysis to test the functional effect of a variant of lamin B1 that we found to affect susceptibility to spina bifida
Impact This data forms part of 2 published manuscripts.
Start Year 2007
 
Description BBC News 24, BBC Online, BBC Regional News 
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 was interviewed for a television piece which focussed on spina bifida, its possible prevention by folic acid and the need for additional therapies as folic acid does not prevent all cases. The interview also introduced our current clinical trial.

Increased recruitment into clinical trial.
Year(s) Of Engagement Activity 2010
 
Description BBC Radio - Five Live 
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 Live radio interview discussing spina bifida, the need for women to take folic acid and the possibility that additional therapies are required. Resulted in increased awareness of clinical trial for prevention of neural tube defects by inositol - additional women entered trial after hearing radio interview.

Additional media interest in clinical trial. Contact from potantial trial participants and clnicians
Year(s) Of Engagement Activity 2010
 
Description BBC Radio Five Live 
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 Increased public awareness of research into neural tube defects.

Increased entry in clinical trial in progress in our laboratory.
Year(s) Of Engagement Activity 2010
 
Description BBC TV - Breakfast 
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 Live TV interview discussing spina bifida, the need for women to take folic acid and the possibility that additional therapies are required. Resulted in increased awareness of clinical trial for prevention of neural tube defects by inositol - additional women entered trial after seeing TV interview.

Increased public awareness of neural tube defects, the need for folic acid supplementation and the possible benefit of additional therapies, such the as one being tested in the clinical trial run by this resaerch group.
Year(s) Of Engagement Activity 2010
 
Description Careers forum 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact 80 undergraduate and post-graduate students attended a careers forum, involving talks and a question and answer session about academic careers

Many students reported that their understanding of the pros and cons of academia was clarified
Year(s) Of Engagement Activity 2009,2010