Adult somatic stem cells in the human parasite Schistosoma mansoni

Abstract

Schistosomiasis is among the most prevalent human parasitic diseases, affecting more than 200 million people worldwide. The aetiological agents of this disease are trematode flatworms (Schistosoma) that live and lay eggs within the vasculature of the host. These eggs lodge in host tissues, causing inflammatory responses that are the primary cause of morbidity. Because these parasites can live and reproduce within human hosts for decades, elucidating the mechanisms that promote their longevity is of fundamental importance. Although adult pluripotent stem cells, called neoblasts, drive long-term homeostatic tissue maintenance in long-lived free-living flatworms (for example, planarians), and neoblast-like cells have been described in some parasitic tapeworms, little is known about whether similar cell types exist in any trematode species. Here we describe a population of neoblast-like cells in the trematode Schistosoma mansoni. These cells resemble planarian neoblasts morphologically and share their ability to proliferate and differentiate into derivatives of multiple germ layers. Capitalizing on available genomic resources and RNA-seq-based gene expression profiling, we find that these schistosome neoblast-like cells express a fibroblast growth factor receptor orthologue. Using RNA interference we demonstrate that this gene is required for the maintenance of these neoblast-like cells. Our observations indicate that adaptation of developmental strategies shared by free-living ancestors to modern-day schistosomes probably contributed to the success of these animals as long-lived obligate parasites. We expect that future studies deciphering the function of these neoblast-like cells will have important implications for understanding the biology of these devastating parasites.

Full text

Adult somatic stem cells in the human parasite, Schistosoma
mansoni
James J. Collins III1,2, Bo Wang1,3, Bramwell G. Lambrus1, Marla Tharp1, Harini Iyer1, and
Phillip A. Newmark1,2,*
1Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University
of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
2Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
3Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801,
USA
Summary
Schistosomiasis is among the most prevalent human parasitic diseases, affecting more than 200
million people worldwide1. The etiological agents of this disease are trematode flatworms
(
Schistosoma
) that live and lay eggs within the vasculature of the host. These eggs lodge in host
tissues, causing inflammatory responses that are the primary cause of morbidity. Because these
parasites can live and reproduce within human hosts for decades2, elucidating the mechanisms that
promote their longevity is of fundamental importance. Although adult pluripotent stem cells,
called neoblasts, drive long-term homeostatic tissue maintenance in long-lived free-living
flatworms3,4 (e.g., planarians), and neoblast-like cells have been described in some parasitic
tapeworms5, little is known about whether similar cell types exist in any trematode species. Here,
we describe a population of neoblast-like cells in the trematode
Schistosoma mansoni
. These cells
resemble planarian neoblasts morphologically and share their ability to proliferate and
differentiate into derivatives of multiple germ layers. Capitalizing on available genomic
resources6,7 and RNAseq-based gene expression profiling, we find that these schistosome
neoblast-like cells express a
fibroblast growth factor receptor
ortholog. Using RNA interference
we demonstrate that this gene is required for the maintenance of these neoblast-like cells. Our
observations suggest that adaptation of developmental strategies shared by free-living ancestors to
modern-day schistosomes likely contributed to the success of these animals as long-lived obligate
parasites. We expect that future studies deciphering the function of these neoblast-like cells will
have important implications for understanding the biology of these devastating parasites.
Although classic studies of cell proliferation in
Schistosoma
focused on reproductive
tissues8,9, Den Hollander and Erasmus did note occasional “undifferentiated” somatic cells
that incorporated tritiated thymidine in adult parasites. Encouraged that these cells could
*Corresponding author: pnewmark@life.illinois.edu.
Full Methods and any associated references are available in the online version of the paper.
Supplementary Information is linked to the online version of the paper at www.nature.com/nature
Author Contributions J.J.C., B.W., B.G.L., M.T., H.I., performed experiments. J.J.C. and B.W. analyzed data. J.J.C. and P.A.N.
designed the study and wrote the paper.
Author Information Experiments with and care of vertebrate animals were performed in accordance with protocols approved by the
Institutional Animal Care and Use Committee (IACUC) of the University of Illinois at Urbana-Champaign (protocol approval number
10035). RNAseq analyses have been deposited in the NCBI Gene Expression Omnibus (Accession number: GSE42757). The authors
declare no competing financial interests.
