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CNS pRECuRSoRS

Roles of small regulatory RNAs

in determining neuronal identity
Xuekun Li and Peng Jin
Abstract | Neurogenesis, the process of generating functional neurons from neural stem cells,
is tightly controlled by many intrinsic and extrinsic mechanisms. Uncovering these regulatory
mechanisms is crucial for understanding the functions and plasticity of the human brain.
Recent studies in both invertebrates and vertebrates point to the importance of small
regulatory RNAs in regulating lineage-specific gene expression and determining neuronal
identity during neurogenesis. These new observations suggest that small regulatory RNAs
could function at many levels to regulate self-renewal of neural stem cells and neuronal fate
specification, implicating small regulatory RNAs in the complexity of neurogenesis.

Self-renewal
The capacity of a cell to
proliferate and produce
identical cells.
Multipotency
The potential of a cell to give
rise to multiple lineage cells.
Neural stem cells, for example
can generate neurons,
astrocytes and
oligodendrocytes.
Department of Human
Genetics, Emory University
School of Medicine, 615
Michael Street, Suite 301,
Atlanta, Georgia 30322,
USA.
Correspondence to P.J.
e‑mail: peng.jin@emory.edu
doi:10.1038/nrn2739
Published online
31 March 2010; corrected
online 30 April 2010
Neurogenesis is the process by which functionally
integrated neurons are generated from neural stem cells
(NSCs). It involves the proliferation and neuronal fate
specification of NSCs, as well as the maturation and
functional integration of the neuronal progeny into neu-
ronal circuits1,2. NSCs exhibit the two essential proper-
ties of stem cells: self-renewal and multipotency. During
embryogenesis, NSCs are located in the ventricular zone
of the neural tube. Embryonic NSCs can give rise to all
the cell types required for the formation of the CNS.
Contrary to the earlier dogma that neurogenesis occurs
only during development, it is now generally accepted
that neurogenesis occurs throughout life in mammalian
brains. Adult neurogenesis has been observed at two
locations under normal conditions: the subventricular
zone (SVZ) of the lateral ventricles and the subgranu-
lar zone (SGZ) of the dentate gyrus in the hippocampus
(FIG. 1). Neurogenesis is regulated at many levels by both
extrinsic factors, such as physiological and pathological
conditions (BOX 1; FIG. 1c), and intrinsic factors, such as
genetic and epigenetic programmes (FIG. 1c). The main-
tenance and differentiation of NSCs are tightly control-
led by intricate molecular networks. Uncovering these
regulatory mechanisms is crucial for understanding the
functions and plasticity of the CNS.
Genome projects have shown that at least 93% of
nucleotides in the human genome are transcribed in dif-
ferent cells, with similar findings in the mouse and other
eukaryotes. However, only around 1.2% of transcribed
RNAs encode proteins, indicating that there may be a
vast reservoir of biologically meaningful non-coding
RNAs, often referred to as the ‘dark matter’ of the cell3.
Non-coding RNAs may regulate gene expression at many
levels and through a range of mechanisms. Uncovering
the functions of these non-coding RNAs could substan-
tially improve our understanding and the treatment of
human diseases.
The small regulatory RNAs identified so far include
microRNAs (miRNAs), endogenous small interfering
RNAs (esiRNAs), Piwi-interacting RNAs (piRNAs) and
promoter-associated short RNAs (PASRs; also known as
transcription start site RNAs (TSS RNAs) and transcrip-
tion initiation RNAs (tiRNAs)). These small RNAs range
from 18 to 30 nucleotides in length and can shape diverse
cellular pathways (BOX 2). Recent studies have shown that
several classes of small regulatory RNAs have impor-
tant roles in stem cell biology; indeed, the stem cell and
miRNA fields have converged with the identification of
stem cell-specific and lineage-specific miRNAs4,5. It has
become clear that miRNAs provide a new dimension to
the regulation of stem cell functions. For example, owing
to their functions in the regulation of translation, miRNAs
can regulate stem cell fate and behaviour by fine-tuning
the protein levels of various factors required for stem cell
proliferation and multipotency. Furthermore, piRNAs
are also important for stem cell self-renewal, as their
partner Piwi proteins are required for stem cell main-
tenance. In this Review, we focus on recent progress in
understanding the roles of small regulatory RNAs
in neurogenesis.
Expression of small RNAs in neurogenesis
Detecting nucleic acids that are only 18–30 nucle-
otides in length is inherently difficult, simply because
of their small size. The past several years have seen the
emergence of new techniques for the identification

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and profiling of small regulatory RNAs (BOX 3). Earlier
expression profiling studies revealed that miRNAs
have spatiotemporal and cell-specific expression
patterns in the brain6–14. The expression of some miRNAs
is brain-specific, such as miR-9, miR-124a, miR-124b
and miR-135. other miRNAs are enriched in the brain,
such as let-7, miR-9* (from the opposite arm of the
miRNA precursor), miR-125a, miR-125b and miR-128
(ReFs 8–10,13,15) . miRNAs also have cell-specific
expression patterns. miR-92b is restricted to neuronal
progenitor cells, whereas miR-23 is expressed mainly in
astrocytes. Selective miRNAs are either exclusively or
preferentially expressed in neurons, including miR-125,
miR-128 and miR-138. Neuronal differentiation of
embryonic stem (ES) cells induced by retinoic acid sub-
stantially alters the expression profile of miRNAs10,16.
Furthermore, expression of let-7 is highly induced in
both early neurogenesis and retinoic acid-induced neu-
ronal differentiation in embryocarcinoma, whereas it is
decreased in adult brains10,13,16. The specific expression

a Neocortical b
neuroepithelium
LV Blood Type B LV
Neocortex vessel

Type A

Neuronal identity
Neurogenesis
E8 E14 P1 Adult

Neurogenesis-related miRNAs Ependymal

Type C cell

Neuronal specification-related
miRNAs SVZ

Hipp
c CB
OB
Extrinsic factors
• Disease and ageing • Physical activity
• Enriched environment • Hormones
• Growth factors • Neurotransmitters
SGZ

