Dicer1 and miR-219 Are required for normal oligodendrocyte differentiation and myelination.
ABSTRACT To investigate the role of microRNAs in regulating oligodendrocyte (OL) differentiation and myelination, we utilized transgenic mice in which microRNA processing was disrupted in OL precursor cells (OPCs) and OLs by targeted deletion of Dicer1. We found that inhibition of OPC-OL miRNA processing disrupts normal CNS myelination and that OPCs lacking mature miRNAs fail to differentiate normally in vitro. We identified three miRNAs (miR-219, miR-138, and miR-338) that are induced 10-100x during OL differentiation; the most strongly induced of these, miR-219, is necessary and sufficient to promote OL differentiation, and partially rescues OL differentiation defects caused by total miRNA loss. miR-219 directly represses the expression of PDGFRalpha, Sox6, FoxJ3, and ZFP238 proteins, all of which normally help to promote OPC proliferation. Together, these findings show that miR-219 plays a critical role in coupling differentiation to proliferation arrest in the OL lineage, enabling the rapid transition from proliferating OPCs to myelinating OLs.
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ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancers in the world with one of the worst outcome. The oncogenic mucin MUC4 has been identified as an actor of pancreatic carcinogenesis as it is involved in many processes regulating pancreatic cancer cell biology. MUC4 is not expressed in healthy pancreas whereas it is expressed very early in pancreatic carcinogenesis. Targeting MUC4 in these early steps may thus appear as a promising strategy to slow-down pancreatic tumorigenesis. miRNA negative regulation of MUC4 could be one mechanism to efficiently downregulate MUC4 gene expression in early pancreatic neoplastic lesions. Using in silico studies, we found two putative binding sites for miR-219-1-3p in the 3'-UTR of MUC4 and showed that miR-219-1-3p expression is downregulated both in PDAC-derived cell lines and human PDAC tissues compared with their normal counterparts. We then showed that miR-219-1-3p negatively regulates MUC4 mucin expression via its direct binding to MUC4 3'-UTR. MiR-219-1-3p overexpression (transient and stable) in pancreatic cancer cell lines induced a decrease of cell proliferation associated with a decrease of cyclin D1 and a decrease of Akt and Erk pathway activation. MiR-219-1-3p overexpression also decreased cell migration. Furthermore, miR-219-1-3p expression was found to be conversely correlated with Muc4 expression in early pancreatic intraepithelial neoplasia lesions of Pdx1-Cre;LSL-Kras(G12D) mice. Most interestingly, in vivo studies showed that miR-219-1-3p injection in xenografted pancreatic tumors in mice decreased both tumor growth and MUC4 mucin expression. Altogether, these results identify miR-219-1-3p as a new negative regulator of MUC4 oncomucin that possesses tumor-suppressor activity in PDAC.Oncogene advance online publication, 10 March 2014; doi:10.1038/onc.2014.11.Oncogene 03/2014; · 8.56 Impact Factor
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ABSTRACT: To understand the involvement of structural maintenance of chromosome 4 (SMC4) in the development and progression of hepatocellular carcinoma (HCC).Journal of experimental & clinical cancer research : CR. 06/2014; 33(1):55.
- Annals of Neurology 03/2014; · 11.19 Impact Factor
Dicer1 and miR-219 Are Required for Normal
Oligodendrocyte Differentiation and Myelination
Jason C. Dugas,1,* Trinna L. Cuellar,2,3Anja Scholze,1,3Brandon Ason,2,4Adiljan Ibrahim,1Ben Emery,1
Jennifer L. Zamanian,1Lynette C. Foo,1Michael T. McManus,2and Ben A. Barres1
1Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305-5125, USA
2Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA
3These authors contributed equally to this work
4Present address: Sirna Therapeutics/Merck & Co. Inc., San Francisco, CA 94158, USA
To investigate the role of microRNAs in regulating
oligodendrocyte (OL) differentiation and myelination,
we utilized transgenic mice in which microRNA pro-
cessing was disrupted in OL precursor cells (OPCs)
and OLs by targeted deletion of Dicer1. We found
disrupts normal CNS myelination and that OPCs
lacking mature miRNAs fail to differentiate normally
in vitro. We identified three miRNAs (miR-219,
miR-138, and miR-338) that are induced 10–1003
during OL differentiation; the most strongly induced
of these, miR-219, is necessary and sufficient to
promote OL differentiation, and partially rescues OL
differentiation defects caused by total miRNA loss.
PDGFRa, Sox6, FoxJ3, and ZFP238 proteins, all of
which normally help to promote OPC proliferation.
Together, these findings show that miR-219 plays
a critical role in coupling differentiation to prolifera-
tion arrest in the OL lineage, enabling the rapid tran-
sition from proliferating OPCs to myelinating OLs.
Myelination has evolved in vertebrates to electrically insulate
axons to promote rapid, energy-efficient action potential propa-
gation. In the central nervous system (CNS), the production of
multilamellar myelin sheaths is performed by oligodendrocytes
(OLs). The location and timing of CNS myelination is regulated
in large part by controlling the progression of OL differentiation,
the initiation of myelination in proximal axon tracts (Baumann
and Pham-Dinh, 2001). Differentiation from a proliferating,
morphologically simple OL precursor cell (OPC) into a postmi-
totic OL capable of myelinating axons is a complex process
requiring the coordinated regulation of many genes. Remark-
ably, the initiation of OL differentiation is very tightly linked to
the cessation of OPC proliferation (Barres and Raff, 1994; Raff
et al., 1998; Temple and Raff, 1985). In fact, tight association
between cessation of proliferation and initiation of differentiation
is observed during the development of many cell types (Buttitta
and Edgar, 2007; Kitzmann and Fernandez, 2001; Politis et al.,
2008; Truong and Khavari, 2007). Simply arresting proliferation
by targeted disruption of cell cycle components, however, is
often insufficient to promote the program of OL differentiation
(Casaccia-Bonnefil and Liu, 2003). How is it, then, that the
repression of proliferative genetic programs and the induction
of terminal differentiation programs are so tightly coordinated?
MicroRNAs (miRNAs) are small, noncoding RNA molecules
expressed by many eukaryotic organisms. These small miRNAs
are generated from longer hairpin-loop RNA sequences, which
are trimmed to functional 19–21 mers by successive cleavages
by the obligate miRNA processing enzymes Drosha and Dicer1,
then incorporated into an RNA-induced silencing complex
(RISC) (Bartel, 2004). When a RISC-loaded miRNA recognizes
a complementary sequence in the 30untranslated region (UTR)
of a protein-coding messenger RNA (mRNA), it generally either
represses RNA translation or directly promotes the degradation
of the associated mRNA (Wu and Belasco, 2008). Because
recognition sequence within a mRNA transcript is sufficient for
miRNA-induced silencing, and because miRNAs can silence
mRNAs to which they are only partially complementary, single
miRNA molecules are predicted to be capable of influencing
the expression of hundreds of different targeted genes (Bartel,
Several labs have demonstrated the importance of miRNAs in
regulating vertebrate development. Transgenic mice that do not
produce mature miRNAs due to the loss of functional Dicer1 die
embryonically (Bernstein et al., 2003). Using Cre-mediated
recombination, lineage-specific disruptions of Dicer1 have also
been performed. These have demonstrated that mature miRNAs
are required for the normal differentiation and function of several
different cell types, including neurons (Cuellar et al., 2008; Davis
et al., 2008; Harfe et al., 2005; Lynn et al., 2007). We were there-
fore interested in determining whether miRNAs play a similarly
important role in regulating OL differentiation.
By utilizing mice in which Cre recombinase was expressed
from either the OL lineage transcription factor 2 (Olig2) or
20,30-cyclic nucleotide 30phosphodiesterase (CNP) gene locus,
we were able to disrupt a floxed Dicer1 allele within cells of the
Neuron 65, 597–611, March 11, 2010 ª2010 Elsevier Inc. 597
in their OPCs and OLs display a shivering phenotype, character-
istic of classic CNS dysmyelination defects (Baumann and
Pham-Dinh, 2001). Normal development of CNS myelination is
disrupted intheseanimalsatboththehistological andultrastruc-
tural levels. Furthermore, OPCs isolated from these animals are
unable to differentiate normally in vitro. To determine which
miRNAs may be critical to OL differentiation, we investigated
the comparative expression of miRNAs in OPCs and OLs. We
found that one of the most highly OL-specific miRNAs,
miR-219, was specifically induced by mitogen withdrawal, was
both necessary and sufficient to promote the program of OL
differentiation, and was also able to partially rescue the differen-
tiation defect caused by the loss of miRNA production in OPCs.
