The development and maintenance of vertebrate tissues depends upon diverse stem cells. Stem cells are functionally distinct from downstream progenitors and likely depend upon regulatory mechanisms that are conserved among stem cells from different tissues but are absent in most downstream progenitors6
. Yet few such mechanisms have been identified. FoxO family transcription factors7–10
, polycomb family (e.g. Bmi-1) epigenetic regulators11
, and DNA repair genes12
are required for the maintenance of stem cells in multiple tissues but are widely expressed within tissues and likely to regulate many cells. Other genes, like Evi113
, are preferentially expressed by stem cells and required for stem cell maintenance in one tissue (in this case the hematopoietic system) but are not known to regulate stem cells in other tissues. Other genes, like Lgr515, 16
, are preferentially expressed by stem cells in multiple tissues but it is unknown whether they regulate stem cell function. The identification of genes that are preferentially expressed by stem cells and required to maintain stem cells in multiple tissues would therefore provide important new insights into stem cell identity and function.
To identify genes that regulate the self-renewal of diverse stem cells we performed a Bmi-1 suppressor screen by transposon mutagenesis (data not shown). We reasoned that by screening for suppressors of the Bmi-1
deficiency phenotype we could identify genes that modulate a widely used self-renewal pathway and that might encode new self-renewal regulators required by diverse stem cells. Our screen revealed that over-expression of Prdm16
partially restored the ability of Bmi-1
deficient hematopoietic cells to reconstitute irradiated mice (data not shown). Prdm16
over-expression can contribute to leukemogenesis1, 17
and can increase the ability of cultured hematopoietic stem cells (HSCs) to reconstitute irradiated mice18
, though no study has addressed whether Prdm16
is required for stem cell function in any tissue. We decided to test whether Prdm16
, like Bmi-1
, is physiologically required for stem cell function in multiple tissues.
We reanalyzed previously published gene expression profile data19
and found that Prdm16
was expressed by highly purified HSCs and transiently reconstituting multipotent hematopoietic progenitors (MPPs) but was not detectable in unfractionated hematopoietic cells (). Quantitative RT-PCR (qPCR) in independent samples yielded similar results as Prdm16
was expressed at 100-fold higher levels in highly purified CD150+
as compared to unfractionated bone marrow cells ().
Prdm16 is preferentially expressed by stem cells and primitive progenitors in the hematopoietic and nervous systems
To evaluate Prdm16
expression at the single cell level we used Prdm16Gt(OST67423)Lex
(further referred to as Prdm16LacZ
) genetrap mice in which LacZ
was inserted into the first intron of Prdm162
. This allele allowed us to assess Prdm16
expression based on β-galactosidase activity but terminated Prdm16
translation after the first exon, causing a loss of Prdm16
. Based on staining with the fluorogenic β-galactosidase substrate fluorescein di-β-D-galactopyranoside (FDG), less than 3% of Prdm16LacZ/+
bone marrow cells had β-galactosidase activity (). Most β-galactosidase+ cells were c-kit+
, markers of HSCs and other primitive progenitors. 61±7% of all c-kit+
bone marrow cells were β-galactosidase+ in Prdm16LacZ/+
mice (). Almost all CD150+
(90±10%) and CD150−
(82±2%) from 2 month-old Prdm16LacZ/+
mice expressed β-galactosidase by flow-cytometry (). In contrast, differentiated B, myeloid, T, and erythroid cells rarely expressed β-galactosidase (). β-galactosidase+ cells from the bone marrow of 2 month-old Prdm16LacZ/+
mice contained nearly all of the colony-forming cells (CFU-Cs; 0.7±0.2% of bone marrow cells) in Prdm16LacZ/+
bone marrow (). Prdm16
is thus preferentially expressed by HSCs and other primitive progenitors in the hematopoietic system.
was also preferentially expressed by stem cells in the nervous system. In forebrain sections from adult Prdm16LacZ/+
mice, β-galactosidase strongly overlapped with the stem/progenitor cell marker, Nestin, in the lateral ventricle subventricular zone (SVZ) but much less β-galactosidase staining was observed among the differentiated cells in the striatum and cortex (). Similar results were observed in the newborn forebrain where β-galactosidase overlapped with Nestin in the lateral ventricle ventricular zone (VZ) (Suppl. Fig. 1
). Virtually all neurospheres cultured from the Prdm16LacZ/+
, but not Prdm16+/+
, forebrain exhibited strong β-galactosidase staining (). Cultured and uncultured neural crest stem cells from the enteric nervous system also expressed Prdm16
(Suppl. Fig. 2
is therefore expressed by neural stem/progenitor cells in the central and peripheral nervous systems but expression declines as these cells differentiate.
