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Figure 1. Expression of skp2 and Xic1. Whole-mount in situ hybridisation at indicated stages show expression of (a,b,d,e,g) skp2 and (c,f) Xic1. (a,c) Dorsal view with anterior toward the bottom. (b,d) Anterior view with dorsal toward the top. (e-g) Lateral view with anterior to the left. (h,i) Expression of skp2 and Xic1, respectively, in a vibratome section of stage 16 embryos; primary neurons are indicated with black arrows. Ep, epidermis; I, intermediate stripe; L, lateral stripe; M, medial stripe; My, myotome; Nf, neural folds; Not, notochord; Np, neural plate; Nt, neural tube; P, placodes.
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Figure 2. 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).
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Figure 3. Over-expression of skp2 blocks primary neurogenesis and reduces cell proliferation. (a-e) Embryos were injected with 1 ng wild-type (Wt) skp2 (a,d), 1 ng FBox skp2 (b,e), or 1 ng Wt MAFbx (c) mRNA, along with ßgal mRNA as a lineage tracer, in one blastomere at the two cell stage. Embryos were analyzed for expression of nßt mRNA (a-c) at stage 15. Dorsal views with injected side to the right. (d,e) Whole mount stage 15 embryos immunostained (red) to detect pH3 after injection of 1 ng Wt skp2 (d), or 1 ng FBox skp2 (e) mRNA, along with ßgal mRNA as a lineage tracer, in one blastomere at the two cell stage. Dorsal views with injected side to the right. (d',e') Detail of pH3-positive cells on the injected side relative to the uninjected side (boxed area in (d,e), dashed line is dorsal mid-line separating injected and uninjected halves). (f) Embryos were injected with 250 pg, 500 pg or 1 ng Wt skp2 mRNA (n = 41, 83, 87 embryos, respectively) in one blastomere at the two cell stage. Embryos were analyzed for expression of nßt mRNA at stage 15. Data shown in (f) are the percentages of embryos with no change, or moderate or substantial reduction of nßt positive cells on the injected side relative to the uninjected side for each injection (see Additional file 1 for photographs of representative embryos).
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Figure 4. Over-expression of skp2 counteracts the effect of skp2 Mo on neural differentiation. (a-e) Embryos were injected with 30 ng skp2 Mo (a), 30 ng Con Mo (d), or 1 ng skp2 (missing first two amino acids) mRNA (b) alone, or together as labelled (c,e), in one blastomere at the two cell stage. ßgal mRNA was co-injected as a lineage tracer. Embryos were analyzed for expression of nßt mRNA at stage 15. Dorsal views with injected side to the right. (f) Percentages of embryos with moderate increase, no change, or moderate or substantial reduction of nßt positive cells on the injected side relative to the uninjected side for each injection.
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Figure 5. skp2 inhibits primary neurogenesis between XNgnr1 and NeuroD. (a-f) Embryos were injected with XNgnr1 (a) or NeuroD (d) mRNA alone, or together with Wt skp2 (b,e) or FBox skp2 (c,f) mRNA, in one blastomere at the two cell stage. ßgal mRNA was co-injected as a lineage tracer. Embryos were analyzed for expression of nßt mRNA at stage 15. Dorsal views with injected side to the right.
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Figure 6. Over-expression of skp2 can degrade p27Xic1. (a) Western blot for embryos at stage 9, either uninjected or injected with 1 ng mRNA encoding wild-type (WT) skp2 or F box skp2, as labelled; tubulin demonstrates equal loading. (b) Western blot for Xic1 levels in embryos injected with 50 pg Xic1 mRNA and increasing doses (100 pg to 1 ng) of skp2 mRNA at the one cell stage. Embryos were harvested at stage 13; tubulin demonstrates equal loading. (c) Western blot for Xic1 levels in embryos harvested at stage 7 that were injected with 1 ng WT skp2 or 1 ng FBox skp2 mRNA at the one cell stage along with 50 pg Xic1 mRNA; tubulin demonstrates equal loading. (d) Western blot for Xic1 levels in neural plates harvested at stage 16 from embryos that were injected with 1 ng WT skp2 or 1 ng FBox skp2 mRNA into the animal pole of a fertilised egg. Tubulin demonstrates equal loading. (e-h) Embryos were injected with 1 ng WT skp2 (e,g) or 1 ng FBox skp2 (f,h) mRNA, along with ßgal mRNA as a lineage tracer, in one blastomere at the two cell stage. Embryos were analyzed for expression of muscle actin (e,f) or heavy chain myosin expression (g,h) at stage 15. Dorsal views with injected side to the right.
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Figure 7. The effect of skp2 over-expression on neural differentiation is partially rescued by extra Xic1. (a-c) Embryos were injected with wild-type skp2 (a) or wild-type Xic1 (b) mRNA alone, or together (c), in one blastomere at the two cell stage. ßgal mRNA was co-injected as a lineage tracer. Embryos were analyzed for expression of nßt mRNA at stage 15. Dorsal views with injected side to the right. (d) 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 (see Additional file 1 for photographs of representative embryos).
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skp2 (S-phase kinase-associated protein 2, E3 ubiquitin protein ligase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, anterior dorsal up.
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skp2 (S-phase kinase-associated protein 2, E3 ubiquitin protein ligase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
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skp2 (S-phase kinase-associated protein 2, E3 ubiquitin protein ligase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33, lateral view, anterior left, dorsal up.
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Additional File 1.
Catagories of neural beta tubulin expression.
Representative embryos shown for the expression catagories of N-tubulin (A-G).
E shows a moderate increase in primary neurons outside the usual stripes (see
Figure 5), while G shows a moderate increase in primary neurons within the
usual stripes (see Figures 4 and 7). Dorsal views with injected side facing
rightwards.
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