DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 1231
Development 137, 1231-1241 (2010) doi:10.1242/dev.042051
© 2010. Published by The Company of Biologists Ltd
The Mi-2-like Smed-CHD4 gene is required for stem cell
differentiation in the planarian Schmidtea mediterranea
M. Lucila Scimone1, Joshua Meisel1 and Peter W. Reddien1,2,*
SUMMARY
Freshwater planarians are able to regenerate any missing part of their body and have extensive tissue turnover because of the
action of dividing cells called neoblasts. Neoblasts provide an excellent system for in vivo study of adult stem cell biology. We
identified the Smed-CHD4 gene, which is predicted to encode a chromatin-remodeling protein similar to CHD4/Mi-2 proteins, as
required for planarian regeneration and tissue homeostasis. Following inhibition of Smed-CHD4 with RNA interference (RNAi),
neoblast numbers were initially normal, despite an inability of the animals to regenerate. However, the proliferative response of
neoblasts to amputation or growth stimulation in Smed-CHD4(RNAi) animals was diminished. Smed-CHD4(RNAi) animals displayed
a dramatic reduction in the numbers of certain neoblast progeny cells. Smed-CHD4 was required for the formation of these
neoblast progeny cells. Together, these results indicate that Smed-CHD4 is required for neoblasts to produce progeny cells
committed to differentiation in order to control tissue turnover and regeneration and suggest a crucial role for CHD4 proteins in
stem cell differentiation.
INTRODUCTION
Stem cells have diverse and important roles in animal development
and are required for the maintenance of cell numbers during tissue
homeostasis in adult organisms. The hallmark properties of stem
cells are long-term self-renewal and differentiation potential
(Wagers and Weissman, 2004; Weissman et al., 2001). Regulation
of these two processes under normal and abnormal conditions is
crucial, although still poorly defined.
Planarians are one of the simplest organisms known to have
robust regeneration capacities and extensive tissue turnover as adults
(Reddien and Sánchez Alvarado, 2004); these properties require
active dividing cells known as neoblasts. Neoblasts have been
identified by their stem-cell-like morphology and their proliferative
capacity (Dubois, 1949; Reddien and Sánchez Alvarado, 2004).
Although neoblasts were first described to have stem-cell-like
properties more than 100 years ago, the mechanisms regulating their
proliferation and differentiation still remain largely unknown.
Neoblasts are constantly dividing to maintain tissue turnover in the
animals. In addition, injury (e.g. amputation) stimulates neoblast
proliferation. Neoblasts are required for the generation of a
blastema, an unpigmented epithelial/mesenchymal bud at the site of
injury, within which missing body parts are formed (Dubois, 1949;
Reddien and Sánchez Alvarado, 2004).
Many newly developed resources and methodologies have made
the study of stem cells in planarians possible (Newmark and
Sánchez Alvarado, 2000; Newmark et al., 2003; Reddien et al.,
2005a; Sánchez Alvarado et al., 2002). For example, whole-mount
in situ hybridizations and high-throughput RNAi screens have
resulted in the identification of genes required for normal neoblast
1
Howard Hughes Medical Institute, Whitehead Institute, and 2Department of
Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge,
MA 02142, USA.
*Author for correspondence ([email protected])
Accepted 4 February 2010
function (Guo et al., 2006; Reddien et al., 2005a; Reddien et al.,
2005b). Because neoblasts are the major category of proliferating
cells in the animal, they can be labeled with the thymidine analog
bromodeoxyuridine (BrdU) and can also be isolated using a DNAlabeling dye by flow cytometry (Newmark and Sánchez Alvarado,
2000; Hayashi et al., 2006; Reddien et al., 2005b). More recently, a
panel of neoblast progeny markers have been identified (Eisenhoffer
et al., 2008). The abundance of neoblasts, the prominent role they
have in regeneration and the newly available experimental tools for
the study of neoblasts, make these cells a powerful new setting for
in vivo stem cell experimentation.
In several stem cell systems, transcription factors regulate
differentiation towards a particular lineage (Bonifer et al., 2008;
Fuchs and Horsley, 2008; Le Grand and Rudnicki, 2007; Orkin and
Zon, 2008). The accessibility of target genes to these transcription
factors can be dictated by chromatin structure; therefore, regulation
of chromatin structure can contribute to changes in gene activity
(Bibikova et al., 2008; Georgopoulos, 2002; Kingston and Narlikar,
1999; Kouzarides, 2007; Li et al., 2007; Lunyak and Rosenfeld,
2008; Narlikar et al., 2002). Changes in chromatin structure can be
accomplished by both post-translational modifications of histone
tails and by ATP-dependent chromatin remodeling proteins (Becker
and Horz, 2002; Kouzarides, 2007).
The Mi-2 protein is a central component of the nucleosome
remodeling deacetylase (NuRD) complex (Tong et al., 1998; Wade
et al., 1998; Zhang et al., 1998). There are two Mi-2 proteins: Mi2, also known as the chromodomain helicase DNA binding protein
3 (CHD3) and Mi-2, also known as CHD4 (Seelig et al., 1996).
The latter is the predominant Mi-2 protein associated with the
mammalian NuRD complex (Zhang et al., 1998). Chromatin
remodeling by the NuRD complex usually results in transcriptional
repression due to the histone deacetylase activity residing in the
complex (Ng and Bird, 2000; Tong et al., 1998). However, Mi-2
can also associate with histone acetyltransferases and methyl
transferases, resulting in these cases in transcriptional activation
(Denslow and Wade, 2007; Shimono et al., 2003; Williams et al.,
2004). The NuRD complex can be recruited to a specific promoter
DEVELOPMENT
KEY WORDS: CHD4, Differentiation, Planaria, Regeneration, Stem cells
1232 RESEARCH ARTICLE
MATERIALS AND METHODS
CHD4 and RNAi experiments
RT-PCR was used to amplify the CHD4 gene. Complete gene sequence was
determined using 5⬘ and 3⬘ RACE PCR (Ambion). CHD4 and the control
gene unc-22 from C. elegans were cloned into the pPR244 RNAi expression
vector using Gateway recombination reactions as described (Reddien et al.,
2005a). RNAi experiments were performed by feeding the animals with a
mixture of liver and bacteria expressing CHD4 dsRNA (Reddien et al.,
2005a). Ten milliliters of bacteria culture was pelleted and resuspended in
100 l of liver or in 30 l of liver. Three feeding protocols were used:
animals were fed on day 0, day 4 and day 7, or on day 0 and day 4, or on day
0 only. For regeneration studies, animals were cut at day 8 (for the threefeedings protocol) or at day 5 (for the two-feedings protocol). For
homeostasis studies, one extra feeding was added to the three-feedings
protocol at day 14.
