Novel approaches to define tissue fusion mechanisms in embryonic development

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

Abstract

Disruptions to how tissues fuse together during human development is a common cause of birth defects, affecting approximately 1 in 500 people in the UK. This research has been prompted because despite our best efforts, most patients born with problems such as cleft palate, spina bifida, and heart defects still don't have the genetic cause of their disorder identified. This often impacts genetic counselling and efforts towards prevention. We also remain unsure what impacts maternal environment have on these conditions (e.g. illness, vitamin deficiency, or substance abuse). I aim to address these by performing transformative research to reveal the key genes, cell behaviours, and molecular systems required for normal tissue fusion, and provide a step-change in our knowledge of how these can be perturbed.
To enhance our understanding of fusion, we require experimentally versatile and appropriate model systems. I will focus on the causes of ocular coloboma, the leading inherited cause of blindness arising from a fusion defect of optic fissure closure (OFC) in the eye in the first 7 weeks of pregnancy. This defect leaves a persistent gap in the retina and optic nerve which cannot be cured or repaired. I have established the chick eye as a powerful and tractable new model for fusion because of a unique combination of features: (i) it closely resembles OFC in humans; (ii) experiments can be performed inside the egg; and (iii) chick eyes are sufficiently large to accurately dissect fusing tissues for further experimentation. At Roslin Institute we have also developed unique transgenic chickens with fluorescent cells whose behaviours I can observe throughout OFC, and from which I can selectively isolate specific cells to reveal their unique gene expression profiles. This work will combine these new technologies with other cutting-edge techniques in DNA sequencing, live-cell imaging, gene-network analysis and cell behaviour modelling to generate the most robust and accurate framework of OFC and tissue fusion information available to date.
The first aim of this project is to reveal gene expression levels and molecular signatures for the cells directly taking part in the OFC fusion process. I will also determine the changes in morphology, movement and organisation of these cells using mathematical and computer simulations, and integrate this with my molecular data. Next, by selectively ablating OFC-specific genes using gene-editing techniques, I will determine the consequence of their loss or dysregulation to learn more about the genetic drivers and modifiers for specific cell behaviours during fusion. Understanding the function of the Netrin-1 gene is a key part of our work, as I have recently shown that this is an essential factor for normal OFC in diverse species, and that it is required for other developmental fusion contexts (eye, ear and palate). I will then go on to reveal how Netrin-1 and other fusion-specific genes are regulated by genome-wide networks, allowing us to understand better the biological consequences of mutations identified in human patients, and to accurately predict new disease-causing candidate genes and the effect of non-genetic factors on the regulation of gene expression in fusion.
This is a powerful combination of tools to transform fusion biology. It will provide valuable information for how specific challenges during embryogenesis can disrupt genetic and cellular programmes, directly or indirectly leading to developmental fusion defects. The information I reveal will help clinicians to uncover new causative mutations and provide evidence to support genetic counselling in affected families. In the longer term, my system will reveal how the maternal environment can influence the causes of fusion defects, and help define strategies to reduce their incidence. Lastly, my data will reveal how gene function links to cell behaviour, providing insight for a broad range of biological contexts in health and disease.

Planned Impact

This work will impact upon the following beneficiaries. See Pathways to Impact for details of how these will be delivered.

Improved health: Patients, affected families, clinicians and health workers. The findings from this study will influence the identification of genetic variants that cause developmental diseases though partnerships with human geneticists. Genes identified as important in optic fissure closure can be easily assessed for mutations in patient cohorts, through working with clinical collaborators and giving direct clinical relevance to patients and their families. Subsequently, these can also be included in genetic testing screens in wider clinical settings, for example in the NHS for patients with inherited eye disorders. These could also have a major impact on the interpretation of disease inheritance for genetic counsellors, to help reduce disease incidence and allow for prenatal or preimplantation diagnostic testing. The factors identified may similarly improve understanding of other fusion defects with the same potential impacts. In the longer-term, this work will provide a platform to understand the influence of maternal environment on disease susceptibility and penetrance, for example exploring the impact of alcohol/drug abuse, vitamin deficiency or viral infection. This can support evidence-based strategies for public health bodies to improve public knowledge of susceptibility and risk factors for reducing the incidence of these birth defects.

Raised awareness of basic research: The public, charities, and research councils. This work will increase the profile, importance and awareness of basic vision research among the UK public through the dissemination and social media strategy, and key findings will also be packaged towards journalists and the media (see Communications Plan). In turn this will create wider awareness of basic research into fusion defects among the public, and will help to maintain a supportive public feeling towards the worthwhile use of research council funding for such studies, indirectly giving support to fundraising efforts for patient charities.

Reduced use of animal models: Animal welfare stakeholders. The use of chick models for developmental biology research and the application of novel avian genetics approaches will promote wider uptake of the chick as a model system among developmental biologists. Use of the chick as an alternative to rodents (e.g. the laboratory mouse where female dams must be sacrificed for embryos) can provide significant impact to reduce the number of animals used in experimentation, as adult laying hens used in research are not sacrificed to produce embryos. This benefit will considerably impact on researchers, members of the public and research councils seeking to reduce or refine animal use in research.

Publications

10 25 50
 
Description Collaboration with Brian Brooks - Director, National Eye Institute. NIH 
Organisation National Eye Institute (NEI)
Country United States 
Sector Academic/University 
PI Contribution Establishing the use of chick as a model for vertebrate structural eye development. This is novel and complements other groups use of alternative models (zebrafish and mouse) for understanding human eye development. In particular, our strengths are the investigation of structural development, such as optic fissure closure and anterior segment and iris development.
Collaborator Contribution Professor Brooks is the Director of The National Eye Institute. (NIH, US). His group is focused on the identification of mutations in genes that cause structural eye defects, and uses zebrafish to model genetic defects.
Impact Currently we have a manuscript in preprint, detailing the identification of a novel coloboma associated gene and the transcriptional landscape of tissue fusion in the embryonic chicken eye.
Start Year 2017
 
Description Donna Fekete. Purdue University, Indiana, US. 
Organisation Purdue University
Country United States 
Sector Academic/University 
PI Contribution I share resources, methodologies and data pertaining to the delivery of exogenous gene constructs into the developing chicken embryo, and details of their impact on embryonic tissue fusion events.
Collaborator Contribution They reciprocally share resources, methodologies and data pertaining to the delivery of exogenous gene constructs into the developing chicken embryo, and details of their impact on embryonic tissue fusion events.
Impact None as yet.
Start Year 2019