Integrative and Comparative Biology
Integrative and Comparative Biology, volume 54, number 4, pp. 700–713
doi:10.1093/icb/icu084
Society for Integrative and Comparative Biology
SYMPOSIUM
Piwi and Potency: PIWI Proteins in Animal Stem Cells and
Regeneration
Josien C. van Wolfswinkel1
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA
From the symposium ‘‘The Cell’s View of Animal Body Plan Evolution’’ presented at the annual meeting of the Society
for Integrative and Comparative Biology, January 3–7, 2014 at Austin, Texas.
1
E-mail: [email protected]
Synopsis PIWI proteins are well known for their roles in the animal germline. They are essential for germline development and maintenance, and together with their binding partners, the piRNAs, they mediate transposon silencing. More
recently, PIWI proteins have also been identified in somatic stem cells in diverse animals. The expression of PIWI
proteins in these cells could be related to the ability of such cells to contribute to the germline. However, evaluation
of stem cell systems across many different animal phyla suggests that PIWI proteins have an ancestral role in somatic
stem cells, irrespective of their contribution to the germ cell lineage. Moreover, the data currently available reveal a
possible correlation between the differentiation potential of a cell and its PIWI levels.
Germ cells have special status as mediators of the
long-term survival of multicellular species, and exhibit several specialized mechanisms for control of
gene expression and for protection of their
genome. In animals, one of these protective mechanisms is established by PIWI proteins (Khurana and
Theurkauf 2010), and loss of PIWI protein expression in animals as diverse as flies and zebrafish results in sterility (Lin and Spradling 1997; Houwing
et al. 2007).
Over the last decade, PIWI proteins have also been
detected in a growing range of somatic stem cells
such as planarian neoblasts and cnidarian interstitial
cells (Reddien et al. 2005; Plickert et al. 2012). These
pluripotent cells retain the potential to generate
germline cells, and therefore the presence of PIWI
proteins in these cells could be related to that potential (Juliano et al. 2011), or, alternatively, PIWI proteins could have an ancestral function in somatic
stem cells, independent of their involvement in the
germline.
Somatic stem cell systems vary considerably in
their spatial distribution, their proliferation rate,
and most importantly, in their differentiation potential. This review will focus mostly on pluripotent and
multipotent adult stem cells. Pluripotent stem cells
are able to make cells of all germ layers, and this
term is here used for stem cell systems that also
contribute to the germline. Stem cell systems that
make many cell types, but do not contribute to the
germline will be referred to as multipotent. Other
lineage-restricted types of stem cells include tissuespecific stem cells, such as epithelial stem cells and
intestinal stem cells in vertebrates, which generate
several different cell types but only within the
tissue in which they reside, and unipotent stem
cells, which make only a single cell type.
Different species employ different strategies with
regard to their somatic stem cells to maintain
tissue homeostasis. The nematode Caenorhabditis elegans, for example, has no adult somatic stem cells,
whereas vertebrates typically have many different
types (Tanaka and Reddien 2011). In this review,
the presence of PIWI proteins in various somatic
stem cell systems is evaluated, and possible roles of
PIWI proteins in stem cells, and more generally, in
potency, will be explored.
A brief introduction to PIWI proteins
and piRNAs
Piwi, or ‘‘P-element induced wimpy testis’’ as it is
named in full, was first discovered in 1997 as a gene
Advanced Access publication June 19, 2014
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PIWI and potency
required for germ cell proliferation in Drosophila
melanogaster (Lin and Spradling 1997). This gene
was soon found to encode a broadly conserved protein (Cox et al. 1998), and became the first-known
representative of an animal-specific clade of the
Argonaute protein family (Carmell et al. 2002).
PIWI proteins in the Argonaute protein family
Argonaute proteins are RNA-binding proteins that
represent the core players in silencing mechanisms
mediated by small noncoding RNAs (Ghildiyal and
Zamore 2009). Argonautes have a bi-lobed structure
(Song et al. 2004), containing binding pockets for
both ends of the small RNA, and a RNaseH-like
domain that establishes endonuclease activity (Tolia
and Joshua-Tor 2007). The small RNA acts as a
guide that by basepairing directs the Argonaute protein to a specific RNA or DNA target. The downstream implications of target recognition depend on
the Argonaute protein and its binding partners, but
can involve inhibition of translation, mRNA degradation, or epigenetic modification (Meister 2013).
Phylogenetic analysis has shown that Argonaute
proteins fall into two major clades: the Argonaute
and the Piwi clade (Carmell et al. 2002). The
Argonaute clade has representatives in all eukaryotes
and some prokaryotes. They typically are cytoplasmic
proteins with ubiquitous expression, and bind 21- to
22-nt long miRNAs and siRNAs. In animals, they
play a role in development, mostly by posttranscriptional regulation. The Piwi clade is found exclusively
in the animal kingdom (with the exception of some
unicellular eukaryotes such as Tetrahymena), and expression of PIWI proteins is highly enriched in the
germline.
