Hajihosseini, Mohammad; (1994) A study of neuronal precursors using retrovirus-mediated gene transfer. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

This study concerns an investigation of factors that may influence the behaviour and development of neuronal precursors derived from embryonic rat cerebral cortex. In this study, single retrovirally-labelled E16 or E14 cortical precursors were cultured amongst unlabelled cells, on monolayers of cortical astrocytes in the presence or absence of basic fibroblast growth factor (bFGF). Several important observation were made when the fate of such cells was analysed after seven days in culture. Most virally-labelled E16 and E14 cortical cells were found to produce clones that were composed of only one of the cell types found in the adult brain; namely neurones, oligodendrocytes, or astrocytes, showing that cortical precursor cells are specified in the phenotypic fate at the time of their isolation from the embryonic cortex. bFGF did not override this specification. However, bFGF was found to act as a survival factor and possibly a mitogen exclusively for neuronal precursor derived from either of the embryonic ages; significantly more and larger neuronal clones were found in the presence of this factor. To test whether bFGF's effects were mediated by cortical astrocytes, on which the embryonic cells were routinely grown, cultures were grown on substrates other than astrocytes in the presence or absence of bFGF. It was discovered that survival of cultures grown in the absence of cortical astrocytes was poor and that bFGF could enhance the survival of neuronal precursors in such cultures, thus arguing for a direct effect by bFGF. However, despite the poor survival of cultures, absence of cortical astrocytes resulted in an increase in the size of neuronal clones even when bFGF was absent. This observation suggested that cortical astrocytes inhibit the proliferation of cortical neuronal precursors. In a second line of pursuit, this study investigated whether retroviral DNA can integrate into post-replication DNA of its host. This was addressed by analysing the distribution of viral genes amongst the progeny of single retrovirally-labelled NIH-3T3 cells. This analysis showed that retroviruses integrate almost exclusively into post-replication DNA of host cells as only half the progeny of single infected NIH-3T3 cells were found to inherit the viral genes, a finding that could be used to explain the high frequency of single-cell neuronal clones generated by retrovirally-labelled neural precursors. Results of this study suggested that single-cell neuronal clones arise when only one daughter of an asymmetrically-dividing neuronal precursor inherits the viral genes.

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