NIH Public Access
Author Manuscript
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. Author manuscript; available in PMC 2013 August 28.
Published in final edited form as:
Nature
. 2013 February 28; 494(7438): 476–479. doi:10.1038/nature11924.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

represent neoblast-like stem cells, we treated adult
S. mansoni
with the thymidine analogue
5-ethynyl-2'-deoxyuridine (EdU)10 to examine the distribution of S-phase cells in the
parasite (Fig. 1a,b). In addition to the expected incorporation in the highly proliferative
reproductive organs (testes, ovaries, and vitellaria) (Fig. 1a,b and Supplementary Fig. 1), we
observed a population of EdU+ cells throughout the soma of male and female parasites (Fig.
1a–d). Similar distributions of EdU-incorporating cells were observed whether parasites
were given EdU during
in vitro
culture or
in vivo
by intraperitoneal injection of
schistosome-infected mice. Analogous to the neoblasts in free-living flatworms11,12, these
proliferating somatic cells (PSCs) were restricted to the mesenchyme of male and female
worms (Fig. 1c,d), not associated with reproductive organs, and were often found in clusters
near the intestine (Supplementary Fig. 2a). We also observed a conspicuous population of
PSCs adjacent to the ventral sucker (Supplementary Fig. 2b). PSCs traversed the cell cycle:
they initially expressed the cell cycle-associated transcript
histone h2b
(Supplementary Fig.
3a–c) and progressed to M-phase within 24 hours following an EdU pulse (Supplementary
Fig. 3d).
Neoblasts are the only proliferating somatic cells in planarians4,11 and they possess a
distinct morphology; they are round-to-ovoid mesenchymal cells with a high nuclear-
tocytoplasmic ratio, a large nucleolus, and they often extend a cytoplasmic projection3,11,13.
To determine if PSCs share similarities with planarian neoblasts, we examined these cells by
dissociating male tissues devoid of germ cells (Fig. 1e). In these preparations we observed a
number of distinct differentiated cell types that failed to incorporate EdU, including cells
with a low nuclear-to-cytoplasmic ratio, neuron-like cells, and ciliated cells (Fig. 1f). By
contrast, we found that EdU incorporation was restricted to a neoblast-like population of
cells with scant cytoplasm (n=136/137 cells) and often a prominent nucleolus (Fig. 1f). We
also inspected PSCs within the mesenchyme using EdU to label nuclei and fluorescent in
situ hybridization (FISH) to detect
histone h2b
mRNA in the cytoplasm of proliferative
cells. Consistent with our results from tissue macerates, EdU+ cells possess a narrow rim of
cytoplasm surrounding their nucleus, and these cells often display a cytoplasmic projection
(Fig. 1g). These observations highlight morphological similarities between proliferating
cells in schistosomes and planarian neoblasts.
Previous studies have exploited the sensitivity of planarian neoblasts to γ-irradiation as a
means to identify neoblast-enriched transcripts14–16. Using this strategy to identify PSC-
expressed genes, we exposed parasites to various dosages of γ-irradiation and determined
that 100–200 Gy were sufficient to block EDU incorporation (Fig. 2a). Because of their high
ratio of somatic tissue to reproductive tissue and their large number of PSCs relative to
female worms (compare insets in Fig. 1a and 1b), our remaining studies, unless otherwise
noted, focused on male parasites. By comparing the transcriptional profiles of irradiated and
non-irradiated parasites by RNAseq (Fig. 2b), we identified 128 genes with significantly
down-regulated expression (≥ 2-fold, p < 0.05) 48 hours post-irradiation (Fig. 2c and
Supplementary Table 1). Highlighting the efficacy of this approach to identify transcripts
specific to proliferating cells, we found that genes expressed in differentiated tissues, such as
the intestine (
Sm
-
cathepsin B
17), were unaffected by irradiation. By contrast, our list of
down-regulated genes was enriched for factors involved in the cell cycle (Supplementary
Fig.4 and Supplementary Table 1).