PIWI-interacting RNAs
As small RNAs guide Argonaute proteins to their
targets, they are central to the understanding of
PIWI proteins. Just like PIWI proteins, piRNAs are
animal-specific and are highly enriched in the germline (Aravin et al. 2006; Girard et al. 2006; Grivna
et al. 2006). Their production, in contrast to
miRNAs and siRNAs, does not require the dsRNAprocessing enzyme Dicer (Vagin et al. 2006;
Houwing et al. 2007). Instead, piRNAs are transcribed as single-stranded precursors from specific
loci or clusters in the genome (Brennecke et al.
2007; Malone et al. 2009; Li et al. 2013), and are
subsequently amplified through the alternated catalytic activity of two different PIWI proteins in a
mechanism known as the ping-pong cycle
(Brennecke et al. 2007; Gunawardane et al. 2007).
PIWI proteins are thus involved in the function as
well as in the biogenesis of piRNAs.
The source of the initial piRNAs that start this
amplification cycle is still enigmatic. In some cases
the initial piRNAs may be maternally deposited
(Brennecke et al. 2008). In other cases they are generated de novo, by the primary pathway of piRNA
biosynthesis, which likely involves nucleases other
than the PIWI proteins (Siomi et al. 2011; Luteijn
and Ketting 2013).
Subcellular localization of PIWI proteins
The ping-pong cycle of piRNA production takes
place in nuage (Lim and Kai 2007; Aravin et al.
2009), and this is also where most PIWI proteins
are found (Fig. 1). Nuage is an RNA-rich, electrondense structure that can take many forms, from a
diffuse cloud to distinct granules. It is found in the
perinuclear region of germline cells and is closely
associated with the nuclear pores (Eddy1975;
Voronina et al. 2011). Nuage contains several other
RNA-binding proteins such as the RNA helicase
Vasa, the Vasa-like protein PL10, and the RNA-binding protein Bruno (Voronina et al. 2011). Tudor
proteins, which bind dimethylated Arginine residues
that are present in both Vasa and PIWI proteins,
function as the scaffold of nuage (Kirino et al.
2009; Huang et al. 2011; Mathioudakis et al. 2012)
(Fig. 1). Nuage-associated Vasa and Tudor proteins
are required for efficient biogenesis and function of
piRNAs (Malone et al. 2009; Reuter et al. 2009;
Vagin et al. 2009; Kuramochi-Miyagawa et al. 2010).
Several PIWI proteins have also been detected in
nuclei of germ cells, often during specific developmental stages. Drosophila Piwi is nuclear in germ cell
precursors as well as in mature germ cells (Cox et al.
2000), mouse Miwi2 (also known as Piwil4) is found
in the nuclei of pre-pachytene spermatocytes (Aravin
et al. 2008), and zebrafish Zili is nuclear in primordial germ cells (PGCs) as well as in maturating oocytes (Houwing et al. 2007, 2008).
Molecular function of PIWI proteins
A prominent and conserved role of PIWI proteins
and piRNAs is to regulate transposon activity. The
majority of Drosophila piRNAs maps to transposon
sequences (Vagin et al. 2006; Brennecke et al. 2007;
Klenov et al. 2007), and interference with the PIWIpiRNA pathway results in elevated levels of transposon activity in the Drosophila germline. In mouse
male gonads, roughly half of the piRNAs is related
to transposable elements, and PIWI proteins are required to maintain transposon repression (Watanabe
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Fig. 1 Subcellular localization and molecular functions of PIWI
proteins. The primary location of PIWI proteins is the perinuclear
nuage, shown as three smaller spheres attached to the nucleus. In
nuage, PIWI proteins associate with Tudor proteins and Vasa, are
involved in the ping-pong cycle of piRNA amplification, and target
transposon RNA (and possibly other mRNAs) for degradation.
Nuage is closely associated with nuclear pores. Some PIWI
proteins are present in the nucleus and function in epigenetic
modification of target DNA, with the help of proteins such as
histone methylase HP1 (Drosophila) or DNA methylase Dnmt3
(mouse).
et al. 2006; Aravin et al. 2007; Carmell et al. 2007;
Aravin et al. 2008; Kuramochi-Miyagawa et al. 2008).
Also in zebrafish (Houwing et al. 2007), Xenopus
(Lau et al. 2009), and C. elegans (Bagijn et al.
2012), piRNAs are enriched for transposon-related
sequences.
PIWI proteins suppress transposon activity by two
complementary mechanisms (Rozhkov et al. 2013)
(Fig. 1). First, as part of the ping-pong cycle they
degrade transposase mRNAs and other transposonderived RNAs that have sequence complementarity
to the piRNA. Second, PIWI proteins that localize
to the nucleus can target the same transposon sequences for epigenetic silencing by recruiting DNAor histone methylase activity (Brower-Toland et al.
