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. 2010 Oct;12(10):999-1006.
doi: 10.1038/ncb2101. Epub 2010 Sep 12.

Prdm16 promotes stem cell maintenance in multiple tissues, partly by regulating oxidative stress

Sergei Chuikov  1 , Boaz P Levi, Michael L Smith, Sean J Morrison
Affiliations

Prdm16 promotes stem cell maintenance in multiple tissues, partly by regulating oxidative stress

Sergei Chuikov et al. Nat Cell Biol. 2010 Oct.

Abstract

To better understand the mechanisms that regulate stem cell identity and function, we sought to identify genes that are preferentially expressed by stem cells and critical for their function in multiple tissues. Prdm16 is a transcription factor that regulates leukaemogenesis, palatogenesis and brown-fat development, but which was not known to be required for stem cell function. We demonstrate that Prdm16 is preferentially expressed by stem cells throughout the nervous and haematopoietic systems and is required for their maintenance. In the haematopoietic and nervous systems, Prdm16 deficiency led to changes in the levels of reactive oxygen species (ROS), depletion of stem cells, increased cell death and altered cell-cycle distribution. In neural stem/progenitor cells, Prdm16 binds to the Hgf promoter, and Hgf expression declined in the absence of Prdm16. Addition of recombinant HGF to Prdm16-deficient neural stem cells in cell culture reduced the depletion of these cells and partially rescued the increase in ROS levels. Administration of the anti-oxidant, N-acetyl-cysteine, to Prdm16-deficient mice partially rescued defects in neural stem/progenitor cell function and neural development. Prdm16 therefore promotes stem cell maintenance in multiple tissues, partly by modulating oxidative stress.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors are not aware of any competing financial interests.

