Callard, N.A.L.; (2008) Time-lapse studies of neural precursor cell divisions in vitro. Doctoral thesis , University of London.

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Abstract

The entire adult central nervous system (CNS) derives from an initially small population of apparently homogeneous neuroepithelial precursor cells (NEPs) which produce specific differentiating cell types in a highly organised fashion with respect to both the time and place at which they are generated. A unifying phenomenon throughout the CNS is that neurons are always generated before glia. To better understand what mechanisms might be involved, the dynamics of precursor lineages need to be described. Here, single NEPs from the murine dorsal embryonic neocortex were cultured at clonal density and filmed using time-lapse microscopy to monitor their divisions over time. The progeny they gave rise to were identified by immunocytochemical methods and expression of the oligodendrocyte lineage-affiliated transcription factor, olig2 was directly observed by using a transgenic mouse that expressed enhanced green fluorescent protein (EGFP) in olig2-expressing cells. (The transgenic mouse line was created by phage artificial chromosome (PAC) transgenesis). This data enabled lineage trees for individual clones to be retrospectively drawn to include the timing of olig2 expression alongside the final identification of the daughter cells produced. In this way, the effects of different growth factors with respect to the induction of glial in preference to neuronal phenotypes were assessed. Using this system it was possible to determine what cell types could be derived from a single precursor and with what pattern within a lineage olig2 might be expressed under different culture conditions. Both FGF-2 and a Sonic Hedgehog agonist were seen to produce mixed clones in which olig2 was transcribed at early branch points within a lineage and later down-regulated in a selection of daughter cells. This means that olig2 expression does not denote commitment to the oligodendrocyte lineage and, furthermore, that induction is a sporadic event which seems to be dictated at the level of the individual progenitor cells rather than by an intrinsic cell-timer dictated within the original NEP.

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