2007; Klenov et al. 2007; Aravin et al. 2008;
Kuramochi-Miyagawa et al. 2008; Huang et al.
2013). Both these processes play a role in transponson silencing, but their relative contributions may
vary depending on the circumstances (Rozhkov
et al. 2013). Interestingly, in C. elegans epigenetic
silencing initiated by PIWI proteins, can subsequently be inherited over many generations even in
the absence of PIWI protein (Ashe et al. 2012;
Luteijn et al. 2012). It is however currently unclear
J. C. van Wolfswinkel
whether this type of inheritance also exists in other
species.
Although transposons are the primary targets of
piRNAs, a significant fraction of the piRNAs has
no apparent relationship to transposons. In certain
contexts, such as in pachytene cells in the mouse
male germline, even the majority of piRNAs is unrelated to repetitive elements (Grivna et al. 2006;
Watanabe et al. 2006). PIWI proteins have been implicated in the regulation of nonrepetitive sequences,
mostly at the posttranscriptional level, for example
during embryonic development in Drosophila and
during spermatogenesis in mouse (Yin and Lin
2007; Robine et al. 2009; Unhavaithaya et al. 2009;
Rouget et al. 2010; Gou et al. 2014). Although these
results await confirmation, the findings suggest that
regulation of coding transcripts could be another
conserved role of PIWI proteins.
PIWI proteins in adult pluripotent stem
cells
Although it is clear that PIWI proteins have important functions in the germline, it is possible that the
prevailing germline-centered view of PIWI proteins is
a consequence of the selection of model organisms
used in developmental biology. Specifically, the ability to regenerate, although widely conserved in the
animal kingdom, is fairly restricted in the primary
model species used to study animal development
(Bely and Nyberg 2009). Newly developed invertebrate models that possess a capacity for whole-body
regeneration (i.e., the ability to reestablish an organism with a mature body plan from small body fragments) are demonstrating conserved features of
regenerating systems and show PIWI proteins in a
new light (Fig. 2).
Planarian neoblasts
Adult flatworms have a legendary ability to repair
extensive wounds, reorganize complex structures
such as the intestinal system, and recreate entire
organs such as the brain and the germline
(Reddien and Sanchez Alvarado 2004; Newmark
et al. 2008). This regenerative capacity is mediated
by a population of small, mitotically active cells
named ‘‘neoblasts’’ that are dispersed throughout
the parenchymal space of the body. Although neoblasts probably are a heterogeneous population, at
least some of these cells are pluripotent (Wagner
et al. 2011). When neoblasts are eliminated by irradiation or RNAi (Dubois and Wolff 1947; Solana
et al. 2012), regeneration and cell turnover are
blocked and the animal invariably dies.
PIWI and potency
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Fig. 2 PIWI proteins in stem cells. Phylogenetic tree indicating the presence of PIWI proteins in various stem cell types in each group.
Presence of a cell type is indicated with the corresponding letter. Black letters correspond to cell types in which PIWI proteins have
been identified; gray letters indicate cell types without PIWI expression. Demonstrated requirement of a PIWI protein for the cell type
is indicated with an asterisk; presence of Vasa or nuage in the same cell type is indicated with a V or N, respectively. Ability for wholebody regeneration or posterior body regeneration is indicated with a W or P.
Neoblasts have several features that previously had
been associated with germ cells. They have an electrondense nuage-related structure called the chromatoid body (Morita et al. 1969; Coward 1974;
Rouhana et al. 2012), and they express transcripts
corresponding to three PIWI proteins (Smedwi-1,
Smedwi-2, and Smedwi-3) (Reddien et al. 2005;
Palakodeti et al. 2008), two Vasa proteins, two
Tudor proteins, and the Bruno homolog Bruli
(Guo et al. 2006; Onal et al. 2012; Wagner et al.
2012). Whereas Smedwi-1 protein is cytoplasmic
(Guo et al. 2006), Smedwi-2 has been detected in
the nucleus (Zeng et al. 2013).
Planarian piRNAs with all hallmarks of a pingpong cycle have been identified (Palakodeti et al.
2008; Friedlander et al. 2009). Inhibition of
smedwi-2 or smedwi-3 (but not smedwi-1) caused
significant reductions in piRNA levels, and resulted
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in regenerative defects and lethality (Reddien et al.
2005; Palakodeti et al. 2008; Friedlander et al. 2009).
Bruli and vasa-1 were also required for regeneration
and animal viability (Guo et al. 2006; Wagner et al.
2012), but their effects on piRNAs are currently
unknown.