To investigate the mechanisms by which miR-219 promotes OL
differentiation, we confirmed that miR-219 directly represses the
expression of four of its predicted targets, platelet-derived
growth factor receptor alpha (PDGFRa), SRY-box containing
gene 6 (Sox6), forkhead box J3 (FoxJ3), and zinc finger protein
238 (ZFP238), all of which are involved in promoting OPC prolif-
eration and inhibiting OL differentiation (Barres et al., 1994a;
Besnard et al., 1987; Stolt et al., 2006). These data provide
evidence that the induction of miRNA expression, and miR-219
in particular, upon the withdrawal of mitogens serves to link
the initiation of OL gene expression with the rapid inhibition of
OPC proliferation, thereby promoting rapid and coordinated
OL differentiation and subsequent myelin formation.
Loss of Dicer1 Function in OPCs and OLs Leads
to a Delay in CNS Myelination
To investigate the ability of miRNAs to regulate CNS myelination,
we used mice in which a floxed Dicer1 exon can be conditionally
deleted by Cre recombinase to generate nonfunctional Dicer1
protein (Harfe et al., 2005). We crossed mice containing the
floxed Dicer1 exon with mice expressing Cre recombinase
from either the Olig2 or the CNP gene locus (Lappe-Siefke
et al., 2003; Schu ¨ller et al., 2008); Olig2 is normally highly
expressed by OPCs, whereas CNP is weakly expressed by
OPCs but strongly induced very early during OL differentiation
(Baumann and Pham-Dinh, 2001; Zhou et al., 2000). Both of
these lines have been used previously to delete floxed gene
expression in the OPC/OL lineage (Emery et al., 2009;
Lappe-Siefke et al., 2003). We observed that mutant Olig2Cre/+
Dicer1flox/floxor CNPCre/+Dicer1flox/floxmice developed a notable
tremor from postnatal day 9–10 (P9–10) (Movies S1 and S2),
reminiscent of previously reported CNS myelin deficits (Bau-
mann and Pham-Dinh, 2001). This phenotype was pronounced
genotype mice, yet appeared to lessen with age in older
Olig2Cre/+Dicer1flox/floxmice (Figure S1), and by P60 these
mutant mice were indistinguishable from littermate controls
(data not shown). Interestingly, in addition to the shivering
phenotype seen early in development, CNPCre/+Dicer1flox/flox
mice developed increasing functional deficits consistent with
peripheral neuropathy at older ages and generally did not live
past 4 weeks. Knowing that CNP, but not Olig2, is expressed
in PNS-myelinating Schwann cells, we stained peripheral nerves
of mutant Olig2Cre/+Dicer1flox/floxand CNPCre/+Dicer1flox/flox
mice for expression of myelin basic protein (MBP), a typical
marker of mature myelin (Baumann and Pham-Dinh, 2001). We
found that peripheral myelination was severely disrupted
specifically in CNPCre/+Dicer1flox/floxmice (Figures 1M–1P) and
concluded that this likely accounted for the differences ob-
served in motor function and lethality between older Olig2Cre/+
We next investigated whether the shivering phenotype
Dicer1flox/floxmice resulted from a disruption of normal CNS
myelination. When we stained sagittal brain sections with
FluoroMyelin, a stain for compact myelin, we found that mature
myelin formation in both the cerebellum and corpus callosum
was significantly reduced through P24 in mutant Olig2Cre/+
Dicer1flox/floxand CNPCre/+Dicer1flox/floxmice relative to litter-
mate controls (Figures 1A–1H and S2A–S2R). Consistent with
observed behavioral recovery in Olig2Cre/+Dicer1flox/floxmice,
at older ages (P60), these mutant mice had levels of compact
CNS myelination that approached control levels (due to strain
lethality, we were unable to assess myelination levels in older
CNPCre/+Dicer1flox/floxmice). We also observed reduced, patchy
myelination early in development in both Olig2Cre/+Dicer1flox/flox
and CNPCre/+Dicer1flox/floxmutant mice when measured byMBP
expression (Figures S2S–S2D0). Interestingly, we found that
MBP expression levels in mutant mice appeared closer to
control littermate levels by P24 (Figures S2A0–S2D0), despite
the fact that compact myelin levels were still low (Figures 1
and S2A–S2R). Therefore, to further investigate the extent of
CNS myelination in older mutant mice, we examined the optic
nerves of mutant P23–24 Olig2Cre/+Dicer1flox/floxmice by
electron microscopy (EM). We found that optic nerves from
Olig2Cre/+Dicer1flox/floxmice contained ?50% less myelinated
axons than littermate controls (Figures 1I–1L). However, we did
not observe any gross morphological defects in the myelin
or paranodes present in the Olig2Cre/+Dicer1flox/floxmice
relative to controls (data not shown). By P60, mutant Olig2Cre/+
Dicer1flox/floxmice and littermate controls had similar numbers
of myelinated axons (data not shown). Cumulatively, these
data suggest that myelin generation is significantly delayed in
Olig2Cre/+Dicer1flox/floxmice, but that, eventually, normal myeli-
nation is achieved in these animals.
Do OLs that lack functional Dicer1, though delayed, eventually
form normal myelin sheaths, or is the myelin observed in older
Olig2Cre/+Dicer1flox/floxand CNPCre/+Dicer1flox/floxmice formed
by OLs that have failed to correctly excise the floxed Dicer1
allele? To address this question, we compared Dicer1 expres-
sion levels between mutant P23 Olig2Cre/+
CNPCre/+Dicer1flox/floxand control genotype littermate OLs. By
qRT-PCR, we found that Dicer1 expression was ?10-fold
reduced in acutely purified mutant Olig2Cre/+Dicer1flox/floxOLs
relative to littermate control OLs, despite the fact that MBP
expression levels were only slightly reduced in the mutant
sample (Figure S4A); similar results were observed with
CNPCre/+Dicer1flox/floxOLs (data not shown). Interestingly,
when we repeated these experiments with P45 mice, we found
that the levels of functional Dicer1 transcripts were significantly
Dicer1 and miR-219 Promote Myelin Development
598 Neuron 65, 597–611, March 11, 2010 ª2010 Elsevier Inc.
50% of control levels, indicating that at this later age as many as
half of the Olig2Cre/+Dicer1flox/floxOLs may be expressing func-
tional Dicer1. These data indicate that Olig2Cre/+Dicer1flox/flox
OLs that fail to disrupt functional Dicer1 expression may signifi-
cantly contribute to the recovery of compact myelin formation in
older mutant animals and that the Olig2Cre/+Dicer1flox/floxOLs
lacking functional Dicer1 expression, which account for the
vast majority of OLs in younger mutant animals, are likely
severely delayed or disrupted in their ability to form mature
Finally, to determine whether axons were disrupted in the
mutant mice, we stained Olig2Cre/+Dicer1flox/floxand CNPCre/+
Dicer1flox/floxbrains for neurofilament expression (NF-200) and
found that axons appeared normal in areas of reduced myelina-
tion (Figures S2E0–S2G0). These data indicate that the reductions
in myelination we have observed in postnatal mice are likely not
secondary to axonal loss in the CNS.
Figure 1. Disruption of Dicer1 in Myelinat-
ing Cells Disrupts Normal Myelin Formation
(A–F) Visualization of compact CNS myelin by
FluoroMyelin (FlMy) staining in the cerebellar
arms (A–C) and splenium regions of the corpus
callosum (D–F) from similar-depth sagittal brain
sections from P24 littermate control Olig2+/+;
Dicer1f/f(A and D), Olig2Cre/+;Dicer1f/+(B and E),
and mutant Olig2Cre/+;Dicer1f/f(C and F) mice.
Scale bar, 500 mm.
(G and H) Percentage area myelinated, as deter-
mined by FlMy staining, in the cerebellar arms
(G) and corpora callosa (H) of mutant Olig2Cre/+;
Dicer1f/fand littermate control mice at P17–18,
P23–24, and P60.