Figures

Figure 1
Figure 1. Prdm16 is preferentially expressed by stem cells and primitive progenitors in the hematopoietic and nervous systems
a) Expression intensity for Prdm16 in HSCs, non-self-renewing multipotent progenitors (MPPs) and CD45+ bone marrow cells from young adult mice (data were extracted from a microarray analysis in an earlier study19). b) Quantitative RT-PCR of cDNA from CD150+CD48−CD41−Sca1+c-kit+ HSCs and unfractionated bone marrow cells from young adult mice in three experiments confirmed Prdm16 is expressed at 100-fold higher levels in HSCs. Samples were normalized using β-actin (*, P=0.029). c) FDG staining for β-galactosidase activity in cells from 2–6 month-old Prdm16LacZ/+ mice indicated that only 3±1% (mean±SD) of bone marrow cells expressed Prdm16. Background staining was observed in 0.6±0.3% of control bone marrow cells. Most of the bone marrow cells from Prdm16LacZ/+ mice that had β-galactosidase activity were c-kit+. d, e) The vast majority of CD150+CD48−CD41−Sca1+c-kit+ HSCs (90±10%; the same surface markers were used to isolate HSCs in subsequent figures) and CD150−CD48−CD41−Sca1+c-kit+ MPPs (82±2%; the same surface markers were used to isolate MPPs in subsequent figures) but few differentiated hematopoietic cells had β-galactosidase activity in Prdm16LacZ/+ mice. f) FDG+ bone marrow cells from Prdm16LacZ/+ adult mice were significantly (p<0.00005) enriched for colony-forming cells (CFU-C) in methylcellulose cultures and contained nearly all colony-forming cells in the bone marrow. The number of independent replicates is indicated in each panel that includes data from multiple independent experiments, and all error bars represent SD. Statistical significance was always assessed by Student’s t-test. g–h) Neurospheres cultured from lateral ventricle VZ cells from newborn Prdm16+/+ (g) or Prdm16LacZ/LacZ (h) mice and stained with X-gal (blue) revealed that virtually all Prdm16LacZ/LacZ neurospheres expressed Prdm16. i–l) Antibody staining for β-galactosidase indicated Prdm16 was expressed in the adult VZ in a pattern that overlaps with the stem/progenitor cell marker Nestin.
Figure 2
Figure 2. Prdm16 is required for survival, cell cycle regulation, and maintenance in fetal and adult HSCs
a) The cellularity of the liver and spleen and b) the frequencies of mature hematopoietic cells in the liver were normal in newborn Prdm16LacZ/LacZ mice (3 independent experiments with a total of 5–12 mice/genotype; the exact number of mice is indicated in the panel legend). c) The frequencies of most colony-forming progenitors were normal in the liver of Prdm16LacZ/LacZ mice, but CFU-GEMM and CFU-GM were significantly depleted. d) HSCs were depleted in the fetal (E14.5) and newborn (P0) liver of Prdm16LacZ/LacZ mice (4 independent experiments with a total of 3–12 mice/genotype; the exact number of mice is indicated under each bar). e) c-kit and Sca-1 staining is shown in the right column for CD150+CD48−CD41− cells gated in the left column for representative mice of each genotype. f) Irradiated CD45.1+ recipient mice were competitively reconstituted with 3×105 CD45.2+ neonatal liver cells from Prdm16+/+ (black lines), Prdm16LacZ/+ (green lines) or Prdm16LacZ/LacZ mice (red lines) along with 3×105 CD45.1+ recipient bone marrow cells. Each line represents average donor cell reconstitution levels (mean±SD; 3 independent experiments with 2–5 recipients/treatment/experiment; the total number of mice transplanted with cells of each genotype is inducted in panel legends for f–h). g) Donor cell reconstitution in an independent experiment in which recipients were transplanted with 3×105 CD45.2+ neonatal liver cells from a Prdm16+/+ donor (black lines), or 3×105 cells from a Prdm16LacZ/LacZ donor (red lines), or 6×106 cells from Prdm16LacZ/LacZ donor (blue lines) along with 3×105 young adult CD45.1+ bone marrow cells (1 experiment with at least 5 recipients/treatment). h) Donor cell reconstitution in 2 experiments in which recipients were transplanted with 20 CD45.2+ HSCs from Prdm16+/+ (black lines), Prdm16LacZ/+ (green lines), or Prdm16LacZ/LacZ donors (red lines) along with 3×105 CD45.1+ bone marrow cells (2 independent experiments). i) Bone marrow HSC frequency in adult Prdm16+/+ and Prdm16LacZ/+ mice (2 experiments with 5 or 6 mice/genotype). j) The frequency of annexin V+/DAPI+ unfractionated newborn liver cells or c-kit+Sca-1+ cells (5 independent experiments with 6 or 9 mice/genotype). k) Cell cycle distribution of cells from newborn liver (3 independent experiments with 3–9 mice/genotype). In panels i–k, the exact number of mice of each genotype is indicated under each bar in each panel (*, P<0.05; **, P<0.01; ***, P<0.001, error bars always represent SD).
Figure 3
Figure 3. Prdm16 is required for survival, cell cycle regulation, and self-renewal in neural stem cells