NSC NPC
proliferation differentiation
MCL
Intrinsic factors
Genetics Epigenetics
• Signalling pathways: • DNA methylation
SHH, WNT and Notch • Histone modification GCL
• Transcriptional regulators
• Translational regulators
• Small regulatory RNAs SGZ

Figure 1 | embryonic and adult neurogenesis. a | Mammalian neurogenesis starts around embryonic day 8 (E8), reaches
its peak around E14 and decreases thereafter. The vertebrate CNS is derived from neuroepithelium. For cortical
Nature Reviews | Neuroscience
neurogenesis, neuroepithelial cells begin to acquire features of radial glial cells. These cells serve as the progenitor
population for neuroblasts that migrate to the cortex and differentiate into mature cortical neurons. During neurogenesis,
the expression levels of a subset of microRNAs (miRNAs) increase and are maintained at high levels in neuronal cells,
whereas the expression patterns of other miRNAs mirror the wave of neurogenesis, suggesting that they have different
roles in neurogenesis. b | Adult neurogenesis is observed at two locations under normal conditions: the subventricular
zone (SVZ) of the lateral ventricles (LVs) and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus (Hipp).
Slowly dividing SVZ astrocytes (type B cells) derived from embryonic radial glial cells are neural stem cells (NSCs) that are
relatively quiescent and can give rise to rapidly dividing immature precursors (type C cells). Type C cells actively proliferate
and can generate immature neuroblasts (type A cells), which migrate in chains along the rostral migratory stream to the
olfactory bulb (OB) and differentiate into interneurons. SGZ astrocytes (type B cells) could also function as NSCs. NSCs in
the dentate gyrus undergo proliferation, fate specification, migration, axon and dendrite targeting and synaptic
integration. c | Neurogenesis is regulated at many levels by both extrinsic factors, such as physiological and pathological
conditions, and intrinsic factors, such as genetic and epigenetic programmes. CB, cerebellum; GCL, granular cell layer;
MCL, molecular cell layer; NPC, neuronal progenitor cell; P1, postnatal day 1; SHH, Sonic hedgehog protein.