Neoblasts with similar features have been identified in several other flatworms. Dugesia japonica neoblasts have chromatoid bodies and express DjPiwi
mRNA and two Vasa-related transcripts (Shibata
et al. 1999; Rossi et al. 2006). In Macrostomum lignano, transcripts macpiwi and macvasa were detected
in the germline and in a subset of the neoblasts
(Pfister et al. 2008; De Mulder et al. 2009b), and
nuage-like perinuclear granules were observed in
neoblasts by visualization of MacVasa protein
(Pfister et al. 2008). Moreover, inhibition of macpiwi
eliminated adult neoblasts, resulting in regeneration
defects and animal lethality (De Mulder et al. 2009b).
Acoel stem cells
Although acoels and flatworms are not closely related, their stem cell systems show remarkable similarities. Acoels have extensive regenerative capacities
and possess small parenchymal cells that are the only
mitotically active cells in the body (De Mulder et al.
2009a). In the acoel Hofstenia miamia, dividing cells
expressed transcripts of two PIWI homologues
(Srivastava et al. 2014), and piwi-1 was found to be
required for regeneration. In another acoel,
Isodiametra pulchra, a subpopulation of the neoblasts
expressed ipiwi-1 and ivasa-1 (De Mulder et al.
2009a). Inhibition of ipiwi-1 by RNAi did not interfere with regeneration (De Mulder et al. 2009a), although this could be due to redundancy or
incomplete inhibition.
Sponge archaeocytes
Sponges belong to an early-diverging animal phylum.
They lack body symmetry and true tissues or organs,
but have the ability to recover from any type of
injury (Simpson 1984; Leys et al. 2007). Even upon
complete cell dissociation, an amoeboid cell type,
named the archaeocyte, was observed to reaggregate
and through mitosis and well-defined differentiation
paths recreate all the characteristic structures of a
mature sponge (De Sutter and Van de Vyver 1979;
Buscema et al. 1980).
Ephydatia fluviatilis archeaocytes expressed
mRNAs EfPiwiA and EfPiwiB, and this expression
was lost upon differentiation into all other cell
types except for the choanocytes (Funayama et al.
2010). The flagellated choanocytes are involved in
J. C. van Wolfswinkel
nutrient entrapment, but maintain mitotic ability
and can produce sperm (Simpson 1984). Moreover,
under specific circumstances, choanocytes have been
proposed to dedifferentiate into archaeocytes
(Simpson 1984). Therefore, although choanocytes
have a clear differentiated morphology, they appear
to maintain certain features and capabilities of the
pluripotent archeocytes.
Stem cells in Botryllus
The urochordate Botryllus is a colonial tunicate,
composed of up to thousands of genetically identical
zooids, which are connected by ‘‘blood vessels’’ and
are embedded in a gelatinous matrix called the tunic.
Each zooid is only a few millimeters large, but has a
variety of organs among which a two-chambered
heart, a neural network, and a gastrointestinal
system (Manni et al. 2007). Tiny fragments of vasculature can regenerate an entire mature organism
(Rinkevich et al. 1995; Brown et al. 2009), leading
to the view that pluripotent stem cells circulate
through the blood, although their identity has remained elusive.
In Botryllus leachi, induction of regeneration revealed a population of BlPiwi positive cells on the
luminar side of the vasculature (Rinkevich et al.
2010). These cells subsequently entered the blood
vessel lumen, proliferated, and moved through the
vasculature, consistent with the notion of circulating
stem cells. Inhibition of BlPiwi expression resulted in
a complete halt of whole body regeneration
(Rinkevich et al. 2010). Surprisingly, these cells appeared to upregulate BlPiwi specifically when activated by injury, suggesting that PIWI proteins
might be absent from these cells for prolonged
time spans.
In the related species Botryllus schlosseri, BsPiwi
positive cells were detected in so called ‘‘Cell
Islands’’, which also expressed mRNA for Vasa and
PL10 (Rosner et al. 2009; Rinkevich et al. 2012).
Transplantation of these CIs resulted in chimerism,
indicating that they contain self-renewing cells that
can give rise to differentiated cell types. Similar to
the situation in Botryllus leachi, RNAi against BsPiwi
abrogated regeneration completely.
Cnidarian i-cells
Cnidarians are aquatic animals with a sac-like body
plan composed of two germ layers (endoderm and
ectoderm). The adult Hydra polyp is radially symmetric and consists of a foot, a cylindrical body
column, and a head with tentacles. Any part of the
body column, when dissected, is able to regenerate a
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PIWI and potency
new head and a foot (Holstein et al. 2003). Even
upon complete dissociation, Hydra cells can reaggregate and form a new animal de novo (Gierer et al.
1972).
The Hydra endoderm and ectoderm each make a
single-layer epithelium and together form the polyp
body. Each epithelium is constantly replenished by
mitosis of its own linage-committed stem cells,
which are located in the body column (Holstein
et al. 1991). Found between these two epithelial
layers are migratory interstitial cells (i-cells). I-cells
are mitotically active and give rise to a constant
supply of neurons, secretory cells, nematocytes, and
germ cells (David and Murphy 1977; David 2012).