In situ hybridizations
In situ hybridizations on whole animals, cell macerates or isolated cells were
performed as described (Reddien et al., 2005b). CHD4 whole-mount in situ
hybridizations and fluorescence in situ hybridizations (FISH) were
performed as described (Pearson et al., 2009). To quantify data from in situ
hybridizations on cells, the percentage of DAPI-positive cells with signal
was determined. To determine the number of Smed-AGAT-1-expressing cells
present following irradiation, the number of Smed-AGAT-1+ cells within 0.3
mm by 0.3 mm tissue from the dorsal side and anterior to the pharynx was
determined and normalized by animal length.
Antibody stainings
Animals were treated with 10% N-acetyl-cysteine in PBS for 3-5 minutes at
room temperature (RT) and fixed in Carnoy’s solution. Animals were then
labeled as previously described (Newmark and Sánchez Alvarado, 2000).
Numbers of mitoses were divided by animal surface area. For SMEDWI-1
antibody stainings, animals were fixed in a 4% formaldehyde, PBST
solution following FISH with the Smed-AGAT-1 riboprobe, blocked and
labeled with 1:2000 -SMEDWI-1.The SMEDWI-1 polyclonal antibody
was raised in rabbits using the peptide previously described (Guo et al.,
2006). Total numbers of Smed-AGAT-1+ cells, SMEDWI-1; Smed-AGAT-1
double-positive cells, or SMEDWI-1-positive cells were determined in
optical sections.
BrdU labeling
Animals were fed with RNAi food at days 0 and 4, and injected at day 6
following initial RNAi feeding with a solution of 5 mg/ml of BrdU (Fluka)
in planarian water. Animals were then killed 4 days later in 5% N-acetylcysteine in PBS for 5 minutes at RT and fixed as described (Pearson et al.,
2009). FISH using the Smed-AGAT-1 riboprobe were performed as
described (Pearson et al., 2009). Following FISH, animals were fixed,
treated with 2N HCl for 45 minutes at RT and labeled with a 1:100 rat antiBrdU antibody (Oxford Biotech) as previously described (Newmark and
Sánchez Alvarado, 2000). Apotome images were obtained from BrdUlabeled and FISH animals using an Axiocam digital camera, a Zeiss
AxioImager with use of an Apotome, and Axiovision software. Total
numbers of Smed-AGAT-1-expressing cells containing a BrdU-labeled
nucleus were determined in optical sections.
FACS
Planarians were cut into small pieces and incubated in calcium-free,
magnesium-free medium plus BSA (CMFB) containing 1 mg/ml of
collagenase for 45 minutes at RT as described (Reddien et al., 2005b). Cells
were centrifuged at 1250 rpm for 5 minutes and resuspended in CMFB
containing Hoechst 33342 (10 g/ml) for 45 minutes at RT. Calcein (0.5
g/ml) was incubated with the cells for 15 minutes at RT. Finally, 5 g/ml
of propidium iodide was added to the cell suspension prior to flow cytometry
analysis or sorting. Analyses and sorts were performed using a MoFlo3
FACS sorter.
CHD4 qPCR
Total RNA was isolated from wild-type or lethally irradiated animals and
from isolated X1 and X2 cells. cDNA was prepared and quantitative PCR
(qPCR) was performed using SYBR Green (Applied Biosystems). Data
were normalized to the expression of UDP (clone H.55.12e, accession
number AY068123). UDP was selected as an internal control as a
constitutively expressed gene. Sets of specific CHD4-primers were used to
evaluate gene expression (5⬘-GTCAGCAAAATGTTTACACTGAGG-3⬘
and 5⬘-ACGTCTTGCTTCATGTCTGATAGT-3⬘). Samples without reverse
transcriptase served as the negative control template.
RESULTS
CHD4 is required for planarian regeneration
We identified the S. mediterranea Smed-CHD4 gene
(chromodomain helicase DNA-binding protein 4) in a screen for
genes expressed in neoblasts after wounding (data not shown).
SMED-CHD4 is highly similar to proteins within the conserved
CHD family of chromatin regulatory proteins; by phylogenetic
analysis it clustered to the CHD3/4/5 members of the family, and
using BLAST we found that it is most similar to the mammalian
CHD4 (see Fig. S1 and Fig. S2 in the supplementary material).
CHD4/Mi-2 is a central component of the nucleosome remodeling
histone deacetylase complex NuRD (Tong et al., 1998; Wade et al.,
1998; Zhang et al., 1998). The Smed-CHD4 gene will be hereafter
referred to in the text in short as CHD4.
CHD4(RNAi) worms were unable to form a blastema following
amputation, demonstrating that CHD4 is required for planarian
regeneration (Fig. 1A). The inability to form a blastema was
noticeable as early as 5 days following initial double-stranded (ds)
RNA delivery (see Fig. S3 in the supplementary material).
Compared with other genes required for regeneration in planarians
(Reddien et al., 2005a), the onset of regeneration failure in
CHD4(RNAi) animals is very fast. The rapid appearance and
strength of the CHD4(RNAi) phenotype is consistent with the
possibility that CHD4 has a primary function in a crucial aspect of
neoblast function.
CHD4 was widely expressed in the planarian parenchyma (a
region containing neoblasts), brain and ventral nerve cords, as
determined with in situ hybridizations (Fig. 1B). CHD4 mRNA
was reduced in CHD4(RNAi) worms following dsRNA feeding
(see Fig. S4A in the supplementary material). A lethal dose of irradiation (6000 rads) depletes planarians of neoblasts,
eliminating the ability to regenerate and to replace aged cells
during homeostasis (Bardeen and Baetjer, 1904; Dubois, 1949;
DEVELOPMENT
directly by one of its subunits or indirectly by interaction with a
sequence-specific DNA-binding protein (Ahringer, 2000; Denslow
and Wade, 2007; Kim et al., 1999). The CHD4 protein belongs to
an evolutionarily conserved family and contains two plant
homeodomain (PHD) zinc-finger motifs, two chromodomains and
an ATPase/helicase domain of the SWI2/SNF family. The NuRD
complex, and in particular Mi-2, is required for normal development
and cell fate control in several organisms such as Caenorhabditis
elegans, Drosophila melanogaster, Arabidopsis thaliana and
mammals (Kehle et al., 1998; Ogas et al., 1997; Ogas et al., 1999;
Unhavaithaya et al., 2002; von Zelewsky et al., 2000; Yoshida et al.,
2008). The general role(s) for Mi-2 in adult stem cell biology has
remained unresolved.