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Deep sequencing of miRNA in the nervous system strongly suggests that
An approach enabled by miRNAs could play key parts in brain development and
next-generation sequencing neuronal fate specification (FIG. 2a).
technology that is particularly Studies in human and mouse ES cells have shown
useful for identifying low-
abundance RNAs or
that the complete repertoire of miRNAs in stem cells
low-frequency mutations. can only be uncovered through deep sequencing , as
some miRNAs are expressed solely in ES cells. Thus, it
Cre–loxP system is important to use this technology to profile the expres-
A site-specific recombination
sion of small regulatory RNAs in both proliferating and
system derived from
Escherichia coli bacteriophage
differentiated NSCs — an approach that has not been
P1. Two short DNA sequences taken so far.
(loxP sites) are engineered to
flank the target DNA. NSC fate determination by miRNAs
Activation of the
Cre-recombinase enzyme
To determine whether miRNAs play a crucial part in
catalyses recombination neurogenesis and the development of the nervous system,
between the loxP sites, leading early studies focused on key components of the miRNA
to excision of the intervening pathway. loss of Dicer (an RNase III enzyme that
DNA sequence.
cleaves the miRNA precursor (pre-miRNA) to pro-
Environmental enrichment duce mature miRNA) in zebrafish was found to lead to
Providing animals under defective neural tubes, reduced ventricle size and loss
managed care with of the midbrain–hindbrain boundary 17. Surprisingly,
environmental stimuli to these defects could be partially rescued by a single
improve the quality of life by
increasing physical activity,
miRNA, miR-430 (ReF. 17). Similarly, the disruption
stimulating natural behaviours of miRNA-induced silencing complex (miRISC) activity
and preventing or reducing due to a mutation in Argonaute 2 (AGo2), an essential
neural disorders including component of the complex, prevented neural-tube clo-
stereotypical behaviours.
sure in mice18. These two studies provide strong in vivo
evidence that the miRNA pathway is important for brain
development and morphogenesis. Further advances were
provided by studies in which Dicer activity was condi-
tionally removed in specific neuronal cell types using
the Cre–loxP system. This led to various defects in neural
development, such as increased cortical apoptosis,
smaller cortex size, enlarged lateral ventricles, profound
astrogliosis, altered dendritic maturation, smaller neural
cells and abnormal neurogenesis19–22. These findings
indicate an essential role for the miRNA pathway in
neural development.
Box 1 | Multiple extrinsic factors regulate neurogenesis
Diverse intrinsic and extrinsic mechanisms are known to regulate neurogenesis1,2. The
following extrinsic factors have been found to be involved: hormones and neural
transmitters, including dopamine, serotonin, acetylcholine and glutamate; growth
factors, such as fibroblast growth factor 2, epidermal growth factor and vascular
endothelial growth factor; exercise and learning; environmental enrichment and
pathological conditions, such as stress, seizure, ischaemia and ageing.
Mounting evidence indicates the essential role of the stem cell niche in the
maintenance and regulation of neurogenesis. In the subventricular zone (SVZ) of adult
mice, the cilia of ependymal cells transform the flow of cerebrospinal fluid into a chemical
signal that directs the establishment of the chemorepulsive gradients, which guides the
migration of newly born neurons88. Recent studies revealed that the vascular system is
also a key component of the stem cell niche and is involved in regulating neurogenesis in
the SVZ89,90. Proliferating neural stem cells (NSCs) are tightly apposed to the blood vessels
through the laminin receptor α6β1 integrin, and neuroblast chains run parallel to blood
vessels in the direction of migration towards the rostral migratory stream89,90.
Vasculature-derived signals can access the SVZ, and most of the proliferating cells that
were recovered from treatment with the anti-mitotic reagent cytosine–arabinofurano-
side were directly coupled to the vasculature90, suggesting that the vasculature provides
signalling cues to NSCs and regulates their function. Given the roles of small regulatory
RNAs in other types of stem cell niche5, it is likely that they also have important roles in
the NSC niche, although this remains to be explored.
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During neurogenesis, neuronal diversity can be
achieved through the asymmetrical cell division of
NSCs, which produces two daughter cells with different
developmental potentials. whereas one differenti-
ates into a neuron, the other maintains a stem cell fate.
Although the mechanism is more complex in verte-
brates than invertebrates, the unequal inheritance of cell
fate determinants (DNA or protein) ensures that each
daughter cell acquires a different fate23–25. The TRIm-
NHl (tripartite motif and NCl1, HT2A and lIN41
domain-containing) protein TRIm32, a previously
identified E3 ubiquitin ligase, was recently found to
enhance the activity of selected miRNAs by interacting
with AGo1, a member of the Argonaute protein family
involved in miRNA-mediated gene regulation26. During
asymmetrical cell division in NSCs and neural progeni-
tor cells (NPCs), TRIm32 is enriched in one of the two
daughter cells. TRIm32 suppresses cell proliferation and
induces neuronal differentiation by degrading the tran-
scription factor myC and enhancing let-7a activity 26.
Because TRIm32 and let-7a are also present in NSCs
and NPCs, it is possible that another pathway might exist
to antagonize the effect of TRIm32–let7a and maintain
their multipotency 27. These findings suggest that the
unequal distribution of a ribonucleoprotein complex-
containing TRIm32 and its target miRNAs could regu-
late the balance between differentiation and proliferation
in neural stem cell lineages, and thus that miRNAs could
participate in cell fate determination and the regulation
of asymmetrical cell division.
Recent studies on individual miRNAs have begun to
shed light on their specific functions in neurogenesis and
neuronal development (see below)4,28–32. Further research
in this area could reveal novel mechanisms regulating
neurogenesis and targets to manipulate this process.
Interplay between miRNAs and transcription factors.
The interplay between miRNAs and transcription
factors was first uncovered in Caenorhabditis elegans.
In C. elegans, two taste neurons, ASE right (ASER) and
ASE left (ASEl), display left–right asymmetrical expres-
sion patterns of putative chemoreceptor genes, which are
important for functional lateralization of taste neurons.
ASEl and ASER were found to express distinct miRNAs,
lsy-6 and miR-273, respectively 33,34. lsy-6 and miR-273
regulate the establishment of neuronal left–right asym-
metry by suppressing the translation of their targets,
cog‑1 and die‑1. CoG-1 and DIE-1 can also inhibit
the expression of lsy-6 and miR-273 to form a double-
negative feedback loop33–35. These studies provide an
excellent example of how miRNAs can specify distinct
neuronal cell fates from a multipotent precursor state.
miR-124 is highly conserved in both invertebrates
and vertebrates and is the most abundant miRNA in
both the embryonic and adult CNS8. Its temporal expres-
sion pattern suggests it may have a function in neuronal
development 7,16. Ectopic expression of miR-124 in cul-
tured non-neuronal cells increased the expression of
neuronal genes and inhibited non-neuronal genes16,36,37.
This is required during early embryonic neurogenesis
to ensure that differentiation occurs at the correct
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Transit-amplifying cells time and that the appropriate number of neurons is
Cells that arise from adult generated. RE1-silencing transcription factor (REST;
stem cells and divide a finite also known as NRSF) suppresses neuronal genes in
number of times until they non-neuronal cells by binding to a conserved repressor
become differentiated. They
are committed progenitor cells.
element (RE1) in neuronal gene loci and recruiting the
co-repressor complex containing histone deacetylases
and the methyl-CpG-binding protein mECP2 (ReF. 38).
Downregulation of REST during the transition from pro-
genitors to post-mitotic neurons allows neuronal gene
expression. Small carboxy-terminal domain phosphatase 1
(SCP1; also known as CTDSP1) is another anti-neural
factor expressed in non-neuronal tissues39. SCP1 can be
recruited to RE1-containing neuronal genes by REST
in non-neuronal cells. The timely downregulation of
the anti-neural REST–SCP1 pathway is thought to be
important in embryonic neurogenesis. REST binds to
RE1 sites within the promoter regions of three miR-124
genomic loci and represses their expression through the
recruitment of two co-repressor complexes, SIN3 and
CoREST40. miR-124 has been shown to directly target
the SCP1 3′-untranslated region (UTR), which sup-
presses SCP1 expression39. In addition to SCP1, compu-
tational approaches have also uncovered miR-124 target
sites in the 3′-UTR of mECP2 and CoREST. These
findings suggest the existence of a negative-feedback
loop between REST–SCP1 and miR-124 that mediates
the rapid transition between neural progenitors and
post-mitotic neurons during embryonic neurogenesis39
(FIG. 2b).
Box 2 | Types of small regulatory RNAs and their biogenesis
With the advance of high-throughput sequencing technology, the field of small
regulatory RNAs is rapidly expanding. Currently, the small regulatory RNAs identified
in mammals include microRNAs (miRNAs), endogenous small interfering RNAs (esiRNAs),
Piwi-interacting RNAs (piRNAs) and promoter-associated short RNAs (PASRs; also
known as transcription start site RNAs (TSS RNAs) and transcription initiation RNAs
(tiRNAs))75,83,91,92.
miRNAs are 18–25-nucleotide non-coding RNAs that can regulate the translation of
target mRNA molecules in a sequence-specific manner. Most endogenous miRNA
genes are transcribed initially as primary transcripts that contain one or more extended
hairpin structures. The nuclear RNase III enzyme Drosha, working with DGCR8, cleaves
both strands near the base of the primary stem loop and yields the precursor miRNA
(pre-miRNA). After being exported to the cytoplasm by exportin 5 (also known as
RANGTP), pre-miRNAs are further cleaved by the RNase III enzyme Dicer into mature
miRNAs, which will be incorporated into the RNA-induced silencing complex. It has
been shown that a single miRNA can simultaneously regulate the expression of many
mRNA targets and thereby fine-tune protein expression. Many miRNAs are expressed
in the nervous system, where they affect numerous neuronal genes.
High-throughput sequencing has recently identified three new classes of small
regulatory RNAs: esiRNAs, piRNAs and PASRs. The biogenesis of esiRNAs starts with
either bidirectional transcription or the transcription of an inverted repeat. The
resulting double-stranded RNA or the hairpin RNA precursor is then processed by the
components of the miRNA pathway. Although miRNA and esiRNA biogenesis is
generally Dicer-dependent, piRNAs are generated from a long single-stranded
precursor independently of Dicer. piRNAs also differ from miRNAs and siRNAs in that
they are usually 26–31 nucleotides long and associated with Piwi subfamily proteins.
Most piRNAs map to the repetitive regions of the genome and are thought to control
the activity of transposons. PASRs map to the 5′ end or promoter region of the
protein-coding genes and are proposed to have a role in the transcription of splice
variants. Whether these small regulatory RNAs are expressed during neurogenesis
remains to be determined.
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In the SVZ of the adult mouse, miR-124 shows cell
type-specific expression. It is expressed in neuroblasts
as well as mature granule and periglomerular neu-
rons, but not in dividing doublecortin-negative transit-
amplifying cells or in oligodendrocytes or astrocytes41.
During the transition from amplifying type C cells to
neuroblasts, the expression of miR-124 increases and is
much higher in mature neurons, suggesting miR-124
might be important for cell lineage progression. Further
gain- and loss-of-function studies suggest that miR-124
regulates the progression of neuronal differentiation by
targeting SoX9, a transcription factor that can maintain
the pluripotent state of NSCs and inhibit neuronal dif-
ferentiation41 (FIG. 2b). whether SCP1 is regulated by
miR-124 in adult NSCs remains to be determined. As
well as the interplay between miR-124 and transcrip-
tion factors, it has also been shown that miR-124 could
promote neuronal differentiation and neuronal line-
age commitment by inducing neuron-specific alterna-
tive splicing of polypyrimidine tract-binding protein 1
(PTBP1; also known as PTB and hnRNP I), a global
repressor of neuron-specific splicing 37. These findings
suggest that miR-124 fine-tunes neurogenesis by target-
ing multiple mRNAs that are involved in different gene
regulatory mechanisms.
miR-9 is also highly conserved and is expressed in
most of the NPCs of zebrafish, chickens and mice6,15,42.
In Drosophila melanogaster, sensory organs develop
from the divisions of single sensory organ precursor
cells (SoPs). Ectopic miR-9a expression led to a signifi-
cant reduction in the number of SoPs, whereas the loss
of miR-9a resulted in extra production of SoPs. This
indicates that miR-9a ensures that the precise number of
neuronal precursor cells are generated by controlling cell
fate specification43. Appropriate expression of the tran-
scription factor Senseless (SENS) is required to control
the formation of the precise number of SoPs: high levels
of SENS in SoPs promote pro-neural gene transcrip-
tion, whereas low levels of SENS in non-SoPs repress the
expression of pro-neural genes. miR-9a inhibits neuronal
fate in non-SoP cells by repressing SENS expression in
the embryonic and adult stages43.
During vertebrate brain development, the midbrain–
hindbrain boundary (mHB) functions as an organizing
centre in the neural tube that is both necessary and suf-
ficient for the ordered development of the hindbrain.
The mHB contains a pool of NPCs that contributes
neurons to the entire mHB. Both gain- and loss-of-
function studies in zebrafish have shown that miR-9 is
required for the normal generation of new neurons in the
mHB by repressing the anti-neurogenic genes her5 and
her9 (ReF. 44). moreover, ectopic expression of miR-9 in
embryonic mouse cortex through in vivo electroporation
led to premature neuronal differentiation in the cor-
tex, produced more neurons in the ventricular zone by
repressing the expression of forkhead box protein G1
(which has been shown to suppress early cortical cell
fate) and disrupted the migration of new neurons in the
cortex45. In adult neurogenesis, miR-9 inhibits NSC pro-
liferation and accelerates neural differentiation by tar-
geting the nuclear receptor TlX, an essential regulator
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Box 3 | Expression profiling of small regulatory RNAs