Hydra PIWI proteins Hywi and Hyli were detected
in the i-cells, as well as in the germline (Juliano et al.
2013; Lim et al. 2013), but were absent from postmitotic differentiated cells. I-cells also contained
transposon-related piRNAs with hallmarks of the
ping-pong cycle (Juliano et al. 2013; Lim et al.
2013), and expressed Vasa, PL10, and the Tudor protein TDRD9, all of which were enriched in perinuclear granules that resemble nuage (Lim et al. 2013).
Interestingly, variations exist between different
cnidarian stem cell systems. Morphology of the
Hydractinia polyp is very similar to that of Hydra,
but Hydractinia i-cells, which expressed piwi, vasa,
and nanos2 (Rebscher et al. 2008; Plickert et al.
2012), were also able to generate epithelial cells,
and therefore are truly pluripotent (Muller et al.
2004).
In another cnidarian, Podocoryna carnea, the transcript for the PIWI protein Cniwi was most highly
expressed in the germline, but lower levels were detected in differentiated epithelio-muscular cells
(Seipel et al. 2004). Upon stimulation, these cells
have been proposed to dedifferentiate and transdifferentiate into various other cell types (Schmid and
Alder 1984), which at least in vitro, included gametes
(Schmid et al. 1982).
In summary, pluripotent stem cells are found in diverse animal species, and in each of the cases studied
so far these stem cells express PIWI proteins (or at
least mRNA), often combined with the presence of
other typical nuage constituents and a nuage-like
structure. Moreover, PIWI expression tends to decrease upon cell differentiation, and to be required
for stem cell function. One could argue that it is
expected that pluripotent stem cells, which can generate germ cells, maintain the same degree of genome
protection that germ cells have, and therefore maintain nuage and PIWI proteins. An alternative possibility, however, is that these proteins have a function
in stem cells that is unrelated to the germ cell lineage. An important question therefore is what the
status of PIWI proteins is in lineage-committed
stem cells that do not generate germ cells.
PIWI proteins in adult lineage-restricted
stem cells
Cnidarians revisited
Although Hydra i-cells can be considered pluripotent, Hydra endoderm and ectoderm are formed
from the lineage-restricted stem cells (Holstein
et al. 1991). These stem cells were found to express
PIWI proteins, albeit at lower levels than the i-cells
(Juliano et al. 2013). Interestingly, elimination of
Hywi specifically from the epithelial lineages resulted
in a loss of epithelial integrity followed by death
within days after hatching (Juliano et al. 2013), suggesting that Hywi has an essential function in these
lineage-committed cells. Similarly, in Clytia hemishaerica medusas, expression of piwi, vasa, pl10,
and nanos1 transcripts was detected in mitotic cells
of the tentacle bulb, which are thought to be lineagecommitted cells that give rise to only nematocytes
(Denker et al. 2008).
Localized stem cell populations in ctenophores
The tentacle root of the ctenophore Pleurobrachia
pileus shows similarities to the Clytia tentacle bulb.
It contains undifferentiated cells that express high
levels of ppiPiwi1, ppiVasa, ppiPL10, and ppiBruno
transcripts (Alie et al. 2010), and are able to regenerate the tentacle structure consisting of muscle cells
and colloblasts throughout the animal’s lifespan, but
are not known to contribute to any other cell types.
Pleurobrachia has several additional lineage-restricted
stem cell types, including a spatially confined proliferative population that replenishes the ciliated polster cells in the combs (which are required for
ctenophore locomotion), and a population of stem
cells that specifically renews sensory or neuronal cells
at the aboral pole. Both of these stem cell populations were found to express ppiPiwi1, ppiVasa,
ppiPL10, and ppiBruno (Alie et al. 2010). The same
set of genes, together with ppiPiwi2, was also expressed in the germline. The germline however is
spatially separated from the other lineages, indicating
that the expression of nuage-related genes in the
other stem cell types is not related to the germline
lineage.
Posterior growth zone in annelids
Juveniles of segmented annelids typically have a limited number of segments, which is expanded over the
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course of their life in a process known as posterior
elongation, by a region called the Posterior Growth
Zone (PGZ) (de Rosa et al. 2005). In addition to its
developmental function, the PGZ also mediates regeneration of posterior segments upon amputation
or injury. In Platynereis dumerilii, transcripts pduPiwi, pdu-Vasa, pdu-PL10, pdu-nanos, and transcripts
for three Tudor proteins were detected in undifferentiated cells of the PGZ (Rebscher et al. 2007;
Gazave et al. 2013). These same genes were also
expressed in germ cell precursors, which during
embryogenesis are located in the same region that
gives rise to the PGZ, and only later migrate anteriorly. For a long time it was believed that PGCs
differentiate from the PGZ cells, however, recent
EdU-labeling experiments showed that although
these populations of cells share gene expression profiles and an anatomical position during embryogenesis, they are separate cell populations formed during
different stages of embryogenesis (Rebscher et al.