We next tested whether Prdm16
is required to regulate HSCs. Prdm16LacZ/LacZ
mice were born at Mendelian frequency but died soon after birth (Suppl. Fig. 3a, b
). Hematopoiesis was grossly normal in newborn Prdm16LacZ/LacZ
mice as liver and spleen cellularity were normal (). No significant differences were detected in the frequency of myeloid, B, or T cells in the livers or spleens of newborn Prdm16LacZ/LacZ
, and Prdm16+/+
mice (, Suppl. Fig. 3c
). Most colony-forming progenitors were present at normal frequencies in the neonatal liver of Prdm16LacZ/LacZ
mice (). However, mixed myeloerythroid (CFU-GEMM) and mixed myeloid (CFU-GM) progenitors were significantly depleted in the livers of neonatal Prdm16LacZ/LacZ
mice (). The frequency of CD150+
HSCs in the embryonic day (E)14.5 liver (), newborn (P0) liver (), and newborn spleen (Suppl. Fig. 3d
) was reduced by approximately 2-fold in Prdm16LacZ/+
mice and by approximately 20-fold in Prdm16LacZ/LacZ
mice compared to Prdm16+/+
littermates. Loss of Prdm16
therefore profoundly depletes HSCs without depleting most downstream hematopoietic progenitors.
Prdm16 is required for survival, cell cycle regulation, and maintenance in fetal and adult HSCs
To assess HSC function we performed competitive long-term reconstitution assays using neonatal liver cells from Prdm16LacZ/LacZ mice and littermate controls. 300,000 donor (CD45.2+) Prdm16+/+ cells or Prdm16LacZ/+ cells gave long-term multilineage reconstitution by myeloid, B, and T cells in all irradiated recipient (CD45.1+) mice (). In contrast, 300,000 Prdm16LacZ/LacZ cells did not give long-term multilineage reconstitution in any recipients. All recipients of Prdm16LacZ/LacZ cells had low levels of transient reconstitution by donor B cells, and most had very low levels of transient myeloid or T cell reconstitution ().
Since Prdm16LacZ/LacZ mice exhibited a 20-fold depletion of HSCs (), we performed an additional experiment in which a 20-fold excess of donor (CD45.2+) cells from newborn Prdm16LacZ/LacZ mice was transplanted into irradiated wild-type recipients (CD45.1+) along with 300,000 recipient bone marrow cells. As in prior experiments, 300,000 Prdm16LacZ/LacZ cells gave poor reconstitution in all lineages while 300,000 Prdm16+/+ cells gave long-term multilineage reconstitution in all recipients (). 6×106 neonatal Prdm16LacZ/LacZ liver cells gave long-term multilineage reconstitution in all recipients, but the levels of donor cell reconstitution were significantly lower than from 300,000 Prdm16+/+ control cells and were declining by 16 weeks after transplantation (). Some HSC activity thus remains in newborn Prdm16LacZ/LacZ mice, and can sustain hematopoiesis, but these HSCs are greatly depleted.
To further test whether the residual Prdm16LacZ/LacZ HSCs were defective we transplanted 20 CD150+CD41−CD48−Sca1+c-kit+ HSCs from Prdm16LacZ/LacZ, Prdm16LacZ/+, or Prdm16+/+ neonates into irradiated recipients along with 300,000 recipient bone marrow cells. Four of 10 recipients of Prdm16+/+ HSCs and 6 of 10 recipients of Prdm16LacZ/+ HSCs, but none of 9 recipients of Prdm16Lacz/LacZ HSCs, were long-term multilineage reconstituted (). The levels of donor cell reconstitution by Prdm16Lacz/LacZ HSCs were significantly lower than from control HSCs in all lineages (). Even highly enriched HSC populations therefore have little reconstituting capacity in the absence of Prdm16. Furthermore, the frequency of HSCs in adult Prdm16LacZ/+ bone marrow was significantly reduced (3-fold) relative to Prdm16+/+ bone marrow (). Prdm16 is therefore required for the maintenance of fetal and adult HSCs.