Here, we show that Smed-CHD4 is essential for regeneration in
the planarian Schmidtea mediterranea. Many of the defects
observed in intact Smed-CHD4(RNAi) animals are hallmarks of
neoblast dysfunction. Our experiments indicate that Smed-CHD4 is
required for the ability of neoblasts to produce progeny cells
committed to differentiation, suggesting that chromatin regulation
by CHD4 is an important aspect of adult stem cell lineage
specification.
Development 137 (8)
Planarian stem cell differentiation
RESEARCH ARTICLE 1233
Fig. 1. CHD4 is required for planarian regeneration and is expressed in neoblasts. (A)As shown in comparison with a control animal, a
CHD4(RNAi) worm did not regenerate 7 days following amputation (41/41 were similar). Worms were fed three times and amputated at day 8
after initial RNAi. White dotted line represents the blastema boundary. Anterior is to the left. (B)Expression of CHD4 in wild-type and irradiated
(6000 rads 5 days prior) animals, dorsal and ventral views (7/7 were similar). Expression of an irradiation-insensitive gene, PC2 (expressed in the
nervous system) served as a control. Anterior is to the left. (C)Expression of CHD4 in neoblast subsets, X1 (1472 cells counted) and X2 (655 cells
counted), and radiation-insensitive cells, Xins (1117 cells counted). Scale bars: 0.1 mm.
CHD4 is required for tissue turnover
Neoblasts constantly proliferate in adult planarians in order to
replace aged tissues (Reddien and Sánchez Alvarado, 2004). To
investigate the role of CHD4 during normal tissue turnover, we
performed RNAi experiments in intact (i.e. non-amputated) animals.
Head regression is a characteristic sign of neoblast dysfunction that
occurs between 10 and 12 days following a lethal dose of irradiation
that eliminates all neoblasts. Head regression is also observed in
animals in which genes required for neoblast function have been
inhibited with RNAi (Guo et al., 2006; Reddien et al., 2005a;
Reddien et al., 2005b). Intact CHD4(RNAi) worms started to display
head regression 14 days after initial dsRNA feeding (Fig. 2A, middle
panel). Head regression in some intact CHD4(RNAi) animals started
in the center of the head instead of being initiated at the tip of the
head, as is typically the case for irradiated animals. Another welldocumented defect of irradiated animals is curling around the ventral
surface of the animal, probably stemming from the failure of
neoblast progeny to replace normally aging differentiated tissue
(Reddien and Sánchez Alvarado, 2004). Curling occurs by
approximately 14 days following irradiation. Following head
regression, intact CHD4(RNAi) animals also displayed curling
around their ventral surface (Fig. 2A, right panel). In addition to
these classic hallmarks of neoblast dysfunction, intact CHD4(RNAi)
worms showed tissue protrusions after 16-18 days following the first
dsRNA feeding (Fig. 2A, right panel). These protrusions will be
discussed below. Shortly after curling, the CHD4(RNAi) animals
died by lysis. Together, these observations demonstrate that CHD4
is required for normal tissue homeostasis.
CHD4 is required for an increase in neoblast
proliferation in response to feeding and
wounding
One simple hypothesis to explain the similarity of the CHD4(RNAi)
phenotype to that of irradiated animals lacking neoblasts would be that
CHD4(RNAi) causes a decrease in the presence of neoblasts or in the
capacity of neoblasts to proliferate. To test this hypothesis, we
quantified the number of mitotic neoblasts (Newmark and Sánchez
DEVELOPMENT
Reddien et al., 2005b). Five days after lethal irradiation,
expression of CHD4 in the nervous system persisted but the
expression in the parenchyma was reduced, suggesting that CHD4
was expressed in neoblasts or in their immediate descendants
(Fig. 1B). Expression of non-neoblast genes remained unchanged
following irradiation (Fig. 1B; see Fig. S4B in the supplementary
material).
Neoblasts are actively dividing cells that are required for the
production of all cells of the adult organism (Reddien and
Sánchez Alvarado, 2004). Studies to uncover their lineage
potential are emerging (Eisenhoffer et al., 2008). Flow cytometry
has been used to isolate cells with neoblast properties (Hayashi et
al., 2006; Reddien et al., 2005b). Using vital dyes, two
populations of irradiation-sensitive cells can be purified. X1 cells
are actively dividing cells (in S and G2/M stages of the cell cycle)
with a higher DNA content (>2n); X2 cells contain postmitotic
neoblast descendants (Eisenhoffer et al., 2008) and might contain
neoblasts in the G1 stage of the cell cycle as well. A population of
irradiation-insensitive cells, Xins, has been used as a control
population in flow cytometry experiments (Hayashi et al., 2006;
Reddien et al., 2005b). To determine whether CHD4 was
expressed in neoblasts, we performed in situ hybridizations with
X1, X2 and Xins cells. CHD4 was indeed expressed in neoblasts
as well as in presumptive differentiated cells: 53% of X1 cells,
78% of X2 cells and 69% of Xins cells were labeled with the
CHD4 riboprobe (Fig. 1C). We further confirmed that CHD4 is
expressed in neoblasts using quantitative PCR (qPCR; see Fig.
S4C in the supplementary material). Moreover, CHD4 was highly
expressed at wound sites 3 days following amputation and this
expression was not detected in irradiated worms that lack
neoblasts (see Fig. S4D in the supplementary material). It is not
known whether CHD4 expression was upregulated following
wounding or whether the high abundance of CHD4 expression
near wounds was due to migration of cells that already expressed
this gene. Because irradiation eliminates neoblasts, this result
suggests that neoblasts and/or their progeny near wounds express
CHD4 during regeneration.