2012; Gazave et al. 2013).
A similar situation has been described for the
annelid Capitella teleta, where transcripts ct-piwi1,
ct-piwi2, ct-Vasa, and ct-nanos were expressed both
in germ cells and the PGZ, but these lineages were
generated at different times during embryogenesis
and remained separate during the remainder of the
animal’s life (Dill and Seaver 2008; Giani et al. 2011).
Tail bud in cephalochordates
Expression of PIWI protein in cephalochordates
shows remarkable resemblance to that in annelids.
During embryonic development of the amphioxus
Branchiostoma floridae, two populations of cells expressed transcripts bf-Piwil1, bf-Vasa, and bf-Nanos:
the PGCs and the cells of the tail bud, which is the
site of posterior elongation where new somites progressively develop (Wu et al. 2011; Zhang et al.
2013). After gastrulation both of these cell types localize to the tail bud, resembling a single population,
from where the germ cell precursors migrate to their
final ventral position later during larval development.
The localization of PGCs to the tail bud has, again,
led to the view that the PGCs develop from the stem
cells in the tail bud. However, closer observations
demonstrated that during the first embryonic divisions germ plasm containing maternal transcript of
Bf-Piwil1, Bf-Vasa, Bf-Nanos, and Bf-TDRD7 was
segregated asymmetrically to one cell, with asymmetric segregation of these components continuing all
the way to the 64-cell stage. The single cell that received maternal transcripts for nuage proteins, was
the only cell that gave rise to the PGCs. The stem
J. C. van Wolfswinkel
cells that form the tail bud expressed similar transcripts zygotically, but were not able to generate
PGCs. An experiment separating the first two embryonic blastomeres showed that the larva developing
from the blastomere lacking the germ plasm still generated a tail bud, but never generated PGCs (Wu
et al. 2011), confirming the independence of the
two lineages.
Together, these studies show that in nonbilaterians,
protostomes, and deuterostomes, multipotent stem
cells that do not appear to give rise to germ cells
do express PIWI proteins, together with several
other nuage-constituents. In fact, most-to-all multipotent adult stem cells involved in wound-induced
regeneration studied thus far show expression of
nuage-associated factors, indicating that PIWI proteins and nuage components likely have some somatic function in addition to their germline roles.
PIWI proteins in vertebrate stem cells
In vertebrates, no cases of whole-body regeneration
are known. Some animals have prominent woundinduced regeneration, such as newts and amphibians,
which can regenerate amputated limbs, or zebrafish
which are able to regenerate their tail fin, heart, and
liver (Tanaka and Reddien 2011). Although such
regenerated structures can consist of many cell
types, the stem cells involved in these processes are
typically restricted in their potential and no expression of PIWI proteins has been reported in dividing
cells of these systems (Gargioli and Slack 2004;
Schebesta et al. 2006; Kragl et al. 2009; Yoshinari
et al. 2009; Jopling et al. 2010; Sleep et al. 2010;
Knopf et al. 2011; Sousa et al. 2011; Tu and
Johnson 2011; Monaghan et al. 2012; Stewart and
Stankunas 2012). Transcriptional profiling showed
that PIWI proteins were also not significantly enriched in two well-studied tissue-specific stem cell
systems: the mammalian intestinal stem cells
(Munoz et al. 2012) and epithelial stem cells
(Kocer et al. 2008).
Two other mammalian stem cell systems, however,
have been reported to express PIWI proteins. Human
hematopoietic stem cells, but not the more lineagerestricted progenitors, were found to express Hiwi
mRNA (Sharma et al. 2001), which rapidly decreased
upon induction of differentiation. Ectopic expression
of Hiwi in the acute leukemic cell line KG1 resulted
in inhibition of proliferation activity and induction
of apoptosis, suggesting that this PIWI protein might
function to keep hematopoietic cell division under
control. Somewhat conflicting results however were
obtained in mouse. First, murine hematopoietic stem
PIWI and potency
cells did not express Miwi or Mili, but instead expressed mRNA for the PIWI protein Miwi2 (also
known as Piwil4) (Nolde et al. 2013). Second, in vitro
overexpression of Mili in murine bone marrow cells
resulted in increased cell proliferation (Chen et al.
2007). Finally, a triple PIWI knockout mouse
showed no major defects in hematopoiesis (Nolde
et al. 2013) casting doubt on the physiological relevance of PIWI protein expression in the hematopoietic system.
