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Fig. 7. The effect of FoxG1 and TLE2 MO on forebrain development. (A) Transverse sections of forebrain of FoxG1 MO-injected (7.5 ng) embryos (st. 28) stained with DAPI (blue), anti-pH3 (green) and anti-FoxG1 (red). Note the reduction of FoxG1 staining and in pH3-positive cells on the MO-injected (left) side in the presumptive FoxG1 expression region. Dashed white line indicates the midline. (B) Schematic representation of the TLE2 splice MO (red) and location of PCR primers in relation to the exon-intron arrangement of TLE2. (C) Semi-quantitative PCR from embryos injected with 10 ng Control MO (lanes 1, 4), 10 ng TLE2 MO (lanes 2, 5) or 20 ng TLE2 MO (lanes 3, 6). Primers used were ornithine decarboxylase (ODC) as control (lanes 4-6) and TLE2 exon 1 and TLE2 exon 3 (lanes 1-3) (see B). (D-G) Nkx2.1 expression in the forebrain of st. 34 control embryos and embryos injected at the one-cell stage with 10 ng Control MO (E), 15 ng FoxG1 MO (F) and 15 ng TLE2 MO (G). The expression in the ventral telencephalon (red arrowhead) is reduced or missing in TLE2 and FoxG1 morphant embryos (F,G). di, diencephalon, dt, dorsal telencephalon, vt, ventral telencephalon. (H-N) Embryos injected in one cell at the two-cell stage with FoxG1 MO (7.5 ng) and TLE2 MO (7.5 ng) processed for in situ hybridisation for elrC (H-K) and TUNEL staining (red nuclei; L-N). Injected side is to the right, as marked by X-Gal staining (H-K) or FITC (L-N). (H,J) Frontal views; (I,K-N) transverse sections through the forebrain of st. 19 (H-K) or st. 32 (L-N) embryos. Reduced forebrain elrC expression on the MO-injected side is indicated by red arrows. There was no statistically significant increase in TUNEL staining. (O) In FoxG1 MO-injected forebrains there was a 40% decrease (P=0.003, paired t-test, n=12 embryos) in pH3-positive cells. (P-T) Transverse sections through the forebrain of FoxG1 MO-injected embryos, subsequently electroporated with wild-type FoxG1 and various FoxG1 mutated versions, as indicated, together with lacZ DNA and analysed for Nkx2.1 expression. Light blue (X-Gal staining) indicates cells that have taken up the electroporated DNA (red arrowheads indicate examples of electroporated cells); dark purple indicates cells expressing Nkx2.1. The experiment was repeated four times with similar results. See Materials and methods for details. Scale bars: 100 μm. |