(I–K) EM of optic nerve cross-sections collected
from P24 littermate control Olig2+/+;Dicer1f/f(I),
Olig2Cre/+;Dicer1f/+(J), and mutant Olig2Cre/+;
Dicer1f/f(K) mice. Yellow asterisks, example
unmyelinated axons. Scale bar, 0.5 mm.
(L) Percentages of axons myelinated in the optic
nerves of P23–24 mutant Olig2Cre/+;Dicer1f/fand
littermate control mice.
(M–P) Sciatic nerve longitudinal sections from P23
littermate control Olig2+/+;Dicer1f/f(M) and mutant
Olig2Cre/+;Dicer1f/f(N) mice, P22 littermate control
CNP+/+;Dicer1f/f(O) and mutant CNPCre/+;Dicer1f/f
(P) mice stained for axons (NF-200, M1–P1) and
myelin (MBP, M2–P2).
Error bars show ±SEM, n = 3–5 animals/condition.
*p < 0.05, **p < 0.001 post-hoc all pairwise SNK
tests, mutant compared to either control condi-
tion. See also Figures S1 and S2 and Movies S1
Loss of Dicer1 Function in OPCs
Delays OL Differentiation In Vivo
Because both Olig2Cre/+Dicer1flox/floxand
behavioral defects and delayed CNS
myelin formation, we hypothesized that
these deficits resulted directly from a
disruption of normal OL differentiation. To test this hypothesis,
we began by quantifying mature OLs in mutant Olig2Cre/+
Dicer1flox/floxand littermate control mice. We performed fluores-
cent in situs against proteolipid protein (PLP), a gene expressed
in mature OLs, and found that OL numbers in mutant Olig2Cre/+
Dicer1flox/floxmice were significantly reduced at various postmi-
totic ages and were still lower than control levels at P60 (Figures
2 and S3A–S3L). Reduced OL numbers were not the result of
a failure to generate OL precursors, as OPC numbers in mutant
Olig2Cre/+Dicer1flox/floxmice were similar to or greater than
numbers observed in control animals (Figure S2M). These data
likely indicate that loss of Dicer1 function in the OPCs of these
mutant mice delays or disrupts the normal program of OL differ-
Surprisingly, when we performed similar experiments with
mutant CNPCre/+Dicer1flox/floxmice, we detected no significant
reductions in mature OL numbers, even at P9, when OL matura-
tion is just commencing in the brain (Figures S3N–S3U). As CNP
Dicer1 and miR-219 Promote Myelin Development
Neuron 65, 597–611, March 11, 2010 ª2010 Elsevier Inc. 599
expression is induced just as OPCs begin to differentiate into
OLs (Baumann and Pham-Dinh, 2001), it is possible that Dicer1
is still functional at the earliest stages of OL differentiation in
the initiation of differentiation, and this is sufficient to disrupt
subsequent maturation and/or myelin sheath formation in these
OPCs Lacking Functional Dicer1 Fail
to Differentiate Normally In Vitro
Is disruption of normal CNS myelination and OL differentiation
a cell-autonomous effect resulting from the loss of Dicer1
function within OPCs and OLs in mutant Olig2Cre/+Dicer1flox/flox
and CNPCre/+Dicer1flox/floxmice? To address this question, we
purified immature OPCs from the brains of P7 mutant CNPCre/+
Dicer1flox/floxand control littermate mice and analyzed their
out the normal program of OL differentiation in vitro when
cultured in differentiation-promoting conditions, such as with-
growth factor a (PDGF) and neurotrophin 3 (NTF3), and presen-
therefore allowed us to directly determine the effects of intrinsic
Figure 2. Mature OL Numbers Are Reduced
in Mice Lacking Dicer1 Function in OPCs
(A–F) In situs for PLP expression (red) to visualize
mature OLs in the cerebellar arms (A–C) and
corpus callosum (D–F) in P24 littermate control
Olig2+/+;Dicer1f/f(A and D), Olig2Cre/+;Dicer1f/+
(B and E), and mutant Olig2Cre/+;Dicer1f/f(C and
F) mice. Scale bar, 500 mm.
(G and H) Quantification of mature OLs/mm2in the
cerebellar arms (G) and corpora callosa (H) of
mutant Olig2Cre/+;Dicer1f/fand littermate control
mice at P10, 17, 24, and 60.
Error bars show ±SEM, n = 3 animals/condition.
*p < 0.01, **p < 0.001, post-hoc Holm-Sidak/
SNK tests, mutant compared to either control
condition. See also Figure S3.
Dicer1 disruption on OL differentiation,
removed from the influence of interacting
heterologous cell types in the CNS.
We first confirmed that OPCs purified
from CNPCre/+Dicer1flox/floxmice had
indeed lost their expression of the func-
tional Dicer1 gene. When purified, OPCs
were induced to differentiate in media
lacking mitogens for 4 days in vitro
(DIV), we found that Dicer1 expression
was almost completely eliminated in
mutant CNPCre/+Dicer1flox/floxOLs rela-
Similar results were obtained with mutant
Olig2Cre/+Dicer1flox/floxOLs (data not
OPCs were cultured for 4 or 7 DIV in mitogen-free media, the
proportion of OPCs able to differentiate into normal, MBP-
expressing OLs was strongly decreased (Figures 3A–3F). When
early (CNP), intermediate (MBP), or late (myelin oligodendrocyte
glycoprotein: MOG) stage OL differentiation (Baumann and
Pham-Dinh, 2001; Dugas et al., 2006), we found that a significant
portion of mutant OPCs initiated differentiation (being CNP+) but
were severely delayed in expressing markers of later OL differen-
tiation (MBP, MOG) (Figures 3G and 3H). Conversely, a small but
significant proportion of mutant OPCs continued to express
chondroitin sulfate proteoglycan 4 (NG2), a marker of undifferen-
tiated OPCs, whereas expression of NG2 was nearly completely
tions in OL protein expression were observed at 4 DIV in mutant
CNPCre/+Dicer1flox/floxOPC cultures when assayed by western
blotting (Figure 3I). Similar reductions in OL differentiation were
noted when comparing cultured Olig2Cre/+Dicer1flox/floxOPCs to
control genotype OPCs (data not shown). These data show that
impairment of mature miRNA production strongly retards
complete OL differentiation in a cell-autonomous manner.
Dicer1 and miR-219 Promote Myelin Development
600 Neuron 65, 597–611, March 11, 2010 ª2010 Elsevier Inc.
Analysis of miRNAs Induced during OL Differentiation
The defects observed in vivo and in vitro as a result of the loss of
Dicer1 function in OPCs and OLs indicate that mature miRNA
production is crucial to normal OL differentiation and CNS
myelination. Which specific miRNAs regulate these processes?
To investigate this question, we used miRNA microarrays to
identify miRNAs regulated during OL differentiation. Several
miRNAs found to be differentially expressed are depicted in
Figure 4A, and the top statistically ranked candidates are listed
change were expressed at a higher level in OLs; very few
miRNAs were strongly repressed as OPCs differentiated into
OLs. qRT-PCR performed on independently obtained OPC and
OL samples confirmed these results (Figure 4B) and indicated
that the top three ranked candidate miRNAs, miR-219,
Figure 3. Purified OPCs Lacking Dicer1 Fail
to Differentiate Normally
(A–F) OPCs purified from P7 control CNP+/+;
Dicer1f/f(A and D), CNPCre/+;Dicer1f/+(B and E),
and mutant CNPCre/+;Dicer1f/fmice (C and F),
cultured for 4 DIV (A–C) or 7 DIV (D–F) in -PDGF-
T3 media, then stained for MBP expression
(green); nuclei stained by DAPI (blue). Scale bar,
the indicated markers (NG2, CNP, MBP, or MOG)
at 4 DIV (G) or 7 DIV (H). Error bars show ±SEM,
n = 3 samples/condition. *p < 0.05, **p < 0.005,
***p < 0.001, post-hoc all pairwise SNK tests,
mutant compared to either control condition.
(I) Western blots showing reduced expression of
CNP, MBP, and MOG in mutant CNPCre/+;
Dicer1f/f(?/?) OLs relative to control CNP+/+;
OLs; protein from purified OPCs cultured 4 DIV
in -PDGF-T3 media. Actin blot shown is stripped
and reprobed MBP blot.