Diverse methodologies have been used to determine the expression of small regulatory RNAs, including northern blot,
in situ hybridization, microarray hybridization-based detection (using locked nucleic acid (LNA) and DNA), reverse
transcription PCR-based detection (TaqMan and SYBR Green), direct sequencing of adaptor-ligated cDNA that is
complementary to the small RNA and solution hybridization to oligonucleotide (LNA and DNA)-conjugated beads.
In recent years, next-generation sequencing technology has been widely used to profile small regulatory RNAs. Currently,
three high-throughput sequencing platforms are commercially available: the Illumina Solexa Genome Analyser, the Roche
454 GS FLX Genome Analyser and Applied Biosystems’s SOLiD system93–95. All three systems require similar preparation of a
small RNA library, including small RNA isolation, reverse transcription and ligation with system-specific adaptors. With the
Roche 454 system, cDNAs are mixed with agarose beads and clonally amplified by emulsion PCR. The products are
randomly deposited to a picotitre plate, and each well holds only one bead, which is pyrosequenced. With the Illumina
Solexa platform, single cDNA strands bind to the surface flow cell channels and are amplified in situ by bridge PCR. Using a
sequencing-by-synthesis approach, each sequencing cycle adds a mixture of four modified deoxynucleotides that are
labelled with four different fluorescence colours. By imaging four channels with laser excitation, the identity of the first base
in each cluster is recorded. After removing the fluorescent label, the next cycle is initiated, and the process is repeated until
the full oligomer is sequenced. With the SOLiD platform, cDNA is clonally amplified by emulsion PCR on the surface of
magnetic beads and subsequently deposited onto the surface of a glass slide. Rather than using polymerase,
ligase-mediated sequencing is performed by introducing a primer to the adaptor sequence of cDNA and then ligating a
fluorescently labelled eight-base oligonucleotide to the sequencing primer. After the fluorescence signal is recorded with
a laser scanner, the octamer is cleaved between the fifth and sixth bases (removing the fluorescent group), and the next
round is repeated until the sequence is completed.

