1 Dipartimento di Morfologia Umana e Biologia Applicata, Sezione di Biologia e
Genetica, Università di Pisa, Pisa, Italy
2 Dipartimento di Fisiologia e Biochimica, Laboratorio di Biologia Cellulare e
dello Sviluppo, Università di Pisa, Pisa, Italy
3 Istituto di Fisiologia Clinica, Laboratorio di Terapia Genica e Molecolare,
CNR, Pisa, Italy
4 Dipartimento di Patologia Sperimentale Biotecnologie Mediche, Infettivologia e
Epidemiologia Università di Pisa, Pisa, Italy
Author for correspondence (e-mail:
gremigni{at}biomed.unipi.it)
Accepted 14 February 2005
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SUMMARY |
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Key words: Planarians, Regeneration, Neoblasts, Stem cells, RNAi, Cell proliferation, Pumilio, PUF proteins, Confocal microscopy, TEM, Cytofluorimetry, In situ hybridization, Post-transcriptional regulation
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Introduction |
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As adult stem cells are rare and difficult to study in vivo in most
organisms, the use of classical models of regeneration for studying stem cell
biology has been recently re-proposed
(Newmark and Sánchez Alvarado,
2002; Pearson,
2001
; Pennisi,
2004
; Tanaka,
2003
; Tsai et al.,
2002
; Weissman,
2000
). Planarians (Platyhelminthes, Tricladida), an invertebrate
group well known for the exceptional regenerative capability, retain a
population of totipotent stem cells, the neoblasts, throughout their life. The
unlimited capability of neoblasts for self-renewal and their ability to
generate all differentiated cell types is crucial for planarian regeneration.
During this process, stem cells proliferate and accumulate beneath the wound
epithelium, giving rise to the regenerative blastema, from which the missing
body parts are reconstructed
(Baguñà, 1998
;
Brønsted, 1969
;
Gremigni, 1981
). These cells
are scattered in the parenchyma with the exception of the most anterior end of
the cephalic region and are preferentially accumulated in the dorsolateral
body area, along the anteroposterior axis
(Newmark and Sánchez Alvarado,
2000
; Salvetti et al.,
2000
). A combination of grafting and X-ray irradiation experiments
has recently demonstrated that neoblast commitment depends on the positional
information signals coming from differentiated cells
(Agata and Watanabe, 1999
;
Kato et al., 2001
).
Here, we report the isolation and characterization of a planarian gene, DjPum, that shares significant sequence similarity with members of the PUF gene family. We demonstrate that this gene is expressed in neoblasts and its inactivation by RNA interference (RNAi) inhibits the formation of the regenerative blastema. Indeed, planarians injected with DjPum dsRNA are unable to regenerate and die, owing to a dramatic reduction of neoblasts. This finding demonstrates that DjPum plays a crucial role in neoblast maintenance and supports the intriguing possibility that PUF proteins play a key function in sustaining mitotic proliferation and self-renewal of both somatic and germline stem cells.
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Materials and methods |
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Cloning of DjPum and sequence analysis
A DjPum cDNA fragment of 440 bp was amplified with two degenerate
oligonucleotides corresponding to the amino acid sequence IQKFFEFG and IGNYVIQ
directed against two conserved regions of the second and sixth repeat,
respectively. The SMART RACE cDNA amplification kit (Clontech) was used to
obtain the full-length DjPum sequence. Amplification of the 5'
region was obtained with the sequence-specific antisense primer
5'-TAATTACTGATCCCTCCAATTCACGCAC-3'. The 3' region was
amplified with the sequence-specific sense primer
5'-GTACACCAGAACAAACCGCTCCAA-3'. The PCR products were TA-cloned
using pGEM-T easy vector (Promega). All clones were sequenced by automated
fluorescent cycle sequencing (ABI). Sequences related to DjPum were
identified with BLAST (Altschul et al.,
1990). CLUSTALW was used to obtain the multiple alignment of the
DjPum PUF repeats and the PUF repeats of human Pum 2 and Drosophila
Pumilio. The sequences of PUF-related proteins used for the phylogenetic tree
construction were obtained from the EMBL/GenBank.
