Figure 3

The relative abundance of Mtss1 comprising either exon 12 or exon 12a changes during neuronal differentiation. A, B: Immature (i) and more mature (m) granule cells were isolated based on their differential expression of Math1-EGFP by flow sorting. Results from 3 independent experiments are shown in panel A. In mRNA from immature granule cells, the band indicative of the exon variant 11/12/13 is most prominent. In contrast, in more mature granule cells, the band indictive of the splice variant 11/12a/13 is strongest. Sorting efficacy was verified by screening for expression of endogenous Math1 (lower part of panel A). B: contamination of granule cell fractions by Purkinje cells was excluded by screening for the Purkinje cell specific Marker, L7/Pcp2. Lanes 8 and Ad positive controls with mRNA prepared from p8 and adult cerebellum. C: In mRNA isolated from P19 cells differentiated by growth in N2 medium (lane N2), the Mtss1-band indicative of the splice variant 11/12a/13 is most prevalent. In mRNA from undifferentiated controls (C), that representing the exon combination 11/12/13 predominates. Upon induction of differentiation with increasing doses of retinoic acid (0.1, 0.5 and 1.0 μM for 4 days), the intensity of the band indicative of usage of exon 12 gradually decreases, whereas that indicative of exon 12a remains unchanged, i.e. there is a relative shift towards the latter. For panel A and C, primers 4 and 5 were used.