Locked nucleic acid
A modified RNA nucleotide
with high stability, which can be
used as a highly sensitive
detection probe.
of NSC proliferation and self-renewal, by activating
wNT–β-catenin signalling and repressing the cyclin-
dependent kinase inhibitor 1A (CDKN1; also known
as wAF1 or CIP1)46. miR-9 suppresses TlX expres-
sion through the 3′-UTR of TlX mRNA47. Introducing
a TlX expression vector lacking the endogenous TlX
3′-UTR rescued the proliferation deficiency induced by
miR-9 overexpression and reversed the miR-9-induced
premature differentiation47. Furthermore, TlX binds to
the 3′ genomic sequences of miR-9 and inhibits miR-9
expression. Thus, miR-9 and TlX form a feedback loop
to regulate the switch between NSC proliferation and
differentiation (FIG. 2b).
In summary, these studies show that transcription
factors can directly regulate the expression of specific
miRNAs, and specific miRNAs can then target other
transcription factors and regulate their expression
post-transcriptionally. The result is a transcription factor
to miRNA to another transcription factor (sometimes
even of the miRNA itself ) paradigm for regulating
neurogenesis. This represents an interesting, evolution-
arily conserved strategy to keep the balance between
miRNAs and their transcriptional regulatory pro-
grammes. However, it has been well documented that
many transcription factors can coordinate the regulation
of a given gene and that one miRNA can regulate many
mRNA targets. Thus, although the current studies focus
on one miRNA and one transcription factor at a time, it
is likely that there is a more complex interplay between
the miRNA network and the transcriptional regulation
network in regulating neurogenesis.
miRNA-mediated regulation of signalling cascades.
miR-9 not only controls neurogenesis at the mHB, but
also regulates the formation of the mHB in embry-
onic zebrafish despite not being expressed at this site.
overexpression of miR-9 in the mHB leads to loss of
the mHB by inhibiting the fibroblast growth factor (Fgf)
signalling pathway components Fgf8, Fgf receptor 1
and canopy 1 (ReF. 44). These results indicate that miR-9
inhibits the Fgf signalling pathway and promotes neuro-
genesis by targeting different mRNAs, which act con-
vergently to negatively delimit the mHB, and suggest a
mechanism whereby organizer activity and neurogenesis
are spatially coordinated in the mHB. It also illustrates
a common theme in miRNA-mediated gene regulation;
namely, that a given miRNA often regulates several com-
ponents within the same biological pathway to fine-tune
the output of this pathway.
The Sonic hedgehog (SHH) signalling pathway acts
through the Patched 1–Smoothened (Smo) receptor
complex to activate downstream effectors and has a piv-
otal function in cell differentiation, survival and prolif-
eration, and in tissue patterning48. For example, during
Xenopus laevis retinal development, blocking the SHH
pathway delayed cell cycle exit and induced the release of
the translational inhibition of two homeobox genes, otx2
and vsx1, which act as key positive regulators of bipolar
neuron generation49. It was shown that four miRNAs
(miR-129, miR-155, miR-214 and miR-222) concertedly
bind to the 3′-UTR of otx2 and vsx1 mRNAs, inhibiting
their translation and thus the generation of bipolar neu-
rons49. Inhibition of these four miRNAs promotes pro-
genitor cell commitment to the bipolar cell fate49. The
SHH pathway also controls the proliferation of cerebellar
granule cell progenitors (GCPs), which respond to SHH
produced by Purkinje cells. Excessive SHH signalling in
GCPs induces neoplastic transformation into medullob-
lastoma. Some miRNAs, including miR-125b, miR-324-5p
and miR-326, are abnormally expressed in medullo-
blastoma, and these miRNAs inhibit medulloblastoma
cell proliferation by repressing components of the SHH
pathway, such as GlI1 and Smo50. Conversely, loss of
these miRNAs has been shown to promote GCP differ-
entiation into granule cells50. A recent study showed that
the miR-17/92 cluster is upregulated by SHH signalling