Murine mesenchymal stem cells, which are multipotent cells that can give rise to cell types as diverse
as osteoblasts and fat cells, were also reported to
express Mili (or Piwil2) (Wu et al. 2010). In this
context, siRNA-mediated inhibition of mili resulted
in increased cell proliferation.
Although these findings are intriguing, and suggest
the presence of PIWI proteins in some vertebrate
stem cells, the relevance of PIWI proteins to these
systems remains to be clarified. Meanwhile, some
further interesting findings along these lines come
from the study of induced stem cell populations in
vertebrates.
iPS cells
Induced pluripotent stem (iPS) cells are formed
when adult differentiated cells (usually fibroblasts)
are reprogrammed to a pluripotent state by stimulation with exogenous factors (Takahashi and
Yamanaka 2006). In a study analyzing gene expression in successfully reprogrammed mouse iPS cells
originating from various cell types, mili (piwil2)
and vasa were consistently upregulated (Mikkelsen
et al. 2008). Another study found that mili (piwil2)
expression increased early in the reprogramming
process, in the same time frame as nanos and the
Tudor protein tdrd7 (Samavarchi-Tehrani et al.
2010), suggesting that this is not a late indirect
effect. The level of piwil2 in iPS cells may differ between species though (Pashai et al. 2012; Marchetto
et al. 2013). Interestingly, the lower piwil2 levels
found in nonhuman primate iPS cells compared
with human iPS cells, corresponded with increased
levels of L1 retrotransposition, which could be suppressed by overexpression of piwil2 in these cells
(Marchetto et al. 2013), suggesting that PIWI proteins in iPS cells could function in transposon
silencing.
Cancer stem cells
Many studies have shown expression of Hiwi
(Piwil1) or Hili (Piwil2) in human cancers, and expression of these PIWI proteins is often associated
707
with adverse long-term prognosis (Sun et al. 2010;
Zhao et al. 2011; Suzuki et al. 2012; Wang et al.
2012; Greither et al. 2012; Liang et al. 2013).
The human PIWI protein Hiwi was even found to
function as an oncogene, increasing the tumorogenic
potential of a cell transplant, whereas inhibition of
hiwi expression in a sarcoma cell line reduced the
efficiency of colony formation and induced differentiation (Siddiqi et al. 2012), suggesting that Hiwi was
required to maintain expansion capabilities.
Interestingly, increased expression of PIWI protein
in the context of tumor formation is not unique to
humans. In the cnidarian Hydractinia echinata, ectopic expression of a POU-domain transcription
factor in epithelial cells resulted in tumor-like
growths, which exhibited high expression of piwi,
vasa, and nanos (Millane et al. 2011). Similarly in
Drosophila, mutation of the transcriptional repressor
l(3)mbt resulted in brain tumors that expressed elevated levels of aub, piwi, and vasa, and each of these
genes was required for tumor growth (Janic et al.
2010).
Together, these results indicate that expression of
PIWI proteins tends to be elevated in vertebrate cells
that acquired an increased differentiation potential.
Although the causal relationship between these
events is currently unclear, it raises the possibility
that PIWI proteins have a connection to pluripotency in this subphylum.
Conclusions and speculations: the role of
PIWI proteins in potency
In summary, PIWI proteins are present in a wide
range of somatic stem cells, and in many cases are
required for stem cell function. PIWI proteins are
found in all stem cell systems that are capable of
generating germ cells, but also in many lineage-restricted systems, indicating that their presence in
stem cells is not directly related to the ability to
contribute to the germline lineage. Rather, expression
of PIWI proteins in somatic stem cells seems to be
the rule throughout most of the animal kingdom
(Fig. 2), involving nonbilaterian phyla, and several
protostomes as well as deuterostomes, suggesting
that this likely reflects the ancestral metazoan state.
Two major clades tend to lack somatic stem cells
with PIWI proteins: ecdysozoans and vertebrates
(Fig. 2). PIWI proteins still function in the germline
in these clades, where they have well-described and
essential roles. Moreover, most members of these
groups have somatic stem cells, although these tend
to be lineage-restricted. Unlike lineage-restricted
somatic stem cells in other phyla however, they have
708
not been found to express PIWI protein, or the PIWI
proteins appear not essential for their function.
Interestingly, ecdysozoans and vertebrates are also
two main groups that lack the ability for whole-body
regeneration (Bely and Nyberg 2009), raising the possibility of a relation between PIWI-expressing stem
cells and regenerative plasticity. Further study of
stem cell systems and regenerative potential in
nonecdysozoan protostome species such as molluscs
and nemerteans, and nonvertebrate deuterostomes
will strengthen or weaken this correlation.