1234 RESEARCH ARTICLE
Development 137 (8)
Alvarado, 2000) in intact animals using an RNAi feeding approach
(see Materials and methods). Food stimulates neoblast proliferation,
and proliferation returns to the previous steady-state levels a few days
later (Baguñà, 1976). The elevation of mitotic numbers immediately
following feeding failed to robustly occur in CHD4(RNAi) animals
(days 7 and 9 after RNAi; Fig. 2B). However, steady-state numbers
of mitotic cells were comparable with those of control animals,
including at time points after RNAi at which animals completely
failed to regenerate (between days 12 and 16 following the first RNAi
feeding), suggesting that neoblasts were maintained in CHD4(RNAi)
animals (Fig. 2B). Much later, when severe head regression and
curling was occurring (approximately between 16-18 days following
the first dsRNA feeding), a significant decrease in mitotic cells was
observed (Fig. 2B). A drop in neoblast population numbers late in the
progression of an RNAi phenotype has also been observed following
RNAi of a different gene (smedwi-2) required for homeostasis but not
for neoblast maintenance (Reddien et al., 2005b). Therefore, this late
mitotic decrease might be a consequence of, rather than cause of,
failed homeostasis.
Because the number of steady-state mitotic neoblasts was normal
at time points at which regeneration failed in CHD4(RNAi) animals,
we sought to determine whether the total number of neoblasts was
unaffected. Indeed, approximately normal numbers of X1 and X2
cells following RNAi were present at all time points examined,
except near the time at which animals were dying (Fig. 2C). A
transient decrease in the number of X2 cells at day 11 following the
first dsRNA feeding was observed. Because X2 cells appear to
include non-dividing neoblasts as well as neoblast progeny
(Eisenhoffer et al., 2008), a defect in neoblast proliferation following
feeding could explain this slight decrease in X2 cells. Moreover, the
gross expression of markers of X1 cells, smedwi-1 and Smed-histone
H2B (Smed-H2B), appeared normal in CHD4(RNAi) animals 10
days after the initial dsRNA feeding (Fig. 2D; see Fig. S5 in the
supplementary material). Together, these observations indicate that
the regeneration defect of CHD4(RNAi) animals is not a
consequence of a loss of neoblasts or a loss of their capacity to
proliferate during homeostasis prior to injury.
In addition to the sustained neoblast proliferation in intact
planarians that is required for replacement of aging cells during
normal turnover, neoblasts undergo an increase in proliferation after
wounding (Saló and Baguñà, 1984). We found that although
neoblasts in CHD4(RNAi) worms were able to respond to wounding
by elevating mitotic numbers at 48 hours and 96 hours following
amputation, the response was diminished compared with control
DEVELOPMENT
Fig. 2. CHD4 is required for homeostasis and normal stimulation of neoblast proliferation. (A)Control and CHD4(RNAi) animals during
normal tissue turnover 14 and 18 days after initial RNAi. Yellow arrowhead, head regression; white arrowhead, lesions; red arrowhead, curling (10/10
were similar). Anterior is to the left. (B)Numbers of mitoses labeled with an anti-phospho histone 3 (H3P) antibody were divided by animal surface
area. Animals were fed three times and fixed at time points after initial RNAi. ‘+ food’ indicates the proliferation response following feedings. Data
are means ± s.e.m. (n2 experiments, >8 worms per time point). *, P<0.05; **, P<0.001, ***, P<0.0001, Student’s t-test. (C)Animals were fed three
times with dsRNA and cell macerates were generated at multiple time points after initial RNAi. Cells were labeled with Hoechst 33342, calcein, and
propidium iodide; X1 and X2 cell percentages (in the total population of Hoechst-labeled cells) were determined by flow cytometry. Data are means ±
s.e.m. (n3 experiments). *, P<0.05; **, P<0.001, Student’s t-test. (D)Fluorescence in situ hybridizations of animals fixed 10 days after multiple
dsRNA feedings using the smedwi-1 riboprobe (>12 worms per condition were similar). pr, photoreceptors. Anterior is up. (E)Numbers of mitoses
labeled with H3P were divided by animal surface area. Animals were fed three times, amputated 8 days after initial feeding and fixed at different
times following wounding. Data are means ± s.e.m.; n2 experiments, >8 animals per time point. ***, P<0.0001, Student’s t-test. Scale bars: 0.1 mm.
Planarian stem cell differentiation
RESEARCH ARTICLE 1235
Fig. 3. CHD4(RNAi) intact animals
die faster following irradiation.
(A)Control and CHD4(RNAi) animals
were lethally irradiated with 6000
rads 8 days following initial RNAi.
Irradiated control(RNAi) animals
showed head regression by 12 days
following irradiation (7/10 animals)
and curling by 14 days (10/10),
whereas irradiated CHD4(RNAi)
animals showed head regression by 6
days following irradiation (10/10) and
curling by 8 days (10/10). Yellow
arrowhead, head regression; red
arrowhead, curling. Anterior is to the
left. (B)Percentages of control or
CHD4(RNAi) animals without head
regression (left) and still alive (right)
following irradiation are shown. Scale
bars: 0.1 mm.
CHD4(RNAi) animals die more rapidly than do
normal animals following lethal irradiation
Lethally irradiated and intact wild-type worms develop the first
signs of defects due to neoblast loss approximately 10-12 days after
irradiation (Reddien et al., 2005b). This phenotype involves
darkening of the tip of the head, followed by head regression. A few
days later, the worms curl around their ventral surface and finally
they lyse. Although actively dividing neoblasts completely
disappear within 1 day after irradiation, the initial 10- to 12-day
window during which no phenotype is apparent might in part rely
on the replacement of normally dying aged differentiated cells by
existing non-dividing progenitors (Reddien and Sánchez Alvarado,
2004). Therefore, the time of survival after irradiation could be
affected by alterations in numbers or function of neoblast progeny
cells that can act as progenitors of differentiated cells. We lethally
irradiated control and CHD4(RNAi) worms 8 days after the initial
dsRNA feeding. As expected, irradiated control(RNAi) worms
showed head regression by 12 days following irradiation (Fig. 3A,B,
left graph), followed by curling and lysis (Fig. 3A,B, right graph).