Prdm16 is required for normal cell cycle regulation and survival in HSCs and other primitive hematopoietic progenitors. Prdm16 deficiency significantly increased the frequency of c-kit+Sca-1+ cells, but not unfractionated liver cells, that stained positively for AnnexinV and DAPI (; we could not evaluate highly purified HSCs in this assay because they were too rare in Prdm16Lacz/LacZ mice). Prdm16 deficiency also significantly increased the frequency of c-kit+Sca-1+ cells, but not unfractionated liver cells, that stained positively for activated caspase-3 (data not shown). Prdm16 deficiency significantly increased the frequency of HSCs, MPPs, and c-kit+Sca-1+ cells, but not unfractionated liver cells, in S/G2/M phase of the cell cycle ().
also regulates neural development. Brain mass was significantly reduced in Prdm16LacZ/LacZ
mice compared to littermate controls (). Prdm16LacZ/LacZ
forebrains had a thinner cortex (see brackets in ), narrower ventricles (see arrowheads in ), and agenesis of the corpus callosum (see arrows in , confirmed by serial sections). Axons in Prdm16LacZ/LacZ
mice formed Probst bundles21
instead of crossing the midline (see * in ; see arrows in ).
Prdm16 is required for survival, cell cycle regulation, and self-renewal in neural stem cells
To test whether Prdm16
regulates neural stem cell function we cultured VZ cells from newborn Prdm16LacZ/LacZ
mice and littermate controls at clonal densities (<1 cell/μl). Prdm16LacZ/LacZ
VZ cells formed neurospheres that underwent multilineage differentiation (). However, the frequency of VZ cells that formed multilineage colonies in culture was significantly reduced in Prdm16LacZ/LacZ
mice (). Prdm16LacZ/LacZ
neurospheres were also significantly smaller than Prdm16+/+
neurospheres (). The self-renewal potential of Prdm16LacZ/LacZ
neural stem cells was significantly less than control stem cells based on the number of multipotent daughter cells that could be subcloned from individual primary neurospheres (). Prdm16
was similarly required for stem cell function in the peripheral nervous system (Suppl. Fig. 2c,d
deficiency thus reduces self-renewal potential and depletes neural stem cells, similar to the defects observed in the hematopoietic system.
To test whether we could observe neural stem/progenitor cell defects in vivo we stained sections through the forebrain of newborn Prdm16LacZ/LacZ mice and littermate controls with an antibody against phospho-Histone3 (pH3) to identify mitotic cells. We observed significantly fewer pH3+ cells in the lateral ventricle VZ of Prdm16LacZ/LacZ mice compared to littermate controls (). We also observed a significantly increased frequency of activated caspase-3+ cells among Prdm16LacZ/LacZ VZ cells (). These effects were more pronounced in the dorsal VZ than in the ventral VZ. Prdm16 is therefore required for normal cell cycle regulation and survival in neural stem/progenitor cells.
To investigate the underlying mechanisms we compared the gene expression profiles of uncultured VZ cells from newborn Prdm16LacZ/LacZ
, and Prdm16+/+
mice (3 independent samples per genotype). Thirteen genes were significantly reduced in expression () and six were significantly increased in expression (Suppl. Fig. 3e
) in the Prdm16LacZ/LacZ
VZ (at least 2.2-fold different (p<0.05) between Prdm16LacZ/LacZ
VZ and at least 1.8-fold different (p<0.05) between Prdm16LacZ/LacZ
VZ). These differences were confirmed by qPCR in 3 independent samples per genotype (; Suppl. Fig. 3e
). Several of these genes regulate the generation of ROS or the response to oxidative stress (see * in ). In particular, Hepatocyte growth factor
), and Metallothienin2
) expression were reduced in Prdm16LacZ/LacZ
VZ (). The reduction in Hgf
expression in the absence of Prdm16
was further confirmed in neurospheres cultured from the newborn VZ (). These changes would be predicted to increase ROS levels as MT2 scavenges free radicals22
and HGF can protect cells from oxidative stress by reducing ROS levels23, 24
) expression was also reduced in the Prdm16LacZ/LacZ
VZ (1.7-fold by microarray and 3.7-fold by qPCR; data not shown). Consistent with these predictions, DCFDA staining (an indicator of ROS levels) significantly increased in newborn Prdm16LacZ/LacZ
VZ cells as compared to littermate controls (). Although Prdm16LacZ/LacZ
VZ cells had increased ROS levels, we did not detect any defects in mitochondrial mass or membrane potential (Suppl. Fig. 4c, d
is therefore required to control the expression of genes that regulate ROS levels in neural stem/progenitor cells and to avoid oxidative stress.
Prdm16 promotes the expression of Hgf and regulates ROS levels in neural stem/progenitor cells
Chromatin immunoprecipitation experiments were conducted to test if Prdm16 directly binds the promoters of Hgf and Mt2. Two of four sequences 5′ of the Hgf start codon were immunoprecipitated from Prdm16+/+, but not Prdm16LacZ/LacZ, neurospheres using anti-Prdm16 antibodies (). In contrast, no Prdm16 binding was detected at the Mt2 promoter. These data suggest that Hgf, but not Mt2, is a direct target of Prdm16.
