(A) Exons are represented as boxes, while horizontal lines represent introns. Inclusion of exon 7 results in the longer Pbx1a protein (top). Skipping of exon 7 results in a frameshift and translation termination at a stop codon (*) in exon 8 to yield the shorter Pbx1b protein isoform (bottom). The DNA-binding homeodomain (HD) is indicated by a blue box. (B) Genome Browser tracks of aligned RNA-seq reads show exon 7 inclusion as ESCs (red) differentiate into NPCs (blue) and GFP+ MNs (yellow). A significant PTBP1 iCLIP cluster is present upstream of exon 7 in NPCs (green). (C) Both Pbx1 exon 7 (top panel, solid line bottom panel, n=3) and Pbx1 mRNA expression (dashed line bottom panel, n=2) are induced with MN differentiation. (D, E) ESCs and NPCs were treated with siControl, siPtbp1, or both siPtbp1 and siPtbp2. (D) RT-PCR of Pbx1 exon 7 splicing following siRNA treatment in ESCs (24 PCR cycles) and NPCs (19 PCR cycles). (E) Bar chart of Pbx1 exon 7 splicing (Mean ± SEM, n=3). Statistical analyses were performed using paired one-tailed Student’s t-test (P-value<0.01**, 0.05*).