a) The brains of neonatal Prdm16LacZ/LacZ mice were significantly smaller than those of Prdm16+/+ or Prdm16LacZ/+ mice (the number of mice of each genotype is shown under each bar). b–c) Hematoxylin and eosin staining of coronal sections showed that Prdm16LacZ/LacZ brains (c) were smaller and that morphology was disrupted relative to control brains (b). Brackets show reduced cortical thickness, arrowheads show narrower lateral ventricle, and arrows point to the lack of a corpus callosum in the Prdm16LacZ/LacZ brain. d–e) Agenesis of the corpus callosum was confirmed by staining with the neuronal marker TuJ1 which revealed axon tracts that crossed the midline in wild-type (arrow, d) but Probst bundles that did not cross the midline in Prdm16LacZ/LacZ brains (*, e). f–j) Some primary neurospheres of all genotypes underwent multilineage differentiation, forming neurons (TuJ1+), astrocytes (GFAP+), and oligodendrocytes (O4+); however, a significantly lower percentage of VZ cells from newborn Prdm16LacZ/LacZ mice formed multipotent neurospheres compared to littermate controls (h) and the diameter (i) and self-renewal potential (j) of Prdm16LacZ/LacZ neurospheres was significantly less than control neurospheres (the number of mice per genotype is indicated in each panel; panels h, i, and j reflect 6, 4, and 3 independent experiments). Self-renewal was quantified as the number of multipotent secondary neurospheres generated upon the subcloning of individual primary neurospheres. k–o) Significantly fewer dividing cells were observed in the VZ of newborn Prdm16LacZ/LacZ mice compared to littermate controls based on phospho-histone3 (pH3) staining (3 mice per genotype and 4 sections per mouse). p–r) Significantly more cells underwent cell death in the lateral ventricle VZ of newborn Prdm16LacZ/LacZ mice compared to littermate controls (* marks lateral ventricle) in sections (p–q), and by flow cytometry (6 mice/genotype from 3 independent experiments). (*, P<0.05; **, P<0.01; ***, P<0.001, error bars always represent SD).
Figure 4
Figure 4. Prdm16 promotes the expression of Hgf and regulates ROS levels in neural stem/progenitor cells
a) The gene expression profiles of VZ cells from newborn Prdm16+/+, Prdm16LacZ/+, and Prdm16LacZ/LacZ mice were compared by microarray (3 independent samples per genotype). The list shows all genes that were significantly (p<0.05) reduced in expression within Prdm16LacZ/LacZ VZ cells as compared to control cells (by at least 2.2 fold between Prdm16+/+ and Prdm16LacZ/LacZ VZ and at least 1.8 fold between Prdm16LacZ/+ and Prdm16LacZ/LacZ cells). Asterisks indicate genes associated with ROS regulation or response to oxidative stress. Differential expression was confirmed by qPCR in 3 independent samples/genotype. Genes that increased in expression in Prdm16LacZ/LacZ VZ cells are shown in Suppl. Fig. 3e. d) Hgf expression was confirmed to decline by qPCR in neurospheres cultured from Prdm16LacZ/LacZ mice. c–d) Newborn Prdm16LacZ/LacZ VZ cells had significantly and uniformly increased ROS levels based on DCFDA staining. e) Prdm16 bound to the promoter of Hgf but not Mt2. Chromatin immunoprecipitation was conducted using anti-Prdm16 antibodies and primary neurospheres from Prdm16+/+ and Prdm16LacZ/LacZ mice. qPCR was used to quantify the immunoprecipitated Hgf and Mt2 promoter regions indicated on the schematics. Data are shown as fold enrichment over control IgG immunoprecipitation. Statistical significance was determined by paired T-tests comparing fold enrichment of target sequences immunoprecipitated by anti-Prdm16/MT2 antibody versus input DNA compared to control IgG versus input DNA. f) HGF treatment reduced ROS levels in neurospheres grown adherently for 5–6 days. HGF was added to the cultures 20 hours before ROS measurement. The number of independent experiments is indicated in each panel, or on each bar in panel c to indicate the number of experiments in which mice of the indicated genotype were used. *, P<0.05; **, P<0.01; ***, P<0.001, error bars always represent SD.
Figure 5
Figure 5. Prdm16 promotes neural stem/progenitor cell function by regulating Hgf expression and ROS levels
NAC treatment significantly increased brain size (a) and mass (b) in Prdm16LacZ/LacZ mice but not in littermate controls (the number of mice per treatment is indicated under each bar; each panel reflects at least 3 independent experiments). c–e) Newborn Prdm16LacZ/LacZ mice (d) had reduced GFAP staining (red) in the midline relative to control littermates (c) but this phenotype was partially rescued by NAC treatment (e). Higher magnification images of the boxed regions are shown to the right. f) NAC treatment in utero significantly (by paired t-test) increased the percentage of newborn Prdm16LacZ/LacZ VZ cells that formed multipotent neurospheres in culture. Addition of NAC (g) or HGF (h) to culture significantly increased the percentage of newborn Prdm16LacZ/LacZ VZ cells that formed multipotent neurospheres. i) NAC treatment of pregnant mice did not rescue the depletion of HSCs in Prdm16LacZ/LacZ mice. (*, P<0.05; **, P<0.01; ***, P<0.001, error bars always represent SD). j) A model of Prdm16 function in neural stem cells.
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References

    1. Morishita K. Leukemogenesis of the EVI1/MEL1 gene family. International journal of hematology. 2007;85:279–286. - PubMed
    1. Bjork BC, Turbe-Doan A, Prysak M, Herron BJ, Beier DR. Prdm16 is required for normal palatogenesis in mice. Human molecular genetics. 2010;19:774–789. - PMC - PubMed
    1. Seale P, et al. PRDM16 controls a brown fat/skeletal muscle switch. Nature. 2008;454:961–967. - PMC - PubMed
    1. Seale P, et al. Transcriptional control of brown fat determination by PRDM16. Cell metabolism. 2007;6:38–54. - PMC - PubMed
    1. Murholm M, et al. Dynamic regulation of genes involved in mitochondrial DNA replication and transcription during mouse brown fat cell differentiation and recruitment. PloS one. 2009;4:e8458. - PMC - PubMed

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