By contrast, irradiated CHD4(RNAi) animals displayed head
regression 6 days following irradiation, and curled and lysed 8 days
after irradiation (Fig. 3). The rate of appearance of defects in
irradiated CHD4(RNAi) animals was faster than that observed in
starved or fed irradiated wild-type or control(RNAi) worms (Reddien
et al., 2005b) (Fig. 3). Most of the non-irradiated CHD4(RNAi)
animals showed head regression by 12 days after the irradiation day
(Fig. 3B, left graph) and they curled and lysed between days 20 and
28 after the irradiation day (Fig. 3B, right graph). Similar results
were obtained with CHD4(RNAi) animals irradiated at 10 days and
12 days after the initial dsRNA feeding, times at which normal
steady-state neoblast numbers were reached following feeding (data
not shown). This indicates that the faster appearance of the
irradiation phenotype in CHD4(RNAi) animals is not a consequence
of the failure to boost proliferation immediately after feeding. The
rapid appearance of head regression following irradiation of
CHD4(RNAi) animals suggests a possible decreased number of nondividing progeny cells that are neoblast descendants or impaired
neoblast differentiation. We therefore directly examined neoblast
progeny cells in CHD4(RNAi) animals (see below).
CHD4(RNAi) animals have decreased numbers of
Smed-AGAT-1-expressing cells
Previous studies identified three irradiation-sensitive, lineagerelated cell subsets (Eisenhoffer et al., 2008) (Fig. 4A). During
neoblast differentiation, Category 1 gene-expressing cells
differentiate into cells expressing Category 2 and 3 genes
(Eisenhoffer et al., 2008) (Fig. 4A). Category 1 gene-expressing
cells are self-renewing neoblasts (i.e. X1 cells) (Eisenhoffer et al.,
2008), which are found in the parenchyma but excluded from the
region anterior to the photoreceptors and the pharynx (Newmark and
Sánchez Alvarado, 2000). Category 1 gene-expressing cells
disappear 1 day after irradiation. Category 1 genes include the
known stem cell genes smedwi-1 and Smed-bruli-1, as well as cell
cycle regulatory genes (Eisenhoffer et al., 2008; Guo et al., 2006;
Reddien et al., 2005b). Genes from Categories 2 and 3 are expressed
in X2 and Xins cells but not X1 cells, and are found anterior to the
photoreceptors and close to the animal margins (regions where no
mitotic cells are found). Similar to the case for Category 1 genes, the
cells expressing Category 2 genes (e.g. the two novel genes SmedNB.21.11e and Smed-NB.32.1g) rapidly decrease in number 1 day
following irradiation (Eisenhoffer et al., 2008). Cells expressing
Category 3 genes are lost within 7 days following irradiation.
DEVELOPMENT
RNAi animals (Fig. 2E). Because neoblast numbers in CHD4(RNAi)
animals were normal in steady-state, a partially defective
proliferative response to injury is probably not sufficient to explain
the complete absence of a blastema in CHD4(RNAi) worms. For
example, prior RNAi screening indicates that animals with reduced,
but not eliminated, mitoses can be capable of making small
blastemas (Reddien et al., 2005a). Thus, given the severe blastema
failure of CHD4(RNAi) animals, it is probable that an additional and
robust defect exists to explain the regeneration phenotype. We
therefore examined the ability of neoblasts in CHD4(RNAi) animals
to produce progeny cells.
1236 RESEARCH ARTICLE
Development 137 (8)
Category 3 genes include those that encode L-arginine:glycine
amidinotransferase (Smed-AGAT)-1, -2 and -3, a ras-related protein
family member (Smed-ras-related), a mitochondrial carrier protein
1 (Smed-MCP-1) and a novel protein (Smed-NB.52.12f), among
others. Based on BrdU labeling experiments, Category 2 and 3 geneexpressing cells are rapidly produced descendants of neoblasts
(Eisenhoffer et al., 2008). Although the precise nature of these cells
is still unclear, they might either represent a population of nonmitotic progenitors that will generate terminal differentiated cells or
a very short-lived population of differentiated cells. Regardless,
these markers provide a useful tool to study neoblast progeny cells
and, therefore, neoblast differentiation.
We utilized expression of these genes as markers for distinct cell
types to determine if CHD4(RNAi) animals have a defect in the
production or maintenance of neoblast progeny cells. We examined
the expression of the Category 2 gene Smed-NB.21.11e and the
Category 3 gene Smed-AGAT-1 by in situ hybridizations.
CHD4(RNAi) worms had approximately normal numbers of Smed-
NB.21.11e-expressing cells (Fig. 4B,C; see Fig. S6 in the
supplementary material). However, there was a marked decrease in
the number of Category 3 Smed-AGAT-1-expressing cells in
CHD4(RNAi) worms, as early as 6 days after initial dsRNA feeding
(Fig. 4B,C; see Fig. S6 in the supplementary material).
We quantified numbers of Category 1, 2 and 3 cell types by
performing in situ hybridizations on total cells from macerated
planarians and counting the fraction of total cells that expressed
smedwi-1 (Category 1), Smed-NB.21.11e (Category 2) or SmedAGAT-1 (Category 3). CHD4(RNAi) animals had significantly
reduced numbers of Smed-AGAT-1-expressing cells at every time
point analyzed and largely normal numbers of Smed-NB.21.11eexpressing cells (Fig. 4C). There were lower numbers of SmedNB.21.11e-expressing cells at 11 days following RNAi (Fig. 4C).
The small defects in the numbers of Category 2-expressing cells
observed were possibly due to defects in responses to feeding used
in the RNAi experiments. smedwi-1-expressing cells were present
in CHD4(RNAi) animals in approximately normal numbers initially,
DEVELOPMENT
Fig. 4. CHD4(RNAi) animals have
reduced numbers of Smed-AGAT-1expressing cells. (A)Lineage-related cells
and the markers expressed in each cell
type. Other models are possible, see
Eisenhoffer et al. (Eisenhoffer et al., 2008).
(B)Head regions of whole-mount in situ
hybridizations using a Category 2 probe
Smed-NB.21.11e (upper row) and a
Category 3 probe Smed-AGAT-1 (lower
row) of control and CHD4(RNAi) animals
at several time points following initial
RNAi (>4 worms/time point displayed
similar results). Anterior is to the left.
(C)Cell in situ hybridizations using a
Category 1 probe (smedwi-1; >823 cells
per time point), a Category 2 probe
(Smed-NB.21.11e; >954 cells per time
point) and a Category 3 probe (SmedAGAT-1; >1157 cells per time point) of
control and CHD4(RNAi) worm macerates
at multiple times after initial RNAi.