In addition to identifying cell cycle-associated factors, systematic comparison of irradiation-
sensitive genes with neoblast-enriched transcripts14,15,18,19 uncovered a number of
interesting similarities (Supplementary Table 1). For instance, homologues of genes known
to regulate planarian neoblasts such as
p53
, a sox-family transcription factor, fibroblast
growth factor receptors, and
argonaute2
14,20–22, were significantly down-regulated in
irradiated schistosomes (Supplementary Fig. 4 and Supplementary Table 1). Another
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distinctive feature of neoblasts3,14,22, and the somatic stem cells of other invertebrates23, is
that they often express post-transcriptional regulators associated with germline development
(e.g.,
vasa, piwi, tudor, and nanos
). Although
vasa-like
genes have been reported in
Schistosoma
, no true
vasa
orthologue has been indentified24. Similarly,
piwi
and
tudor
genes
appear to be absent from schistosomes (data not shown). However, we identified a
nanos
orthologue (
Sm-nanos-2
) that was down-regulated in somatic tissue following irradiation
(Supplementary Fig. 4, and Supplementary Table 1). Since these genes represented potential
regulators of PSC behavior and could serve as useful markers for these cells, we examined
their expression by whole-mount in situ hybridization (WISH). We detected
Sm-ago2-1
,
Sm-nanos-2
, and
Sm-fgfrA
transcripts in cells scattered throughout the mesenchyme (Fig.
2d and Supplementary Fig. 5) in a pattern similar to that of cells incorporating EdU (Fig.
1A). This mesenchymal expression was radiation sensitive (Fig. 2d), suggesting these genes
are expressed in proliferating cells. Consistent with this idea, we found that following an
EdU pulse, >99% of EdU-incorporating somatic cells also expressed
Sm-fgfrA
(Fig. 2e).
To determine if PSCs are stem cells, we assessed their ability both to self-renew and to
produce differentiated cell types. To examine self-renewal, we administered sequential
pulses of EdU and 5-Bromo-2'-deoxyuridine (BrdU) to parasites
in vitro
. Because nearly all
PSCs that incorporate EdU are
Sm-fgfrA
+, the ability of EdU+ cells to incorporate BrdU in
subsequent cell cycles would suggest that
fgfrA
+ PSCs self-renew (i.e., divide and produce
more
fgfrA
+ PSCs). For these experiments we chose a chase period of 44 hours, since this
time frame should give many EdU+ PSCs sufficient time to divide (Supplementary Fig. 3d).
Consistent with PSCs possessing the capacity for self-renewal, we find that 41% of cells that
initially incorporate EdU are BrdU+ 3 days following an initial EdU pulse (Fig. 3a).
Furthermore, we observed that many EdU+ cells were distributed in pairs, or “doublets”
(Fig. 3a); we suggest a majority of these doublets are the products of cell division
(Supplementary Discussion and Supplementary Fig. 6). In these EdU+ doublets, a
disproportionately large fraction displayed asymmetric BrdU incorporation (i.e. one nucleus
is EdU+BrdU+, while the other is EdU+BrdU−) (Fig. 3b and Supplementary Discussion).
This observation suggests that division progeny have an asymmetric capacity to proliferate.
Whether this represents stem cell-like asymmetric division or temporal differences in the
ability of these cells to reenter the cell cycle requires further experimentation. Nevertheless,
these data are consistent with PSCs (or some PSC subpopulation) being capable of self-
renewal.
To examine the capacity of PSCs to differentiate, we performed EdU pulse-chase
experiments
in vivo
. For these experiments, schistosome-infected mice were injected with
EdU and the distribution of EdU+ cells was monitored at early (D1) and late (D7) time
points (Fig. 3c). We successfully used this pulse-chase approach to monitor the
differentiation of schistosome germ cells (Supplementary Fig. 7). Visualizing the syncytial
epithelium of the schistosome intestine at D1, we did not observe EdU+ intestinal nuclei in
male or female parasites (Fig. 3d, 0 EdU+/3151 DAPI+ nuclei, 14 mixed sex parasites, n=5
mice), confirming that cells in the intestine do not proliferate. Following a 7-day chase,
however, ~2.5% of the intestinal nuclei were EdU+ (Fig. 3e, 56 EdU+/2189 DAPI+ nuclei,
10 mixed sex parasites, n=3 mice). This observation suggests that cells initially labeling
with EdU have the capacity to migrate into the intestine and differentiate into new intestinal
cells. Similarly, we were able to monitor the differentiation of new cells in the body wall
muscles. At D1 no EdU+ nuclei were observed in the male body wall musculature (Fig. 3f, 0
EdU+/1882 DAPI+ nuclei, 13 male parasites, n=6 mice), whereas at D7 ~10% of the muscle
cell nuclei were EdU+ (Fig. 3g, 55 EdU+/584 DAPI+ nuclei, 6 male parasites, n=3 mice).
