PMC

(a) Dlk1 transcripts in whole brain. Proteolytic cleavage domain (grey box) is shown. (b) Semi-quantitative PCR of Dlk1 isoforms in SVZ, NSCs and niche-astrocytes at postnatal 7 and 60. (c) Neurospheres generated from Dlk1^−/− or Dlk1^+/+ cultures nucleofected with pIRES-GFP, pIRES-GFP-S-Dlk1 (secreted isoform) or pIRES-GFP-MB-Dlk1 (membrane-bound isoform) after DLK1 treatment or from wild-type NSCs co-cultured with transduced Dlk1^−/− astrocytes. (d) Semi-quantitative PCR of Dlk1 isoforms in nucleofected NSCs. **p<0.01, ***p<0.001. Error bars: s.e.m; triplicate cultures (n=6 animals per genotype).

(a) Secondary spheres formed from wild-type and Dlk1^−/− primary neurospheres at P7 and P60. (b) Primary spheres from Dlk1^+/+ and Dlk1^−/− SVZ, after DLK1 treatment. (c) Quantification of unipotent (astrocytes), bipotent (astrocytes/neurons) and tripotent (astrocytes/ neurons/oligondendrocytes) clones derived from Dlk1^+/+ and Dlk1^−/− neurospheres grown in the presence or absence of DLK1 (left panel). Immunocytochemistry for GFAP (red), βIII-tubulin (green) and O4 (yellow) in differentiated neurospheres (right panel). (d) Primary neurospheres co-cultured with wild-type or Dlk1 mutant niche-astrocytes (upper panels). Immunocytochemistry for GFAP (red) and DLK1 (green) in niche-astrocyte cultures (lower panels). (e) Quantification of P7 and P60 co-culture experiments shown in d. (f) Primary spheres co-cultured with Dlk1^+/+ or Dlk1^−/− astrocytes and treated with DLK1. Dashed-lines indicate non co-cultured spheres. *p<0.05, **p<0.01, ***p<0.001. Error bars, s.e.m of triplicate cultures (3-6 samples per genotype). Scale bars: in c, 50 μm; in d (upper panel, 100 μm; lower panel, 30 μm).

A) Volcano plot detailing the genes with altered expression between DLK1⁺ and DLK1⁻ areas of ACC tumors (adj. p <0.01, fold change >/< 2). B) Geneset ANOVA showing the most differentially regulated pathways in DLK1⁺ and DLK1⁻ tumor areas. The most upregulated pathway was of steroid C) PCR analysis of DLK1 isoforms’ expression in H295R, MUC-1, TVBF7, CU-ACC1 and human adrenal. D) TaqMan analysis of DLK1 mRNA expression in the indicated ACC lines. E) Western blotting analysis of DLK1 protein expression in the indicated ACC lines. F) Western blotting analysis of DLK1 protein expression in the indicated ACC lines grown in 2D and 3D (spheroids). G-I) Example of colony forming units (CFU) in DLK1⁺ and DLK1⁻ FAC-sorted H295R cells (G) and analysis of CFU (H). I) TaqMan analysis of DLK1 mRNA expression in DLK1⁺ and DLK1⁻ FAC-sorted H295R cells after sorting, and after 7 and 30 days. **p<0.01, ***p<0.001, ****p<0.0001.

(a) Immunohistochemistry for GFAP (green) and Ki67 (red) within the postnatal 7 (P7) SVZ of wild-type and Dlk1^−/− mice. (b) Percentage of cell-types in the P7 SVZ from wild-type and Dlk1^−/− mice. (c) Immunohistochemistry for GFAP (blue), SOX2 (red) and BrdU (green) within the adult SVZ of wild-type and Dlk1^−/− mice. (d) Number of GFAP⁺/SOX2⁺/BrdU-label retaining cells (LRC)⁺ in the SVZ of mice with deletions of maternally-inherited (Dlk1^−/+), paternally-inherited (Dlk1^+/−), or both alleles (Dlk1^−/−). Light-green bar represents paternal transmission mutant mice in a Dlk1 transgenic background (Dlk1^+/−/Dlk1^tg/+). (e) Whole-mount for DCX+ migrating neuroblasts within the SVZ. (f) Primary spheres from wild-type and Dlk1 mutant SVZs at different developmental stages. (g) Primary spheres from adult SVZ of different Dlk1 mutants. *p<0.05, **p<0.01, ***p<0.001. Error bars, s.e.m. of five experiments (n=4 cultures per genotype). V, ventricle. Scale bars; in a, c, 20 μm (inset 10 μm).