In situ hybridization
Whole-mount in situ hybridization was carried out according to the protocol
described by Agata et al. (Agata et al.,
1998). Sense and antisense DIG-labelled RNA probes were obtained
using the DIG-RNA labelling kit (Roche). The clone DjPum 440
(Fig. 1A; 1900 bp to 2340 bp),
containing the coding region from the second to the sixth PUF repeat and the
clone DjPum 550 (Fig.
1A; 2446 bp to 2996 bp), containing the seventh PUF repeat and the
3' terminus, were used to obtain sense and antisense DIG-labelled RNA
probes. The clone DjPum 440 was also used to obtain the antisense
biotin-labelled RNA probe. The clone DjMCM2 was used to obtain the
antisense DIG-labelled RNA probe (Salvetti
et al., 2000
). Dissociated cells were prepared as described by
Hwang et al. (Hwang et al.,
2004
). Double fluorescent in situ hybridization was carried out
using a TSA-indirect kit (NEL Life Science Products). After hybridization the
biotin-labelled probe was revealed by SA-HRP and the signal was amplified
using TSA-tetramethylrhodamine. DIG-labelled RNA was revealed by
FITC-conjugated anti-DIG antibody (Roche). Dissociated cells were also
hybridized with DjPum 440 DIG-labelled RNA probe as described by
Salvetti et al. (Salvetti et al.,
2000
).
|
Analysis of endogenous transcripts by RT-PCR
Total RNA was extracted from fragments injected with Djeya
dsRNA, DjPum 440 dsRNA, DjPum 550 dsRNA or water,
respectively, using the NucleoSpin RNAII kit (Macherey-Nagel). cDNA was
generated from 1 µg of total RNA using Superscript First Strand Synthesis
System for RT-PCR (Invitrogen). To assess the reduction of DjPum
endogenous transcripts in the injected specimens, we used the following
primers: DjPum, forward 5'-TCGGGAACACCTGAGCAA-3' and
DjPum reverse 5'-CTGGAGGAACACATTCTAC-3'.
The primers utilized to investigate the expression level of stem cell markers were: DjMCM2, forward 5'-CAGGCGAATTCCAGAACTTG-3' ann reverse 5'-TTCGGAAAGAATTGGAACAAT-3' DjFGFR1, forward 5'-TGAGCTATTGATACTACTTGGG-3' and reverse 5'-TAGATTAATTGAAATTGGTGAGA-3'.
The primers utilized to investigate the expression level of differentiated cell markers were: DjMHC-B, forward 5'-CAACATCATCAACGTGAATTGG-3' and reverse 5'-AGCTCATTAAGTTTATCAACGG-3'; DjMHC-A, forward 5'-CAAGAACGATTGCAAGATTTAG-3' and reverse 5'-TAGATGCAGACACCGATAGAG-3'; DjIFb, forward 5'-CAAGTAACAAGTATTGTCAAAGG-3' and reverse 5'-TCCGTATCCCCAATTTGATTCT-3'; Djsix-1: forward 5'-GTTAGCGCATTTAGTACAAG-3' and reverse 5'-ATTTGGCGTTTGATCTGTTG-3'; Djops, forward 5'-ATTATCAAATCGTGAAAGCC-3' and reverse 5'-ATATAAAGGGATTGTACATAG-3'.
The primers utilized to investigate the expression level of the apoptotic cell marker DjClg3 were: forward, 5'-GGGAATCAGGATATTGTTGCT-3' and reverse, 5'-CTTCCGTCAAACCCAGATCA-3'.
Control reactions were performed in the absence of reverse transcriptase. The constitutively expressed elongation factor gene DjEF2 was amplified as an internal control using forward 5'-TTAATGATGGGAAGATATGTTG-3' and reverse 5'-GTACCATAGGATCTGATTTTGC-3' primers. For each PCR reaction, the concentration of cDNA and the number of cycles used were optimized to observe a quantifiable signal within the linear range of amplification, according to the putative abundance of each mRNA amplified and the size of the corresponding PCR product. The analysis was performed in duplicate with RNA extracted from at least two independent samples.
TUNEL assay
The TUNEL assay was performed according to Hwang et al.
(Hwang et al., 2004). Intact
planarians were injected (as described in the RNAi experiments section) with
DjPum dsRNA or water and sacrificed 1, 3, 5 or 7 days after the first
injection.
