Loss of skp2 protein promotes primary neurogenesis. (a) Embryos were injected with wild-type (Wt) skp2, skp2 1–2 alone or in combination with skp2 Mo or Con Mo as indicated. Protein (30 μg) from a stage 15 embryo was western blotted to determine skp2 levels; tubulin was used as a loading control. (b) Western blot for endogenous skp2 protein levels on stage 15 embryos that were injected with 20 ng, 30 ng, or 40 ng skp2 Mo or 40 ng Con Mo at the one cell stage, arrow to skp2 protein band. In vitro translated (IVT) skp2 protein is run in lane 6, and alpha-tubulin was used as a loading control. (c) The percentages of embryos with mild increase, no change, or moderate or substantial reduction of nßt positive cells on the injected side relative to the uninjected side for 20 ng, 30 ng, and 40 ng skp2 Mo, or 30 ng and 40 ng Con Mo (see Additional file 1 for photographs of representative embryos). (d,e) Embryos were injected with 30 ng skp2 Mo (d) or 30 ng Con Mo (e) in one blastomere at the two cell stage, along with ßgal mRNA as a lineage tracer, and analyzed for nßt mRNA expression at stage 15 ((d) arrow to show expansion of primary neurons). The view is dorsal with injected side to the right. (f,g) In situ hybridisation sections, which are transverse across the centre of the embryo, with injected side to the right (f) Section of an early neurula embryo injected with 30 ng skp2 Mo, indicating nßt upregulation by skp2 protein depletion. (g) Section of a mid neurula embryo injected with 30 ng Con Mo showing no difference in nßt distribution. Arrows (f, g) denoting staining of nßt in primary neurons. (h,i) Whole mount stage 15 embryos immunostained (red) to detect pH3 after injection of 30 ng skp2 Mo (h) or 30 ng Con Mo (i) in one blastomere at the two cell stage. ßgal mRNA was co-injected and X-Gal staining (blue) was performed to reveal injected side. Dorsal views with injected side to the right. (h',i') Detail of pH3 cells on the injected side relative to the uninjected side of representative embryos (boxed area in (h,i), dashed line is dorsal mid-line separating injected and uninjected halves).