Since virtually all cells that initially incorporate EdU are
Sm-fgfrA
+, we suggest that these
double-positive cells are likely to represent the only source of new intestinal and muscle
cells and, thus, represent a collectively multipotent population of neoblast-like stem cells.
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Whether all
Sm-fgfrA
+ PSCs are multipotent or whether they exist as lineage-restricted
progenitors remains unclear.
While progress has been made in identifying transcriptional14 and post-transcriptional22
regulators of planarian neoblasts, little is known about the signal transduction networks
functioning within these cells. Since the expression of FGF receptor family members in
proliferative cells is conserved between planarians14,21 and schistosomes, we speculated that
FGF signaling could regulate these cells in
S. mansoni
. To examine this idea, we disrupted
Sm-fgfrA
in
in vitro
-cultured adult parasites using RNA interference (Supplementary Fig.
8). We found that inhibition of
Sm-fgfrA
resulted in reduced EdU incorporation (Fig. 4a,b
and Supplementary Table 2) and down-regulation of cell cycle-associated transcripts (Fig.
4c and Supplementary Fig. 8). To resolve whether this effect is due to reduced cell
proliferation or a failure to maintain neoblast-like cells, we monitored the expression of PSC
markers
Sm-ago2-1
and
Sm-nanos-2
in
Sm-fgfrA(RNAi)
parasites.
Sm-fgfrA
RNAi
treatment resulted in a dramatic reduction in the number of cells expressing
Sm-nanos-2
(Fig. 2c) as well as significantly reduced mRNA levels for
Sm-ago2-1
and
Sm-nanos-2
(Supplementary Fig. 8b). Together, these results suggest that
Sm-fgfrA
promotes the long-
term maintenance of neoblast-like cells in
S. mansoni
. FGF signaling is known to influence
multiple processes, such as cell proliferation, differentiation, and survival; furthermore, it
plays key roles in various stem cell populations25. Our results suggest a conserved role for
FGF signaling in controlling stem cell behavior in these parasites and demonstrate the
feasibility of using RNAi to abrogate adult gene expression and manipulate neoblast-like
cells in
S. mansoni
.
Adult schistosomes can modulate growth in response to host immune signals26 and male-
female pairing status2,27 and they can regenerate damaged tissues following sub-lethal doses
of the anti-schistosomal drug praziquantel28. These observations reveal the developmental
plasticity of schistosomes, and suggest that these parasites can utilize distinct developmental
programs in response to a range of external stimuli. Future studies characterizing the role of
neoblast-like cells in diverse contexts could address long-standing gaps in our knowledge of
schistosome biology and may reveal novel therapeutic strategies for treating and eliminating
schistosomiasis.
Methods
Parasite Acquisition and Culture
Adult
S. mansoni
(6–8 weeks post-infection) were obtained from infected mice by hepatic
portal vein perfusion31 with 37°C DMEM (Mediatech, Manassas, VA) plus 5% Fetal Calf
Serum (FBS, Hyclone/Thermo Scientific Logan, UT). Parasites were rinsed several times in
DMEM + 5% FBS and cultured (37°C/5% CO2) in Basch's Medium 16932 and 1×
Antibiotic-Antimycotic (Gibco/Life Technologies, Carlsbad, CA 92008). Media was
changed every 1–3 days.