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REST photoreceptor cells. Expression is switched when EGFR

a b
SCP1 signalling transiently triggers yAN degradation (FIG. 2c).
NSC NPC Mature neuron
A major question in signal transduction research is how
miR-124
weak or transient activation of signalling pathways can
SOX9
lead to robust and long-term switches in gene expres-
sion. These studies suggest that, during neurogenesis,
miRNAs may act through feedback loops to reinforce
and stabilize changes in gene expression in response to
let-7 miR-92b miR-124a signalling input.
miR-125 miR-124
miR-9 miR-128
miR-138
NSC NPC Neuron Crosstalk between miRNAs and epigenetic regulation.
Epigenetic regulation, including DNA methylation and
histone modification, is known to be involved in the
GFAP+ Nestin+ NeuN+ TLX modulation of stem cell proliferation and differentia-
Nestin+ DCX+ miR-9 tion, including the proliferation and differentiation of
s100β– PSA–NCAM+
NSCs53,54. Recent genome-wide analyses have shown a
role for DNA methylation and chromatin remodelling,
particularly by the polycomb group proteins, in defining
c the properties and regulating the functions of stem
miR-7 ? Target cells55. Epigenetic regulation and chromatin remodel-
PNT-P1 miR-7 gene
genes
ling are also thought to have important roles in neuro-
Target genesis56–58; however, the identification of downstream
YAN targets has proved elusive59–61.
genes
ORF AAAA Functional interactions between small regulatory
EGFR YAN mRNA RNAs and epigenetic mechanisms in gene silencing
were initially demonstrated in plants and yeast 62, and
Figure 2 | role of mirNAs in fine-tuning neurogenesis. a | Distinct microRNA
(miRNA) expression profiles characterize the transition fromNature
neuralReviews
stem cells (NSCs) to recently in mammals63. on one hand, epigenetic regu-
| Neuroscience
neural progenitor cells (NPCs) and neurons. Marker genes for each of these cell types are lation can modulate small RNA expression and, on
listed below the miRNA profiles. b | Schematic illustration of a transcription factor to the other hand, small regulatory RNAs can induce
miRNA to another transcription factor (sometimes even of the miRNA itself) paradigm for epigenetic changes that lead to mitotically heritable
regulating neurogenesis. This negative-feedback loop keeps the balance between transcriptional silencing 62,64. The crosstalk between
miRNAs and their transcriptional regulatory programmes during neurogenesis (the blue epigenetic regulation and miRNAs in brain develop-
triangles indicate the expression levels of transcription factors and miRNAs). ment and function is now beginning to be investigated.
c | Schematic illustration of the feedback loop between miR-7 and its target, YAN. During A neuronal activity-dependent miRNA, miR-132, was
eye development in Drosophila melanogaster, progenitor cells express YAN, which shown to regulate mECP2 expression in neurons65, and
directly inhibits miR-7 transcription. When progenitor cells differentiate into
the level of mECP2-regulated miR-184 was increased
photoreceptor neurons, epidermal growth factor receptor (EGFR) signalling is activated,
which results in the degradation of YAN. Thus, miR-7 expression is initiated and promotes in neurons following depolarization66. mECP2 belongs
photoreceptor neuron differentiation by directly repressing YAN. Transcription activator to a family of DNA methyl-CpG-binding proteins
pointed P1 (PNT-P1) can also activate miR-7 expression and contribute to YAN that interpret DNA methylation as a signal to sup-
repression. This reciprocal feedback ensures mutually exclusive expression of YAN in press gene expression67. De novo mutations in MECP2
progenitor cells and miR-7 in photoreceptor cells. DCX, doublecortin; GFAP, glial cause neurodevelopmental disorders, including
fibrillary acidic protein; NeuN, neuronal nuclei; ORF, open reading frame; PSA–NCAM, Rett’s syndrome67–69. How the loss of functional mECP2
polysialylated neural cell adhesion molecule; REST, RE1-silencing transcription factor; protein leads to neurodevelopmental deficits is still
SCP1, small carboxy-terminal domain phosphatase 1. under investigation.
Some studies are now revealing a link between miRNAs
and epigenetic regulation in neurogenesis. For example,
in medulloblastoma and promotes GCP proliferation51, gliomas retain many features of NPCs, including the
suggesting that a feedback loop might exist here as well. ability to grow as neurospheres in culture, self-renew
This feedback loop has been shown more clearly in and migrate in the brain. moreover, profiling studies
D. melanogaster. In the eye of the fruitfly, expression of have shown that gliomas express many genes that are
miR-7 is activated in retinal progenitor cells as they begin expressed by NPCs. miRNA expression profiling of
to differentiate into photoreceptors52. This process is human glioblastoma specimens revealed a marked
dependent on epidermal growth factor receptor (EGFR) reduction of miR-128 in tumour samples. miR-128 could
signalling, which triggers extracellular signal-regulated reduce glioma cell proliferation by suppressing BmI1, a
kinase-mediated degradation of the transcription factor member of the polycomb repressive complex 70. It will
yAN. In non-stimulated cells, yAN represses miR-7 tran- be interesting to further examine the role of miR-128 in
scription, whereas miR-7 RNA represses yAN protein neurogenesis.
expression in photoreceptors by binding to sequences NSC differentiation into neurons is accompanied by a
within its mRNA 3′-UTR52. This reciprocal negative feed- switch of neuronal progenitor-specific BRG1-associated
back between yAN and miR-7 ensures mutually exclusive factor (npBAF) to neuron-specific BAF (nBAF) through
expression, with yAN in progenitor cells and miR-7 in ATP-dependent chromatin-remodelling mechanisms.