See also Figure S4.
miR-138, and miR-338, were induced
100-, 30-, and 30-fold, respectively, in
differentiating OLs. As further indepen-
dent confirmation, the strong induction
of thesemiRNAs during OLdifferentiation
has also recently been reported by Lau
et al. (2008).
strongly induced during OL differentia-
tion, we next wanted to investigate
whether expression of these candidate
miRNAs is specific to cells of the OL
lineage within the CNS. Previous reports
had already indicated that expression of
these three miRNAs in studied verte-
brates and invertebrates is highest in
the CNS (Kim et al., 2004; Landgraf
et al., 2007; Ruby et al., 2007; Wienholds
et al., 2005). To investigate the identity of
the neural cell types that express these
miRNAs, we utilized previously established protocols in our lab
(Cahoy et al., 2008) to acutely isolate pure populations of
OPCs, immature GC+MOG–OLs, mature MOG+OLs, and also
young (P5) and older (P17) astrocytes and neurons. qRT-PCR
analyses of miRNA expression in these samples indicated that
expression of all three candidate miRNAs is highest in acutely
purified OLs relative to other CNS cell types (although miR-138
expression is weaker than miR-219 and -338) and also
confirmed that all three miRNAs are induced in OLs relative to
OPCs in vivo as well as in vitro (Figure 4C). We also investigated
the most highly expressed miRNA, miR-219, by northern blot-
ting. We found that purified OLs expressed detectable levels of
fully processed miR-219 and that this expression was strongly
reduced in mutant Olig2Cre/+Dicer1flox/floxOLs (Figure 4D). To
further investigate the in vivo expression of these miRNAs, we
Dicer1 and miR-219 Promote Myelin Development
Neuron 65, 597–611, March 11, 2010 ª2010 Elsevier Inc. 601
performed in situ hybridizations on WT P17 brain sections with
LNA probes for all three miRNAs. We found that miR-219 is
expressed specifically and robustly in the white matter areas of
the brain, in a pattern similar to PLP expression (Figures 4E–4G).
miR-138 was also expressed in the white matter, but expression
was weaker than miR-219, and was also detected in other areas
of the brain (Figures S5A–S5F). Expression of these miRNAs
persists in mature OLs, as consistent expression patterns were
observed at P24 (data not shown) and P60 (Figures S5G–S5O).
In contrast, we detected miR-338 expression only at later devel-
opmental ages (Figures S5H, S5K, and S5N). In vivo expression
of miR-219 and miR-338 in OLs of the murine CNS was also
Figure 4. miR-219, -138, and -338 Are Most Highly Expressed by OLs in the CNS and Are Induced Specifically by Mitogen Withdrawal
(A) miRNA expression was compared between OPCs and OLs. Averaged results from 43 two-color microarrays are shown. ‘‘Cand’’ = candidate miRNAs at the
time of microarray printing. Hsa, mmu, and dre = human, mouse, and zebrafish annotated miRNAs.
(B) qRT-PCR to determine relative OL/OPC expression level of individual miRNAs. Error bars show ±SEM; n = 3 samples, 43 independent qRT-PCR runs
OLs, and P14 MOG+mature OLs (left), or in OPCs cultured for 7 DIV in indicated media: ±PDGF ±T3 (right). Error bars show ±SEM, n = 2–8 samples.
(D) Northern blot showing mature miR-219 expression in acutely purified P23 Olig2Cre/+;Dicer1f/+OLs (+/?), which is strongly reduced in Olig2Cre/+;Dicer1f/fOLs
(?/?). Blots were stripped and reprobed for U6 snRNA as loading control.
(E–G) In situ hybridizations showing expression of PLP (E) and miR-219 (F) in sagittal sections of a WT P17 mouse brain, compared to short LNA negative control
probe (G). Boxed regions in (E1)–(G1) shown at higher magnification in (E2)–(G2); corpus callosum outlined by dashed lines.
See also Figure S5 and Table S1.
Dicer1 and miR-219 Promote Myelin Development
602 Neuron 65, 597–611, March 11, 2010 ª2010 Elsevier Inc.
independently confirmed by Zhao et al. (2010 [this issue of
Several groups have previously established that OL differenti-
ationcan bestimulated bytwodistinct pathways. OPCscultured
in the absence of proliferation-promoting mitogens immediately
leave the cell cycle and initiate differentiation (Barres et al., 1993;
Raff et al., 1983). Alternatively, in the presence of mitogens, T3
can trigger OPCs to differentiate (Barres et al., 1994a; Temple
and Raff, 1986). To investigate whether miR-219, miR-138, and
miR-338 expression is induced equally by these two distinct
expression in OPCs cultured either in the presence of T3 (plus
saturating amounts of mitogens) for 7 DIV or in the absence of
Normal OL Differentiation
sion (white, A1–H1) that express the indicated
markers (green, MBP: A2–D2; MOG: E2–H2) are
indicated by yellow arrows; transfected GFP+cells
negative for marker expression are indicated by
blue arrows; untransfected GFP–cells positive for
marker expression are indicated by green arrows.
Nuclei marked by DAPI (blue, A2–H2). Scale bar,
200 mm. (A and B) OPCs transfected with control
mimic (A) or miR-219 mimic (B) cultured for 7 DIV
in +PDGF-T3 media. (C and D) OPCs transfected
with control inhibitor (C) or miR-219 inhibitor (D)
cultured for 3 DIV in -PDGF-T3 media. (E and F)
OPCs transfected with control mimic (E) or miR-
138 mimic (F) cultured for 4 DIV in -PDGF-T3
media. (G and H) OPCs transfected with control
inhibitor (G) or miR-138 inhibitor (H) cultured for 4
DIV in -PDGF-T3 media.
(I–L) Percentages of transfected, GFP+cells
expressing the indicated markers (CNP, MBP, or
MOG). OPCs transfected with control, miR-138,
and miR-219 mimics (I–K) or control, miR-138,
and miR-219 inhibitors (L). Transfected cells
cultured for 7 DIV in +PDGF-T3 (I) or +PDGF+T3
(J) media, or 3–4 DIV in -PDGF-T3 (K-L) media.
(L). *p < 0.05, **p < 0.01 post-hoc Holm-Sidak test
versus control. See also Figure S6.
mitogens for 4 DIV. All three miRNAs
were reproducibly induced by mitogen
withdrawal, but not by T3 (Figure 4C).
These data indicate that miR-219, miR-
138, and miR-338 are primarily induced
in response to mitogen-withdrawal in
miR-219 and miR-138 Promote
miRNAs play a functional role in regu-
lating OL differentiation? To find out, we
transfected purified OPCs with nucleo-
tide reagents homologous to the mature
miRNAs (mimics) and cultured trans-
fected cells in media that should maintain OPCs in an immature,
the most strongly induced miRNA, into OPCs increased by 3- to
4-fold the number of cells that differentiated in proliferative
conditions (Figures 5A, 5B, and S6A–S6D). OPCs induced to
differentiate by miR-219 mimic expressed markers of both early
and late OL differentiation (Figure 5I), whereas OPCs induced to
differentiate by miR-138 mimic expressed markers of the earlier
stages of OL differentiation (CNP, MBP), but not later differenti-
ation (MOG). When OPCs were induced to differentiate by
mitogen withdrawal, transfecting cells with miR-219 and miR-
138 mimics had very little ability to further increase OL differen-
tiation (Figure 5K). This result is consistent with the previous
Dicer1 and miR-219 Promote Myelin Development
Neuron 65, 597–611, March 11, 2010 ª2010 Elsevier Inc. 603
finding that expression of these miRNAs is induced endoge-
nously in OPCs by mitogen withdrawal. In contrast, addition of
miR-219 or miR-138 mimics to OPCs cultured in mitogens and
induced to differentiate by T3 greatly increases the rate of OL
differentiation (Figure 5J). Consistent with the findings in
+PDGF-T3 media, miR-219 is the stronger inducer of OL differ-
entiation in the combined presence of mitogens and T3
(+PDGF+T3), and only miR-219 increases expression of the
late differentiation marker MOG. Interestingly, we found that
miR-138 significantly repressed expression of the late differenti-
ation marker MOG during differentiation promoted by either
mitogen withdrawal (-PDGF-T3) or T3 (+PDGF+T3) (Figures 5E,
5F, 5J, and 5K). These data indicate that both miR-219 and
miR-138 are sufficient to induce OL differentiation, but only
miR-219 induced both the early and late stages of OL differenti-
ation. Incontrast,miR-138appears tohavetheinterestingroleof
promoting the early phase of OL differentiation (CNP+, MBP+)
while delaying the later phase of OL differentiation (MOG+).