To test whether HGF regulates oxidative stress in neural stem/progenitor cells we added recombinant HGF to neurospheres cultured from the VZ of newborn Prdm16LacZ/LacZ mice and littermate controls. The Prdm16LacZ/LacZ cells exhibited significantly higher levels of DCFDA staining but HGF treatment significantly reduced DCFDA staining in these cells (). These data suggest that HGF regulates ROS levels in neural stem/progenitor cells and that Prdm16 regulates oxidative stress in neural stem/progenitor cells partly by regulating Hgf expression.
To test whether oxidative stress contributed to the defects in neural stem/progenitor cell function and neural development we administered the anti-oxidant N-acetyl-cysteine (NAC) to pregnant Prdm16LacZ/+ mice. NAC significantly increased brain size in newborn Prdm16LacZ/LacZ mice () but did not affect brain size in Prdm16LacZ/+ or Prdm16+/+ mice (). In wild-type mice, glial fibrillary acidic protein (GFAP) expressing astrocytes were evident around the midline and the corpus callosum at birth () but many fewer GFAP+ cells were observed around the midline of Prdm16LacZ/LacZ mice (). Prdm16LacZ/LacZ mice treated with NAC in utero had more GFAP+ cells around the midline in the forebrain (), though the corpus callosum still did not develop in these mice. NAC treatment in utero () or in culture () also significantly increased the frequency of newborn Prdm16LacZ/LacZ VZ cells, but not Prdm16LacZ/+ or Prdm16+/+ VZ cells, that formed multipotent neurospheres in culture. Oxidative stress therefore contributes to the defects in neural development in Prdm16LacZ/LacZ mice.
Prdm16 promotes neural stem/progenitor cell function by regulating Hgf expression and ROS levels
Since Prdm16 promotes Hgf expression in neural stem/progenitor cells (), HGF regulates ROS levels in these cells (), and increased ROS levels contribute to the defects in neural stem/progenitor cell function in Prdm16LacZ/LacZ mice (), we tested whether addition of HGF to culture could partially rescue the defects in Prdm16LacZ/LacZ neural stem cell function. Addition of HGF significantly increased the frequency of Prdm16LacZ/LacZ, but not Prdm16LacZ/+ or Prdm16+/+, VZ cells that formed multipotent neurospheres ().
To test whether loss of Prdm16
changes ROS levels within primitive hematopoietic progenitors, we stained c-kit+
stem/progenitor cells from newborn Prdm16LacZ/LacZ
mice with DCFDA. Surprisingly, we observed a significant decline in ROS levels within Prdm16LacZ/LacZ
liver cells compared to control cells while ROS levels remained unchanged in unfractionated liver cells (Suppl. Fig. 4a–b
). Thus, Prdm16
is also required to regulate ROS levels in primitive hematopoietic progenitors, though loss of Prdm16
appeared to decrease ROS levels in these cells, in contrast to the nervous system. Furthermore, NAC treatment of pregnant mice did not rescue the depletion of CD150+
HSCs in Prdm16LacZ/+
mice (). Prdm16 might promote stem cell maintenance by different mechanisms in different tissues. Alternatively, there could be an increase in ROS levels in Prdm16LacZ/LacZ
HSCs that is masked by other changes that occur among the heterogeneous c-kit+
cells. Unfortunately, this is impossible to test because too few HSCs can be recovered from Prdm16LacZ/LacZ
mice () to assess DCFDA staining. In either case, the failure of NAC to rescue the depletion of Prdm16LacZ/LacZ
HSCs suggests that Prdm16 has other critical functions in HSCs beyond regulating oxidative stress. Finally, compensatory changes might also be induced in response to oxidative stress25
in either HSCs or neural stem/progenitor cells that alter the observed effects of Prdm16
deficiency on ROS levels.
Our results demonstrate that Prdm16 is required for stem cell function in the nervous and hematopoietic systems (, ). In the central nervous system, Prdm16 appears to promote neural stem/progenitor cell function partly by promoting HGF expression () and regulating ROS levels (). However, Prdm16 likely regulates ROS levels and stem cell maintenance by other mechanisms as well. Our results emphasize the critical and diverse mechanisms required by stem cells to regulate oxidative stress. Bmi-1
also regulates ROS levels in stem cells26
, but Prdm16 does not appear to regulate Bmi-1
expression or vice versa (data not shown). Considerable additional work will be required to fully elucidate the mechanisms by which Prdm16 regulates oxidative stress and stem cell maintenance.