Percentages of smedwi-1+, SmedNB.21.11e+, and Smed-AGAT-1+ cells
within the total DAPI-positive cell
population are shown (means ± s.e.m.,
n3 experiments). *, P<0.05; **,
P<0.001, Student’s t-test. (D)Wholemount in situ hybridizations with Category
1 probes (Smed-H2B and smedwi-1), a
Category 2 probe (Smed-NB.21.11e) and
a Category 3 probe (Smed-AGAT-1) of
control and CHD4(RNAi) animals 20 days
following initial RNAi. At this late time
point, CHD4(RNAi) animals displayed head
regression, had lesions and began to curl
(>7 worms displayed similar results).
Anterior is to the left. (E)Whole-mount in
situ hybridizations with the Smed-AGAT-1
riboprobe of control and CHD4(RNAi)
head, trunk and tail pieces fixed 3 days
following amputation. Animals were
amputated 7 days following initial RNAi
(>11 regenerating pieces displayed similar
results). Arrows indicate the site of
amputation. Anterior is to the left. Scale
bars: 0.1 mm.
elevated in number at day 11 after the first dsRNA feeding, and low
in number late following RNAi (Fig. 4C). The increased number of
smedwi-1-expressing cells in CHD4(RNAi) animals at day 11,
considered together with the associated decrease in Category 3expressing cells, is consistent with a possible accumulation of
smedwi-1-expressing cells because of failure to differentiate.
Overall, the earliest, most prominent and most constant abnormality
observed in CHD4(RNAi) animals was the pronounced low number
of Smed-AGAT-1-expresssing cells.
Twenty days after the initial dsRNA feeding, when the
CHD4(RNAi) animals displayed head regression and were dying,
the animals were mostly depleted of Smed-NB.21.11e-expressing
cells and entirely lacked Smed-AGAT-1-expressing cells (Fig. 4D).
At this late time point, similar to the observed decrease in mitotic
cells (Fig. 2B), smedwi-1-expressing cells and Smed-H2Bexpressing cells were greatly reduced as well (Fig. 4D). Because this
defect occurred late following RNAi, it probably occurred as a
consequence of an earlier primary defect in the number of neoblast
progeny cells. At this 20-day time point, CHD4(RNAi) animals had
protrusions; however, we saw no accumulation of dividing cells,
Smed-NB.21.11e or Smed-AGAT-1-expressing cells into foci that
could correspond to protrusions (Fig. 4D).
Smed-AGAT-1-expressing cells normally accumulate at the
wound site and in blastemas after amputation, providing evidence
for an important role for this population in regeneration (Eisenhoffer
et al., 2008). Perhaps as a consequence of the reduced number of
Smed-AGAT-1-expressing cells, CHD4(RNAi) animals failed to
display accumulation of Smed-AGAT-1-expressing cells at wound
sites to the extent observed in the control (Fig. 4E). Our data suggest
that a failure to produce sufficient numbers of neoblast progeny cells
committed to differentiation, such as Smed-AGAT-1-expressing
cells, results in insufficient neoblast progeny for blastema formation
in CHD4(RNAi) animals.
CHD4 is required for the formation of Smed-AGAT1-expressing cells
The decreased numbers of Smed-AGAT-1-expressing cells in
CHD4(RNAi) animals suggests that CHD4 is required either for the
normal generation or maintenance of these cells. First, in order to
determine if the CHD4(RNAi) phenotype is explained by a defect in
the maintenance of these cells, we determined the rate at which
Smed-AGAT-1-expressing cells disappeared following irradiation.
Irradiation kills neoblasts and therefore blocks the generation of new
Smed-AGAT-1-expressing cells. Animals were fed only once with
CHD4 dsRNA or control food and irradiated the same day (Fig. 5A,
left graph), or fed 3 times over 7 days and irradiated 7 days following
the initial feeding (Fig. 5A, right graph). The numbers of SmedAGAT-1-expressing cells were determined at different time points
following irradiation. In both experiments, Smed-AGAT-1expressing cells disappeared at similar rates following irradiation in
both control and CHD4(RNAi) animals, suggesting that the
CHD4(RNAi) phenotype is not explained by a defect in the
maintenance of these cells. Therefore, these data point to a defect in
the generation of Smed-AGAT-1-expressing cells.
To determine if CHD4 is required for the generation of SmedAGAT-1-expressing cells directly, two different experiments were
performed. First, the formation of Smed-AGAT-1-expressing cells in
CHD4(RNAi) or control animals following one pulse with the
thymidine analog BrdU was observed (see Fig. S7 in the
supplementary material). BrdU is incorporated into neoblasts within
a few hours following injection (Newmark and Sánchez Alvarado,
2000), and 4 days later, double-labeled BrdU-Smed-AGAT-1-
RESEARCH ARTICLE 1237
expressing cells are found (Eisenhoffer et al., 2008). Four days after
the BrdU pulse, most of the Smed-AGAT-1-expressing cells were
labeled with BrdU (see Fig. S7 in the supplementary material). We
found that the number of newly formed Smed-AGAT-1-expressing
cells was significantly lower in CHD4(RNAi) animals compared
with control animals 4 days after the BrdU pulse and 10 days
following the initial RNAi feeding (see Fig. S7 in the supplementary
material).
Second, we determined the number of SMEDWI-1; Smed-AGAT1 double-positive cells in CHD4(RNAi) and control animals. The
SMEDWI-1 protein has been demonstrated to be present not only in
neoblasts, which also express smedwi-1 mRNA, but also in nonmitotic neoblast progeny that no longer express smedwi-1 mRNA
(Guo et al., 2006). This is presumably due to perdurance of the
SMEDWI-1 protein into immediate neoblast descendants.
Moreover, three days following lethal irradiation, all SMEDWI-1expressing cells disappeared from the animal, indicating that only
neoblasts or their immediate progeny cells express this protein (Guo
et al., 2006). We found that CHD4(RNAi) animals have a decreased
number of SMEDWI-1; Smed-AGAT-1 double-positive cells at 7
days following initial RNAi and an even greater defect at 10 days
following the initial RNAi feeding (Fig. 5B). In summary, these
experiments demonstrate that the CHD4(RNAi) phenotype is
explained by a defect in the generation of Smed-AGAT-1-expressing
cells rather than by a defect in their perdurance.
