The clusterin gene in mouse inner ear development: expression analysis and generation of reporter constructs

Yadollahi, Faranak (2013) The clusterin gene in mouse inner ear development: expression analysis and generation of reporter constructs. Doctoral thesis (PhD), University of Sussex.

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Abstract

Clusterin has previously been identified as a gene potentially involved in development of the cochlear sensory epithelium. In order to be able to predict the cellular roles that clusterin may play in the development of this organ, an understanding of the spatiotemporal expression pattern is required. Therefore, clusterin gene expression during mouse inner ear development was studied using riboprobes from the mouse gene. Clusterin mRNA demonstrates a dynamic expression pattern within the developing cochlear sensory epithelium. Clusterin mRNA expression is initiated at 12.5dpc (days post coitum) along the entire length of the cochlear sensory epithelium. Throughout in utero development, expression is maintained but becomes progressively restricted in this sensory epithelium. Postnataly expression resolves to specific cellular regions, but clusterin expression ceases at some time point between P2-P17.

The analysis of clusterin protein expression revealed this was not restricted to the developing sensory epithelium alone, but also was detected transiently in the periotic mesenchye, otic capsule, as well as Reissner’s membrane, a non-sensory epithelium. The detailed localisation of clusterin was compared to other inner ear markers. Using α and β tectorin mRNA markers, clusterin mRNA was shown to localise to the developing inner and outer sulcus and spiral prominence. Clusterin expression also overlaps with both Myosin VIIA and Prox1, markers for hair cells and supporting cells respectively. Clusterin mRNA and protein was absent from the developing mouse vestibular system.

In order to study the regulatory mechanisms underlying inner ear clusterin expression, a clusterin BAC was modified by insertion of ZsGreen reporter gene into clusterin genomic regions using recombineering technique. The pronuclear injection of this construct has not been successful so far although these studies are ongoing. Finally in order to determine the fate of clusterin expressing cells after the expression is ceased in the inner ear, clusterin BAC was modified by insertion of Cre recombinant gene at the same location as ZsGreen gene for the generation of Cre transgenic mouse. This transgenic mouse will be crossed with a silent reporter mouse for future clusterin fate mapping studies.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biology and Environmental Science
Subjects: Q Science > QL Zoology > QL0605 Chordates. Vertebrates > QL0700 Mammals > QL0737 Systematic divisions. By order and family, A-Z > QL0737.R6 Rodentia
Depositing User: Library Cataloguing
Date Deposited: 21 Jun 2013 10:32
Last Modified: 15 Sep 2015 12:05
URI: http://sro.sussex.ac.uk/id/eprint/45293

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