Aside from the phylogenetic trends, there appears
to be a correlation between cell plasticity and PIWI
protein expression. All pluripotent stem cell systems
studied to date express PIWI protein. In additition,
even in ecdysozoans and vertebrates, when plasticity
of a cell is artificially increased (e.g. in iPS cell induction or tumorogenesis) levels of PIWI proteins
tend to increase. It remains to be determined
whether this increase in PIWI expression is a prerequisite or a side-effect of reprogramming, but it nevertheless suggests that PIWI proteins could be part of
a conserved transcriptional program that is activated
when the differentiation potential, or potency, of a
cell is increased.
What could the role of PIWI proteins in stem cells
be? The main known molecular functions of PIWI
proteins involve piRNAs, and the structural features
of PIWI proteins support piRNAs as their primary
interactors. Indeed, piRNAs, typical nuage-components such as Vasa, and structures that resemble
nuage have been detected alongside PIWI proteins
in many pluripotent cells (Fig. 2). PIWI proteins
and piRNAs could be involved in genome protection
in somatic stem cells. As stem cells are self-renewing
and constantly give rise to new descendants, they
may require more protection than terminally differentiated cells, including tighter protection against
transposon activity. In addition, the high plasticity
of pluripotent cells may involve a more ‘‘open’’
chromatin structure, which could make the cells vulnerable to transposon activation. Pluripotent embryonic stem cells are thought to have such an open
chromatin state which is lost upon differentiation
(Gaspar-Maia et al. 2011) (although the role of
PIWI proteins in these stem cells is currently unknown). Lineage-restricted intestinal stem cells on
the other hand were found to undergo little change
in overall DNA methylation upon differentiation
(Kaaij et al. 2013), suggesting that their chromatin
was already mostly in the ‘‘closed’’ differentiated
state. In planarians, chromatin remodeling is thought
to play a role during differentiation of stem cell
J. C. van Wolfswinkel
progeny (Scimone et al. 2010; Hubert et al. 2013;
Jaber-Hijazi et al. 2013), but it is currently unclear
whether differentiation coincides with a global increase in heterochromatin, and whether neoblast
chromatin is in an ‘‘open’’ state.
Speculating further along these lines, it is possible
that the role of PIWI proteins in the stabilization of
the stem cell genome extends beyond the control
of repetitive sequences, and includes the maintenance
of this ‘‘open’’ chromatin structure. The role of RNA
in chromatin structure is gaining appreciation
(Caudron-Herger and Rippe 2012), and mostly
long noncoding RNAs have been proposed to function in the formation of nuclear domains. Similarly,
the interaction of PIWI-piRNA complexes with nascent transcripts or genomic regions could assist in
shaping and maintaining the three-dimensional architecture of the chromatin.
PIWI proteins could also play a role in the regulation of nonrepetitive transcripts in stem cells. PIWI
proteins have been reported to regulate specific genes
or gene groups during specific developmental phases
(Yin and Lin 2007; Robine et al. 2009; Unhavaithaya
et al. 2009; Rouget et al. 2010; Gou et al. 2014). The
fact that modification of PIWI protein levels can
affect expansion efficiency in some vertebrate cell
systems is consistent with a function in regulation
of gene expression beyond repetitive transcripts.
PIWI proteins could play a role in inhibition of differentiation genes, thereby maintaining stem cells in
an undifferentiated state, or they could be required
to regulate genes involved in mitotic activity.
Much remains to be discovered on the presence and
function of PIWI proteins in somatic stem cells.
First, many stem cell systems have not been
probed, and the data indicating absence of PIWI
proteins from several stem cell systems is not conclusive. Some stem cell systems have large numbers
of amplifying progenitor cells, which may mask the
expression signature of the rare stem cells if the analysis is done on a population basis. Studies of expression with single cell resolution, such as in situ
hybridization or single cell RNA sequencing, may
therefore help to further clarify the presence or absence of PIWI expression. Second, functional studies
are essential to determine whether the presence of
PIWI in any stem cell system has physiological relevance for stem cell function. And third, closer analysis of the effects of the loss of PIWI proteins in
pluripotent stem cells will be required to determine
the molecular underpinnings of their requirement.
If their main role is in transposon silencing, the
loss of stem cells upon inhibition of PIWI protein
PIWI and potency
expression should correlate with increased transposon activity and genomic damage, possibly accompanied by apoptosis. Alternatively, if their role lies in
inhibition of differentiation, loss of PIWI protein
should result in activation of differentiation markers.
Many other cellular outcomes are possible however.
As stem cells provide an opportunity to study PIWI
proteins without the complicated cell biology of
germ cells, further investigations along these lines
could increase our understanding of both stem cells
and the functions of PIWI proteins.
Acknowledgments
Thanks to Irving Wang for illustrations, and to
Michelle Carmell, René Ketting, Peter Reddien, and
Mansi Srivastava for helpful comments on the
manuscript.
Funding
JCvW is supported by a fellowship from the Human
Frontier Science Program (HFSP). Support for attendance at the symposium was provided by the Society
for Intergrative and Comparative Biology (SICB).
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