To determine whether these miRNAs are necessary for
OPCs with competitive inhibitors of miR-219 and miR-138 and
cultured the cells for 4 DIV in -PDGF-T3 media. Inhibition of
miR-219 strongly repressed OL differentiation (Figures 5C, 5D,
5L, and S6E–S6H). In contrast, inhibition of miR-138 did not
repress the early stages OL differentiation, but did promote
expression of the late-phase gene MOG (Figures 5G, 5H, and
5L). Consistent with the finding that miR-219 and miR-138 are
not induced by T3 presentation, T3-promoted OL differentiation
is not significantly affected by inhibition of either of these
miRNAs (Figure S6I). In contrast to the findings for miR-219
and miR-138, neither mimics nor inhibitors of the third OL-in-
duced miRNA, miR-338, had any significant effect on OL myelin
gene expression tested in any of these assays (data not shown).
However, miR-338 may still play an important role in regulating
CNS myelination, as disruption of miR-338 function has been
shown to promote OL differentiation in other vertebrate model
system assays (Zhao et al., 2010).
Cumulatively, these data indicate that miR-219 is both neces-
sary and sufficient to promote rapid OL differentiation promoted
by mitogen withdrawal, whereas miR-138 is necessary and
sufficient to delay the later stage of OL differentiation. We have
previously noted that OL differentiation appears to progress in
at least two distinct stages of gene expression (Dugas et al.,
2006). As only immature, MOG–OLs are able to robustly initiate
the process of axon wrapping that leads to compact myelin
formation (Watkins et al., 2008), it could be that miR-138 plays
a role in elongating the immature phase of OL differentiation,
entiating OL can select and correctly myelinate nearby axons.
miR-219 Partially Rescues OL Differentiation
Based on our finding that miR-219 strongly induces OL differen-
tiation, we next investigated whether miR-219, on its own, could
rescue differentiation disrupted in OLs lacking functional Dicer1
expression. To address this question, we purified both mutant
CNPCre/+Dicer1flox/floxOPCs and control genotype CNPCre/+
Dicer1flox/+OPCs, and then transfected them with either
miR-219 or negative control mimic. These cells were then
cultured for 4 DIV in mitogen-withdrawal media to determine
the effect of adding miR-219 back to OPCs lacking mature
miRNA expression. As expected, loss of Dicer1 function strongly
repressed myelin gene expression; however, when miR-219
mimic was transfected into these mutant OPCs, the number of
transfected cells expressing MBP (Figures 6A–6E), as well as
the overall expression of CNP, MBP, and MOG (Figure 6F),
robustly increased relative to control transfected mutant OPCs.
These data implicate miR-219 as a key miRNA regulator of
mitogen-withdrawal-mediated OL differentiation.
OL-Expressed miRNAs Preferentially Target Genes
Repressed during OL Differentiation
miRNAs generally function by altering the expression or transla-
tion of ‘‘targeted’’ mRNAs, which are recognized by miRNAs via
complementary sequences in their 30UTRs. Therefore, identifi-
cation of miRNA targets often reveals the mechanisms by which
miRNAs exert their influence. To analyze the genes targeted by
miR-219, miR-138, and miR-338, we obtained the lists of their
predicted targets from TargetScan 5.1 (http://www.targetscan.
org/). We then ascertained the expression patterns of these
targeted genes by consulting genomic expression data obtained
from acutely purified OPCs, immature GC+OLs, and mature
MOG+OLs (Cahoy et al., 2008). In this way, we were able to
determine the percentages of miRNA-targeted genes that were
either induced or repressed during OL differentiation (Fig-
ure S7A). Interestingly, when compared to the percentage of
total characterized, unique genes that are induced or repressed
during OL differentiation, we found that all three OL-expressed
miRNAs preferentially targeted genes that are repressed as
OLs differentiate. To determine whether the enriched targeting
of genes repressed during OL differentiation is restricted to the
miRNAs that are highly expressed in OLs, we also analyzed
two unrelated miRNAs, miR-142 and miR-196, which are most
strongly expressed outside of the nervous system (Landgraf
et al., 2007), and found that they do not similarly target such
a high percentage of genes repressed during the full course of
OL differentiation (miR-142 targets a higher percentage of genes
repressed in GC+OLs, but not in the more mature MOG+OLs).
Cumulatively, these data indicate that miR-219, miR-138, and
miR-338 may function by targeting OPC-expressed genes
whose repression is necessary to promote the rapid cessation
of proliferation coupled to OL differentiation.
miR-219 Directly Represses the Expression
of Several Genes that Inhibit OL Differentiation
To further investigate the mechanisms by which miR-219
promotes OL differentiation, we next wanted to determine
whether miR-219 directly targets any genes that functionally
regulate OL differentiation. We looked for predicted miR-219
targets that were repressed >2-fold in OLs relative to OPCs
that were also either (1) computationally determined to be very
likely targets of miR-219 (Table S2), (2) very highly repressed
during OL differentiation (Table S3), or (3) very highly expressed
in OPCs (Table S4). From these analyses, we selected
four candidate genes for further study: PDGFRa, the receptor
for the OPC mitogen PDGF (Besnard et al., 1987), Sox6, a
Dicer1 and miR-219 Promote Myelin Development
604 Neuron 65, 597–611, March 11, 2010 ª2010 Elsevier Inc.
transcription factor previously reported to repress terminal OL
differentiation (Stolt et al., 2006), and also FoxJ3 and ZFP238
(a.k.a. RP58), two transcription factors with no previously re-
ported functions in OPCs. ZFP238 is a transcriptional repressor
expressed in neuronal precursors and distinct subsets of mature
neurons that has been implicated in regulating neuronal differen-
tiation (Aoki et al., 1998; Okado et al., 2009), and FoxJ3 expres-
sion has been detected in various developing tissues, including
facts, coupled with the observed expression of these transcrip-
tionfactors inOPCs andsubsequent repression inOLs(TableS2
and FigureS7B), implicated themas potential novel regulators of
the OPC-OL transition.
We first confirmed that these genes were in fact targets of
miR-219. We subcloned the 30UTRs of these candidate genes,
which contain the predicted miR-219 target sites, into a reporter
construct that fused these 30UTRs to the end of a constitutively
expressed luciferase gene (Figure 7A). These constructs were
then cotransfected into HEK293 cells along with miR-219 mimic
to determine the extent to which miR-219 could repress expres-
sion of mRNAs physically linked to these candidate 30UTRs.
When compared to the negative control, nontargeting mimic,
miR-219 reproducibly repressed, by ?50%, the translation of
luciferase linked to the 30UTRs of FoxJ3, PDGFRa, and
ZFP238 (Figure S8A). To further investigate targeting of these
Figure 6. miR-219 Partially Rescues OL Differenti-
ation in Dicer–OPCs
(A–D) Transfected OPCs (GFP+= white, A1–D1), stained
for MBP expression (green) and costained with DAPI
(blue, A2–D2). OPCs purified from P7 control littermate
(A and B) and mutant CNPCre/+;
Dicer1f/fmice (C and D) were transfected with control
mimic (A and C) or miR-219 mimic (B and D) and cultured
4 DIV in -PDGF-T3 media. Scale bar, 200 mm.
(E) Percentages of healthy control (cont) or miR-219 (219)
mimic transfected CNPCre/+;Dicer1f/+(WT) and mutant
CNPCre/+;Dicer1f/f(mut) OLs expressing MBP. Error bars
show ±SEM, n = 9. **p < 0.005, n.s. = not significant,
t test control versus miR-219 transfections.
(F) Western blots to determine CNP, MBP, and MOG
expression levels in control CNPCre/+;Dicer1f/+(+/?) and
mutant CNPCre/+;Dicer1f/f(?/?) OPCs transfected with
miR-219 or control mimic miRNA and cultured 4 DIV
in -PDGF-T3 media. Actin blot shown is stripped and
reprobed MBP blot.
