(A) Sagittal sections of the cortex of embryos at E18.5 were stained for Ki67 (upper panels) and counterstained with DAPI (lower panels). Quantification of Ki67⁺ cells normalized by area (150 μm²) is shown in the graph. Bars indicate Mean±SD. n = 3 for Huwe1^F/Y and 3 for Huwe1^F/YNes; p = 0.0008. (B) Cortical sections at E18.5 were stained for Ki67 (red) and counterstained with DAPI (blue). Clusters of Ki67⁺ cells are detected in Huwe1^F/YNes basal ganglia (upper panel) and mesencephalic area (lower panel). Arrowheads indicate Ki67⁺ clusters of cells. IV, IV ventricle. (C) Immunofluorescence for Ki67 (red) and BrdU (green) 24 h after a single pulse of BrdU in E15.5 embryos. The fraction of cells labeled only with BrdU (BrdU⁺/Ki67^-, no longer dividing), as compared with BrdU⁺/Ki67⁺ cells (re-entering cell cycle), was scored. Bars indicate Mean±SD. n = 3 for Huwe1^F/Y and 3 for Huwe1^F/YNes; p = 0.016. (D) Immunofluorescence for Ki67 (red) and BrdU (green) after 1 h pulse of BrdU in E18.5 embryos. The fraction of cycling progenitor cells (Ki67⁺) labeled with BrdU was scored. Bars indicate Mean±SD. n = 3 for Huwe1^F/Y and 3 Huwe1^F/YNes; p = 0.006. Arrows indicate BrdU⁺/Ki67⁺.

(A) Sagittal sections of the cortex of embryos at E15.5 stained for HuC/D (upper panel) and GAP43 (lower panel). Arrows point to regions in which GAP43 expression is reduced. (B) Staining for Pax6 (green, upper panels) and Tbr2 (red, lower panels) in E18.5 brains. Nuclei were counterstained with DAPI (blue). Note the expansion of the Pax6 domain with superficial displacement of the Tbr2 positive region in the Huwe1-null cortex. (C) Immunofluorescence for MAP2 (green), Tbr1 (red) (upper panels) and Ctip2 (lower panels) in E18.5 cortices. Nuclei were counterstained with DAPI (blue). (D) Staining for Cux1 (upper panels) and p27^Kip1 (lower panels) in E18.5 cortices. Nuclei were counterstained with DAPI (blue). (E) Immunofluorescence for BrdU (red) and Tbr1 (green) on cortical sections of P0 pups after injection of BrdU at E15.5. (F) Quantification of BrdU⁺ neurons in the superficial layers (II-III) marked by dashed lines in E. Bars indicate Mean±SD. n = 4 for Huwe1^F/Y and 4 Huwe1^F/YNes; p = 0.0005.

(A) E18.5 cortical sections were stained for N-Myc (upper panel) and cyclin D1 (lower panels). LV, lateral ventricle. (B) The N-Myc subnetwork in GBM. Positively and negatively connected genes are shown in pink and green, respectively. (C) Ranking of the top 30 transcription factors showing changes in their regulon expression based on the Huwe1 expression status in 236 human GBM. Positive and negative normalized enrichment score (NES) values indicate positive and negative association of transcription factor's activities with the expression of Huwe1, respectively. (D) GSEA² plot for N-Myc regulon genes. All genes are sorted on the horizontal axis according to their differential expression between two extreme expression states of Huwe1, with the most down-regulated gene on the left and the most upregulated gene on the right. Red/blue bars indicate overlaps between the ranked list and the individual positive/negative N-Myc targets (p<1×10^-5).

(A) Chromatin immunoprecipitation shows that the N-Myc protein binds the promoter of DLL3 but not OLR1 in neuroblastoma cells. (B) Quantitative RT-PCR for DLL3 after ectopic expression of N-Myc in MEFs. Values were normalized for β-actin. Bars indicate Mean±SD of triplicate samples, p = 4.5×10^-6. (C) Western blot for cleaved Notch1, N-Myc and β-actin. (D) Correlation of the expression of N-myc and DLL3 in human GBM cells cultured in NBE (blue dots) and serum (red dots). (E) RNA was prepared from E18.5 and P0 brains and analyzed by qRT-PCR for the indicated genes. Values are normalized for β-actin. Bars indicate mean±SD, n = 6 for Huwe1^F/Y and 6 for Huwe1^F/YNes; p = 0.00027 for DLL3. (F) Brain sections from E15.5 (upper panels) and E18.5 (lower panels) embryos were stained for cleaved Notch1.

(A) Ex vivo electroporation of MSCV-GFP or MSCV-GFP-Cre plasmid together with control, N-myc, or Dll3 siRNA into E14.5 mouse cortices followed by organotypic slice culture for 2 days. Cortical sections were stained for GFP (green) and BrdU (red). Arrowheads indicate GFP⁺/BrdU⁺ cells; arrows indicate GFP⁺/BrdU^- cells. (B) Cortical slices were stained with the neuronal marker βIII-tubulin (red) and GFP (green). Arrowheads indicate GFP⁺/βIII-tubulin⁺ cells; arrows indicate GFP⁺/βIII-tubulin^- cells. (C) Immunofluorescence with GFP (green) and DLL3 (red) antibodies. Arrowheads indicate GFP⁺/DLL3⁺ cells. Double positive cells were scored and quantitative results are shown as Mean±SD from at least three electroporated embryos per group from a minimum of two experiments. (**p < 0.01, Student's t-test).

(A) Ex vivo electroporation of pcDNA3.1/v5-His/LacZ, pcDNA3.1/v5-Huwe1-WT and pcDNA3.1/v5-Huwe1-CA plasmid into E14.5 mouse cortices followed by organotypic slice culture for 2 days. Cortical sections were double-stained with V5 (green) and BrdU (red). Arrowheads indicate V5⁺/BrdU⁺ cells; arrows indicate V5⁺/BrdU^- cells. (B) Cortical sections were stained with V5 (green) and DLL3 (red). Arrowheads indicate V5⁺/DLL3⁺ cells. Double positive cells were scored and quantitative results are shown as Mean±SD from at least three electroporated embryos per group from a minimum of two experiments. (** p<0.01)

(A) 8 of 129 GBM contain amplification of the N-myc gene and two of them (#178 and #289) have focal deletions of Huwe1. (B) The two Huwe1 deletions occur in male (XY) patients and define a focal minimal common region of deletion of 65 Kb, which is entirely contained within the Huwe1 gene. The blue, dashed lines define the limits of the deletions for each tumor. (C) The expression of Huwe1 is significantly down regulated in 77 samples from human GBM (class 2, red) compared with 23 samples from non-tumor human brain (class 1, blue) p = 9.3×10^-10.