Chrysostomou, E.; (2012) Investigating the roles of Notch signalling during inner ear development: new insights into the dynamics of Notch activity and the functions of Delta1. Doctoral thesis , UCL (University College London).

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Abstract

The Notch signalling pathway has ubiquitous roles during inner ear development. An early phase of Notch activity, via the ligand Serrate 1 mediates lateral induction and promotes the early formation of inner ear sensory patches. In addition, Notch signalling mediated by the ligand Delta1 regulates otic neurogenesis and hair cell formation by lateral inhibition. To get new insights into the dynamics of Notch-activity during these processes, a fluorescent reporter consisting of the cis-regulatory element of the mouse Hes5 gene was introduced in the embryonic chick inner ear. In ovo electroporation was used to transfect the otic cup and to demonstrate the sensitivity of the Hes5 reporter to Notch activity. Using the retroviral vector RCAN and the Tol2 transposon system this reporter was stably integrated into the cells genome following electroporation of the otic cup. At late stages of inner ear development, the reporter was specifically activated in sensory progenitors and supporting cells but not in differentiated hair cells. The reporter was also shown to be sensitive to artificial gain- and loss-of-Notch activity. Time-lapse imaging of transfected sensory epithelia showed intercellular differences in fluorescent levels and variations over time suggesting endogenous variations of Notch activity occur within progenitor cells. Extensive proliferation and cell rearrangements could also be directly visualised at the time of hair cell differentiation. In order to test the role of lateral induction during sensory patch formation, the Hes5 regulatory element was next used to drive Delta1 expression within Notch-active cells. This created an artificial positive feedback loop mimicking the endogenous lateral induction via Serrate1. This resulted in abnormal inner ear morphogenesis and disrupted hair cell differentiation within the sensory epithelia. The inner ears transfected with the Hes5- Delta1 construct had a smaller vestibular region, cochlear defects and ectopic hair cell formation, suggesting abnormal boundary formation between sensory and non-sensory regions. Furthermore expression of Delta1 inhibited hair cell formation in trans but promoted hair cell differentiation in cis. The analysis of these effects at the single cell level provided new insights into the function of Delta1 during lateral inhibition.

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