Numb alternative splicing in the chick neural tube. (A) Diagrams of chicken Numb splice isoforms. Locations of the regions encoded by two alternative exons, exon 6 and exon 12, are shown. Numb-12in and Numb-12ex includes and excludes exon 12, respectively. Two protein domains, phosphotyrosine-binding domain (PTB) and PRR, are indicated. P3–P6 indicate primers used for RT-PCR. P7 and P8 indicate in situ hybridization probes. (B) Developmental change in RNA splicing of Numb exon 6 and exon 12. The Numb mRNAs from embryos at the indicated times were analyzed by RT-PCR using primers P3 and P4 (A) for exon 6 (top) and P5 and P6 (A) for exon 12 (bottom). Numb-6in, Numb-exon 6 included; Numb-6ex, Numb-exon 6 excluded. (C) Developmental change in Numb protein isoform. Embryonic extracts harvested at the indicated times were analyzed by immunoblotting for Numb proteins. The top and bottom bands represent Numb-12in and Numb-12ex proteins, respectively. Each band most likely represents two proteins that include and exclude the 11 aa encoded by exon 6 but are not resolved under these electrophoresis conditions. (D–G) Restricted expression of the Numb-12ex splice variant to the MZ. In situ hybridization detecting total Numb mRNAs (D) using the common probe P7 (A) and that detecting the Numb-12ex splice variant (E) using the specific probe P8 (A) are shown. Hybridization-positive signals can be seen as dark blue staining. Sense strand probes complementary to P7 (F) and P8 (G) serve as negative controls. The spinal cord is outlined by a broken line. Bars, 50 µm.