If neoblasts are defective in production of Smed-AGAT-1-positive
cells, where is the process of differentiation affected? We found a
transient increase in the number of SMEDWI-1 protein-expressing
cells in animal head regions in CHD4(RNAi) animals at day 7
following RNAi (Fig. 5C). These SMEDWI-1 protein-positive cells
in head tips represent immediate non-mitotic neoblast progeny.
Therefore, coincident with a decrease in Smed-AGAT-1-positive
cells, there existed an increase in number of immediate neoblast
progeny. This observation indicates that immediate neoblast
progeny might accumulate because of a failure to differentiate.
CHD4(RNAi) animals have a decreased number of
neoblast progeny cells
To more definitively determine the role of CHD4 in neoblast
differentiation, and to exclude any potential role of CHD4 in
specifically regulating Smed-AGAT-1 expression, we examined the
expression of three additional Category 3 markers in CHD4(RNAi)
animals (Eisenhoffer et al., 2008). CHD4(RNAi) animals also had a
robust decrease in the number of cells expressing the Smed-rasrelated, Smed-MCP-1 and Smed-NB.52.12f genes (Fig. 6A,B).
However, the existing cells that expressed these genes displayed no
obvious defect in expression levels for these genes. Previous data
have shown that not all Smed-AGAT-1-expressing cells co-express
the Smed-ras-related gene (Eisenhoffer et al., 2008). In addition,
almost no Smed-AGAT-1-expressing cells co-express the SmedMCP-1 gene (Eisenhoffer et al., 2008), indicating that there is a
heterogeneous population of neoblast progeny cells expressing
Category 3 genes. Our results demonstrate that CHD4 is required for
the generation of a heterogeneous population of neoblast progeny
cells committed to differentiation.
DISCUSSION
Planarians are an emerging in vivo model system for the study of
mechanisms that regulate stem cell proliferation, maintenance and
differentiation because of their remarkable regenerative capacities
and extensive tissue turnover. These two processes are driven by the
numerous neoblasts that reside throughout the animal (Reddien and
DEVELOPMENT
Planarian stem cell differentiation
1238 RESEARCH ARTICLE
Development 137 (8)
Sánchez Alvarado, 2004). The use of adult stem cells to replace aged
or damaged cells and tissues is essential for most metazoans but is
still poorly understood at the molecular level.
To date, very few planarian genes have been described to have a
function crucial for neoblast biology. Two examples are the Smedbruli-1 and smedwi-2 genes (Guo et al., 2006; Reddien et al., 2005b).
Smed-bruli-1 is not needed for neoblast proliferation in response to
wounding and differentiation. Instead, Smed-bruli-1 has been shown
to play a role in neoblast self-renewal. Smed-bruli(RNAi) animals
are gradually depleted of neoblasts and are therefore unable to
regenerate and survive during homeostasis (Guo et al., 2006). By
contrast, smedwi-2 is not required for neoblast maintenance but
instead is needed for the production of cells capable of replacing
aged differentiated cells. In smedwi-2(RNAi) animals, aged
differentiated cells are not replaced during homeostasis and
regeneration, resulting in the incapacity of these animals to survive
and regenerate missing tissues (Reddien et al., 2005b). Few genes
have yet been described to have a crucial role in the production of
differentiation-committed neoblast progeny cells (Pearson and
Sánchez Alvarado, 2010).
The usage of RNAi and lineage markers helps to establish S.
mediterranea as a powerful in vivo model system for the study of
stem cell proliferation and lineage differentiation during tissue
homeostasis and regeneration. Here, we show the existence of a
DEVELOPMENT
Fig. 5. CHD4 is required for the formation of Smed-AGAT-1-expressing cells. (A)Smed-AGAT-1+ cell numbers normalized by animal length
from whole-mount in situ hybridizations are shown at time points following irradiation. Animals (>4 worms per group) were fed once and lethally
irradiated the same day (left) or fed three times and lethally irradiated at day 7 (right). Data are means ± s.e.m. (B)Percentage of double-labeled
SMEDWI-1; Smed-AGAT-1-expressing cells in the total number of Smed-AGAT-1+ cells in control and CHD4(RNAi) animals (upper graph), and the
percentage of double-labeled SMEDWI-1; Smed-AGAT-1-expressing cells normalized by the area of the head region (lower graph). Data are means
± s.e.m. (>5 worms per group). *, P<0.05; **, P<0.001; ***, P<0.0001, Student’s t-test. Animals were fed twice and fixed at time points following
the initial RNAi. Fluorescence in situ hybridizations using Smed-AGAT-1 riboprobe (green) and SMEDWI-1 antibody (red) are shown. Arrowheads
indicate cells that co-express Smed-AGAT-1 and SMEDWI-1. (C)SMEDWI-1-expressing cell numbers in control and CHD4(RNAi) animals.
pr, photoreceptors. Circled area, region counted. Data are means ± s.e.m. (>8 worms per group). **, P<0.001, Student’s t-test. A second
experiment had similar results. Animals were fed twice and fixed at time points following initial RNAi. Anterior is up. Scale bars: 0.1 mm.
Planarian stem cell differentiation
RESEARCH ARTICLE 1239
gene that is required for the transition of neoblasts from selfrenewing stem cells into more committed non-dividing cells during
regeneration and homeostasis.
We found that a CHD4-like gene is required for planarian
regeneration and neoblast differentiation. Numerous abnormalities
were observed in CHD4(RNAi) animals. These included a robust
block in regeneration, failure in the maintenance of differentiated
tissues (which normally requires the neoblasts) and ultimately
lesions/protrusions in the body of the RNAi animals before animal
death. The explanation for the protrusions is unknown. Our data
indicate that these protrusions are not explained by an accumulation
of proliferating cells or any known neoblast progeny cells. Therefore,
this defect might reflect a requirement for CHD4 in differentiated
tissue or the aberrant action of unidentified neoblast progeny cells.
