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Methods to isolate and culture adult zebrafish CNS neurons: Role of various neurotrophic factors
Author(s) -
Navneet A. Vasistha,
Jamin DeProto,
Sheena Lee,
Zoltán Molnár,
Kavita Babu,
Zhitao Hu,
Shih-Chieh Chien,
Gian Garriga,
Joshua M. Kaplan
Publication year - 2012
Publication title -
international journal of developmental neuroscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.761
H-Index - 88
eISSN - 1873-474X
pISSN - 0736-5748
DOI - 10.1016/j.ijdevneu.2012.03.316
Subject(s) - zebrafish , neuroscience , neurotrophin , neurotrophic factors , brain derived neurotrophic factor , biology , genetics , gene , receptor
E-mail address: navneet.vasistha@dpag.ox.ac.uk (N.A. Vasistha). Recent research has conclusively shown that the sub-ventricular zone (SVZ) is an important site of neurogenesis while also playing a role in the evolutionary growth and development of the neocortex. During neurogenesis, radial glia lining the ventricular epithelium divide to generate a neuron while also renewing themselves. In addition, the radial glia also generate intermediate progenitor cells (IPCs) that migrate to the embryonic SVZ and further proliferate to generate neurons. In this manner, IPCs are able to amplify neuronal output of the ventricular epithelium without increasing the ventricular surface area. Intermediate progenitors are marked by the expression of Tbr2 (also Eomesodermin); a transcription factor that separates it from the ventricular progenitors. However, despite the widespread use of Tbr2 as a marker, its function remains unknown. Loss of function studies using conditional knockouts show a decrease in the thickness of the cortical layers with both upper and lower layers affected equally. Conversely, over-expression studies show an increase in pH3+ cells with an expansion of the VZ/SVZ thickness and also a shift towards basal progenitor cell identity. In order to understand the molecular role of Tbr2 in IPCs we undertook an overexpression microarray experiment. We show that overexpression of Tbr2 triggers an up-regulation of Cdk4 and Aspm, 2 genes shown to have important roles in cell-cycle progression. We also show that the regulation of these by Tbr2 is by binding to their upstream sequences. Additionally, we also observe an up-regulation of Wnt5a in response to Tbr2 overexpression. Cumulatively, this suggests that Tbr2 has an important role in regulating the cell cycle and hence can influence the rate of neurogenesis. We aim to probe further on the effect of these changes and the pathway by which such regulation is brought about.