factors, significantly more and larger neuronal clones were detectable (figure 4.8).
4.3.5 Analysis of bFGF's effects in the absence of cortical astrocvte monolaver It was conceivable that bFGF's effects on embryonic cortical cells may have been mediated by cortical astrocytes, cells on which the precursors were routinely cultured. To investigate this possibility, parallel experiments were conducted without astrocytes. In preliminary experiments, embryonic cortical cells were grown on a monolayer of air-dried cortical astrocytes (referred to as 'astrocyte matrix') in the presence or absence of bFGF. In most such experiments, however, all embryonic cortical cells died within 48 hours of plating on matrix alone. Although cultures grown in the presence of bFGF did survive for slightly longer, death of control cultures meant that a quantitative clonal comparison could not be made between the two treatments. In order to test whether a different matrix/ substrate may enhance the survival of cultures, identical experiments were carried out by plating embryonic cortical cells on laminin (50 pg/ml) or poly-D-lysine coated coverslips or air-dried monolayers of NIH-3T3 cells (3T3 cell matrix). The degree of survival obtained on these substrates, however, was similar to that obtained on astrocyte matrix. Moreover, astrocyte conditioned medium consistently failed to enhance the survival of embryonic cells grown in the absence of cortical astrocytes.
In another approach to boost the survival of cultures in the absence of astrocytes, embryonic cells were plated at a slightly higher density: 3.5 xlO^ cells per coverslip compared to 2.0-2.5 x 10^ cells in the initial experiments. Only in 6 out of 20 such experiments, however, did the cultures survive the seven day period in the absence of astrocytes. Nonetheless, the few cultures that did survive made it possible to analyse and compare the effects of bFGF on cortical precursor cells grown in the presence or absence of cortical astrocytes. The results of this analysis are described below.
4.3.5.1 Analvsis of clones found in E16 cultures grown in the absence of cortical astrocvtes
Several important observations were made when clones derived from E l6 precursors that were grown in the absence of cortical astrocytes were analysed. For the sake of analysis, these observations will be divided into three different groups: observations made both in the presence and absence of bFGF; those made exclusively in the presence of bFGF; and those that were exclusive to the absence of cortical astrocytes.
4.3.5. 2 Observations made in the presence and absence of bFGF 4.3.5.2.1 Clone tvpe restrictions and proportions
The types of clones found in cultures that were grown in the absence of astrocytes, either in the presence or absence of bFGF, were found to be identical to those seen in E16
I l l
cultures grown on cortical astrocytes. i.e. four types of clones, each containing a single cell type were noted. These were clones of neurones, astrocytes, oligodendrocytes, or cells that could not be identified (Table 4.2). Of these four clone types, neuronal clones were found to be the most frequent clone type, both in the presence and absence of bFGF. For example, in cultures that were grown on astrocyte matrix, neuronal clones made up 83% of all clones in the absence, and 81% of all clones in the presence of bFGF.
4.3.5.S Effects exclusive to the presence of bFGF 4.3.5.3.1 Direct survival effect on precursor cells
The poor survival of cultures grown in the absence of cortical astrocytes was reflected in the number of clones obtained in such cultures. In parallel experiments, for example, on average only 8.6 clones per coverslip were found when E16 cells were cultured on PDL alone, compared to 27.6 clones per coverslip when grown on cortical astrocytes (Table 4.2). Similarly, in separate sets of experiments, an average of 17.2 clones per coverslip were found in cultures that were grown on astrocyte matrix, compared to 28.6 clones in cultures grown on cortical astrocytes. In the presence of bFGF, however, dramatically more clones could be found in cultures that were grown in the absence of astrocytes. In cultures grown on PDL, for example, the average number of clones detected per coverslip was increased from 8.6 to 32.5 by the addition of bFGF. A similar increase in clone numbers was also observed in cultures that were grown on astrocyte matrix in the presence of bFGF; from 17.2 clones per coverslip to 30.6. Importantly though, it was always the number of neuronal clones that showed the greatest increase in the presence of bFGF (Table 4.2). These observations suggested, therefore, that even in the absence of cortical astrocytes, bFGF could act as a survival factor for E 16 cortical neuronal precursors. To investigate whether bFGF may have also acted as a mitogen for neuronal precursors, the size distribution of neuronal clones found in the presence and absence of bFGF was analysed (figures 4.9 and 4.10). However, this analysis revealed an important effect on the size of neuronal clones, not due to the presence of bFGF per se^ but due to the absence of cortical astrocytes. This effect is considered below.
4.3.5.4 Effects exclusive to the absence of cortical astrocvtes
Two striking effects were noted when E l6 cultures were grown in the absence of
astrocytes. First, more and larger clones of unidentified cells were recorded in cultures that were grown in the absence of astrocytes, either on astrocyte matrix or PDL-coated
coverslips (Table 4.2). Cells in some of these clones were similar in appearance to those found in cultures grown on astrocytes; these cells had an astrocytic morphology, and stained with anti-GFAP. A proportion of unidentified clones, however, were clearly different from those found in the presence of cortical astrocytes. Cells in these clones.
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