Metabolic refinement of sensory cell development

Our ability to understand speech, listen to music or hear the high pitch of a mosquito, relies on highly specialised cells in our inner ears called hair cells (HCs). These cells are named so because of the presence of hair-like bundles on their apical surfaces made of a special protein called Actin. Every sound we hear is composed of many different frequencies. The primary job of HCs is to break down complex sounds, like speech and music, into their individual frequency components. This process takes place along the length of the hearing organ, the cochlea. One way to envisage this, is to picture HCs in the cochlea much like the keys on a piano, where each cell, or key, responds to a specific frequency or note. Developmental errors in how HCs form or damage and loss of HCs causes permanent hearing loss. There are currently no biological therapies available to replace these cells once they are lost. Successfully identifying the factors needed to generate functionally viable HCs requires a detailed understanding of the basic cell biology and developmental programs that drive their formation during development. Recent advances in the field mean it is now possible to make hair cells in a dish from both human and mouse stem cells. However, although these cells look like hair cells and possess a number of their physiological properties, they never reach functional maturity and they don't survive long-term. HCs generated in this way also do not display the same level of functional diversity seen in a normal cochlea. It is not clear at present what goes wrong during their formation to cause these problems.

Each cell type uses a different method to generate and burn energy substrates (known as their "metabolism"). These different metabolic pathways are important for regulating how cells form during development and if they are perturbed this can cause errors how the cells function during later life. In an attempt to identify novel factors that fine tune the functional properties of cochlear HCs, this project will explore how metabolism could be used as a tuneable tool with which to direct functional branch-points during their development. We will characterise the metabolic profiles of developing HCs, and determine how they change with frequency position along the cochlea. We will then explore how metabolism might be exploited to generate these functionally distinct types of sensory cell. We have already found that if metabolism is disrupted during development, HCs do not form properly. We now want to understand why this happens. Why does metabolic disruption cause these errors in normal HC development? We are addressing this novel question by investigating its role in the specification of sound frequency coding by HCs.

The complex network of developmental pathways important for cochlear cell fate specification is well defined. We want to understand how metabolic and developmental signalling molecules interact during development to specify cell fate. Understanding how metabolic and developmental signalling cues regulate the basic cell biology in developing HCs has significant future impact not only for hearing loss but also any tissue such as they eye, the skin or even teeth, where there is great need to replace lost or damaged cells.

Hearing relies on specialised sensory receptors in the cochlear neurosensory epithelium known as hair cells (HCs). Along the basal-to-apical axis HCs are tuned to different sound frequencies ("tonotopy"). Basal HCs respond to high frequencies and apical HCs to low frequencies. Understanding how these properties are specified requires a detailed understanding of the developmental programs and basic cell biology involved in their formation. As the programs driving cochlear cell fate are highly conserved in vertebrates, this project capitalises on the experimental advantages presented from a combined species approach. Birds are born hearing with tonotopic gradients at both the physiological and morphological level. It is easy to access and manipulate the chick cochlea throughout the entirety of cochlear development making it a more tractable model to test the role for metabolism in HC development. The aim is to identify critical time windows for metabolic re-programming in the chick and translate these to mouse.

Metabolic pathways can determine cell identity. Switching between pathways of energy metabolism, known as metabolic-reprogramming, regulates the activity of cell-specific transcription programs that specify different fates. We will characterise the metabolic profiles of different cochlear cells as they develop using fluorescence lifetime imaging of cellular NAD(P)H, live Airyscan confocal imaging of mitochondria and CLEM microscopy to study mitochondrial network morphology.

Once we know the metabolic profiles of basal and apical cells, and when reprogramming of their metabolic pathways takes place, we will perturb these pathways and determine the effects on tonotopy and the developmental signalling pathways that establishing it. We will use pharmacological and genetic approaches, and the effects on tonotopy will be determined using antibody staining for known markers combined with in situ and SEM to confirm expression patterns and morphology.