EdU labeling
For
in vitro
labeling, parasites were cultured in Basch's Medium 169 supplemented with 10
μM EdU (Invitrogen, Carlsbad, CA) diluted from a 10 mM stock in DMSO. Unless
otherwise noted, animals were pulsed for 18–24 hours. For
in vivo
labeling, schistosome-
infected mice (6–8 weeks post-infection) were given a single intraperitoneal injection (100–
200 mg EdU/kg bodyweight) with 5 mg/ml EdU dissolved in PBS and then harvested at
various time points after injection.
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In situ hybridization
Male and female parasites were separated by incubation (2–3 minutes) in a 0.25% solution
of the anaesthetic ethyl 3-aminobenzoate methanesulfonate (Sigma-Aldrich, St. Louis, MO)
dissolved in Basch's Medium 169 or Phosphate Buffered Saline (PBS). Relaxed parasites
were then killed in a 0.6 M solution of MgCl2 and fixed for 4.5 hrs in 4% Formaldehyde
dissolved in PBSTx (PBS + 0.3% Triton X-100). Following fixation, parasites were
dehydrated in MeOH and stored at −20°C. Samples were rehydrated by incubation in 1:1
MeOH:PBSTx followed by incubation in PBSTx. Rehydrated samples were bleached for 1–
2 hours in formamide bleaching solution (0.5% Formamide, 0.5% SSC, and 1.2% H2O2),
rinsed with PBSTx, treated with Proteinase K (2–10 μg/mL, Invitrogen, Carlsbad, CA) for
20–30 minutes at room temperature and post-fixed for 10–15 minutes in 4% formaldehyde
in PBSTx. Samples were hybridized at 52–55°C and otherwise processed as previously
described29,33. Plasmids used for riboprobe synthesis were generated as described
previously29 using oligonucleotide primers listed in Supplementary Table 3.
Immunofluorescence, histological staining, and EdU detection
Parasites were relaxed, killed, fixed, dehydrated and rehydrated as described above and
bleached in 6% H2O2 dissolved in PBS for 0.5–2 h. Dehydration and bleaching were
omitted for samples labeled with phalloidin. Samples were then treated with Proteinase K
and post-fixed as described above. Immunofluorescence, lectin, and phalloidin staining were
performed as described previously30. Rabbit anti-Phospho-Histone H3 Ser10 (anti-pH3)
(D2C8, Cell Signaling, Danvers, MA), rhodamine-conjugated sWGA (Vector Laboratories
Burlingame, CA), and Alexa Fluor 568 phalloidin (Invitrogen, Carlsbad, CA), were used at
1:1000, 1:100, and 1:100, respectively. EdU detection was performed essentially as
previously described10,34 with 100 μM Alexa Fluor 488 or Alexa Fluor 594 azide
conjugates. All imaging was performed as described previously29,30. To quantify intestinal
cell differentiation, the number of EdU+ and DAPI+ intestinal nuclei were determined from
12 consecutive confocal sections imaged from the intestine. To quantify muscle cell
differentiation, the number of EdU+ and DAPI+ nuclei were determined from 4 to 9
consecutive confocal sections through the dorsal muscle layer of male parasites.
Tissue dissociation and EdU detection
Following an overnight pulse with 10 μM EdU, the heads and testes of adult male
S.
mansoni
were removed and the remaining tissue added to dissociation solution (Hanks
Balanced Salt Solution with 3.5 × Trypsin-EDTA (from 10× stock, Sigma-Aldrich, St.
Louis, MO)) and minced with a razor blade. These tissue fragments were incubated in ~4
mL of dissociation solution for 45–60 min at room temperature on a rocker and gentle
pipetting was used to break up large tissue fragments. This mixture was passed over two sets
of cell strainers (100 and 40 μm, BD, Franklin Lakes, New Jersey) and dissociated cells
were collected by centrifugation (250 × g for 5 m). Pelleted cells were fixed in 4%
formaldhyde in PBS for 30 min, spotted on Superfrost Plus microscope slides (Fisher
Scientific), permeabilized for 30 min with PBSTx, and EdU was detected as described above
with 10 μM Alexa Fluor 488 azide.
To quantify the ratio of EdU+ to total DAPI+ nuclei in RNAi knockdowns, 8 male parasites
were processed as above and tiled images of EdU and DAPI labeling were captured on a
Zeiss LSM 710 (Plan-Apochromat 20×/0.8). Numbers of EdU+ and DAPI+ nuclei were
quantified using the Image-based Tool for Counting Nuclei (ITCN) plugin for ImageJ35.
