Wylie LA , Hardwick LJ , Papkovskaia TD , Thiele CJ , Philpott A .

T32 GM007309 NIGMS NIH HHS , Intramural NIH HHS, Medical Research Council

             sympathetic nervous system development

Xla Wt + Mmu.ascl1 (Fig.7.a,b)
Xla Wt + Mmu.ascl1 + ccna2 + cdk2 (Fig.6.a,b)
Xla Wt + Mmu.ascl1 + mycn (Fig.7.a,b)
Xla Wt + Mmu.ascl1{S62A,S88A,S185A,S189A,S202A,S218A} (Fig.6.a,b)
Xla Wt + Mmu.ascl1{S62A,S88A,S185A,S189A,S202A,S218A} + ccna2 + cdk2 (Fig.6.a,b)
Xla Wt + Mmu.ascl1{S62A,S88A,S185A,S189A,S202A,S218A} + mycn (Fig.7.a,b)
Xla Wt + ccna2 + cdk2 (Fig.6.a,b)
Xla Wt + cdknx (Fig.3.a,b)
Xla Wt + cdknx MO (Fig.3.a,b)
Xla Wt + mycn (Fig.7.a,b)
Xla Wt + {dn}snai2 (Fig.1.a,b)

	Fig. 1. Migration of neural crest is required for expression of noradrenergic markers in the anteroventral region Xenopus embryos were injected in one of the two-cell stage with beta-galactosidase (light blue) as a tracer along with either GFP as a control, or mRNA encoding a dominant negative form of Slug (dnSlug) to inhibit neural crest migration, injected side to the right. (A) Embryos were fixed at stage 17/18 and subject to in situ hybridization for the neural crest markers Slug, Snail, and FoxD3 and noradrenergic markers Phox2a and Hand2. (B) Embryos were scored compared to the uninjected side based on inhibition of migration for neural crest markers and reduced expression of noradrenergic markers (See Supplementary Fig.2). For scoring data, [n=28-45]. Kruskal-Wallis non-parametric ANOVA was performed comparing GFP control with dnSlug-injected embryos (p-values of <0.05 = *, <0.0005 = **, and <0.000005 = ***).
	Fig. 2. Ascl1 is expressed transiently during noradrenergic development in the anteroventral region Xenopus embryos were fertilized and allowed to develop to the developmental stage indicated before fixing and staining by ISH for NA neuron markers Ascl1, Hand2, Phox2a, and TH along with the neural crest marker Sox10. Embryos are all orientated with the ventral side imaged and head to the top. The anteroventral region where AVNA cell markers are expressed is expanded in the lower panel.
	Fig. 3. p27Xic knock-down by morpholino injection inhibits neurogenesis Xenopus laevis embryos were injected in one of the two-cell stage with beta-galactosidase (light blue) as a tracer along with either Xic1 mRNA or Xic1 antisense morpholino (MO) as indicated, injected side to the right. (A) At stage 19-20 embryos were subject to in situ hybridization to detect neural-β-tubulin, Phox2a, Hand2 and TH. (B) Embryos were scored for marker expression on the injected side relative to the uninjected side. For scoring data, [n=12-34]. Kruskal-Wallis non-parametric ANOVA was performed on control MO compared to Xic1 MO and uninjected compared to Xic1 mRNA (p-values of <0.05 = *, <0.0005 = **, and <0.000005 = ***).
	Fig. 4. Neuroblastoma expresses the proneural protein Ascl1 and noradrenergic markers Relative expression of Ascl1 in neuroblastoma primary tumors and normal tissue (A) and across tumor types (B) were determined by analyzing publicly available microarray data (see Materials and Methods). Ascl1 expression in neuroblastoma cell lines and Ewing Sarcoma line, TC-71, as a negative control, was determined by western blot (C) and qPCR (D).
	Fig. 5. Ascl1 is phosphorylated on multiple sites in NB cells (A) Protein extracts from Fig. 4 were normalized based on Ascl1 expression and treated with and without lamda phosphatase. Samples were then separated by SDS PAGE and subject to western blotting for Ascl1 protein. (B) Human Ascl1 or phosphomutant S-A Ascl1 were over-expressed in SY5Y cells, and treated with and without lamda phosphatase. Slowed migration observed on SDS PAGE can be attributed to phosphorylation on serine-proline sites.
	Fig. 6. CDK activity inhibits AVNA cell differentiation induced by WT Ascl1 but not SA Ascl1 Xenopus embryos were injected in one of the two-cell stage with beta-galactosidase (light blue) as a tracer along with mRNA encoding Cyclin A/CDK2 and/or mouse WT/S-A Ascl1, as indicated, injected side to the right. (A) Embryos were subject to in situ hybridization for AVNA markers at stage 18-19, as labeled, (see Supplementary Fig. 2 for scoring scheme). (B) Embryos were scored for marker expression comparing the injected and ininjected side. For scoring data, [n=20-32]. Kruskal-Wallis non-parametric ANOVA was performed comparing WT Ascl1 with S-A Ascl1 without and with Cyclin A/CDK2 (p-values of <0.05 = *, <0.0005 = **, and <0.000005 = ***).
	Fig. 7. N-Myc inhibits AVNA cell differentiation induced by WT Ascl1 but not S-A Ascl1 Xenopus embryos were injected in one of the two-cell stage with beta-galactosidase (light blue) as a tracer along with mRNA encoding N-Myc with or without mouse WT/S-A Ascl1, as indicated. (A) Embryos were subject to in situ hybridization for AVNA markers as indicated at stage 18-19. (B) Embryos were scored for marker expression comparing the injected and ininjected side. For scoring data, [n=24-32]. Kruskal-Wallis non-parametric ANOVA was performed comparing WT Ascl1 and S-A Ascl1 with and without N-Myc (pvalues of <0.05 = *, <0.0005 = **, and <0.000005 = ***).
	Figure. S1. Sox10 expression on the dorsal region of Xenopus embryos Xenopus embryos were fertilised and allowed to develop to the indicated developmental stage before fixing and staining by in situ hybridisation for neural crest marker Sox10. Embryos are orientated with the dorsal side imaged and head to the top.

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