See also Figure S7 and Tables S2–S4.
candidate 30UTRs by miR-219, we performed
similar transfection experiments in purified
OPCs. We observed robust miR-219-mediated
translational repression of luciferase linked to
all four candidate 30UTRs when expressed in
OPCs (Figure 7B). This level of repression is
similar to or greater than previously reported
repression of protein expression by miRNA
(Cheng et al., 2007; Kocerha et al., 2009; Wu
and Belasco, 2008), and therefore we believe
that PDGFRa, Sox6, ZFP238, and FoxJ3 all
represent true functional targets of miR-219 repression in vivo.
To further confirm the repression of these genes by direct inter-
action with miR-219, we utilized site-directed mutagenesis to
specifically disrupt each of the predicted miR-219 targeted
sequences in our reporter constructs. This mutagenesis
completely abolished the ability of miR-219 to repress transla-
tion from each of our candidate gene 30UTRs in both HEK293
cells and OPCs (Figures 7B and S8A). To test the role of endog-
enously expressed miR-219 in regulating PDGFRa, Sox6,
ZFP238, and FoxJ3, we cotransfected our reporter constructs
into OPCs along with miR-219 inhibitor and immediately
induced OL differentiation. We found that expression of all four
reporter constructs was strongly derepressed (2- to 6-fold
increases) relative to control transfected cells (Figure 7C), indi-
cating that sufficient miR-219 is produced in newly differenti-
ating OLs to repress translation of PDGFRa, Sox6, ZFP238,
To further confirm the targeting of endogenous PDGFRa and
Sox6 by miR-219, we compared the expression of PDGFRa
and Sox6 protein in OPCs transfected with either miR-219 or
control mimic. PDGFRa and Sox6 protein expression, normally
repressed in OLs, was high in OPCs transfected with control
mimic and then cultured for 2 DIV in +PDGF-T3 media. In
contrast, in OPCs transfected with miR-219 mimic, both
PDGFRa and Sox6 expression was strongly repressed,
Dicer1 and miR-219 Promote Myelin Development
Neuron 65, 597–611, March 11, 2010 ª2010 Elsevier Inc. 605
consistent with our luciferase assay data (Figures 7D, 7E, and
7H). Also consistent with our luciferase assay data was the
finding that both PDGFRa and Sox6 expression was increased
in OLs transfected with miR-219 inhibitor (Figures 7F–7H). These
data indicate that miR-219 directly represses endogenous
PDGFRa and Sox6 protein expression, and likely does so in
response to mitogen withdrawal in OPCs. Interestingly, the
repression of PDGFRa would set up a positive feedback loop,
wherein reduced levels of mitogen stimulation would induce
miR-219 expression, leading to a repression of PDGFRa expres-
sion and a subsequent further reduction of mitogen stimulation
and a more rapid initiation of OL differentiation. This effect would
be further enhanced by the loss of repressors of OL differentia-
tion, such as Sox6.
ZFP238 and FoxJ3 Repress OL Differentiation
After confirming that miR-219 targets and represses the 30UTRs
of FoxJ3 and ZFP238, we next wanted to determine whether
these OPC-enriched transcription factors functionally regulate
OL differentiation. To determine whether the repression of these
genes was required for normal OL differentiation, we drove the
constitutive expression of FoxJ3 or ZFP238 in transfected
OPCs, and then induced differentiation by culturing these cells
for 3 DIV in mitogen-withdrawal (-PDGF-T3) media (Figures
S8B–S8D). We found that overexpression of either of these
OPC-expressed transcription factors significantly inhibited
normal OL differentiation and also increased expression of the
OPC marker NG2 (Figure S8E). We also found that overexpres-
sion of either of these genes did not strongly affect the survival
of immature OPCs cultured in PDGF, but did reduce survival
when mitogens were withdrawn (Figure S8F). These data indi-
cate that ZFP238 and FoxJ3, both previously uncharacterized
in OPCs, are each sufficient to repress OL differentiation and
that failure to inhibit either of these transcription factors leads
to decreased differentiation and survival in the absence of prolif-
eration-supporting mitogens. It is interesting to note that one
of the unexpected results we obtained from characterizing
OL-induced miRNAs was the identification of novel proteins
involved in regulating OL differentiation. Similar analyses aimed
at correlating expressed miRNAs with expressed miRNA targets
gene functions in future studies.
Figure 7. miR-219 Represses the Expres-
sion of PDGFRa, Sox6, FoxJ3, and ZFP238
(A) Regions of the FoxJ3, Sox6, ZFP238, and
PDGFRa 30UTRs cloned into the luciferase
reporter construct are depicted (black bars).
Green, coding regions of depicted genes; red,
locations of predicted miR-219-targeted sites.
(B) Luciferase activity at 1–2 DIV in OPCs cotrans-
fected with the indicated luciferase reporter
constructs and either control (green) or miR-219
(red) mimic. Blue, cotransfections of miR-219
mimic with reporter constructs containing mu-
tated 219 binding sites; data normalized to control
mimic cotransfection luciferase activity.
(C) Luciferase activity at 4 DIV in OLs cotrans-
fected with the indicated luciferase reporter
constructs and either control (green) or miR-219
(D–G) Western blot showing the levels of PDGFRa
(D and F) or Sox6 (E and G) expression in OPCs
transfected with control or miR-219 mimic, 2 DIV
+PDGF-T3 (D and E), or transfected with control
or miR-219 inhibitor, 1–3 DIV -PDGF+T3 (F and
G). All blots stripped and reprobed for actin as
(H) Actin-normalized PDGFRa and Sox6 expres-
sion, relative to control transfection levels, in
OPCs transfected with miR-219 mimic (red) or
Error bars show ±SEM, n = 8–12 (B and C) or
3–4 (H). *p < 0.001 (B and C) or < 0.05 (H), t test
control versus miR-219 mimic/inhibitor transfec-
tions (t tests 219 site mutants versus control not
done). See also Figure S8.
Dicer1 and miR-219 Promote Myelin Development
606 Neuron 65, 597–611, March 11, 2010 ª2010 Elsevier Inc.
Dicer1 Is Required for Normal OL Differentiation
Our findings show that miRNA processing isrequired to promote
normal OL differentiation and myelination in vitro and in vivo. The
role of miRNAs specifically in promoting myelin formation is
further confirmed in the CNPCre/+Dicer1flox/floxmice, in which
the initial stages of OL differentiation commence normally, but
complete compact myelin formation is still significantly delayed.
The myelin that does eventually form in the absence of
OL-expressed miRNAs appears to be functionally normal, as
evidenced by both the formation of normal paranodal structures
in the optic nerve and by alleviation of behavioral symptoms in
older Olig2Cre/+Dicer1flox/floxmice. However, at older ages,
when compact myelin levels are recovering, a significant portion
functional Dicer1, raising the possibility that mature OLs that fail
to express functional Dicer1 are less healthy or are otherwise at
a competitive disadvantage compared to OLs that continue to
express Dicer1 and that these mutant OLs that fail to excise
Dicer1 eventually generate the majority of healthy compact
myelin sheaths in Olig2Cre/+Dicer1flox/floxmice. Future experi-
ments with strains of mice that more completely disrupt miRNA
processing in the OL lineage will be required to clarify this issue.
Several factors lead us to believe that the myelination deficits
we have observed result directly from a loss of Dicer1 function
in the OPCs and OLs, rather than from an alternative defect
in a secondary cell type. First, we observe an identical CNS
dysmyelination phenotype when either Olig2 or CNP drives
Cre-recombinase-mediated disruption of Dicer1. In particular,
CNP expression is restricted to cells of the OL lineage in the
CNS (Yuan et al., 2002), and we therefore would not predict
that Dicer1 disruption would extend to other CNS cell types in
the CNPCre/+Dicer1flox/floxmice. Second, OLs purified from
either Olig2Cre/+Dicer1flox/floxor CNPCre/+Dicer1flox/floxmice
both fail to fully differentiate in vitro, indicating that complete
OL differentiation is directly impaired by the disruption of miRNA
processing. Third, axons present in dysmyelinated areas appear
normal and comparable to littermate control genotype mice,
implying that observed myelination deficits are not the result of
disrupted axon tracts. All of these data implicate OL-expressed
One distinction between the Olig2Cre/+Dicer1flox/floxand
CNPCre/+Dicer1flox/floxmice is the loss of PNS myelin. In the
CNPCre/+Dicer1flox/floxmice, but not the Olig2Cre/+Dicer1flox/flox
mice, we observe a near-complete ablation of sciatic nerve
myelination at P22–23. This phenotype likely indicates a require-
ment for Dicer1 function in the myelinating Schwann cells of the
thieu et al., 1980; Zhou et al., 2000). In addition, the axons of the
sciatic nerve are still present in the CNPCre/+Dicer1flox/floxmice,
implying that ablation of PNS myelin precedes loss of PNS
axons. Also, PNS myelin and axons both appear normal in
Olig2Cre/+Dicer1flox/floxmice, despite the fact that Olig2 (and
therefore Cre recombinase) is transiently expressed in the
myelinated motor neurons of the sciatic nerve (Takebayashi
et al., 2000; Zhou and Anderson, 2002). Loss of mature miRNAs
appears to severely disrupt the ability of Schwann cells to
produce myelin, and therefore the role of miRNAs in PNS
myelination will be an important area of future study.