Preparation of dissociated cell samples and FACS analysis of neoblast-enriched fractions
Five days after the second transection, planarians injected with
DjPum dsRNA and water-injected controls were dissociated into
individual cells according to Baguñà and Romero
(Baguñà and Romero,
1981). Cell suspensions (50 µl), prepared from three
DjPum dsRNA-injected planarians and from three water-injected
controls were placed on glass slides, air-dried and stained with Giemsa. Three
slides for each sample were examined and a total number of 100 cells for slide
were analyzed. Cells morphologically referred to as neoblasts (round or
pear-shaped cells of 5-8 µm in diameter, with a large nucleus and scanty
cytoplasm) were counted. The experiment was repeated twice.
For FACS analysis, enriched fractions of neoblasts were obtained according
to Asami et al. (Asami et al.,
2002) and Baguñà et al.
(Baguñà et al.,
1989
). Briefly, DjPum dsRNA-injected planarians,
water-injected controls and X-ray-irradiated specimens were dissociated into
single cells by gently pipetting in a Ca2+/Mg2+-free
solution (CMF: NaH2PO4.H2O 2.56 mM; KCl 10.21
mM; NaCl 14.28 mM; NaHCO3 9.42) containing 30 µg/ml trypsin
inhibitor type II-O (Sigma). Neoblast-enriched fractions were obtained by
serial filtration through nylon meshes of decreasing pore sizes (150, 50, 20
and 8 µm, Millipore). For morphological analysis, the neoblast-enriched
fraction was fixed in 4% paraformaldehyde for 30 minutes, placed on glass
slides, air-dried and stained with Methylene Blue and Toluidine Blue. For FACS
analysis the fractions enriched in neoblasts were fixed in 70% ethanol and
incubated for 30 minutes at room temperature in PBS containing propidium
iodide (PI, 50 µg/ml, Roche) to stain DNA, RNAse (6.25 µg /ml, Roche) to
eliminate RNA that could contribute to the fluorescence, and IGEPAL CA-630
(0.5% v/v Sigma-Aldrich) to permeate the cells. FACS analysis was performed by
using a FACScalibur cytofluorimeter (Becton Dickinson) and the data were
analyzed by CELL Quest analysis software (Becton Dickinson). For comparative
RT-PCR analysis, total RNA was extracted from cell fractions obtained by
sequential filtration through nylon meshes of 50, 20 and 8 µm pore
size.
Transmission electron microscopy
Transmission electron microscopy (TEM) was performed on either
DjPum dsRNA- or water-injected planarians. Fragments were fixed with
2.5% glutaraldehyde solution in 0.1 M cacodylate buffer, pH 7.2, for 1 hour at
4°C and postfixed with 2% osmium tetroxide in 0.1 M cacodylate buffer for
2 hours at room temperature. After rapid dehydration in a graded series of
ethanol and a final dehydration in propylene oxide, specimens were embedded in
an `Epon-Araldite' mixture. Ultra-thin sections, obtained with a diamond knife
on an Ultracut Reichert-Jung ultramicrotome, were placed on Formvar-carbon
coated nickel grids, stained with uranyl acetate and lead citrate and observed
with a Jeol 100 SX transmission electron microscope.
Confocal microscopy
Planarians were prepared for confocal microscopy according to Newmark and
Sánchez Alvarado (Newmark and
Sánchez Alvarado, 2000). Polyclonal rabbit anti-phospho
histone H3 antibodies (anti-H3P; Upstate Biotechnology) were used at 1:700
dilution to mark mitotic cells. For primary antibody detection,
rhodamine-conjugated donkey anti-rabbit antibody was purchased from Santa Cruz
Biotechnology and used at 1:200 dilution. After incubation, the specimens were
mounted in Vectashield (Vector Laboratories, Burlingame, CA) and observed
under epifluorescence using a Radiance Plus confocal microscope (BioRad). The
negative control was performed omitting the primary (anti-H3P) antibody.
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Results |
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Expression of DjPum in intact and regenerating planarians
Whole-mount in situ hybridization of intact planarians showed a complex
expression pattern of DjPum transcripts. A detectable expression of
DjPum was observed at the level of the cephalic ganglia (the
planarian brain). In addition, DjPum expression was also found
throughout the parenchyma, where it was preferentially arranged in
anteroposterior dorsal cords (Fig.
