Isolation and propagation of enteric neural crest progenitor cells from mouse embryonic stem cells and embryos

DEV046896 Supplementary Material

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  • Supplemental Figure S1 -

    Fig. S1. Schematic representations and genomic analysis of Sox10 targeting strategies. (A) GFP knock-in at the Sox10 locus. Southern analysis using 5′ and 3′ probes is shown in the left and right panel, respectively. Targeted clones are named S10G. (B) GFP-IRES-BLS knock-in at Sox10 locus. Southern analysis using 5′ and 3′ probes is shown in the left and right panel, respectively. Targeted clones are named S10GB.

  • Supplemental Figure S2 -

    Fig. S2. Stable constitutive expression of Sox10 transgene in ES cells. Immunostaining of parental (S10G) and S10G-S ES cells for Oct4 and Sox10.

  • Supplemental Figure S3 -

    Fig. S3. ES cell-derived neural crest precursors can colonise the foetal gut. Purified Sox10-GFP-positive S10G-SR cells were injected into cultured embryonic gut. Four representative fluorescence images of gut immunostaining for DsRed (red) and TuJ1 (Green) are shown. Arrows indicate both DsRed (red) and TuJ1 (green) positive cells.

  • Supplemental Figure S4 -

    Fig. S4. Metaphase analysis of neural crest progenitor cultures in NWLE plus blasticidin. (A) Chromosome counts from metaphase spreads. (B) Representative metaphase spreads containing 39 or 40 chromosomes. (C) Representative metaphase spreads containing 40 chromosomes.

  • Supplemental Figure S5 -

    Fig. S5. Heterozygosity for Sox10 does not cause any major perturbation of neural crest development but delays colonisation of the hindgut. (A) Typical fluorescent and immunostaining images of wild-type (+/+) and heterozygous (+/−) littermate embryos (E12.5). At this stage, TuJ1-positive cells extended throughout the caecum, as shown by immunostaining of the wild-type embryo. In heterozygous embryos, TuJ1 and Sox10-GFP expression indicated that neural crest cells are present at the intestinal level but have not reached the caecum. The wave fronts of enteric neurons are indicated by arrows. Although there is some variability between embryos, heterozygous embryos showed a consistent trend of retarded migration of neural crest cells to the hindgut when compared with wild-type embryos from the same mating. (B) Scoring of individual E12.5 embryos for presence of TuJ1 staining in the caecum.