Integration of BMP and Wnt signalling in the developing zebrafish ear

We are fascinated by the inner ear and the generation of its beautiful and intricate structure during embryonic development. In the adult, this complex sensory organ detects sound, gravity and motion, enabling us to hear and to maintain balance. In this project, we aim to uncover the mechanisms by which cells signal to one another in the embryo to ensure that the inner ear develops correctly.

The inner ear develops in the embryo from the otic vesicle, a simple ball of cells that forms in close proximity to the developing brain. During embryonic development, cells of the otic vesicle and of the immature brain send and receive molecular signals, relaying instructions about when to activate the expression of different genes. This signalling activity results in cascades of gene activity, leading to the correct formation of different structures in the inner ear. We are particularly interested in the signals that instruct the otic vesicle to form the semicircular canals, curved ducts that function in the vestibular (balance) part of the ear.

We will focus on two sets of signalling components, known as the BMP and Wnt pathways. We will disrupt individual proteins in these pathways either in ear or in developing brain tissue to examine the effects on ear development. Our preliminary studies indicate that a protein known as Bmper acts as a hub to integrate signalling information from both pathways in the developing ear. This is a new idea that has not yet been tested in any system, to our knowledge.

Recent studies in the lab of our collaborator at the University of Exeter and others have shown that many signals are delivered to and received by cells on cytonemes, dynamic thin extensions of the cell membrane. The signalling molecules themselves are thought to be localised to the tips of these membrane protrusions. We hypothesise that cytonemes are involved in signalling to the developing ear, and part of our study will involve searching for these structures, and the signalling molecules within them, using high-resolution microscopy.

We will use the zebrafish embryo as our model system, for two main reasons. Firstly, the zebrafish embryo is optically transparent, meaning that we can see internal organs such as the brain or ear in the live embryo under the microscope, without any need for dissection. We can label cells with fluorescently-tagged proteins, lighting up different structures as they develop. Secondly, a new technique known as CRISPR interference will allow us to block gene function in a very precise manner in either the ear or developing brain tissue, without affecting other tissues in the embryo. Our colleagues at the University of Sheffield are developing this technique as a community resource for those working on the zebrafish model system, and we will exploit this in our project.

Our work aims to uncover fundamental developmental principles about how signalling molecules instruct the normal formation of a developing organ system in the embryo. However, the findings are likely to be of interest and relevance to medicine: in humans, mutations in BMP pathway genes are causative of congenital genetic disorders, while later in life, inappropriate activation of the Wnt signalling pathway underlies many cancers.

In this proposal, we aim to elucidate the signalling pathways that are required for correct morphogenesis of the zebrafish inner ear. We have identified a zebrafish mutant, bmper, with truncated anterior and posterior semicircular canal ducts. Bmper is known both to promote and inhibit BMP signalling in different contexts, but its role in the ear was not previously known. Interestingly, we also see the same specific canal defect in zebrafish ears when we mis-express dkk, a Wnt inhibitor, revealing that Bmper may act to integrate BMP and Wnt signalling in otic tissue.

We will characterise BMP and Wnt signalling pathway activity in the developing zebrafish ear by imaging transgenic fluorescent reporter lines using light-sheet and high-resolution confocal microscopy. A key goal is to generate new tools for imaging and conditional manipulation of the signalling pathways in the ear and hindbrain using CRISPR interference. Recent evidence has shown that signalling molecules, including Wnts, are transmitted via cytonemes, specialised signalling filopodia, that make specific contacts between cells. These structures have not yet been described in the ear. We will collaborate with Steffen Scholpp (Exeter) and use fast light-sheet microscopy and fluorescence cross-correlation spectroscopy to image cytonemes and signalling complexes in the developing zebrafish otic epithelium and surrounding tissues.

The proposed work is timely, building on our previous work on imaging semicircular canal development in the zebrafish embryo, and benefitting from recent BBSRC investment. Although we aim to understand normal processes during development, the work will underpin and inform our knowledge of vestibular disorders, congenital disease and cancers. Our findings are likely to be of significant interest and relevance to those working on organogenesis, cell signalling and imaging.

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 including those working on the ear and epithelial morphogenesis. The work will also be of relevance to those working on signalling pathways, cytonemes, imaging and all aspects of zebrafish developmental biology. We envisage that the tools and imaging datasets we generate will contribute to improvements and developments in these fields and will inform further technology development in the field of bioimaging. 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. NvH will benefit from gaining further experience in computational image analysis, and the Research Technicians will benefit from training and further experience 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; however, it is possible that findings from the BMP and Wnt signalling pathway will relevant to the identification of new targets for drug discovery. We will investigate any opportunities for commercial exploitation via the University's commercialisation partner, Fusion IP.

Health sector:
In the longer term, our work will benefit patients with vestibular disorders, cancer and other diseases associated with dysregulation of the Wnt and BMP signalling pathways. Our immediate 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. A high-profile example is the fundamental work done on the Hedgehog signalling pathway in the fly, which has ultimately led to the development of new pharmaceutical treatments for various cancers. Our work may therefore have indirect benefits for both clinicians and patients. We will look to engage with relevant charities and healthcare professionals where opportunities arise (see Pathways to Impact).

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 in 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.