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γ-irradiation and transcriptional profiling
Parasites (D43 post-infection) were harvested from mice, suspended in Basch medium 169,
and exposed to 200 Gy of γ-irradiation using a Gammacell-220 Excel with a Co60 source
(Nordion, Ottawa, ON, Canada). Control parasites were mock irradiated. Parasites were
cultured in Basch Medium 169 and 48 hours post-irradiation males were separated from
female parasites using ethyl 3-aminobenzoate methanesulfonate. Following separation, the
head and testes of males were removed and purified total RNA was prepared from the
remaining tissue from pools of 14–18 parasites using Trizol (Invitrogen, Carlsbad, CA) and
DNase treatment (DNA-free RNA Kit, Zymo Research, Irvine, CA). Three independent
biological replicates were performed for both control and irradiated experimental groups.
Individually tagged libraries for RNAseq were prepared (TruSeq RNAseq Sample Prep Kit,
Illumina, San Diego, CA), pooled in a single lane, and 100 bp reads were generated using an
Illumina HiSeq2000. Library preparation and Illumina sequencing were performed at the
W.M. Keck Center for Comparative and Functional Genomics. The resulting reads were
mapped to the annotated
S. mansoni
genome6 (v5.0) and differences in gene expression
were determined using CLC Genomics Workbench (CLC bio, Aarhus, Denmark). Statistical
enrichment of Gene Ontology terms was determined in CLC Genomics Workbench using a
hyper geometric test that is similar to the GOstat test described in previous studies36. To
examine similarities between proteins encoded from irradiation-sensitive transcripts in
S.
mansoni
and genes expressed in planarian neoblasts, we compared our schistosome dataset
with both neoblast-enriched and “whole” transcriptomes14,15,18,19 using standalone
tBLASTn. Schistosome proteins sharing no similarity to translated planarian mRNAs (e-
value cut-off > 1e-5) were omitted from analysis. Assignment of whether protein pairs were
orthologous, homologous, paralogous, or unrelated was assessed manually on an individual
basis. Data and evidence supporting protein similarity is provided in Supplementary Table 1.
EdU/BrdU double labeling
Parasites labeled with 10 μM EdU and BrdU were fixed in Methacarn (6:3:1
methanol:chloroform:glacial acetic acid) or processed for in situ hybridization. Following a
45 min 2N HCl treatment, EdU was detected and parasites were processed for anti-BrdU
immunofluorescence (anti-BrdU 1:500, clone MoBU, Invitrogen). We observed no cross-
reactivity between this antibody and EdU.
To quantify the level of BrdU/EdU overlap and measure center-to-center distances between
nuclei, 3D confocal stacks from EdU/BrdU labeled animals were resampled to give isotropic
voxels, and subjected to Gaussian filtering and background-subtraction. Labeled nuclei were
segmented with Imaris (Bitplane Inc., South Winsor, CT) using parameters empirically
determined to minimize the need for manual corrections; typically, fewer than 5% of the
total nuclei required correction. The 3D coordinates of the nuclei were exported and
analyzed with MATLAB. Overlapping EdU and BrdU labeled nuclei were defined as nuclei
with center-to-center distances < 1 nuclear size (~4 um). Statistical analyses were performed
in Origin (OriginLab, Northampton, MA).
