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Fig. 5. Retroviral overexpression of Pax6(5a) reveals a distinct influence on cell
proliferation without effects on cell fate. (A) Corresponding micrographs of a
clone of cells (indicated by arrow), isolated from the embryonic day (E) 14
cortex, infected with the BAG control retrovirus (upper row), Pax6-containing
retrovirus (middle row) and Pax6(5a)-containing virus (lower row), cultured
for 7 days in vitro and immunostained against ß-galactosidase
(ß-gal, green), NeuN (red). Scale bar: 50 µm. (B) Schematic drawings
of the retroviral constructs used for control, Pax6 (canonical form of PD) and
Pax6(5a) overexpression together with the marker gene lacZ. (C)
Histogram depicting the clone type of either control (BAG), Pax6- or
Pax6(5a)-infected cells isolated from E14 WT (bars to the left) or
Pax6Sey–/– mutant cortex (bars to the right)
and cultured for 7 days. Note that the percentage of pure neuronal clones
(blue bars) increases significantly (compared to WT control, t-test,
see Materials and methods; error bars=s.e.m.) in the cells transduced with
virus containing Pax6 with the canonical form of the PD, at the expense of the
mixed (yellow bars) and pure non-neuronal clones (red bars). In contrast,
Pax6(5a) exerted no effect on the clone type, even in the absence of
functional Pax6 in Pax6Sey–/– cortical cells.
(D) Histogram depicting the mean size of clones (=the number of
ß-galactosidase-positive cells per clone, i.e. the number of cells
generated by a single infected precursor) in the cultures described in C. Note
that the clone size was reduced after transduction of cortical cells with Pax6
and Pax6(5a). Pax6(5a) was sufficient to reduce cell proliferation, suggesting
that this effect is mediated by the 5aCON site. Numbers of clones analyzed: WT
Ctrl: 395, WT Pax6: 243, WT Pax6(5a): 268,
Pax6Sey–/– Ctrl: 353,
Pax6Sey–/– Pax6: 229,
Pax6Sey–/– Pax6(5a): 219.
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