01: SUPPLEMENTARY MATERIAL
Supplementary figure 1: Hmga2-deficient mice exhibit progressive growth retardation and reduced brain mass. Hmga2+/+ and Hmga2−/− mice appeared grossly indistinguishable at E14.5 (A), and body masses were not significantly different (B; N=23 wild-type mice and 25 Hmga2−/− mice). At P0 however, Hmga2−/− mice were noticeably smaller (C), and their masses were significantly reduced relative to wild-type littermates (+/+) (D; N=21 wild-type and 16 Hmga2−/−, *P<0.01). By P49-56, this growth retardation became more apparent (E), and the masses of Hmga2−/− mice (−/−) were approximately one third of wild-type littermates (F; N=18 wild-type and 16 Hmga2−/−, *P<0.01). This reduced body mass remained evident at P570-600 (F; N=6 wild-type and 6 Hmga2−/−, *P<0.01). Brain masses were not significantly different between wild-type and Hmga2−/− mice at P0 (H; N=14 wild-type and 12 Hmga2−/−). However, at P49-56, the brains of Hmga2−/− mice were noticeably smaller (G), and their masses were significantly reduced compared to wild-type at both P48-56 (H; N=18 wild-type and 16 Hmga2−/−, *P<0.01) and at P570-600 (H; N=6 wild-type and 6 Hmga2−/−, *P<0.01). All statistical comparisons were done using unpaired T-tests in this figure.
Supplementary figure 2: Gut neural crest stem cell (NCSC) frequency and self-renewal potential are reduced in the absence of Hmga2. A) Typical neurospheres that formed after 10 days in non-adherent cultures from E14.5 gut cells. B) The percentage of cells from E14.5 gut, P0 plexus/outer muscle layer, and P49-56 plexus/outer muscle layer of Hmga2−/− mice and littermate controls that gave rise to multipotent neurospheres, the diameter of these neurospheres, and their self-renewal potential (number and percentage of cells from individual primary neurospheres that gave rise to multipotent secondary neurospheres upon subcloning). Hmga2 deficiency significantly reduced neural stem cell self-renewal at all stages and frequency at P0 and P49-56 (5–7 independent experiments per stage, all statistics represent mean±SD, *P<0.01, **P<0.05). C) P0 gut cells were dissociated and plated in adherent cultures at clonal density and the numbers of cells per colony were counted after 6 and 9 days. Only colonies with the appearance of multilineage NCSC colonies were counted. At each time point, Hmga2−/− (−/−) colonies contained significantly fewer cells than wild-type (+/+) colonies (3 independent experiments, *P<0.01). The percentage of cells that incorporated a pulse of BrdU in P0 NCSC colonies (D) was significantly reduced within Hmga2−/− colonies as compared to wild-type colonies (E; 3 independent experiments, *P<0.01). All T-tests were paired.
Supplementary figure 3: Hmga2−/− neurospheres from the CNS and PNS undergo multilineage differentiation in a manner indistinguishable from wild-type neurospheres. CNS neurospheres were routinely cultured from dissociated E14.5 telencephalon, or P0 VZ (A), or P49-56 lateral ventricle SVZ at very low cell density and then transferred individually to adherent cultures before being stained for markers of differentiation. At E14.5, 95% of Hmga2+/+ neurospheres and 94% of Hmga2−/− neurospheres formed Tuj1+ neurons, GFAP+ astrocytes and O4+ oligodendrocytes. At P0, 94% of Hmga2+/+ and 95% of Hmga2−/− neurospheres formed neurons, astrocytes and oligodendrocytes. At P49-56, 94% of Hmga2+/+ and 93% of Hmga2−/− neurospheres formed neurons, astrocytes and oligodendrocytes. PNS neurospheres were routinely cultured from dissociated E14.5 gut, P0 gut plexus/outer muscle layers (B), or P49-56 gut plexus/outer muscle layers at very low cell density and then transferred individually to adherent cultures before being stained for markers of differentiation. At E14.5, 95% of Hmga2+/+ and 93% of Hmga2−/− neurospheres formed peripherin+ neurons, GFAP+ glia, and smooth muscle actin+ myofibroblasts. At P0, 93% of Hmga2+/+ and 92% of Hmga2−/− neurospheres formed neurons, glia, and myofibroblasts. At P49-56, 94% of Hmga2+/+ and 92% of Hmga2−/− neurospheres formed neurons, glia and myofibroblasts.