CHD4(RNAi) animals cannot regenerate as early as 5 days
following the initial dsRNA feeding (see Fig. S8 in the
supplementary material). This failure to regenerate occurs rapidly
following RNAi when compared with worms where neoblast
function has been disrupted by RNAi of other genes (Reddien et al.,
2005b). The inability of CHD4(RNAi) animals to regenerate and to
replace normally aging differentiated tissue cannot be explained by
a loss of neoblasts because these animals initially have normal or
slightly higher numbers of actively dividing neoblasts (smedwi-1expressing cells). By contrast, our data indicate that the failure to
regenerate and to maintain normal tissue turnover is due to a marked
reduction in numbers of neoblast progeny cells (such as cells
expressing Category 3 genes). Furthermore, our results indicate that
the reduced number of neoblast progeny cells found in CHD4(RNAi)
animals was due to a defect in the generation of these cells as
opposed to a defect in their maintenance. The neoblasts of
CHD4(RNAi) animals produced immediate progeny cells (not
expressing Category 3 genes) that transiently accumulated,
presumably as a consequence of the differentiation failure. Because
no other markers besides the Categories 2 and 3 markers used in this
study have been identified to be expressed in neoblast progeny cells,
and no accumulation of germ cells has been observed in
CHD4(RNAi) animals (not shown), it remains unresolved if
neoblasts in CHD4(RNAi) animals differentiate into a particular
lineage different from the ones tested here. At least some neoblast
progeny cells have been shown to localize to the blastema
(Eisenhoffer et al., 2008); however, perhaps because of the limited
number of these cells in CHD4(RNAi) animals, neoblast progeny
cells did not localize to the wound sites in appropriate numbers.
These observations, together with the finding that CHD4 is
expressed near the wound site in neoblasts or neoblast progeny cells
3 days following amputation, suggest a crucial role for CHD4 early
in the regeneration process.
Changes in chromatin structure have been implicated in the
regulation and maintenance of many cell fate decisions. Chromatin
modifiers can be essential to keep stem cells in a pluripotent stage
or to drive them into a differentiation pathway. For example, in the
case of mouse and human embryonic stem (ES) cells, components
of the polycomb complex can silence developmental regulatory
genes that are preferentially expressed during differentiation (Boyer
et al., 2006; Lee et al., 2006). These results suggest that polycomb
proteins maintain the pluripotent state of ES cells. Ezh, a component
of the PRC2 polycomb complex, is also active in epidermal
progenitors (Ezhkova et al., 2009). However, in general, how the
regulation of differentiation in ES cells corresponds to what occurs
in adult stem cell types in vivo remains unresolved. In the case of the
NuRD complex component CHD4, our data indicate a role in
promoting, rather than repressing, stem cell differentiation. The
NuRD complex has been shown to regulate cell fate decisions and
development in multiple contexts. In D. melanogaster for example,
Mi-2 can function with polycomb genes to maintain repression of
homeotic genes during embryonic patterning (Kehle et al., 1998). In
C. elegans, the CHD4 homolog let-418, is required for vulval cell
fate determination and for maintenance of germline-soma
distinctions (Unhavaithaya et al., 2002; von Zelewsky et al., 2000).
Specifically, in let-418-defective animals, somatic cells express
germ cell markers (e.g. the P granule component PGL-1 and Vasa
homologs), indicating that a CHD4 protein in C. elegans is required
to repress germline cell identity in differentiated cells. Interestingly,
a CHD4 homolog in the plant A. thaliana, pickle, is required for
repression of embryonic-like characteristics after germination (Ogas
et al., 1997; Ogas et al., 1999). In pickle mutants, the fatty acid
composition of roots was shown to resemble that of seeds, and roots
formed callus growths and embryo-like physical structures.
DEVELOPMENT
Fig. 6. CHD4(RNAi) animals have
decreased numbers of several
Category 3-expressing cells.
(A)Whole-mount in situ hybridizations
using Category 3 probes 10 days
following initial RNAi (>4 worms per
riboprobe were similar). Anterior is to
the left. (B)Fluorescence in situ
hybridizations of animals fixed 10 days
after multiple dsRNA feedings using
the Smed-NB.21.11e, Smed-AGAT-1,
Smed-MCP-1 and Smed-NB.52.12f
riboprobes (>12 worms per condition
were similar). The head region is
shown. pr, photoreceptors. Anterior is
up. Scale bars: 0.1 mm.
Therefore, in both C. elegans and Arabidopsis, these data suggest a
function in repression of germ cell/embryonic-like features in
differentiated tissue. It will be interesting to determine in the future
how these roles compare with the action of CHD4 in stem cells in
other organisms. For instance, it is possible that CHD4 represses
stem cell/embryonic-like genes in differentiated tissues in these
organisms and in planarian neoblasts to promote neoblast
differentiation.
In the mouse, knockout of Mi-2 (CHD4) has complex impacts
on the functioning of hematopoietic stem cells (HSCs) (Yoshida et
al., 2008). Mi-2-deficient HSCs displayed increased proliferation
and were able to begin differentiation into erythroid, but not myeloid
and lymphoid, lineages. Specifically, conditional knockout Mi-2
animals showed depletion of granulocytes (myeloid) and newly
formed B cells (lymphocytes). Furthermore, although proerythroblasts were formed, the differentiation of these proerythroblasts into basophilic and other cells was aberrant in these
mice. Therefore, in the case of this stem cell type, Mi-2 appears to
be required for lineage decisions that could reflect a role in
differentiation. Expression analyses indicated that Mi-2 did not
play a global role in gene expression regulation but affected specific
transcripts. The gene expression defects in these Mi-2-deficient
HSCs also indicated a potential additional role for Mi-2 in selfrenewal. A different component of the NuRD complex, Mbd3, was
found to be required for ES cells to differentiate in vitro (Kaji et al.,
2006); Mbd3 was also required for proliferation of epiblast cells in
culture and derivation of ES cells from inner-cell-mass cells (Kaji et
al., 2007). The data from adult planarian neoblasts described here,
taken in the context of the results describing the function of CHD4
and other NuRD components in other organisms, suggest that a
major and broadly utilized role for this complex is in promoting stem
cell differentiation.
Acknowledgements
We thank Laurie Boyer, Ezequiel Alvarez-Saavedra, Daniel Wagner and all
members of the Reddien lab for manuscript comments, support and
discussions. We thank Danielle Wenemoser for collaboration with microarray
experiments and the SMEDWI-1 antibody. This work was supported by NIH
R01GM080639. We acknowledge support by the Keck, Smith, Searle and Rita
Allen Foundations. M.L.S. was supported by a Jane Coffin Childs Memorial
Fund Fellowship. P.W.R. is an Early Career Scientist of the Howard Hughes
Medical Institute and holds a Thomas D. and Virginia W. Cabot Career
Development Professorship. Deposited in PMC for release after 6 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.042051/-/DC1
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DEVELOPMENT
Planarian stem cell differentiation