Non-cell autonomous effects on integrin signalling on neurogenesis in the chick embryonic CNS
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A fundamental characteristic of neural stem cells (NSCs) is their ability to divide symmetrically, producing daughter cells of different fates. This requires the regulation of proliferation verses differentiation, which could occur in two different ways; at a cellular or environmental level. Although there is some evidence for each model, little is known about how this regulation occurs. One candidate is the integrin family. Integrins are known to regulate key aspects of stem cell behaviour. The integrin subunits α6 and β1, which heterodimerise into a laminin receptor, are highly expressed within the NSCs of the embryonic ventricular zone and loss of integrin β1 (itgβ1) function within the ventricular zone of the embryonic mouse results in NSC detachment and apoptosis. The aim of this thesis is to investigate the role of itgβ1 in the regulation of proliferation verses differentiation in the NSC of the chick embryonic neuroepithelium. To test the function of itgβ1 within this system, we are using transfection of a constitutively active (CA*), wild type (WT) and extracellular portion only (EC only) integrin-β1, via electroporation of the chick embryonic CNS, resulting in a patchwork of expression within the NSCs of the midbrain neuroepithelium. This system allows both expressing cells and their non-expressing neighbours to be studied within the same environment. We predicted that if integrins are acting to regulate NSC behaviour via an intrinsic mechanism, only the cells expressing the CA* integrin will alter their behaviour. If the second model is correct and integrins act via an extrinsic mechanism, we predicted that the neighbouring non-expressing cells will also alter their behaviour. We observed a significant increase in the number of neurons generated upon expression of CA*β1. This increase in neurons was a non-cell autonomous effect; the neurons were GFP and human itgβ1 negative, supporting the second model of extrinsic signals and cell-cell interactions in the regulation of proliferation and differentiation. The increase in neurogenesis was only observed in the midbrain upon CA*β1 expression for 48hrs. A significant increase in mitotic cells was observed 12hrs after electroporation, the earliest time point and by E4 (48hrs) a significant proportion of mitotic cells were abnormally located by 48hrs, resulting in basal mitoses. Investigation of signalling between cells was carried out using microarray analysis of the two populations of cells, CA*β1 positive and negative. One candidate from the microarray results was the bHLH transcription factor Tal2. Tal2 has previously been shown to be specifically expressed within the midbrain neuroepithelium at the time of electroporation and to play a role in the regulation of neurogenesis. In summary, this thesis has showed an important role of itgβ1 in the regulation of proliferation and differentiation of NSCs within the chick embryonic neuroepithelium in a non-cell autonomous manner.