OL-Induced miRNAs Link Initiation of OL
Differentiation to Cessation of OPC Proliferation
In several vertebrate cell types, particularly in the nervous
system, cessation of proliferation and initiation of differentiation
are temporally linked (Buttitta and Edgar, 2007; Kitzmann and
Fernandez, 2001; Politis et al., 2008; Truong and Khavari,
2007). How is the timing of the induction of one genetic program
specifying a postmitotic cell fate so tightly associated with the
repression of a distinct genetic program that drives the prolifer-
ation of the immature precursor cell? In OLs, miRNAs induced
by the withdrawal of OPC mitogens, and miR-219 in particular,
appear to be crucial to linking the cessation of proliferation
to the initiation of OL differentiation. Indeed, all three of the
miRNAs highly induced by mitogen withdrawal in OPCs prefer-
entially target genes that are expressed by OPCs and repressed
as OLs differentiate. A similar role for miRNAs in repressing
proliferation has already been postulated in other cell types
(Carleton et al., 2007). In fact, one of the additional miRNAs
induced during OL differentiation, miR-192, has been directly
implicated in arresting the cell cycle by coordinately repressing
several G1-S and G2-M cell cycle checkpoint control genes
(Georges et al., 2008), and another OL-induced miRNA,
miR-181a, functions as a tumor suppressor in gliomas (Shi
et al., 2008). Of equal importance may be the repression of
inhibitors of OL differentiation, as several immature cell types,
including OPCs, have been shown to express not only genes
that promote the cell cycle, but also genes (e.g., transcription
factors) that actively inhibit adoption of a differentiated cell
fate (Dugas et al., 2006; Lang et al., 2007; Stolt et al., 2006;
Wang et al., 2001).
Several labs have demonstrated that OL differentiation can be
induced by distinct pathways. When OPC mitogens become
limiting, proliferation ceases and OL differentiation rapidly com-
mences (Barres et al., 1993; Besnard et al., 1987). In contrast,
tiation: OPCs exposed to T3 in the presence of saturating
amounts of mitogens continue to proliferate and only differen-
tiate once they have become sufficiently ‘‘mature’’ enough to
respond to T3 (Barres et al., 1994a; Durand and Raff, 2000;
Temple and Raff, 1986). T3 exposure does not simply recapitu-
as distinct sets of genes are induced by mitogen withdrawal
and T3 exposure (Tokumoto et al., 2001). The fact that several
miRNAs appear to be specifically induced by mitogen with-
drawal, and not T3, in OPCs is consistent with their proposed
role in inhibiting proliferation, as exposure to T3 alone in the
presence of mitogens is not sufficient to trigger the immediate
arrest of the cell cycle. In addition, the finding that the loss of
OL-expressed miRNAs, which are specifically induced by
mitogen withdrawal, leads to a dysmyelination phenotype
in vivo implies that, consistent with previous reports (Calver
et al., 1998), limiting mitogen levels in vivo play an important
role in regulating the timing of OL differentiation.
Dicer1 and miR-219 Promote Myelin Development
Neuron 65, 597–611, March 11, 2010 ª2010 Elsevier Inc. 607
Model of miR-219 Function in OL Differentiation
Our data indicate that miR-219 in particular plays an important
role in promoting OL differentiation. Not only does inhibition of
miR-219 strongly impair OL differentiation, but miR-219 alone
can both instigate OL differentiation in OPCs immersed in mito-
gens and can also partially rescue the differentiation deficit
caused by the loss of mature miRNA production in OLs. We
have identified several genes directly targeted by miR-219
whose repression accelerates the program of OL differentiation.
mitogens, OL-induced miRNAs, and Dicer1 expression in
general, are low (Figure S4C). In contrast, targets of miR-219,
such as the mitogen receptor PDGFRa and several transcription
factors that repress OL differentiation, are highly expressed
(Figure 8A). Later in development or when mitogens are with-
drawn, genes that inhibit OL differentiation are downregulated.
The reduced expression/residual transcripts of these genes are
then further repressed by miR-219, whose expression is
induced, at least in part, by Dicer1 expression promoted by
mitogen withdrawal (Figure 8B). The importance of this
compounded repression is demonstrated in instances where
miR-219 function is blocked either by introduction of miR-219
inhibitor or disruption of Dicer1, as both of these manipulations
lead to increased repression of OL differentiation (Figures 1–3
and 5). In contrast, introduction of miR-219 into proliferating
OPCs reduces the expressed levels of miR-219-targeted inhibi-
tors of differentiation, leading to a derepression of OL differenti-
ation (Figures 5 and 7).
We hypothesize that the OL-induced miRNAs, and miR-219 in
particular, help to coordinate proliferation and differentiation.
When mitogens are withdrawn, these miRNAs are induced as
part of the program of OL differentiation, at which point they
rapidly repress several genes that normally promote the imma-
ture OPC phenotype. The importance of rapidly repressing all
of these inhibitors of OL differentiation is demonstrated in
a number of experiments. If the normal repression of a single
inhibitor of OL differentiation is blocked by transfection with an
overexpression plasmid, OL differentiation is impaired (Figures
S8C–S8G). Conversely, loss of a single inhibitory transcription
factor in proliferating OPCs does not instantly promote differen-
additional inhibitors of OL differentiation (Stolt et al., 2006).
miR-219 in the CNS and Glioblastoma
Our data indicate that miR-219 is expressed at the highest
level in the OLs of the developing CNS, and the strong degree
of miR-219 induction we observe is consistent with previous
findings (Lau et al., 2008). However, other labs have reported
biological rolesformiR-219inmature neuronsofthesuprachias-
matic nuclei (Cheng et al., 2007) and prefrontal cortex (Kocerha
et al., 2009). Potentially, miR-219 expressed at a lower level
in these neurons may still be able to affect neuronal function.
Alternatively, miR-219 may be expressed at a high level in
a few distinct populations of neurons. As we had assayed the
expression level of miR-219 in the pooled sample of all telence-
phalic neurons, a high level of expression in <5% of the total
population of neurons would be masked by the remaining
>95% of the nonexpressing cells. Nevertheless, OLs are the first
mammalian cells in which a developmental role for miR-219 has
The potential role of miR-219 as an inhibitor of cellular prolifer-
ation has been indicated in previous oncogenetic research.
Wong et al. (2008) identified miR-219 and miR-138 as being
downregulated in squamous cell carcinomas, and Izzotti et al.
(2009) identified both miR-219 and miR-192 as being downregu-
lated in potentially cancerous lung tissue. In CNS tumors,
miR-219,miR-138, andmiR-192 arealldownregulated inmedul-
loblastomas relative to normal cerebellar tissue (Ferretti et al.,
2009). Thesedata, in combination with our current findings, raise
the intriguing possibility that loss of miR-219 expression, along
with other OL-enriched miRNAs, may contribute to the aberrant
proliferation observed in some types of glioblastomas, particu-
larly those derived from or expressing genes of the OL lineage.
In fact, overexpression of PDGF in glial progenitor cells is
sufficient to induce glioblastoma in mice (Assanah et al., 2006,
2009; Uhrbom et al., 2000). Therefore, reintroduction of miRNAs
identified here, such as miR-219, into glioblastomas could be
investigated as a potential means of slowing tumor progression.