2A,B). DjPum parenchymal expression resembles that of
DjMCM2, a member of the minichromosome maintenance gene family, which
represents a molecular marker to detect proliferating neoblasts in planarians
(Salvetti et al., 2000). X-ray
irradiation, a treatment that destroys mitotically active cells and the
regenerative capability (Lange,
1968
), caused a dramatic reduction in the number of
DjMCM2-expressing neoblasts
(Salvetti et al., 2000
). This
treatment also produced a general loss of DjPum hybridization signal,
with the exception of that localized at the brain level
(Fig. 2C). As DjPum
expression in the brain was unaffected by irradiation, we hypothesize that, at
this level, DjPum transcripts are present in nerve cells.
|
DjPum is involved in the formation of the regenerative blastema
We analyzed the effect of RNAi-mediated gene silencing of DjPum
during planarian regeneration. After transection, we observed that about 10%
(6/57) of DjPum dsRNA-injected animals did not have a visible
blastema and were unable to regenerate. This peculiar phenotype was seen only
in anterior fragments injected with DjPum dsRNA. However, when the
injected specimens were transected again, 95% (104/110) of them resulted
devoid of blastema, independently of the level and the orientation of the cut
(Fig. 3A-F). No significant
difference in the type and percentage of phenotypes was found by using dsRNA
obtained from two independent clones, DjPum 440 and DjPum
550, which target different regions of DjPum. Both water or
ß-Gal dsRNA-injected fragments always regenerated a well-formed
blastema (Fig. 3B-D).
DjPum dsRNA-injected fragments did not show a blastema even 14 days
after transection (Fig. 3E,F)
and died within 3-4 weeks. At the same time, the water-injected controls had
completely regenerated the missing body parts (data not shown). The
specificity of DjPum RNAi was further supported by the observation
that, in our experimental conditions, the RNAi-mediated inactivation of a
planarian homologue of eyes absent (Djeya) never inhibited
blastema formation also after the second transection. The specimens injected
with Djeya dsRNA regenerated phenotypes devoid of eyes, as previously
demonstrated by Mannini et al. (Mannini et
al., 2004). As the introduction of a specific dsRNA is expected to
selectively produce the degradation of cognate mRNA
(Fire, 1999
;
Bosher and Labouesse, 2000
),
we analyzed the silencing of DjPum expression in DjPum
dsRNA-injected animals by comparative RT-PCR. We observed that, although
DjPum RNAi drastically decreased endogenous DjPum RNA, no
detectable reduction in the expression level of endogenous DjPum mRNA
was found in planarians injected with Djeya dsRNA or with water
(Fig. 3G). TEM analysis of some
DjPum dsRNA-injected fragments, which were unable to regenerate,
confirmed the absence of unspecialized, neoblast-like cells between the wound
epidermis and the stump region. In particular, RNAi-induced phenotypes devoid
of a visible blastema had few neoblasts (see
Morita et al., 1969
),
intermingled with differentiated cells
(Fig. 4A). By comparison, many
unspecialized, neoblast-like cells were observed in corresponding
water-injected controls (Fig.
4B).
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Discussion |
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DjPum is expressed in planarian stem cells
DjPum-positive cells are distributed throughout the planarian
parenchyma and are also detected at the level of the cephalic ganglia. In
Drosophila the Pumilio/staufen pathway of translational
repression prevents ubiquitous expression of protein products in the neurons
(Dubnau et al., 2003). The
presence of DjPum mRNA in cells of the planarian brain suggests DjPum
to be part of a translational repression complex specific for nerve cells, an
hypothesis that deserves further investigation. Parenchymal cells that express
DjPum have all the requirements to be considered neoblasts. Indeed,
although X-ray irradiation does not eliminate DjPum transcripts from
the planarian brain, this treatment dramatically affects the parenchymal
expression of this gene. In the parenchyma of intact and regenerating
planarians, DjPum hybridization signal resembles that of DjMCM2.
DjPum and DjMCM2 transcripts appear to be preferentially
arranged in longitudinal dorsal cords. During regeneration, both these
transcripts are preferentially accumulated in the postblastema, a region
characterized by intense mitotic activity
(Saló and Baguñà,
1989
). DjPum-expressing cells appear similar to neoblasts
in shape and some of the neoblasts expressing DjPum were also found
positive for DjMCM2 transcripts. These findings suggest a function
for DjPum in proliferating neoblasts.
