Fig 2: TBXT-driven programming of a posterior neural crest (NC) axial identity is an early event.(A) Differentiation/tetracycline (Tet) treatment scheme. The temporal Tet treatment regimens employed were: no Tet control (− −); Tet treatment between days 3 and 7 (− +); Tet treatment between days 0 and 7 (+ +); Tet treatment between days 0 and 3 (+−). (B) qPCR expression analysis of representative lineage identity/posterior markers in early spinal cord/NC cells generated following culture in ‘neutral’ basal media in the presence of Tet during the time windows shown in A. Error bars represent SD (n=3). Only statistically significant changes are indicated. *p<0.05, **p<0.005 (paired t-test). (C) Immunofluorescence analysis of the expression of HOXC9, SOX1, and SOX10 in early spinal cord/NC cells generated following culture in ‘neutral’ basal media in the presence of Tet during the time windows shown in A. Scale bar = 100 µm. Quantification of the cell populations expressing these proteins in relation to the different Tet treatment regimens is shown in (D). Error bars represent SD (n=3). H: HOXC9; S1: SOX1; S10: SOX10. (E) qPCR expression analysis of indicated HOX genes in early spinal cord/NC cells generated following culture in ‘neutral’ basal media in the presence of Tet during the time windows shown in A. Error bars represent SD (n=3). Only statistically significant changes are indicated. *p<0.05, **p<0.005 (paired t-test).

Fig 3: ACTL6a is detected in the developing sciatic nerve and its ablation in Schwann cells induces a progressive motor phenotype(A) The scatterplot represents the average transcript levels ±SEM of Actl6a and Actl6b in sciatic nerves at postnatal day 3, 5, 10, 30 and 60 (n = 3 per age), normalized to the geometric mean of three reference genes (Gapdh, Wdr33, and 18s). Statistical analysis was performed using one-way ANOVA with multiple comparisons.(B) Western blot of ACTL6a protein levels in the developing sciatic nerve at postnatal day 3, 5, 10, 15, 30 and 60. GAPDH levels were used as loading control.(C) Confocal images of postnatal day 5 mouse sciatic nerve sections stained for ACTL6a (red) and SOX10 (white). DAPI (blue) as nuclear stain. Scale bar, 5μm.(D) Tail suspension test showing abnormal hindlimb clasping reflex in Cnp1-cre;Actl6afl/fl mice, compared to controls.(E) The curve represents the average clinical score ±SEM in mutantCnp1-cre;Actl6afl/f (n = 6), heterozygous Cnp1-cre;Actl6afl/+(n = 4), and wildtype Actl6afl/fl (n = 12) controls. Motor scoring was performed starting at P15 and conducted for 14 weeks, until mice were 16 weeks old. Scoring was as follows: 1 – hindlimb clasping, 2 – unable to hold on to inverted grid, 2.5 – can only grip upright grid with front paws, 3 – not able to hold on to upright grid, 3.5 no forelimb resistance when pulled across grid, 4 – hindlimb paralysis, 5 – dead. Statistical analysis was performed using two-way ANOVA.(F) Average grip strength values ±SEM obtained in Cnp1-cre;Actl6afl/fl mice (n = 5) and control littermates (n = 7) at age 12 weeks. Statistical analysis was performed using Student’s t-test.(G) Tail suspension test results in Mpz-cre;Actl6afl/fl mice and relative controls.(H) Motor scoring represents the average clinical score ±SEM over time (from 2 to 16 weeks) for mutant Mpz-cre;Actl6afl/f (n = 4), compared to heterozygous Mpz-cre;Actl6afl/+(n = 3), and Actl6afl/fl (n = 4) controls. Statistical analysis was performed using two-way ANOVA.(I) Average grip strength values ±SEM for Mpz-cre;Actl6afl/fl mice (n = 4) and control littermates (n = 4) at 12 weeks of age. Statistical analysis was performed using Student’s t-test. ∗∗p< 0.01, ∗∗∗p< 0.001, ∗∗∗∗p< 0.001.

Fig 4: ALK-F1174L affects the differentiation of sympathetic progenitors. (A) The fractions of Sox10+/Phox2b−, Sox10+/Phox2b+, and Sox10−/Phox2b+ cell populations in WT and Sox10-Cre;LSL-ALK-F1174L embryos at E10.5 (top) and E11.5 (bottom) are shown in Box-plots (Mann Whitney test or unpaired t-test depending on data distributions, ****p < 0.0001, **p < 0.01). (B) The evolution from E10.5 to E11.5 of the three cell populations in WT (top) and Sox10-Cre;LSL-ALK-F1174L (bottom) embryos are illustrated in Box-plots (Krukal-Wallis and One-way Anova respectively, paired comparisons, ****p < 0.0001, **p = 0.0043, ns comparisons are not shown). Numbers of SG sections analyzed at E10.5 and E11.5, respectively: WT n = 22 and n = 23, Sox10-Cre;LSL-ALK-F1174L n = 14 and n = 16. (C) The mean percentages of each cell population are plotted in grouped representations at E10.5 and E11.5 for WT (top) and Sox10-Cre;LSL-ALK-F1174L (bottom) embryos.

Fig 5: SOX10 overexpression rescued the anti‐stemness phenotypes caused by miR‐637. (A) The expression of miR‐637 was measured by qRT‐PCR in indicated LN229 cells. (B) The expression of SOX10 was measured by qRT‐PCR in indicated LN229 cells. (C,D) The expression of SOX10 was measured by Western blotting in indicated LN229 cells. (E,F) The stemness of indicated cells was examined by neurosphere formation assay, with representative images of formed neurospheres shown in (E) and the quantification results in (F). (G,H) The expression of CD133 on the surface of indicated cells was examined by flow cytometry in (G) and the quantification results are shown in (H). (I,J) The expression levels of stem‐cell markers Nestin, Oct4, Nanog, CD133 and CD44 were measured by Western blotting in indicated cells (I) and are quantified as the ratio to GAPDH (J). (K) The viability of indicated LN229 cells was measured by MTT assay for 1, 2, 3, 4 and 5 days, respectively. LN229 cells were overexpressed miR‐637 (miR‐637 mimics), SOX10 (OE‐SOX10) or both (miR‐637 mimics + OE‐SOX10). Data are presented as mean ± SD from three independent experiments. Comparison between three or more groups was performed using one‐way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001.