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Title: Analysis of Wt1 expression in neural crest cells
Author: Allardyce, Joanna Marie
ISNI:       0000 0004 2734 4604
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2012
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The neural crest is a transient collection of cell, termed neural crest cells (NCCs), which develop during neurulation at the outer extremities of the neural folds between surface ectoderm and the developing neural tube. NCCs del aminate from the crest and migrate throughout the developing embryo and differentiate into many cell types such as melanocytes, peripheral neurons, osteocytes, muscle cells and enteric neurons and glia. With the use of a lineage tracing system (Wtl-Cre X Rosa26R mouse line) it was previously found that cells derived from Wtl-expressing cells have contributed to the post-natal enteric nervous system (ENS), indicating that Wtl must have been expressed in progenitor cells of the ENS during embryonic development. The goal of this project was therefore to identify when and where Wtl is expressed during this process. Data from immunofluorescence studies revealed that Wtl is transiently expressed in Sox10-expressing NCCs when they first begin their migration from the neural crest, at E8.5 in the mouse. Wt1 is then down-regulated in NCCs before they enter the foregut at E9.5. This data has been supported by in situ hybridisation studies, where Wtl has been found in cells of the neural crest at the same time point (E8.5) but Wtl mRNA was not shown to be present at any embryonic stage later than this. In vitro investigations were carried out in order to characterise Wtl in vagal level NCCs, as it is NCCs from this region (opposite somites 1-7) and the sacral neural crest (caudal to somite 28) which have been established as the origin of the ENS. NCCs were characterised by morphology and marker expression over a time-period of 7 days. The results from immunofluorescence experiments revealed co- expression of Wtl and NCC markers, SoxlO, up to 96 hours in culture. After this time-point it was no longer possible to detect these proteins in cultured explants. The neuronal marker ~III Tubulin was detected from 48 hours and was still found to be expressed at high levels after 96 hours when Wtl and NCC markers had ceased to be expressed, suggesting differentiation ofNCCs. Migration assays whereby the rate of migration was determined in NCCs in culture over a period of 48 hours revealed a mean migration rate of oum/hour. These data are relevant for future siRNA Wtl knock-down experiments in NCCs in vitro to investigate the effect of loss of Wtl function on migration rates, cell morphology, and expression patterns following preliminary experiments carried out on kidney stem cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral