Small non-coding RNAs, including microRNAs (miRNAs),
endogenous small interfering RNAs (endo-siRNAs) and Piwi-
interacting RNAs (piRNAs), play essential roles in mammalian
development. The function and timing of expression of these
three classes of small RNAs differ greatly. piRNAs are expressed
and play a crucial role during male gametogenesis, whereas
endo-siRNAs are essential for oocyte meiosis. By contrast,
miRNAs are ubiquitously expressed in somatic tissues and
function throughout post-implantation development.
Surprisingly, however, miRNAs are non-essential during pre-
implantation embryonic development and their function is
suppressed during oocyte meiosis. Here, we review the roles of
small non-coding RNAs during the early stages of mammalian
development, from gamete maturation through to
Key words: siRNA, MicroRNA, Stem cells, Oocyte, Embryonic
The processes of growth and differentiation are kept in balance
during the development of multicellular organisms. Post-
transcriptional control of gene expression plays a key role in this
balance by coordinating the expression of selected genes at specific
times and places. The role of post-transcriptional regulation is
particularly apparent in early mammalian development, from
maturation of the germ line to initiation of gastrulation, when
controls on mRNA localization, stability and translation are the
fundamental means of gene regulation. Indeed, from the fully
grown oocyte stage until zygotic genome activation (ZGA), the
genome is transcriptionally silent (Abe et al., 2010). Therefore, all
mRNA regulation must occur post-transcriptionally. Following
ZGA, the embryo establishes populations of early stem cells (SCs)
within the inner cell mass (ICM, the collection of cells that
eventually will form the fetus) that begin to proliferate rapidly,
ensuring that stocks of unspecialized cells are established for future
differentiation into the three germ layers. This rapid growth and the
subsequent switch from unspecialized cells into specific cell types
is a highly regulated process involving much post-transcriptional
regulation (Lu et al., 2009). Similar regulation also occurs in the
extra-embryonic tissues. This review will focus on one group of
post-transcriptional regulators, the small non-coding RNAs. These
RNAs range in size from 18 to 32 nucleotides (nt) in length and
have emerged in the past decades as major players in post-
transcriptional regulation across many, if not most, multicellular
Classes and biogenesis of mammalian small RNAs
Three major classes of functional small non-coding RNAs have
been found in mammals: microRNAs (miRNAs), endogenous
small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs
(piRNAs) (Babiarz and Blelloch, 2009; Kim et al., 2009; Thomson
and Lin, 2009). These classes differ in their biogenesis, i.e. their
maturation from transcribed forms to the active form of the RNA
miRNAs can be divided into two subclasses: canonical and non-
canonical miRNAs. Canonical miRNAs are initially transcribed as
long RNAs that contain hairpins (Fig. 1A). The 60-75 nt hairpins
are recognized by the RNA-binding protein Dgcr8 (DiGeorge
syndrome critical region 8), which directs the RNase III enzyme
Drosha to cleave the base of the hairpin (Denli et al., 2004;
Gregory et al., 2004; Han et al., 2004; Han et al., 2006; Landthaler
et al., 2004; Lee et al., 2003). Following cleavage by the Drosha-
Dgcr8 complex, also called the microprocessor, the released
hairpin is transported to the cytoplasm, where Dicer, another
RNase III enzyme, then cleaves it into a single short 18-25 nt
dsRNA (Bernstein et al., 2001; Hutvagner et al., 2001; Ketting et
al., 2001; Knight and Bass, 2001). Non-canonical miRNAs bypass
processing by the microprocessor by using other endonucleases or
by direct transcription of a short hairpin. The resulting pre-
miRNAs are then exported from the nucleus and cleaved once by
Dicer (Babiarz et al., 2008; Okamura et al., 2007; Ruby et al.,
By contrast, siRNAs are derived from long dsRNAs (Fig. 1B) in
the form of either sense or antisense RNA pairs or as long hairpins,
which are then directly processed by Dicer consecutively along the
dsRNA to produce multiple siRNAs (Chung et al., 2008; Czech et
al., 2008; Ghildiyal et al., 2008; Kawamura et al., 2008; Okamura
et al., 2008a; Okamura et al., 2008b). Therefore, canonical
miRNAs, non-canonical miRNAs and endo-siRNAs all involve
Dicer processing and are ~21 nt in length. Furthermore, in all three
cases, one strand of the Dicer product associates with an Argonaute
protein (Ago 1-4 in mammals; also known as Eif2c1-4) to form the
active RISC (RNA-induced silencing complex, Fig. 1D)
(Filipowicz, 2005). These ribonucleoprotein complexes are able to
bind to and control the levels and translation of their target mRNAs:
if the match between the small RNA and its target is perfect, the
target is cleaved; if not, the mRNA is destabilized through as yet
unresolved mechanisms (Doench et al., 2003; Fabian et al., 2010;
Zeng et al., 2003).
The piRNAs differ from the miRNAs and endo-siRNAs in that
they do not require Dicer for their processing (Houwing et al.,
2007; Vagin et al., 2006). Indeed, how piRNAs are produced and
their mechanism of action remains poorly characterized (for a
review, see Klattenhoff and Theurkauf, 2008). piRNAs are 25-32
nt in length, and are expressed predominantly in the germline in
mammals (Aravin et al., 2006; Grivna et al., 2006; Watanabe et al.,
2006). They are defined by their interaction with the Piwi proteins,
a distinct family of Argonaute proteins (including Miwi, Miwi2
Development 138, 1653-1661 (2011) doi:10.1242/dev.056234
© 2011. Published by The Company of Biologists Ltd
Small RNAs in early mammalian development: from gametes
Nayoung Suh and Robert Blelloch*
The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research,
Center for Reproductive Sciences, and Department of Urology, University of
California San Francisco, San Francisco, CA 94143, USA.
*Author for correspondence (Blellochr@stemcell.ucsf.edu)
and Mili in mouse; also known as Piwil1, Piwil4 and Piwil2,
respectively). piRNAs are generated from long single-stranded
RNA precursors that are often encoded by complex and repetitive
intergenic sequences. One proposed model for their biogenesis is
the ‘ping-pong mechanism’ (Fig. 1C) (Aravin et al., 2007;
Brennecke et al., 2007; Gunawardane et al., 2007). In this model,
the Argonaute protein Mili cleaves the primary piRNA to define
the 5? end of piRNAs, which is subsequently recognized by Miwi2.
Miwi2 then cleaves the other strand of the precursor, thereby
generating a 5? end of the piRNA that can then bind to Mili, thus
forming a positive amplification loop. Many of the details of this
model remain to be uncovered. Furthermore, the ping-pong model
is likely to explain the biogenesis of only a subset of mammalian
piRNAs, those that are derived from repetitive sequences, such as
transposons, and that are associated with Miwi2 and Mili in the
early stages of spermatogenesis. The mechanism of biogenesis of
piRNAs derived from complex intergenic sequences, associated
with Miwi and Mili in the later stages of spermatogenesis, is
Small RNAs in gametes
Most animals, including vertebrates, reproduce sexually and have
the ability to form gametes. The two types of gametes, the egg and
the sperm, arise from immature germ cells, undergo extensive
differentiation and ultimately fuse to create their progeny. One
fascinating aspect of this process is that, upon fertilization, the
highly specialized sperm and egg unite to produce the zygote,
which is totipotent, having the potential to produce all the cells of
the body. This switch from a singular function to a totipotent cell
involves massive molecular rewiring (Hemberger et al., 2009).
Based on recent findings, small RNAs are likely to play a very
important role during this transition.
Small RNAs in spermatogenesis
Spermatogenesis is a highly regulated process, during which
diploid spermatogonia differentiate into haploid spermatozoa
within the seminiferous epithelium of the testis. During the course
of differentiation into sperm, numerous mRNAs are regulated
post-transcriptionally (Lee et al., 2009). Recent studies using both
Development 138 (9)
A miRNA pathway
D Effector pathway
B endo-siRNA pathway
C piRNA pathway
Translational repression or
Fig. 1. Biogenesis of microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs).
(A)Canonical miRNAs are processed from long primary miRNAs (pri-miRNAs) into short hairpin precursor miRNAs (pre-miRNAs) by the
microprocessor, a complex consisting of the RNA binding protein Dgcr8 and the RNase III enzyme Drosha. By contrast, non-canonical miRNAs
are transcribed directly as short hairpins (shRNAs) or derive from introns that can refold into shRNAs (mirtrons). (B)Precursors of endo-siRNAs
are derived from long stem-loop structures (inverted repeat), opposing strand transcription (cis endo-siRNAs), or gene-pseudogene pairs (trans
endo-siRNAs). Both miRNAs and endo-siRNAs are then processed by the RNase III enzyme Dicer to produce double-stranded RNAs of ~21
nucleotides. (C)piRNAs are processed from single-stranded RNA precursors that are often encoded by intergenic repetitive elements or
transposons. The mechanisms that drive piRNA biogenesis are not well understood, although a ‘ping-pong mechanism’ has been described
for a subset of piRNAs. In this model, Mili cleaves the primary piRNA, which is subsequently recognized by Miwi2. Miwi2 cleaves the other
strand of the precursor that can then bind to Mili, thus forming a positive amplification loop. (D)Following their processing, miRNAs and
endo-siRNAs are assembled into ribonucleoprotein (RNP) complexes called RNA-induced silencing complexes (RISCs). The key components of
RISCs are proteins of the Argonaute (Ago) family. In mammals, four Ago proteins (Ago 1-4) function in miRNA repression but only Ago2
functions in siRNA repression. The fate of piRNAs is unknown. On the DNA, blue represents the positive strand and red represents the
Development 138 (9)
genetics and miRNA profiling on different populations of
spermatogenetic cells have identified an important role for
miRNAs during spermatogenesis (Hayashi et al., 2008; Tang et al.,
2007; Yu et al., 2005; Bouhallier et al., 2010; Yan et al., 2009).
Deletion of Dicer, for example, results in a loss of sperm (Hayashi
et al., 2008; Maatouk et al., 2008). This could be attributed to the
loss of either miRNAs or endo-siRNAs. However, the deletion of
Argonaute 2 (Ago2), a protein that is essential for cleavage of
mRNA targets by endo-siRNAs, has no obvious testis phenotype,
suggesting that the Dicer phenotype is predominantly an miRNA-
based phenotype (Hayashi et al., 2008). Roles for individual
miRNAs during spermatogenesis have also been described. For
example, Mir122a regulates Tnp2, a testis-specific gene involved
in chromatin remodeling during spermatogenesis (Yu et al., 2005).
In addition, Mir34c is highly expressed in germ cells and its
overexpression enhances spermatogenesis (Bouhallier et al.,
2010). Furthermore, Dead end 1 (Dnd1), an RNA-binding protein
that is implicated in prevention of miRNA access to cell cycle-
related target mRNAs, is essential for fetal male germ cell
development (Cook et al., 2010; Kedde et al., 2007). Recently, it
has been also shown that Mir18, a member of the Oncomir-1
cluster of miRNAs, directly targets heat shock factor 2 (Hsf2), a
transcription factor involved in spermatogenesis (Bjork et al.,
Genetic studies imply that, like miRNAs, piRNAs are also
essential for spermatogenesis. In mouse, piRNAs have been
separated into two classes based on the timing of their expression,
their repetitive versus nonrepetitive nature, and the Piwi proteins
with which they are associated (Aravin et al., 2006; Girard et al.,
2006; Grivna et al., 2006; Lau et al., 2006; Watanabe et al., 2006).
The first class is highly repetitive and is expressed before meiotic
pachytene. This class of piRNAs interacts with Mili and Miwi2
(Aravin et al., 2008; Aravin et al., 2007). The second class of
piRNAs is nonrepetitive, becomes abundant during the pachytene
stage and is associated with Mili and Miwi proteins (Aravin et al.,
2008; Aravin et al., 2007; Girard et al., 2006). Consistent with their
timing of expression, deletion of Mili and Miwi2 results in early
arrest in meiosis I (at the primary spermatocyte stage), whereas
deletion of Miwi results in arrest following meiosis II (the round
spermatid stage) (Carmell et al., 2007; Deng and Lin, 2002;
Kuramochi-Miyagawa et al., 2004).
The repetitive piRNAs are associated with the repression of
transposable elements during spermatogenesis (Malone and
Hannon, 2009) but exactly how repression is achieved is unclear.
For example, it is unclear whether this repression occurs
transcriptionally or post-transcriptionally. There is evidence to
suggest that repetitive piRNAs promote de novo demethylation of
the transposons (Aravin et al., 2008; Kuramochi-Miyagawa et al.,
2008); however the mechanism by which these small RNAs can
direct the DNA methylation machinery is unclear.
Small RNAs in oogenesis
Mammalian oogenesis is distinct from spermatogenesis (Matova
and Cooley, 2001). The oocyte pool is largely fixed by the end of
mouse embryogenesis and oocytes are then induced to mature in
response to cyclic waves of hormones, including follicle
stimulating hormone and luteinizing hormone. In the mouse, each
cycle induces a small pool of oocytes to mature before they are
released into the fallopian tube. During maturation, the oocytes
undergo germinal vesicle breakdown (GVBD), meiosis I and
finally meiosis II, which is only completed following fertilization.
Deep sequencing of small RNAs in mouse oocytes has uncovered
not only miRNAs and piRNAs, but also a large population of endo-
siRNAs (Tam et al., 2008; Watanabe et al., 2006; Watanabe et al.,
An essential role for endo-siRNAs in the mouse oocyte has been
inferred by comparing the knockout phenotypes of Dicer and Dgcr8
mutant mice. Dicer loss in the oocyte results in meiotic arrest with
severe spindle and chromosomal segregation defects (Murchison et
al., 2007; Suh et al., 2010; Tang et al., 2007). Furthermore, thousands
of mRNAs are misregulated. By contrast, Dgcr8 loss has no
phenotype and mRNA levels remain unchanged (Suh et al., 2010).
As Dicer processes both miRNAs and endo-siRNAs, whereas Dgcr8
is essential only for miRNA processing, these findings imply that
endo-siRNAs, and not miRNAs, underlie the meiotic defect of Dicer
knockout oocytes. The loss of Ago2 results in a similar phenotype to
that observed in Dicer knockouts, further supporting the role of
endo-siRNAs in regulating meiosis in oocytes (Kaneda et al., 2009).
The mechanism of action of endo-siRNAs in oocytes is unclear. In
Schizosaccharomyces pombe, endo-siRNAs are crucial for
heterochromatin formation in repeat regions of the genome (Moazed,
2009). However, no such function has been shown convincingly in
mammals. It seems more likely that endo-siRNAs in mammals are
acting post-transcriptionally (Tam et al., 2008).
It was surprising to find that in the absence of Dgcr8 in mouse
oocytes, mRNA levels are unchanged. miRNAs are present in
oocytes, as determined by both deep sequencing and multiplex
quantitative PCR-based profiling (Murchison et al., 2007; Tam et
al., 2008; Tang et al., 2007; Watanabe et al., 2006; Watanabe et al.,
2008). For example, Let-7, Mir22, Mir16-1 and Mir29 are all highly
expressed in the oocyte. miRNA profiling following Dgcr8 deletion
confirmed that these and all other miRNAs tested were indeed lost
in knockout oocytes (Suh et al., 2010). Furthermore, mRNA
profiling and bioinformatic analyses showed that many targets for
the expressed miRNAs are present in the oocyte. Therefore,
everything is in place for miRNA-based destabilization to occur, but
mRNA levels remain unchanged (Suh et al., 2010). Consistent with
these findings, reporter assays show robust siRNA activity in mature
oocytes, but little to no miRNA function (Ma et al., 2010). Even
with artificial 3? untranslated regions (3? UTRs) carrying multiple
target sites, and the introduction of supraphysiological doses of
miRNAs, little suppression in terms of mRNA stability or
translation was seen (Ma et al., 2010). Together, these surprising
results show that miRNA function is suppressed in fully grown
oocytes even though miRNA biogenesis is unaffected and miRNA
targets are present. Although the mechanism of suppression is
unknown, one hint comes from the finding that P-bodies (processing
bodies, see Box 1), in which miRNA destabilization normally
occurs (Parker and Sheth, 2007), are lost in maturing oocytes and
only reform at the blastocyst stage (Flemr et al., 2010; Swetloff et
al., 2009). Whether the loss of P-bodies is a primary or secondary
consequence of miRNA functional loss is unclear.
piRNAs are also expressed in mouse oocytes (Watanabe et al.,
2008), but the deletion of the Piwi proteins do not produce an
oocyte phenotype (Carmell et al., 2007; Deng and Lin, 2002;
Kuramochi-Miyagawa et al., 2004). Therefore, it is unclear
whether they play any role during oogenesis.
Small RNAs in early embryogenesis
Small RNA function during pre-implantation development
Zygotic deletion of Dgcr8 or Dicer in mice leads to embryonic
arrest shortly after implantation between embryonic day (E) 6.5 and
E7.5 (Bernstein et al., 2003; Morita et al., 2007; Wang et al., 2007).
However, development to E3.5, the blastocyst stage, occurs
normally. Indeed, maternal and zygotic loss of Dgcr8 leads to no
discernable phenotype in E3.5 embryos (Suh et al., 2010).
Therefore, miRNA function must become essential sometime
between E3.5 and E7.5. Recently, careful characterization of the
zygotic Dicer knockout phenotype suggested that an epiblast forms
and there is an initiation of gastrulation with expression of the early
mesoderm maker brachyury in the posterior epiblast (Spruce et al.,
2010). However, the primitive streak fails to elongate and there is
a loss of expression of the definitive endoderm markers Hex
(Hhex – Mouse Genome Informatics) and Cerl1 (Cer1). A major
caveat of these findings is that it is unclear whether all miRNAs
were lost in the Dicer knockout embryos. Indeed, in situ
hybridization suggested equal levels of miRNAs from the Mir290
cluster in Dicer versus wild-type embryos. Expression of the
Mir290 cluster is initiated with zygotic gene activation (Tang et al.,
2007). Hence, their presence in the zygotic Dicer knockout
blastocyst suggests the perdurance of maternal Dicer rather than
the miRNAs themselves. Because of this caveat, the earliest roles
of miRNAs in mouse development remain unknown. However, the
strong proliferation defects seen in Dicer and Dgcr8 knockout
mouse embryonic stem (ES) cells (Kanellopoulou et al., 2005;
Murchison et al., 2005; Wang et al., 2008; Wang et al., 2007),
suggests that miRNAs are likely to play an important role in the
expansion of the epiblast. Interestingly, the loss of maternal
miRNAs alone results in a decrease in the average number of
progeny produced following fertilization by wild-type males (Suh
et al., 2010). Together with a lack of pre-implantation phenotype,
this finding suggests a role for maternally contributed miRNAs
during the peri- and/or post-implantation stages of development,
multiple days after fertilization.
The lack of phenotypes following miRNA removal in early
mammalian development parallels findings observed in zebrafish:
the loss of both maternal and zygotic miRNAs in zebrafish first
manifests phenotypes relatively late in development (Giraldez et
al., 2005). Indeed, following maternal-zygotic knockdown of Dicer,
zebrafish gastrulate and only begin to manifest clear morphogenetic
defects during organogenesis. Zygotic loss alone allows
organogenesis to proceed normally (Wienholds et al., 2003).
Although the phenotypes are more severe in mouse, with zygotic
deletion of Dicer resulting in arrest prior to gastrulation (Bernstein
et al., 2003; Morita et al., 2007; Wang et al., 2007), these studies
show how early development can proceed normally in the absence
of miRNAs. A striking difference between the mouse and zebrafish
phenotypes is the lack of Dicer requirement in the maturing fish
oocyte. It will be important to determine whether this difference is
secondary to a lack of endo-siRNA function in the zebrafish egg.
An interesting possibility is that the loss of miRNA function
during pre-implantation development is a key component of the
dramatic reprogramming that occurs during this stage (Hemberger
et al., 2009). Indeed, recent profiling experiments suggest a shift
from a dominant presence of endo-siRNAs and piRNAs in the
oocyte to an miRNA majority as pre-implantation development
proceeds (Fig. 2) (Ohnishi et al., 2010). The transition occurs with
zygotic gene activation, which follows resetting of the epigenome.
This resetting occurs in a remarkably short window of time,
between fertilization and E2.5 in mice and slightly later in humans
(de Vries et al., 2008). Therefore, it is tempting to speculate that the
suppression of miRNA function enables this massive epigenomic
reprogramming in preparation for new gene expression.
miRNAs and siRNAs use distinct silencing
A surprising conclusion arising from the comparison of the Dicer
and Dgcr8 knockout phenotypes in mouse oocytes along with the
miRNA versus siRNA reporter assays is that the effector pathways
Development 138 (9)
Box 1. P-bodies
P-bodies (processing bodies) are discrete cytoplasmic foci that
contain proteins involved in mRNA degradation. They are found in
eukaryotic cells as well as in somatic cells in plants and yeast
(Bashkirov et al., 1997; Cougot et al., 2004; Sheth and Parker,
2003; Xu et al., 2006). P-body proteins are required for diverse
post-transcriptional processes: mRNA decay, translational repression,
nonsense-mediated mRNA decay and RNAi-mediated repression. In
particular, all four Ago proteins (Eystathioy et al., 2003; Liu et al.,
2005; Sen and Blau, 2005), GW182 (Eystathioy et al., 2003) and
two RNA helicases RCK/p54 (Chu and Rana, 2006) and MOV10
(Meister et al., 2005) have been found in P-bodies, suggesting that
miRNA suppression is localized to the P-body. However, it has been
proposed that P-body formation is a consequence rather than the
cause of miRNA-mediated gene silencing (Eulalio et al., 2007), as
when siRNA or miRNA silencing pathways are blocked, P-bodies are
not formed (Eulalio et al., 2007). Interestingly, it has been shown
that P-body foci are dynamic, increasing or decreasing in size and
number depending on the global state of RNA turnover in yeast
(Sheth and Parker, 2003). Indeed, recent studies in mouse oocytes
and of early mouse embryonic development demonstrated that
these foci are regulated developmentally (Flemr et al., 2010;
Swetloff et al., 2009) In particular, it was shown that P-bodies are
lost in fully grown oocytes and during
development (Flemr et al., 2010; Swetloff et al., 2009). It will thus
be interesting to know how and when P-bodies are lost or re-stored
during early development.
Fig. 2. Small RNA functions during germ cell and
early embryonic development. Piwi-interacting
RNAs (piRNAs) and microRNAs (miRNAs) are essential
in the developing male germline, whereas endogenous
small interfering RNAs (endo-siRNAs) play their most
crucial role in oocyte maturation. There is a transition
from endo-siRNAs or piRNAs to miRNAs during pre-
implantation development. PGCs, primordial germ
Development 138 (9)
for miRNA and endo-siRNA activity are separable (Fig. 1). That
is, although miRNA-based destabilization (by translational
inhibition) function is lost, siRNA cleavage function remains. In
Drosophila, Ago1, together with Loquacious, is the primary driver
of miRNA function, whereas Ago2, together with its partner R2D2,
is primarily responsible for siRNA function (Ghildiyal and Zamore,
2009). Mammals, by contrast, have four Argonaute proteins: Ago1-
4 (Siomi and Siomi, 2009). Ago2 is the only mammalian
Argonaute with slicer activity and hence the only Argonaute
protein able to perform siRNA cleavage. Indeed, deletion of Ago2
in oocytes produces a phenotype very similar to that of Dicer
(Kaneda et al., 2009). However, the Argonautes are highly
redundant in terms of miRNA activity. Although deletion of all four
Argonautes in ES cells results in complete loss of miRNA function,
re-introduction of any one of the four Argonautes can fully rescue
miRNA activity (Su et al., 2009). Therefore, unlike the situation in
Drosophila, siRNA and miRNA function in mammalian cells is
unlikely to be compartmentalized at the level of the Argonaute
proteins. Instead, proteins associated with or mechanisms
downstream of Argonautes must be influencing the specific loss of
miRNA function. It is unclear what these mechanisms might be. P-
bodies are lost concurrently with miRNA functional loss (Flemr
et al., 2010; Swetloff et al., 2009); therefore, studies of the
components of the P-body might provide hints.
miRNA versus endo-siRNA activity in other tissues
Microprocessor components have also been knocked out in other
tissues and the resulting phenotypes compared with corresponding
Dicer phenotypes. In particular, Dgcr8 has been knocked out in
skin and cardiomyocytes (Rao et al., 2009; Yi et al., 2009), and
Drosha has been knocked out in T cells (Chong et al., 2008). In
these cases, the microprocessor-null phenotypes were very similar
to those of the corresponding Dicer-null phenotypes. An exception
appears to be in the adult brain, where the deletion of Dicer in post-
mitotic neurons produces a more severe phenotype than does
Dgcr8 loss (J. E. Babiarz, R. Hsu, C. Melton, E. M. Ullian and
R.B., unpublished). However, deep sequencing failed to uncover
any evidence of endo-siRNAs in the brain (J. E. Babiarz, R. Hsu,
C. Melton, E. M. Ullian and R.B., unpublished). Instead, many
non-canonical miRNAs were found, suggesting that these small
RNAs might underlie the differences observed. These results
suggest that endo-siRNAs might be specific to oocytes and early
Small RNAs in stem cells
Embryonic stem cells
Three self-renewing cell types can be derived from the late mouse
blastocyst: ES cells, which represent the pluripotent epiblast
lineage; trophoblast stem (TS) cells, which represent the
trophoblast lineage; and extra-embryonic endoderm (XEN) cells,
which represent the primitive endoderm lineage (Fig. 3) (Rossant,
2008). Insights into small RNA regulation of stem cell maintenance
and differentiation have been gained mostly from studies of ES
cells lacking either Dgcr8 or Dicer (Kanellopoulou et al., 2005;
Murchison et al., 2005; Wang et al., 2007). Both Dgcr8- and Dicer-
deficient ES cells exhibit proliferation and differentiation defects.
The proliferation phenotype is associated with accumulation of
cells in the G1 phase of the cell cycle, whereas the differentiation
defect is associated with an inability to silence the self-renewal
machinery (Wang et al., 2007). By adding back individual miRNAs
into Dgcr8 knockout ES cells, a large family of miRNAs, including
members of the Mir290 and Mir302 clusters, were found to rescue
the prolonged G1 phenotype (Wang et al., 2008). These miRNAs
were termed the ESCC miRNAs (ESC cell cycle regulating
miRNAs). Using a similar add-back strategy, the Let-7 (also known
as Mirlet7) miRNA family was shown to rescue the ability to
silence self-renewal (Melton et al., 2010). Specifically, the addition
of Let-7 into Dgcr8 knockout cells led to the loss of expression of
multiple markers of ES cells and blocked the ability of cells to
reform colonies, functionally proving a loss of their self-renewal
capacity. However, Let-7 only silenced self-renewal in the Dgcr8
knockout, not wild-type ES cells, suggesting that miRNAs
normally expressed in ES cells suppress the capacity of Let-7 to
induce differentiation. Indeed, simultaneous introduction of the
ESCC miRNAs into the Dgcr8 knockout ES cells suppressed the
capacity of Let-7 to induce differentiation. Microarray profiling of
mRNAs and bioinformatic analyses showed that these two
antagonizing miRNA families function by having opposing effects
on members of the pluripotency regulatory network, including
Myc, Lin28 and Sall4 along with others (Fig. 4) Taken together,
these findings show that the two miRNA families, the Let-7 and
ESCC miRNAs, play opposing roles in controlling the balance
between ES cell self-renewal and differentiation (Melton et al.,
Endo-siRNAs have also been identified in ES cells (Babiarz et
al., 2008). Interestingly, there is no overlap between the specific
endo-siRNAs expressed in oocytes and those expressed in ES cells,
showing that they are developmentally regulated and likely to have
distinct functions (Babiarz et al., 2008). However, the role of ES
cell endo-siRNAs is unclear. A hint comes once again from the
deletion of members of the biogenesis and effector pathways.
Deletions of Dicer or of all four Argonaute genes have more severe
phenotypes than deletion of Dgcr8 (Kanellopoulou et al., 2005;
Murchison et al., 2005; Su et al., 2009; Wang et al., 2007). In
particular pan-Argonaute-deficient ES cells undergo apoptosis.
Dicer-null ES cells can survive, but during their derivation they go
through a phase of arrest, with escaper cells eventually
proliferating. By contrast, Dgcr8-null ES cells survive and show
Stem cells Gene deleted
Kanellopoulou et al., 2005;
Murchison et al., 2005; Wang et al., 2007
Spruce et al., 2010
Spruce et al., 2010
Fig. 3. Small RNA functions in stem cells derived from the mouse blastocyst. By the time of implantation, the mammalian blastocyst has
developed three different cell lineages: trophectoderm, primitive endoderm and epiblast (shown on left). Three distinct self-renewing cell lines can
be derived from these lineages: trophoblast stem (TS) cells, extra-embryonic endoderm (XEN) cells and embryonic stem (ES) cells. Studies of stem
cell lines lacking either Dgcr8 or Dicer provide insights into small RNA-mediated regulation of stem cell maintenance, proliferation and
no evidence of arrest during derivation. These differences in
phenotype suggest a likely role for endo-siRNAs in ES cells. By
contrast, a role for piRNAs in the embryonic stem cells is doubtful
as their levels are greatly diminished relative to those observed in
the germline (Ohnishi et al., 2010), and the knockout of the Piwi
genes in mice show no embryonic phenotypes (Carmell et al.,
2007; Deng and Lin, 2002; Kuramochi-Miyagawa et al., 2004).
Trophoblast stem cells
During the course of early embryogenesis, the separation of the
trophectoderm and ICM lineages is the first known definitive
differentiation event. Fundamental insights into the molecular
control of trophectoderm determination and differentiation have
been made in the past decade (Chen et al., 2010; Douglas et al.,
2009; Ralston and Rossant, 2005). A number of transcription factors
and signaling pathways have been identified as crucial players. Even
before the formation of the blastocyst, two transcription factors,
Oct4 (Pou5f1 – Mouse Genome Informatics) and Cdx2, act
antagonistically to establish the boundaries between the
trophectoderm and inner cell mass (Niwa et al., 2005). Oct4 is
expressed throughout pre-implantation development whereas Cdx2
is expressed starting around the time of morula compaction
(Dietrich and Hiiragi, 2007). At first, Cdx2 is co-expressed with
Oct4 in the cells of the morula, but its expression is then seen to
segregate to the future trophectoderm cells. Cdx2 protein binds
directly to Oct4 protein resulting in reciprocal inhibition of their
target genes (Niwa et al., 2005). Dominance of one protein over the
other eventually leads to the choice between the two lineages: inner
cell mass versus trophectoderm. A second transcription factor,
Eomes, which acts independently of Cdx2, is also essential early in
trophectoderm determination and maintenance, but little more is
known about its function (Russ et al., 2000). Following the
formation of the blastocyst, the inner cell mass produces fibroblast
growth factor 4 (Fgf4), which signals through the FGF receptor 2
(Fgfr2), to promote proliferation of the overlying polar
trophectoderm, thereby allowing the polar trophectoderm to provide
an ongoing source of trophoblasts both to the mural trophectoderm
and future placenta (Nichols et al., 1998; Tanaka et al., 1998).
In contrast to the ES cell studies, little is known about the role
of small RNAs in trophectoderm specification. miRNA expression
profiling of ES cells, ES cell-derived TS cells and progressive
stages of pre-implantation embryos, has identified a subset of
miRNAs that might play a role in trophectoderm specification:
Mir297, Mir96, Mir21, Mir29c, Let-7, Mir214, Mir125a, and
Mir424 (Viswanathan et al., 2009) Moreover, studies analyzing the
phenotype of Dicer-deficient embryos during early post-
implantation stages have shown an essential role for small RNAs
in trophectoderm development (Spruce et al., 2010). In particular,
expression of the TS cell markers Eomes, Cdx2 and Esrrb was
greatly downregulated in Dicer knockout embryos. Similar to ES
cells, Dicer knockout TS cells show proliferation defects, with an
accumulation of cells in G1. This finding is consistent with the fact
that the Mir290 cluster is also highly expressed in TS cells
(Houbaviy et al., 2005). Indeed, as seen in ES cells, a number of
inhibitors of the cyclin E/Cdk2 pathways were upregulated in TS
cells following miRNA loss (Spruce et al., 2010). Dicer removal in
XEN cells also influenced self-renewal and proliferation, but
potentially through different pathways. In particular, regulation of
ERK activity appears to be an important player in the phenotype.
Taken together, early experiments in TS and XEN cells suggest
overlap in miRNA roles across the three stem cell populations of
Induced pluripotent stem cells
In 2006, Yamanaka and co-workers showed that somatic cells
could be reprogrammed into induced pluripotent stem (iPS) cells
by retroviral introduction of genes encoding four transcription
factors: Oct3/4 (Pou5F1 – Mouse Genome Informatics), Klf4, Sox2
and Myc (Takahashi and Yamanaka, 2006). With improvements in
the methods, these iPS cells have become increasingly similar to
ES cells both in their self-renewal and differentiation potential (for
a review, see Amabile and Meissner, 2009). The realisation of the
importance and therapeutic potential of iPS cells has opened a new
era in regenerative medicine (Yamanaka, 2009).
A role for miRNAs in iPS cell production has recently been
uncovered. In particular, ESCC miRNAs can promote the de-
differentiation of somatic cells to iPS cells. They can replace Myc
and, based on chromatin immunoprecipitation (ChIP) sequence
data, function downstream of Myc (Judson et al., 2009).
Interestingly, ESCC miRNAs also upregulate Myc, albeit indirectly
(Melton et al., 2010). Therefore, ESCC miRNAs and Myc form a
self-reinforcing loop that maintains ES cell self-renewal and even
promotes de-differentiation. Furthermore, inhibition of Let-7
function, either through overexpression of Lin28, which blocks
Let-7 biogenesis, or through antagomirs, which directly target
mature Let-7, is able to promote iPS cell production (Melton et al.,
2010; Yu et al., 2007). This result is consistent with Let-7’s
capacity to suppress Myc and many of the downstream targets of
the pluripotency network of transcription factors. These findings
further emphasize the role of these miRNAs in regulating the
switch between self-renewal and differentiation.