Complete protocols available upon request from firstname.lastname@example.org.
Purification and Culturing of Cells
Unless otherwise indicated, all in vitro culturing and transfection experiments
were performed with OPCs purified from P7–P8 Sprague-Dawley (Charles
Rivers) rat brains as described previously (Dugas et al., 2006). For acutely
Figure 8. Model of Dicer1 and miR-219
Promotion of OL Differentiation
sion, and bars indicate inhibition of pathway or
expression. Stronger activity/expression is de-
noted by bold lines/text, and weaker activity/
expression is denoted by gray lines/text.
(A) OL differentiation is inhibited by several OPC-
expressedgenesinthe presence of large amounts
of PDGF/early development under normal condi-
(B) When mitogens become limiting, Dicer1 and
accelerated repression of several OPC-expressed
genes and a derepression of OL differentiation.
See text for additional details. See also Figure S4.
Dicer1 and miR-219 Promote Myelin Development
608 Neuron 65, 597–611, March 11, 2010 ª2010 Elsevier Inc.
purified samples, OPCs, OLs, astrocytes, and neurons were isolated from
wild-type C57Bl6 (Charles Rivers) or transgenic S100b-eGFP mice as
described previously (Cahoy et al., 2008). See Supplemental Experimental
Procedures for additional details.
Immunohistochemistry and Histology
Immunostaining of OPC and OL cultures for CNP, MBP, MOG, and GFP
expression was performed as described previously (Dugas et al., 2006).
Immunostaining for NG2 expression was performed with 1/400 rabbit-anti-
NG2 antibody (Millipore AB5320).
For tissue sections, mice were perfused with DPBS followed by 4% parafor-
maldehyde (PFA), then immersion fixed in 4% PFA, equilibrated in 30%
sucrose, and mounted in 2:1 30% sucrose:O.C.T. (Tissue-Tek) to generate
8 mm longitudinal (nerves) or 10 mm sagital (brains) sections. Sections were
blocked in 50% goat serum with 0.4% Triton X-100 in PBS, then stained
with 1/100 rat-anti-MBP (Abcam ab7349) and 1/1000 rabbit-anti-NF200
(Sigma N4142) antibodies. In all cases, Alexa-dye-conjugated antibodies (Invi-
trogen) were used to visualize staining, and sections/cells were mounted in
Vectashield (Vector Labs) + DAPI to allow identification of healthy nuclei.
To stain tissue sections for compact myelin, brain sections were rehydrated
in PBS, then incubated in a 1/300 dilution FluoroMyelin (Invitrogen F-34652) in
PBS for 20 min, rinsed extensively in PBS, then mounted in Vectashield +
To quantify cells expressing OPC/OL markers, slides were scored blind,
and only cells with healthy nuclei were counted. To quantify CNS myelination,
cerebellar arms and corpora callosa were outlined, and the percentage area
myelinated was determined as number of pixels +10 arbitrary units (0–255
scale in 8 bit images) brighter than background/total number of pixels. For
each animal, two sagittal sections at different depths (?200–300 mm apart)
were analyzed; the two depths analyzed were the same across all littermates.
miRNA Microarrays and RT-PCR
For miRNA microarrays, OPC samples were collected from purified rat OPCs
cultured for 4 DIV in +PDGF-T3 media, OL samples were collected from
purified rat OPCs cultured for 3 DIV in +PDGF-T3 media, then 4 DIV in
-PDGF+T3 media. Total RNA samples (including small RNA molecules) were
isolated using QIAGEN miRNeasy Mini Kit and were labeled and hybridized
to microarrays as described in Supplemental Information.
ForqRT-PCRto analyze miRNA expression, RNA samples were obtained as
described above from purified rat OPCs cultured for 4 DIV in ±PDGF ±T3, or
from acutely purified neural cell samples as described in Cahoy et al. (2008).
Taqman miRNA qRT-PCR analysis was performed on 10 ng of RNA for
and normalized totheU6 snRNA. Sybrgreen incorporation qRT-PCRtodetect
rat/mouse mRNA expression was performed on an ABI StepOnePlus Real
Time PCR system following manufacturer’s instructions, normalized to
GAPDH mRNA levels. Data were analyzed using the ddCt method (Livak
and Schmittgen, 2001).
For RT-PCR to analyze gene expression, RNA was purified from OPC/OL
samples using the QIAGEN RNeasy Mini Kit, then 400 mg total RNA was
reverse transcribed using SuperScriptIII (Invitrogen). Equivalent volumes
from RT samples to be compared were then amplified using Platinum Taq
(Invitrogen). All primers are in Supplemental Information.
In Situ Hybridization
In situ hybridizations were performed as described previously (Cahoy et al.,
2008). 50-end DIG-labeled LNA probes were obtained from Exiqon for
miR-138, miR-219, miR-338, and scramble-miR (negative control). As a posi-
tive control, a 747 bp DIG-labeled antisense fragment encompassing the
coding region of the DM-20 isoform of the PLP gene and recognizing both
isoforms (gift from Prof. William Richardson) was transcribed as described in
the manufacturer’s protocol (Roche T7/SP6 In Vitro Transcription Kit). All
LNA hybridizations/initial washes were performed at 50?C; all PLP at 72?C.
For fluorescent in situs, the same DIG-labeled PLP probe was used with the
TSA-Plus Cyanine 3 labeling system (PerkinElmer) according to manufac-
turer’s instructions, and slides were mounted in Vectashield + DAPI.
Protein samples were collected from cultured cells in RIPA buffer + Complete
Protease Inhibitor (Roche) at 4?C. Equivalent total protein amounts were
loaded onto 4%–15% polyacrylamide gels (Bio-Rad) and then transferred to
Immobilon-P (Millipore) membranes. Blots were probed with 1/500 rat-anti-
MBP (Millipore MAB386), 1/500 mouse-anti-CNP, 1/100 mouse-anti-MOG,
1/1000 mouse-anti-PDGFRa (Abnova H00005156-M02), 1/10,000 guinea
pig-anti-Sox6 (gift M. Wegner), 1/5000 mouse-anti-b-actin (Sigma A5441),
then HRP-linked goat-anti-mouse, -rabbit, or -guinea pig (Millipore) or -rat
(Jackson Immunology). Blots were developed with ECL western blotting
detection reagent (Amersham) and exposed to film. Blots were quantified
with NIH ImageJ 10.2 Gel Analyzer and normalized to in-lane actin levels as
OPCs were transfected as described previously (Dugas et al., 2006), using
Lonza/Amaxa nucleofector kit; 2–3 3 106rat OPCs or 4–5 3 106mouse
OPCs per transfection. For luciferase assays, 6 mg test firefly luciferase
plasmid, 0.6 mg pRL-TK Renilla luciferase control plasmid (Promega), and
0.4 nmol miRNA mimic reagent were combined in transfections. OPCs were
then cultured 24–48 hr in +PDGF-T3 media. For other miRNA transfection
experiments, 0.4 nmol miRNA mimic or inhibitor was used and OPCs were
cultured ±PDFF ±T3 as indicated. In immunostaining experiments, 3.5 mg
pC1-eGFP was included to allow identification of transfected cells. In FoxJ3/
ZPF238 experiments, 2.5 mg pSPORT6-FoxJ3 or -ZFP238 was cotransfected
with 1.5 mg pC1-eGFP, and OPCs were cultured ±PDFF ±T3 as indicated.
In all immunostained transfection experiments, slides were scored blind,
and data were quantified as the percentage of GFP+transfected cells with
healthy nuclei that coexpressed the indicated marker protein (NG2, CNP,
MBP, or MOG); >150 cells/coverslip were quantified.
Supplemental Information includes Supplemental Experimental Procedures,
references, eight figures, four tables, and two movies and can be found with
this article online at doi:10.1016/j.neuron.2010.01.027.
WethankProf. Klaus-ArminNave forkindlyproviding theCNP-Cre mouseline,
Prof. David Rowitch for providing the Olig2-Cre mouse line, and Prof. Chang-
Zheng Chen for providing the floxed-Dicer mouse line. We would also like to
thank Prof. Michael Wegner for generously providing the Sox6 antibody.
This work was funded by the Myelin Repair Foundation and by NIH grant
Accepted: January 25, 2010
Published: March 10, 2010
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