Supplementary figure 4: Hmga2 knockdown in CNS stem cells using shRNA leads to increased p16Ink4a expression and reduced self-renewal potential. A) P0 VZ cells were dissociated from wild-type mice and infected with either GFP-only or Hmga2 shRNA+GFP lentiviral vectors. Multipotent neurospheres were then allowed to develop from these cells at clonal density. Acute knockdown of Hmga2 by shRNA significantly decreased their self-renewal relative to uninfected neurospheres in the same cultures (3 experiments: **P<0.05) as well as relative to GFP-only infected neurospheres in control cultures. B) Protein was extracted from the primary CNS neurospheres examined in (A) and subjected to Western blot for Hmga2, p16Ink4a, and β-actin (loading control). Hmga2 shRNA decreased Hmga2 protein expression and increased p16Ink4a protein expression in P0 CNS neurospheres. Un: uninfected, In: infected.
Supplementary figure 5: Deletion of p16Ink4a/p19Arf, p16Ink4a alone, or p19Arf alone, partially rescues the defects in NCSC frequency and self-renewal potential as well as gut neurogenesis in Hmga2−/− mice. Images show typical primary PNS neurospheres formed after 10 days culture of P49-56 gut plexus/outer muscle layer cells. p16Ink4a/p19Arf deficiency (A; 4–6 mice per genotype in 4 independent experiments), p16Ink4a deficiency (B; 4–5 mice per genotype in 3 independent experiments), or p19Arf deficiency (C; 3–5 mice per genotype in 3 independent experiments) did not affect the percentage of wild-type gut cells that formed multipotent neurospheres or their self-renewal potential (absolute number or percentage of primary neurosphere cells that gave rise to multipotent secondary neurospheres upon subcloning of individual neurospheres) but did significantly increase the percentage of Hmga2−/− gut cells that formed multipotent neurospheres, the diameter of these neurospheres, and their self-renewal potential. All data represent mean±SD (*, significantly different (P<0.05) from wild-type; §, significantly different from Hmga2+/+p16Ink4a/p19Arf−/− mice (A) or Hmga2+/+p16Ink4a−/− mice (B) or Hmga2+/+p19Arf−/− mice (C); #, significantly different from Hmga2−/−p16Ink4a/p19Arf+/+ mice (A) or Hmga2−/−p16Ink4a+/+ mice (B) or Hmga2−/−p19Arf+/+ mice (C)). D) Gut sections from Hmga2/p16Ink4a/p19Arf mutant mice in which myenteric plexus neurons are indicated with brackets. E) p16Ink4a/p19Arf deficiency partially rescued the reduction in HuC/D+ neurons per transverse section through the distal ileum in young adult Hmga2−/− mice without affecting the numbers of neurons in wild-type littermates (3 mice per genotype, 6–10 sections per mice). All T-tests were paired.
Supplementary figure 6: p16Ink4a/p19Arf deficiency, or p16Ink4a deficiency, or p19Arf deficiency increases the brain mass but not the overall body mass of Hmga2−/− mice. Body masses (A,C,E) or brain masses (B,D,F) of Hmga2/p16Ink4a/p19Arf (A,B; 8–10 mice per genotype), Hmga2/p16Ink4a (C,D; 7–9 mice per genotype), or Hmga2/p19Arf (E,F; 9–11 mice per genotype) compound mutant mice were examined at P49-56. In each case, Hmga2 deficiency significantly reduced body mass. p16Ink4a/p19Arf deficiency, p16Ink4a deficiency, or p19Arf deficiency did not affect the body mass of wild-type or Hmga2−/− mice. p16Ink4a/p19Arf deficiency or p16Ink4a deficiency did not affect the brain mass of wild-type mice but did partially rescue the brain mass reduction observed in Hmga2−/− mice. p19Arf deficiency showed a trend toward rescuing brain mass but the effect was not statistically significant. All error bars represent SD (*, significantly different (P<0.05) from wild-type; §, significantly different from Hmga2+/+p16Ink4a/p19Arf−/− mice (A,B), Hmga2+/+p16Ink4a−/− mice (C,D) or Hmga2+/+p19Arf−/− mice (E,F); #, significantly different from Hmga2−/−p16Ink4a/p19Arf+/+ mice (A,B), Hmga2−/−p16Ink4a+/+ mice (C,D) or Hmga2−/−p19Arf+/+ mice (E,F)). Statistical comparisons were done using unpaired T-tests in this figure.
