Ink4a and Arf differentially affect cell proliferation and neural stem cell self-renewal in Bmi1-deficient mice

Abstract

The Polycomb group (PcG) gene Bmi1 promotes cell proliferation and stem cell self-renewal by repressing the Ink4a/Arf locus. We used a genetic approach to investigate whether Ink4a or Arf is more critical for relaying Bmi1 function in lymphoid cells, neural progenitors, and neural stem cells. We show that Arf is a general target of Bmi1, however particularly in neural stem cells, derepression of Ink4a contributes to Bmi1(-/-) phenotypes. Additionally, we demonstrate haploinsufficient effects for the Ink4a/Arf locus downstream of Bmi1 in vivo. This suggests differential, cell type-specific roles for Ink4a versus Arf in PcG-mediated (stem) cell cycle control.

Figure 1.

Differential effects of Ink4a and Arf dosage and derepression in Bmi1^-/- lymphoid organs. (A) Splenocyte counts are dramatically reduced in Bmi1^-/- mice. Heterozygosity for Arf or Ink4a/Arf partially rescues this phenotype (p < 0.01) but complete loss of Arf or Ink4a/Arf results in a better partial rescue. p53 loss also induces a partial rescue. (B,D) Neither Bmi1^-/- splenocyte nor thymocyte counts are significantly restored by loss of Ink4a. (C) Thymocyte counts are dramatically reduced in Bmi1^-/- mice. Loss of the complete Ink4a/Arf locus gives a substantially better rescue than loss of Arf alone (p < 0.05). Note that heterozygosity does not lead to a rescue. p53 loss induces a minor rescue in lymphocyte counts.

Figure 2.

Ink4a/Arf derepression upon Bmi1 loss prevents efficient Shh-induced CGNP proliferation. (A) qRT-PCR shows up-regulation of Ink4a and Arf mRNA expression in Bmi1^-/- CGNPs and cerebella. (B) Bmi1; (Cb) cerebellum. (B) Reduced BrdU incorporation in Bmi1^-/- CGNPs upon Shh treatment (p < 0.001) is rescued upon subsequent Arf loss (p < 0.01) but not by Arf heterozygosity. (C,D)In wild-type CGNPs, Shh treatment induces an increase in Bmi1 protein levels and a decrease in Ink4a (p < 0.005) and Arf (p < 0.02) mRNA expression. (Tub) Tubulin. (E) Reduced BrdU incorporation in Bmi1^-/- CGNPs upon Shh treatment is rescued by Ink4a/Arf loss (p < 0.05) and Ink4a/Arf heterozygosity (p < 0.03).

Figure 3.

Ink4a and Arf differentially contribute to histological abnormalities of the Bmi1^-/- cerebellum. (A) Morphological analysis of wild-type (left panels), Bmi1^-/- (middle left panels), Bmi1^-/-;Arf^-/- (middle right panels), and Bmi1^-/-;Ink4a/Arf^-/- (right panels) adult cerebellum. Haematoxylin and eosin (H&E, top and middle panels) staining shows rescue of overall cerebellar size and granular layer thickness in Bmi1^-/-;(Ink4a)Arf^-/- mice. (Bottom panels) Aberrant arborization of basket neurons (NF200 staining) is observed in all cerebella lacking Bmi1. Final magnification, 5× and 60×. (IGL) Internal granular layer; (ML) molecular layer. (B) IGL area measurements reveal similar significant rescues in Bmi1^-/-;Arf^-/- and Bmi1^-/-;Ink4a/Arf^-/- mice. (C) Ink4a/Arf loss induces a complete rescue of the number of Bmi1^-/- granule neurons, whereas Arf loss leads to a partial rescue (p < 0.01). (D) The partial rescue in number of Bmi1^-/- molecular layer neurons is significantly better in an Ink4a/Arf deficient background (p < 0.05) (HPF, high-power field; n = 4 mice).

Figure 4.

Accurate repression of Ink4a and Arf is required for neurosphere self-renewal. (A,D) Phase-contrast pictures (A, left panel) and diameter measurements show that Bmi1^-/- neurospheres are much smaller than wild types. Loss of either Arf or Ink4a/Arf completely rescues this Bmi1^-/- phenotype. (A, right panel) All primary neurospheres are multipotent. Note that Bmi1^-/- spheres differentiate less efficiently (GFAP staining in green and β-tubulin-III in red). (B) Western blot analysis reveals increased p16^ink4a expression in Bmi1^-/- cerebrum. (Tub) Tubulin. (C) qRT-PCR shows increased Ink4a and Arf mRNA expression in Bmi1^-/- cerebrum. (Cb) Cerebrum. (E) P7- and P30-derived Bmi1^-/- neurospheres are severely impaired in their self-renewal capacity. Arf loss alone gives a partial rescue of this phenotype (p < 0.05). Removal of the complete Ink4a/Arf locus fully restores the self-renewing ability of Bmi1^-/- neurospheres. Note that loss of Arf or Ink4a/Arf in a Bmi1^+/+ background enhances self-renewal (p < 0.01). (1°) Primary; (2°) secondary. (F) Bmi1^-/- SVZ adherent colonies incorporate less BrdU than control colonies, which is completely rescued upon loss of either Arf or Ink4a/Arf.