The initial induction and subsequent patterning of the central nervous system (CNS) are both mediated by morphogenetic signals emanating from some transient cell populations referred to as signalling or organizing centres. Following induction of the CNS, global signals mediate the establishment of its gross mediolateral and anteroposterior axes at gastrulation and early neural plate stages. Subsequent to this, local signals refine these specifications. Local signalling centres usually lie at boundaries of the tissues they pattern. Also, they are rich sources of morphogens, and are capable of ectopically inducing cell fate changes in adjacent tissues when transplanted. The eminentia thalami (EmT) forms a boundary between the diencephalon and telencephalon during embryonic forebrain development. In amphibians and fishes, the EmT continues to be a prominent structure in the postnatal diencephalon though its role in these is still largely unknown. However, in mammals, it appears transient, being identifiable between embryonic days 11 (E11) and 17 (E17) in the mouse, but not discernible at other ages. Though its function is yet to be determined, available experimental evidence suggests that it might act as a signalling centre in forebrain patterning. Therefore, this study aimed at identifying in the EmT members of the Wnt, Fgf and Bmp families of morphogens implicated in patterning elsewhere, and their spatial and temporal patterns of expression; and also determining whether the EmT is able to induce ectopically cell fate changes in adjacent tissues when transplanted. To address the first aim, Reverse-transcription Polymerase Chain Reaction (RTPCR) and In Situ Hybridization were used to determine the spatial and temporal expression of some members of the Wnt, Fgf and Bmp signalling systems in the EmT in both wild-type mice and Pax6 (Sey/Sey) mutants. The results indicate nested expression of some Wnts, Fgfs and Bmps mRNA in the ventricular zone of the wildtype EmT till E14.5, after which they appear down-regulated. Also, Wnt7b and Wnt8b show the strongest expression at these ages and this may indicate a key role for these genes in the function of the EmT in mammals. In Sey/Sey mutants, the EmT was malformed as shown by the mis-expression of its markers. Additionally, the Wnt, Fgf and Bmp genes normally expressed in the wild-type EmT were either not expressed in the mutant or were mis-expressed. The second aim was addressed by transplanting the EmT into the ventral telencephalon, and using immunohistochemistry and in situ hybridization to analyse for the expression in the ventral telencephalon of Lef1, a transcriptional activator in the Wnt/β-catenin signalling pathway, which is not expressed here, as well as Foxg1, Mash1 and Islet1, three transcription factors normally expressed here. EmT explants induced ectopically Lef1 expression in the ventral telencephalon. Also, while the Lef1-expressing ventral telencephalic cells did express Foxg1, a telencephalic marker, they either did not express or sparsely expressed Mash1 and Islet1, which are specific markers of the ventral telencephalon. These results suggest that the EmT may possess some ability to induce cell fate changes in the ventral telencephalon. The role of Wnt/β-catenin signalling in the function of the EmT was also investigated by analysing Lef1, Foxg1 and Mash1 expression in the ventral telencephalon of cultured E13.5 brains in which Wnt signalling had been activated in the ventral telencephalon through small molecule inhibition of GSK3β, a member of the β- catenin destruction complex. Lef1 expression in the ventral telencephalon increased with an increased inhibition of GSK3β activity. Also, while Foxg1 was expressed normally in the ventral telencephalon irrespective of the level of GSK3β inhibition, there was a significant dose-dependent reduction in Mash1 expression here. These results show that up-regulation of Wnt signalling in the ventral telencephalon may result in cell fate changes here, and also suggest that ventral telencephalic cells are competent to respond to Wnt signalling at this stage of development.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:693576 |
| Date | January 2013 |
| Creators | Adutwum-Ofosu, Kevin Kofi |
| Contributors | Price, David ; Fotaki, Vassiliki |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/16247 |
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