Supplementary figure 7: Hmga2 is not required for the proliferation or self-renewal of gut NCSCs or CNS stem cells from old mice, and Hmga2 protein expression is regulated post-transcriptionally in CNS neurospheres from old Hmga2−/− mice. A) Cells were isolated from wild-type or Hmga2−/− gut plexus/outer muscle layers at P570-600, and cultured to generate PNS neurospheres (A; 10 days in culture). The frequency of PNS cells that formed multipotent neurospheres, the diameter of these neurospheres, and their self-renewal potential upon subcloning were unaffected by Hmga2 deficiency (A; 3 independent experiments). (B–E) Hmga2 deficiency did not affect the numbers of cells per colony within adherent cultures of CNS SVZ cells (B) or gut cells (D) from P570-600 mice. Only colonies with the appearance of stem cell colonies were counted (3 independent experiments). Hmga2 deficiency did not affect the percentage of cells within adherent colonies formed by SVZ cells (C) or gut cells (E) from P570-600 mice that incorporated a pulse of BrdU (3 independent experiments). F) P600 SVZ cells from Hmga2−/− animals were infected with GFP-only control lentivirus, Hmga2+GFP lentivirus, or 3′-UTR truncated Hmga2 (lacking let-7 binding sites)+GFP lentivirus, and allowed to form neurospheres. Neither over-expression of GFP nor wild-type Hmga2 altered the size or self-renewal of neurospheres. In contrast, over-expression of 3′-UTR truncated Hmga2 significantly increased the size and self-renewal of neurospheres (3 experiments: **P<0.05). All T-tests were paired.
Supplementary figure 8: Hmga2 protein binds to the junB locus in CNS neurospheres, and junB expression is increased within neurospheres in the absence of Hmga2 or within wild-type SVZ cells in vivo as Hmga2 expression declines during aging. A) Chromatin immunoprecipitation (ChIP) of Hmga2 protein in P0 CNS neurospheres. P0 SVZ cells from wild-type animals were infected with Hmga2-Flagx3 retrovirus and allowed to form neurospheres. Genomic DNA was then extracted from the neurospheres and subjected to ChIP with anti-FLAG or with anti-mouse IgG (control) antibody. junB locus amplification was detected in the FLAG pull-down fraction (FLAG), but not in the IgG pull-down fraction (IgG). Neither p16Ink4a nor p19Arf locus amplification were detected after FLAG pull-down. We also did not detect Hmga2 binding at other loci that encode proteins that can regulate p16Ink4a or p19Arf expression, including Bmi-1, tbx2, cbx8, or E2F3a. Input is the fraction before immunoprecipitation. (B) CNS neurospheres were cultured from wild-type or Hmga2−/− E14.5 telencephalon, P0 VZ, P49-56 SVZ, or P570-600 SVZ. (C) PNS neurospheres were cultured from wild-type or Hmga2−/− E14.5 gut, or P0, P49-56, or P570-600 plexus/outer muscle layer. RNA was extracted from primary CNS (B) or PNS (C) neurospheres and the levels of junB, bmi-1, and cbx8 were determined by qPCR. Each bar shows the fold-increase in Hmga2−/− as compared to wild-type neurospheres (error bars represent SD, 3–4 independent experiments per stage; **P<0.05). junB expression was increased in CNS and PNS neurospheres, from fetal but not from old mice, in the absence of Hmga2. D) Bmi-1, junB and Pten expression were compared by qPCR in freshly dissected E14.5 telencephalon, P0 VZ, P30 SVZ, P360 SVZ, and P720 SVZ (expressed as fold change relative to P0 SVZ; each bar represents mean±SD for 3–4 mice per stage). junB expression significantly increased with age (*P<0.01,**P<0.05), as Hmga2 expression declines and p16Ink4a/p19Arf expression increase. These data are consistent with the possibility that JunB may mediate the effect of Hmga2 on p16Ink4a/p19Arf expression. All T-tests were unpaired.