DjPum is essential for neoblast maintenance
RNAi-mediated gene silencing has been proven to successfully suppress
specific gene activity in planarians. DjPum RNAi produces a strong
reduction of endogenous mRNA level and results in the loss of regenerative
capability. A high number of phenotypes lacking a visible blastema were
observed after the second transection, being only a small percentage of them
detected during the first regeneration. A more effective dsRNA-mediated
interference during the second regeneration has already been observed in
planarians (Mannini et al.,
2004). The detection of few phenotypes lacking a blastema after
the first transection is probably due to the presence of the high number of
neoblasts in the planarian parenchyma. Head fragments resulted highly
sensitive to DjPum RNAi, and some of them were unable to form a
blastema even after the first transection, probably because a lower number of
neoblasts are localized in the head parenchyma
(Newmark and Sánchez Alvarado,
2000
; Salvetti et al.,
2000
). DjPum dsRNA-injected planarians did not regenerate
even after 3 weeks from transection and died after a short time. This may be
due to a drastic and irreversible reduction in the number of neoblasts.
Consistent with this conclusion is the observation that X-ray irradiation also
produces planarian fragments that are unable to regenerate, and die within 3-4
weeks. Ultrastructural investigations demonstrated that DjPum RNAi
does not interfere with wound closure, because this process occurred normally.
However, under the TEM, no accumulation of unspecialized cells was detected
beneath the wound epithelium in DjPum dsRNA-injected fragments. The
DjPum RNAi-induced loss of regenerative capability may result from a
failure of local neoblasts to migrate and accumulate beneath the wound
epithelium. Alternatively, neoblasts might have a reduced proliferative
capability and not be activated by local signals to resume proliferation after
the cut. As a further possibility, we hypothesize that, after the second
transection, the neoblasts in the parenchyma of DjPum dsRNA-injected
planarians were reduced in number because of DjPum dsRNA interference
and no new neoblasts had been produced. As part of the evidence that the
number of these cells was drastically decreased, we observed that two stem
cell markers, associated (DjMCM2) or not associated
(DjFGFR1) with the cell cycle, had a reduced expression in
DjPum dsRNA-injected specimens. The use of anti-H3P antibodies after
DjPum RNAi confirmed a dramatic reduction in the number of mitoses.
Counting the number of neoblasts after cell dissociation, as well as FACS
analysis, demonstrated a substantial reduction in the number of these cells.
In conclusion, our data indicate that downregulation of DjPum
transcripts does not allow regeneration, because planarian stem cells are not
maintained. Literature data support the hypothesis that an ancestral function
of PUF proteins is that of promoting the mitotic proliferation of stem cells
by controlling the translation of mRNAs known to encode for regulators of cell
cycle and cell differentiation (Chen and
McKearin, 2005
; Gerber et al.,
2004
; Kennedy et al.,
1997
; Lin and Spradling,
1997
). In Dictyostelium and yeast, it has been proposed
that PUF proteins support mitoses by repressing the expression of
cAMP-dependent protein kinase (PKA-c)
(Souza et al., 1999
). It has
been speculated that PUF proteins might also act through a similar mechanism
in Drosophila and C. elegans
(Wickens et al., 2002
). In
order to assess whether DjPum RNAi induces cell differentiation, we
investigated the expression levels of representative genes of a number of
specialized cell types. However, no significant variation was observed in
DjPum dsRNA-injected planarians with respect to the controls.
Interestingly, a significant increase in the expression level of the apoptotic
cell marker DjClg3, as well as a slight increase in the number of
TUNEL-positive cells was observed in DjPum dsRNA-injected animals.
These results indicate that apoptotic cell death occurs as a consequence of
DjPum RNAi. Knockdown of an essential player for stem cell
maintenance, such as DjPum, could be sufficient to trigger apoptosis.
As a further possibility, DjPum-induced arrest of neoblast
proliferation, could represent an altered stem cell condition leading to the
activation of apoptotic cell death pathways.
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ACKNOWLEDGMENTS |
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Footnotes |
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