Characterising the regulatory landscape during inner ear development

Hearing impairment is a debilitating condition that influences many aspects of normal life. The ability to hear is important in different contexts including for the development of speech and cognitive skills in children, for performance at work and normal social interactions in adults. Worldwide, about one of every 800 babies is born with hearing problems, while more than 50% of adults over 60 suffer from some form of hearing deficit. Hearing loss in children is often associated with defects of the inner ear, the part of the ear that houses the cells that perceive sound and balance (hair cells) and generates the neurons that transmit information to the brain. In older people, hair cells die gradually, and cannot be replaced either from other cells within the ear or through cell-based therapies. This leaves hearing aids or cochlear implants in the profoundly deaf as the only solutions.

Although we have made much progress in understanding the genetic and other causes for hearing loss, there are still many conditions where the origin remains unknown. This is largely due to the complexity of the ear, but also to the fact that we still do not fully understand the dynamic genetic programme that controls ear formation in the embryo. The latter is important because mutations in many developmental genes are associated with deafness. On the other hand, many patients that present with deafness or syndromes associated with hearing impairment do not have any mutations in known genes.

Here we propose to establish a complete road map for how progenitor cells, which have the potential to contribute to all sense organs, are committed to ear fate and prevented from taking up other identities. In other words, we will produce a genetic network containing the 'driving instructions' for ear formation.

We have already defined distinct steps during this process, and all the players that help cells to make the correct choice at each crossroad. However, we now need to establish the sequence in which they work, how they control each other and how this process is regulated on a genome-wide level. To this end we have adapted modern molecular biology techniques to a well-studied amniote model system, the chick. The chick embryo lends itself to experimental manipulation, and importantly to rapid testing of our findings in the living embryo. We will now determine:

- the genomic control regions that direct when and where each of the players is used

- where and when these genomic control regions are active in inner ear cells

- the 'master regulators' that interact with these genomic control regions, and hence act upstream of the players

- the way in which one of the key players, the transcription factor Six1, interacts with its genomic surrounding to control the road towards ear, but away from lens fate

We will assemble this information into a large interactive network, which will be made publicly available. This network will allow us and other researchers i) to model the process of how cells become definitive ear cells by providing the detailed instructions encoded in the genome; ii) to predict the functional outcome (phenotype) caused by mutation or deletion of any of its components and vice-versa.

In addition, the project will identify new candidate genes and candidate regulatory control regions for human deafness and their potential functions, provide important information for reprogramming cells into ear progenitors and for re-activating cells in the ageing ear, and provide better tools for diagnosis. Finally, because changes in genetic control regions are the critical drivers of evolutionary change and therefore for biological diversity, our data will also be exploited to study the evolution of complex sense organs.

This project will generate a definitive gene regulatory network that models how sensory progenitors acquire ear identity. We have adapted new, modern molecular biology technologies to an easily tractable in vivo system, the chick. In this project we will:

- Characterise enhancer changes during ear commitment using i) ChIPseq for H3K4me1, H3K4me3, H3K27ac, H3K27me3 to identify enhancers genome-wide and ii) ATAC-seq, to determine nucleosome-bound and -free regions.

- Validate enhancer activity in vivo using a newly developed approach for assessing 10 enhancers or more in a single embryo.

- Use motif enrichment algorithms for enhancers to identify 'master regulators'.

- Assess 'master regulator' requirement for enhancer activity by combining loss-of-function with our new strategy to monitor activity, for target mRNA expression and for otic development.

- Determine the 3D chromatin landscape using 4Cseq with the Six1 promoter as a view point

The network will provide a mechanistic view of the ear programme, have the power to predict phenotypic consequences of gene mutations and of changes in regulatory interactions due enhancer alteration and the functional outcome thereof. Our data will provide a complete, genome-wide view of the dynamic changes of enhancer activity in a developing system.

Who will benefit from this research?
In the long term, this research will benefit children with congenital hearing loss, their parents and adults with age-related hearing impairment, translational researchers engaged in stem cell therapy, medical professionals, like paediatricians, otolaryngologist and genetic counsellors.
The project focuses on ear research, but the general principles will be applicable to other topics. Thus, our research will spark interest of the general public interested in science, with our collaborative arts/science project making it widely accessible. We will also contribute to the UK economy and scientific reputation by training highly skilled researchers in scientific methods and transferable skills, and by engaging with schools, 6th form students etc..

How will they benefit?
Hearing impaired and clinicians:
Hearing impairment is an ever-increasing problem in adults, often leading to social isolation, depression and inability to hold or perform well in a job. In children, hearing loss seriously affects normal development, development of speech, cognitive skills, communication and literacy. Thus, hearing defects have an enormous impact on quality of life of the affected individuals, their family and the community they live in.

Although this project asks basic science questions, they are highly relevant to human deafness. This research will identify new candidates for human deafness, and enhancer regions associated to deafness loci; together this will complement GWAS. This will in the long-term lead to better diagnosis, new strategies for drug design and a better understanding of the causes for deafness.

Currently, no cell-based strategies are available to cure deafness. We will contribute to the development of new strategies for cell re-programming, for generation of ear cells from stem cells or for re-activating differentiated cells in the adult ear.

Overall, our research will therefore benefit individuals affected by hearing loss, as well as the clinicians and medical professionals engaged in the design of new treatments.

General public:
This project will generate an interactive model for early ear development; while this is complex the use of BioTapestry makes it simple to use and understand. Interested individuals can navigate the network, zoom into small circuits and play with the data. This will help the general public to understand the causes of deafness, but also teachers to demonstrate the complexity of biological systems. Our planned photography/video collaboration will generate a lot of interest, and thus actively engage the public in science. This will lead to better understanding of ear related problems, but hopefully also attract more students to study science related subjects. In the long-term this will therefore benefit the UK economy.

The project will contribute to PDRA training in cutting edge technology, relevant to human disease, and in transferrable skills. I am personally involved with training incoming new scientists, and actively engaged in career mentoring to equip them for future challenges far beyond the technical aspects of research. The project will therefore generate highly trained scientist with a large spectrum of transferrable skills. In turn, they will contribute to the UK economy and reputation. In addition, the project will involve junior scientists during summer projects, work experience, or from abroad to encourage engagement in science and enhance international collaborations.