RNA interference
Although procedures have been previously described37,38, RNAi experiments with adult
parasites were based on methods optimized for schistosomula39. Briefly,
in vitro
cultured
parasites were soaked with 20–30 μg of dsRNA freshly added on days 1–3 and every 5–6
days thereafter. As a negative control, animals were soaked with dsRNA synthesized from
the
ccdB
and
camR
-containing insert of pJC53.229. dsRNA synthesis was performed as
previously described29. Sequences used to generate dsRNAs are provided in Supplementary
Fig. 9. To measure mRNA levels, total RNA from control and knockdown parasites (~8
male posterior somatic fragments) was reverse transcribed (iScript cDNA Synthesis Kit,
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Bio-Rad, Hercules, CA) and quantitative real time PCR was performed on an Applied
Biosystems Step One Plus instrument using GoTaq qPCR Master Mix with SYBR green
(Promega, Madison, WI). Transcript levels were normalized to the mRNA levels of
proteasome subunit beta type-4
(smp_056500). Relative quantities were calculated using the
ΔΔCt calculation in the Step One Plus software. Oligonucleotide primer sequences are
listed in Supplementary Table 3.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank Rachel Roberts-Galbraith, Melanie Issigonis, and Labib Rouhana for comments on the manuscript; Ryan
King for sharing the
Cathepsin B
plasmid and unpublished protocols; and Alvaro Hernandez for assistance with
Illumina sequencing. Schistosome-infected mice were provided by the NIAID Schistosomiasis Resource Center and
the Biomedical Research Institute (Rockville, MD) through NIAID Contract N01-A1-30026. This work was
supported by: NIH F32 HD062124 (J.J.C.) and NIH R21 AI099642 (P.A.N.). P.A.N. is an investigator of the
Howard Hughes Medical Institute.
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Figure 1. Proliferation of somatic cells in adult schistosomes
a–b, EdU labeling in (a) male and (b) female parasites.
c–d, Distribution of mesenchymal PSCs in (c) male and (d) female parasites. Phalloidin
staining for actin shows male enteric and dorso-ventral muscles and female enteric and
uterine muscles.
e, Strategy to characterize PSC morphology.
f, The morphology of EdU− and EdU+ cells. Arrowhead indicates a nucleolus.
g, FISH for
histone h2b
with EdU labeling. Arrowhead indicates a cytoplasmic projection.
(a–d, g) are confocal projections; (a–b) are derived from tiled stacks. Scale bars: (a–b) 500
μm, (c–d) 20 μm, (f–g) 5 μm.
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Figure 2. Transcriptional profiling identifies genes expressed in proliferative cells
a, EdU incorporation is abrogated at D3 following irradiation.
b, Strategy to identify PSC-expressed genes.
c, Volcano plot showing expression differences in control versus irradiated parasites. n = 3
for each group.
d, WISH for various transcripts in unirradiated and D5 post-irradiation parasites. n > 3
parasites.
e, EdU labeling and FISH for
Sm-fgfrA
. 1988/2000 EdU+ PSCs were
Sm-fgfrA
+ following
a 20–22 hour pulse (n = 20 male parasites). (a, e) are confocal projections. Scale bars: (a, e)
20 μm, (d) 100 μm.
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Figure 3. PSCs self-renew and differentiate
a, EdU-BrdU double labeling. Arrowheads, EdU+BrdU+ nuclei. Arrows, EdU+ “doublets”.
b, Percentage (±s.d.) EdU+ doublets (green) that are BrdU−-BrdU− (top), BrdU+-BrdU+
(middle, BrdU is magenta), or BrdU+-BrdU− (Bottom). n = 21 parasites.
c, Strategy to monitor cellular differentiation.
d–g, EdU and sWGA labeling showing EdU+ cells in (d,e) male intestine or (f,g) dorsal
musculature at (d,f) D1 and (e,g) D7 following a pulse. (d,e) Insets, intestinal basal surface
(dashed lines) and lumen (green). Arrowheads, EdU+ (e) intestinal cells or (g) muscle cells.
Images are confocal projections. Scale bars: (a,d–g) 20 μm (b) 5 μm.
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Figure 4. Sm-fgfrA is required for the maintenance of somatic stem cells
a, EdU labeling and DIC images in control and
Sm-fgfrA(RNAi)
at RNAi D17.
b, Percentage EdU+ nuclei/total nuclei in dissociated tissues from control(RNAi) (n = 3002
nuclei) and
Sm-fgfrA(RNAi)
(n = 3642 nuclei) parasites. Error bars, 95% confidence
intervals, p < 0.0001 χ2.
c, WISH for
histone h2b
(top row) and
nanos2
(bottom row) transcripts in control (left
column) versus
Sm-fgfrA(RNAi)
(right column) parasites at RNAi D20-21. n > 5 parasites/
experiment. Scale bars: (a) 100 μm (c) 200 μm.
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