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© 2010 Macmillan Publishers Limited. All rights reserved


A2B5
A cell surface ganglioside
epitope expressed in
developing thymic epithelial
cells, oligodendrocyte
progenitors and
neuroendocrine cells.
Long interspersed nuclear
(L1) elements
Full-length active L1 elements
are ~6 kb long, consisting of a
5′-untranslated region that has
promoter activity, two open
reading frames (encoding a
nucleic acid-binding protein
and an endonuclease), a
reverse transcriptase protein
and a poly(A) tail.
Retrotransposon
Genetic elements that can
amplify themselves in a
genome through an RNA
intermediate. They are
ubiquitous components of the
DNA of many eukaryotic
organisms.
NATURE REVIEwS | NeuroscieNce
The BAF complex in mammals is functionally related
to the SwI–SNF complex in Saccharomyces cerevisiae
and D. melanogaster, which is thought to facilitate tran-
scriptional activation of specific genes by antagonizing
chromatin-mediated transcriptional repression. npBAF
has two subunits, BAF53A and BAF45A, which are
essential for NSC proliferation; BAF53B and BAF45B,
by contrast, are subunits of nBAF that are crucial for
dendritic development 71. The repression of BAF53A by
miR-9* and miR-124 was found to mediate the switch
between npBAF and nBAF, suggesting that miRNAs
could target chromatin remodelling factors during
neurogenesis71. However, whether there is an epige-
netic circuitry with a feedback regulatory mechanism
mediated by miRNAs in the regulation of neurogenesis
remains to be determined.
miRNAs in the specification of glia
Profiling studies have also identified a range of miRNAs
that help to establish the lineage specificity for both
astrocytes and oligodendrocytes. The phosphorylation
of signal transducer and activator of transcription 3
(STAT3) can inhibit the differentiation of NPCs
into neurons and promote differentiation into glia.
Knockdown of miR-124a and miR-9 increased the level
of phosphorylated STAT3, whereas overexpression of
miR-124a and miR-9 decreased STAT3 expression,
suggesting that these miRNAs could have a role in
modulating glial cell fate determination by regulating
the levels of STAT3 (ReF. 16). In addition, the expression
of miR-23, a miRNA previously implicated in neural
specification, was found to be restricted in astrocytes10;
however, whether miR-23 is involved in the specification
of astrocytes requires further study.
To delineate the regulation of oligodendrocyte differ-
entiation by miRNAs, oligodendrocyte progenitor cells
(oPCs) and oligodendrocyte lineage cells (olCs) were
isolated based on their differential expression of cell sur-
face markers by fluorescence-activated cell sorting72. There
are dynamic changes in the expression of 43 miRNAs
during the transition from A2B5-positive oPCs to pre-
myelinating olCs. Combined expression profiling of
miRNA and mRNA revealed that miR-9 is downregulated
during oligodendrocyte differentiation, which inversely
correlates with the expression of its predicted targets,
particularly peripheral myelin protein 22 (ReF. 72). Recent
studies have identified specific miRNAs involved in the
differentiation of oPCs into oligodendrocytes (see Note
added in proof).
other small regulatory RNAs in neurogenesis
Besides miRNAs, numerous piRNAs and esiRNAs have
been identified in genomes73–75. piRNAs in particular
have been implicated in controlling the mobilization of
transposable elements in mice and D. melanogaster 76.
Although they were initially discovered only in repro-
ductive systems, mounting evidence suggests that
piRNAs and esiRNAs are present in both germline and
somatic tissues73–75. In mammals, nearly 15% of piRNAs
have been mapped to long interspersed nuclear (L1)
elements (also known as lINE-1 elements). l1 elements
© 2010 Macmillan Publishers Limited. All rights reserved
REVIEWS
Small regulatory RNAs
?
?
?
L1 RNA
L1 gene
Retrotransposition
L1 gene Neuronal gene
Altered neuronal
gene expression
Neuronal
diversity
Figure 3 | A model of neuronal diversity generated by
L1 retrotransposition. During Nature Reviews
neuronal | Neuroscience
differentiation,
long interspersed nuclear (L1) element transcription is
activated. This allows subsequent retrotransposition of L1
elements into neuronal genes, which could alter the
expression of adjacent neuronal genes and contribute to
neuronal diversity. L1 RNAs could be processed into small
regulatory RNAs, which could potentially regulate L1
transcription and retrotransposition.
are abundant retrotransposons that make up ~17% of the
human genome. most l1 elements are retrotransposition
defective; nonetheless, there are approximately 100
full-length l1 elements that are potentially capable of
retrotransposition in the diploid genome. Intriguingly,
recent data indicate that l1 element retrotransposition
occurs in rodent and human NPCs, suggesting that
l1 elements can have an effect on the expression of
neuronal genes and therefore influence neuronal cell
fate and contribute to neuronal diversity 77–80 (FIG. 3). In
addition to the piRNAs that have been mapped to l1
elements, the bidirectional transcripts derived from the
l1 5′-UTR can also be processed to siRNAs that sup-
press retrotransposition81. It is unclear whether these
piRNAs or l1-derived siRNAs can modulate l1 element
retrotransposition in adult NSCs and thereby modulate
neurogenesis.
PASRs are thought to be involved in the regulation
of gene expression82,83. Although the exact biological
roles of these small RNAs remain to be determined, the
involvement of small RNA-mediated transcriptional
regulation in neurogenesis has been established. In this
case, a nuclear double-stranded small RNA contain-
ing the RE1 sequence that can be recognized by REST
was found to be expressed in NSCs. This small RNA
induced the neuronal differentiation of NSCs by acti-
vating the expression of RE1-containing genes84. It can
switch the action of REST from a repressor to an acti-
vator, possibly through chromatin modification, and
its absence blocks neuronal differentiation, suggesting
that it is necessary and sufficient to direct NSCs to the
neuronal lineage84.
VolUmE 11 | mAy 2010 | 335
REVIEWS

