Fig. 2.

LEF1 splicing is regulated during thymic development coincident with TCR-alpha expression. (A) Percent skipping of LEF1 exon 6, as determined by low-cycle RT-PCR, in isolated thymic populations as indicated. The isolated thymic populations were early thymic precursors (ETP), CD4⁻ CD8⁻ double-negative (DN) DN2, DN3a, DN3b, and DN4 developmental populations, the subsequent intermediate CD8 single-positive (ISP), and CD4⁺ CD8⁺ double-positive (DP) cells. Thymocytes were sorted from 10 mice and pooled for analysis. The bar above the graph indicates normal timing of pre-TCR signaling and TCR-alpha expression in thymocyte development. (B) Total TCR-alpha mRNA, relative to the amount in DN2 set at 1, for subsequent thymic developmental states. For all data shown, similar results were obtained from an independent sort. (C) Graph of TCR-alpha mRNA (left) and LEF1 exon 6 inclusion (right) in cells transfected with a morpholino oligomer (MO) complementary to the 3′ splice site (3′ss) of LEF1 exon 6 to specifically block inclusion of this exon, grown under resting (without PMA) or stimulated (with PMA) conditions. Error bars indicate standard deviations from 2 or 3 independent experiments. Contl, control. (D) Schematic of experimental approach and representative RT-PCR gel showing switch in splicing induced by the 3′ss morpholino oligomer. Cont'l, control.