Emerging mechanisms of miRNA regulation
In the past decade, much progress has been made in identifying
miRNAs, understanding miRNA biogenesis and predicting
miRNA targets. Furthermore, it is becoming evident that miRNAs
can exert their effects through single or multiple targets, allowing
them to regulate development, normal physiology, and
pathological processes. However, there remains much to be
learned about miRNA biology. A question of increasing interest is
Development 138 (9)
(e.g. Lin28, Myc)
(e.g. Lin28, Myc)
Fig. 4. The opposing roles of ESCC and Let-7 microRNAs (miRNAs)
in the switch between self-renewal and differentiation. In mouse
ES cells (left panel), ESCC miRNAs (green) and stemness factors are
highly expressed. ESCC miRNAs are regulated by the core ES cell
transcription factors such as Oct4, Sox2, Nanog, Tcf3 and Myc. Upon
differentiation (right panel), the expression of Let-7 miRNAs (red)
increases and helps to repress stemness factors such as Lin28, Myc and
Development 138 (9)
how miRNAs themselves are regulated. However, the answers to
this question are almost as diverse as the miRNAs identified and,
therefore, below we discuss only those mechanisms that regulate
small RNAs with known roles in germ cells and in the gastrulating
embryo [for a broader review on the topic, see Krol et al. (Krol et
Most miRNAs are expressed through an RNA polymerase II
mechanism, and regulation of their expression thus occurs through
the common mechanisms that regulate developmental genes. For
example, the expression of ESCC miRNAs is regulated by the core
ES cell transcription factors Oct4, Sox2, Nanog, Tcf3 and Myc
(Judson et al., 2009; Marson et al., 2008). Furthermore, and as
observed for other pluripotency genes, the expression of ESCC
miRNAs is regulated by epigenetic modifications, which include
activating and suppressive histone marks (Judson et al., 2009;
Marson et al., 2008).
miRNAs are also regulated post-transcriptionally at the level of
their biogenesis and stability. For example, Lin28, an RNA binding
protein, regulates the biogenesis of the Let-7 family of miRNAs
(Heo et al., 2008; Newman et al., 2008; Piskounova et al., 2008;
Rybak et al., 2008; Viswanathan et al., 2008). Lin28 inhibits Dicer
cleavage and destabilizes the pre-miRNA form of Let-7 (Heo et al.,
2008; Rybak et al., 2008). The latter is achieved through Lin28
interacting with the loop region of Let-7 and directing a terminal
uridyl transferase (TUTase) to polyuridylate the 3? end of the pre-
Let-7 miRNA, leading to pre-Let-7 degradation (Hagan et al.,
2009; Heo et al., 2009).
miRNA function can also be regulated by interactions with their
downstream targets. For example, the RNA binding protein dead
end (DND1) blocks the ability of MIR221 and MIR372 to
suppress p27 (CDKN1B – Human Gene Nomenclature Database)
and LATS2, respectively, in human cells (Kedde et al., 2007).
DND1 is essential for germline development in zebrafish and
mouse, where it probably plays a similar role to that seen in the
human cell lines (Kedde et al., 2007; Slanchev et al., 2009).
Another germline RNA binding protein, Dazl (deleted in
azoospermia-like), has also been shown to have a potential role in
regulating the interactions between miRNAs and mRNA targets in
zebrafish (Takeda et al., 2009). In particular, Dazl appears to
inhibit miRNA induced de-adenylation by binding to the 3?UTRs
of specific miRNA targets.
Over the past decade we have learned a great deal about the nature
of small RNAs, their biogenesis and their expression across tissues.
However, we are just beginning to appreciate how these RNAs fit
in to the overall molecular network of the cell. Indeed, we have a
very poor understanding of how the many mRNA targets of
individual miRNAs work together to influence a specific cellular
outcome. To date, the field has mostly limited the analysis of
miRNA targeting in biological processes to a small number of
targets, probably not reflecting the true nature of miRNA function.
Future studies involving more systematic approaches should teach
us about how miRNAs are involved in the control of development,
tissue physiology and disease.
Furthermore, there remains much to be learned about the
transcriptional, epigenetic and post-transcriptional regulation of
miRNAs. miRNA targets are likely to be heavily influenced by
cellular context. The influence of cellular context can, in part, be
explained by differences in expression of those targets. However,
a large part will probably be attributable to the regulation of
miRNA biogenesis and the interaction of miRNAs with their target
mRNAs. The most extreme example is the global suppression of
miRNAs in the oocyte. The reason and mechanistic details of this
global suppression remain unclear. How the global or focused
regulation of miRNA maturation and targeting fit in with the timing
and regulation of cell fate decisions and their conservation across
species should be a fruitful area of research. Finally, it will be
important to study the relationship of these regulatory mechanisms
to the biology and treatment of disease, including within the field
of cellular reprogramming.
We thank Matthew Cook, Raga Krishnakumar and Ronald Parchem for helpful
comments on the manuscript. We apologize to those colleagues whose work
could not be cited directly owing to space constraint. R.B. is funded by the
National Institutes of Health, California Institute of Regenerative Medicine
(CIRM), the American Health Assistance Foundation (formerly Stem Cell
Research Foundation) and the Pew Charitable Trust. Further funds to R.B. came
from the NICHD/NIH through a pilot project funded within a cooperative
agreement as part of the Specialized Cooperative Centers Program in
Reproduction and Infertility Research. Deposited in PMC for release after 12
Competing interests statement
The authors declare no competing financial interests.
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