Table 1 | MicroRNAs (miRNAs) involved in neurogenesis and their mRNA targets

mirNA Function in neurogenesis cells and tissues Targets refs
let-7 Promotes neuronal lineage commitment NSC LIN28 (mouse) 87
Promotes neural tube closure LIN41 (mouse) 96
miR-7 Promotes neuronal lineage commitment Photoreceptor cell YAN (Drosophila melanogaster) 52
miR-9 Inhibits proliferation NSC and NPC Her5 and Her9 (zebrafish) 44
Promotes neuronal lineage commitment ES cell STAT3 (mouse) 16
Inhibits glial lineage commitment Developing telencephalon FOXG1 (mouse) 45
TLX (mouse) 47
BAF53A (mouse) 71
FGF8 and FGFR1 (mouse) 44
PMP22 (rat) 72
miR-9a Inhibits neuronal fate Epithelial cell Senseless (D. melanogaster) 43
miR-17 Inhibits neural differentiation SH-SY5Y cell Unknown 97
miR-23 Promotes glial lineage commitment ES cell and P19 cell Unknown 10
Promotes neuronal differentiation NT2 cell HES1 (human) 98
miR-124 Inhibits NSC and NPC proliferation Chicken neural tube REST–SCP1 (chicken and mouse) 40
Promotes neuronal lineage commitment P19 cell PTBP1 (mouse) 39
NSC and NPC SOX9 (mouse) 37
BAF53A (mouse) 41
71
miR-125(b) Inhibits NSC and NPC proliferation Medulloblastoma cell and GCP GLI1, SMO and LIN28 (mouse and human) 50
Promotes neuronal lineage commitment SH-SY5Y cell Multiple targets (human) 99
miR-128 Inhibits NSC and NPC proliferation Glioblastoma BMI1 (human) 70
miR-132 Regulates homeostasis Neuron MECP2 (rat) 65
miR-200 Promotes neuronal differentiation Olfactory progenitor cells Notch and TGFβ signalling pathways and FOXG1 100
Maintains olfactory progenitor cells (zebrafish and mouse)
miR-279 Promotes neuronal differentiation Sensory organ precursor Nerfin 1 (D. melanogaster) 101
miR-324-5p Inhibits NSC and NPC proliferation Medulloblastoma cell and GCP GLI1 and SMO (mouse and human) 50
miR-326 Inhibits NSC and NPC proliferation Medulloblastoma cell and GCP GLI1 and SMO (mouse and human) 50
ES cell, embryonic stem cell; FGF8, fibroblast growth factor 8; FGFR1, fibroblast growth factor receptor 1; FOXG1, forkhead box protein G1; GCP, granule cell
progenitor; MECP2, methyl-CpG-binding protein 2; NPC, neural progenitor cell; NSC, neural stem cell; PMP22, peripheral myelin protein 22; PTBP1, polypyrimidine
tract-binding protein 1; REST, RE1-silencing transcription factor; SCP1, small carboxy-terminal domain phosphatase 1; SMO, Smoothened; STAT3, signal
transducer and activator of transcription 3; TGFβ, transforming growth factor-β.

Summary and perspectives with the emergence of powerful deep-sequencing

The discovery of different small regulatory RNAs reveals
a new layer of complexity in the regulation of gene expres-
sion28,85,86, and it is becoming increasingly clear that small
regulatory RNAs are bona fide regulators of neurogenesis.
Several principles have emerged from these studies. First,
small regulatory RNAs usually function as a fine-tuning
mechanism, not as an on–off switch, to ensure the proper
progression through each stage of neurogenesis (from NSC
self-renewal, to lineage specification following differentia-
tion, to the integration of new neurons into existing neural
circuits) and to coordinate their action with other biologi-
cal regulators such as transcription factors, signalling mol-
ecules and epigenetic effectors. Second, several miRNAs
can regulate one mRNA, and a single miRNA is capable of
regulating many mRNA targets, which could be different
components within the same pathway (TABLe 1). Finally, sev-
eral feedback regulatory loops exist between miRNAs and
their targets, which could ensure the precision of miRNA-
mediated gene regulation. This regulatory circuit involves
not only the transcription factors, signalling cascades and
epigenetic factors discussed above, but also components
or regulators of the miRNA pathway itself, such as the
feedback loop between let-7 and lIN28 (ReF. 87).
technologies, there is little doubt that discoveries to
date represent the ‘tip of the iceberg’, with many more
small regulatory RNAs involved in regulating neuro-
genesis awaiting identification. Eventually, the interplay
between these small RNAs and different gene regula-
tory mechanisms will be systematically unravelled.
These discoveries should paint a more complete pic-
ture of the intricate and complex web of regulatory net-
works through which small regulatory RNAs regulate
neurogenesis.
Note added in proof
Two recent studies have shown that the miRNA path-
way acts as a ‘micro-brake’ on proliferating oPCs and
promotes the switch of oPCs into post-mitotic oligo-
dendrocytes102,103. Dicer-knockout mice display severe
defects in CNS myelination. miR-219 and miR-338 were
found to promote differentiation into oligodendrocytes
by repressing several mRNA targets, including those
encoding platelet-derived growth factor, SoX6 and
HES5. These studies provide compelling evidence that
miRNAs are essential for regulating oligodendrocyte
differentiation and myelination.

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Acknowledgements
We thank C. Strauss for critical reading of the manuscript. We
apologize to those whose works are not cited here owing
space limitations. The work in our laboratory was supported
in part by the grants from the National Institutes of Health
and International Rett Syndrome Foundation. P.J. is a recipi-
ent of the Beckman Young Investigator Award and the Basil
O’Connor Scholar Research Award, as well as an Alfred P.
Sloan Research Fellowship in Neuroscience. X.L. is supported
by a FRAXA Fellowship.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
OMIM: http://www.ncbi.nlm.nih.gov/omim
Rett’s syndrome
UniProtKB: http://www.uniprot.org
MECP2 | PTBP1 | REST | SCP1 | STAT3 | TLX | TRIM32
FuRTHER INFoRMATIoN
Peng Jin’s homepage: http://genetics.emory.edu/labs/jinlab
ALL LiNks Are AcTive iN The oNLiNe pdF
www.nature.com/reviews/neuro