Development and function of the zebrafish vestibular system across the life course

The inner ear is the sensory organ that detects sound, gravity and motion, enabling us to hear and to balance correctly. Within the ear, sensory hair cells detect gravity together with body movement in all directions. This sensory information is relayed, via the brain, to muscular reflexes to enable an organism to maintain balance.

The inner ear is rightly also called the labyrinth. The vestibular (balance) part of the ear consists of three smoothly curved tubes-the semicircular canal ducts-together with several interlinked sensory chambers, which house the sensory cells of the ear. The whole organ is one continuous fluid-filled cavity. This beautiful and intricate structure is essentially similar in all vertebrate organisms, from the fish (which we use here as a model system) through to humans.

The inner ear develops in the embryo from a simple ball of cells, and the cell and tissue rearrangements that convert this rudimentary structure into the complex labyrinth of the mature ear are truly remarkable. Sheets of cells must grow, bend, fuse and rearrange. Different cells take on widely differing sizes and shapes to achieve this. These events must be tightly controlled to ensure that the correct cell types and tissue shapes are produced in the right place and at the right time.

Our primary aim in this proposal is to use cutting-edge imaging technology to describe and understand these cellular rearrangements. We use the zebrafish as our model system, as it is beautifully suited to this imaging approach. The zebrafish embryo is optically clear, meaning that we can see internal organs in the live animal under the microscope, without any need for dissection. Secondly, we can label cells with fluorescent proteins, lighting up different structures as they develop. By using specialised microscopes, we can measure dynamic changes in cell shapes and tissue movements as the whole organ develops in the live embryo. We will undertake these studies across the life course of the fish, including events during embryogenesis, metamorphosis and adulthood.

A major goal of the project is to develop and forge new links with engineers, who will use our imaging data to develop computer models of how cells change shape, move, fuse and rearrange to form the elaborate structures of the ear.

We also wish to understand the genetic factors that control ear development during embryogenesis. In our previous work, we have identified a number of genes that are critical for development of the semicircular canal system. A number of our fish strains carry specific genetic mutations in these genes: we will use these mutant fish in the imaging and computer modelling experiments described above, to gain new insights into how gene function affects cell movement, cell division and cell shape as the inner ear develops.

A final aim is to correlate our imaging data with vestibular behaviour in our fish. Several of our genetic strains develop with anatomical defects in the semicircular canal system, and have mild balance defects. We will use these fish to understand the contribution of the ear to balance function, using automated tracking of swimming behaviour. This will give us new insights into the function of the balance system of the ear, and its relative importance compared with visual and other functions.

Research on the vestibular (balance) system is hugely under-represented compared with that on the auditory (hearing) part of the ear, despite the fact that vestibular disorders are common and cause significant clinical problems, especially in the elderly. This project will help to redress that discrepancy and will contribute to the knowledge base that underpins our understanding of the human inner ear in both health and disease. We also aim to uncover fundamental developmental principles that will improve our understanding of how organ systems are built from sheets of cells in the developing embryo.

Morphogenesis of the vestibular system illustrates some fascinating underlying biology, but is an under-represented research area, lagging far behind studies on other morphogenetic processes such as neural tube closure. In this proposal, we aim to elucidate the dynamic cellular and tissue behaviour that underlies morphogenesis of the semicircular canals in the zebrafish inner ear. We plan to study the ear at four critical developmental stages, spanning embryogenesis to adulthood. We will use an interdisciplinary combination of cutting-edge imaging technology, computational modelling and behavioural analysis, exploiting genetic, transgenic and molecular tools to understand the dynamic cellular behaviours that underlie vestibular morphogenesis.

We are particularly interested in exploring the events that drive epithelial outgrowth, fusion and rearrangement to form pillars of tissue in the ear, which form the hubs of the developing semicircular canal ducts. We will use both confocal and light-sheet microscopy to image this process in the live embryo, utilising transgenic lines that express GFP specifically in the otic epithelium. We will use and develop computational methods to analyse our imaging data. We also aim to correlate our anatomical findings with behavioural data over the lifecourse of the zebrafish, exploiting a unique set of adult viable mutant lines that display vestibular deficits. We will use the Viewpoint tracking system to quantify these balance defects.

The proposed work is timely, building on our previous work on the genetic control of semicircular canal development in the zebrafish embryo. Although we aim to understand normal processes during development, the work will underpin and inform our knowledge of vestibular disorders, heart valve formation and palate fusion, all of which are of enormous clinical interest. We fully expect that the project will generate data to support future grant proposals with a clinical or translational element.

Who might benefit from this research, and how?
Academics:
In contributing to the scientific knowledge base, our work will benefit a wide range of academic researchers. These include colleagues within the University of Sheffield, the rest of the UK and worldwide. We expect that the findings will be of especial interest to other developmental biologists working on the ear and other aspects of epithelial morphogenesis. The work will be of relevance to those working on the auditory as well as the vestibular system; to understand how the ear functions as an integrated whole, it is important to know how it develops in the embryo.
We envisage that our imaging and computational image analysis work will contribute to improvements and developments in these approaches and will thus contribute to technology development in the field of bioimaging. Our data may also help to inform bioengineers for the development of synthetic scaffolds for tissue engineering. We will work with existing collaborators and establish new collaborations for the development of further projects and proposals.
A goal of the project is to train skilled and enthusiastic personnel. SB will benefit from the career development opportunities that this project offers in terms of practical skills, project management and presentation. The PDRA will benefit from training in computational image analysis, and the technician will benefit from training in a wide variety of laboratory techniques, including zebrafish techniques, molecular biology, compound and confocal microscopy. At the end of the project, all personnel will be well equipped to seek further employment in the academic or commercial sectors.

Commercial sector:
We do not expect this work from this basic science proposal to generate immediate benefits to the commercial private sector, but it is possible that development of software packages for image analysis, for example, will have wider applications. We will investigate any opportunities for commercial exploitation via the University's commercialisation partner, Fusion IP.

Health sector and charities:
Our work should benefit ENT clinicians and patients with vestibular disorders. Our goal is to understand more about how the ear develops, not to identify new treatments. However, such knowledge underpins the development of new diagnostic and therapeutic tools, and is an important part of the process. The work therefore has indirect benefits for both clinicians and patients. We have links with relevant charities (e.g. Action on Hearing Loss) and will aim to develop outreach activities with these charities in mind (see Pathways to Impact for more details).

The public and students:
We feel that one of our strengths is in the quality of the outreach programmes that we deliver. We are regular and active contributors to a range of outreach activities for children, teenagers and adults. Some activities are targeted at particular key stages of the curriculum; others are open to all. Our goals are to inform the public about what we do, encourage and develop their interests and curiosity in science, and attract the younger generation to pursue scientific careers. We also do this through our undergraduate teaching and by hosting undergraduate summer students to the lab. Benefits to society include a better-informed and educated public, who will take their knowledge and understanding with them as they take up careers in science or other influential professions, including industry, politics, arts or education.