1 Department of Histology and Neurobiology, Dokkyo University School of
Medicine, Mibu, Tochigi 321-0293, Japan
2 Department of Physiology, Keio University School of Medicine, Shinjuku-ku,
Tokyo 160-8582, Japan
* Author for correspondence (e-mail: i-oka{at}dokkyomed.ac.jp)
Accepted 15 April 2003
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Summary |
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Key words: Stem cells, Musashi-1, Thyroid hormone, Gastrointestinal remodeling, Xenopus laevis
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Introduction |
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Musashi-1 (Msi-1), an RNA-binding protein, was initially reported in the
sensory organ of Drosophila
(Nakamura et al., 1994) and
has been shown to serve as a marker for proliferative neural precursor cells
including stem cells in the central nervous system (CNS)
(Sakakibara et al., 1996
;
Sakakibara and Okano, 1997
).
Msi-1 expression in neural precursor cells is evolutionally conserved in
different species of vertebrates such as humans, mice and Xenopus
laevis (Kaneko et al.,
2000
). Although its precise mechanisms have not yet been
clarified, Msi-1 is supposed to be involved in the early asymmetric divisions
that generate differentiated cells from neural stem cells
(Okano, 1995
). Interestingly,
Msi-1 is also expressed in the murine intestine other than the neural tissues
(Sakakibara et al., 1996
).
Moreover, it has been shown recently that Msi-1 is preferentially expressed in
the predicted stem cell region of murine intestinal crypts, suggesting that
Msi-1 may be a natural marker for intestinal stem cells and their immediate
descendants (Booth and Potten,
2000
; Potten et al.,
2003
). These recent reports led us to study the expression of
Msi-1 during amphibian gastrointestinal remodeling.
In the present study, to clarify whether the adult progenitor cells in the amphibian gastrointestine express Msi-1 as in the mammalian intestine, we examined Msi-1 expression in the X. laevis small intestine and the stomach during metamorphosis at mRNA and protein levels. We show that expression profiles of Msi-1 coincide well with active proliferation of the adult progenitor cells in both organs, suggesting that Msi-1 serves as a marker for the stem cells common to the amphibian and mammalian intestines. Moreover, we demonstrate that cell-specific Msi-1 expression can be reproduced in vitro by the inductive action of TH in the presence of the connective tissue but not in its absence.
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Materials and Methods |
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RNA isolation and reverse transcription-polymerase chain reaction
(RT-PCR) analysis
Tissue fragments were isolated from the small intestine and the stomach of
tadpoles at various stages, and of stage 57 tadpoles treated with or without 5
nM 3, 5, 3'-L-triiodo-thyronine (T3). Total RNA was purified
from these organs using Trizol reagent (Gibco-BRL, Grand Island, NY). RNAs
were prepared from a mixture of more than three tadpoles at each stage. For
each reaction, 1 µg RNA was reverse transcribed to oligo(dT)-primed
first-strand cDNA by using a cDNA synthesis kit (Amersham Pharmacia Biotech,
Little Chalfont, UK), and the resulting cDNA was subjected to 30 cycles of PCR
with Msi-1-specific primers (5'-ATGGAGACAGAAGCGCCCCAGCCCGGACTG-3'
and 5'-TCAGTGGTAGCCGTTGGTGAAAGCAG-3')
(Kaneko et al., 2000) and to
25 cycles with EF-1
-specific primers
(5'-CCTGAATCACCCAGGCCAGATTGGTG-3' and
5'-GAGGGTAGTCTGAGAAGCTCTCCACG-3')
(Suzuki et al., 1993
). The PCR
products (5 µl) were electrophoresed through a 2% agarose gel and
visualized by ethidium bromide staining.
Organ culture
Tissue fragments were isolated from the anterior part of the small
intestine at stage 57 and were slit open lengthwise with fine forceps. Some
fragments were treated with dispase (1000 units/ml; Godo, Tokyo, Japan), and
their epithelial components were isolated and put on a growth factor-reduced
matrigel (Becton Dickinson, Bedford, MA). They were then cultured as described
previously (Ishizuya-Oka and Shimozawa,
1991). Briefly, they were placed on membrane filters (type HAWP;
Millipore, Bedford, MA) on stainless steel grids and were cultured in 60%
Leibovitz-15 medium supplemented with 100 IU/ml of penicillin, 100 µg/ml of
streptomycin (Gibco-BRL), and 10% charcoal-treated fetal bovine serum
(Gibco-BRL). To induce metamorphosis, T3, insulin and
hydrocortisone (Sigma) were added to the medium at 10 nM, 5 µg/ml and 0.5
µg/ml, respectively. The culture medium was changed every other day for 7
days at 26°C.
In situ hybridization
A cDNA fragment of nucleotides (nt) 74-1117 was amplified by PCR with the
forward primer (5'-ATGGAGACAGAAGCGCCCCAGCCCGGACTG-3'; nt 74-103),
the reverse primer (5'-TCAGTGGTAGCCGTTGGTGAAAGCAG; nt 1117-1092) and the
template nrp1-pSP36T, containing Xenopus Msi-1 full-length cDNA. The
cDNA fragment was subcloned into pCRII vector with TOPO TA cloning kit
(Invitrogen, Carlsbad, CA). Procedures for in situ hybridization were the same
as those described previously
(Ishizuya-Oka et al., 1994).
In brief, digoxigenin (DIG)-11-UTP-labeled antisense and sense probes were
prepared with DIG RNA-labeling kit (Roche Diagnostics, Mannheim, Germany)
according to the manufacturer's protocol. Tissue fragments were fixed with 4%
paraformaldehyde in phosphate-buffered saline (pH 7.4) at 4°C for 4 hours,
frozen on dry ice, and cut at 7 µm. Sections were treated with 0.2 N HCl,
digested with 1 µg/ml proteinase K (Wako, Osaka, Japan), and then fixed
again with 4% paraformaldehyde. Hybridization buffer containing DIG-labeled
antisense RNA probe (200 ng/ml) was applied to the pretreated sections. After
hybridization at 40°C for 18 hours, the sections were treated with 20
µg/ml RNase A (Wako) at 37°C for 30 minutes to remove excess
unhybridized probes. They were then washed, processed for immunological
detection of the hybridized DIG probes according to the manufacturer's
instructions (DIG-probe Detection Kit, Roche Diagnostics), and examined
microscopically. As controls, some sections were hybridized with DIG-labeled
sense RNA probe (200 ng/ml).
Immunohistochemistry for IFABP, Pg and PCNA
Tissue fragments and cultured explants were fixed with 95% ethanol at
4°C for 4 hours, embedded in paraffin and cut at 5 µm. The sections
were incubated at 4°C overnight with the rabbit anti-mouse Msi-1
polyclonal antibody (diluted 1:100)
(Sakakibara et al., 1996) or
with the rat anti-mouse Msi-1 monoclonal antibody (diluted 1:100), which has
been shown to recognize the amino acid sequences highly conserved between
Xenopus and mouse (Kaneko et al.,
2000
). They were then incubated with the biotinylated secondary
antibody, followed by incubation with avidin-conjugated horseradish peroxidase
(HRP) (Vector Labs, Burlingame, CA). HRP reactions were developed using 0.02%
3, 3'-diamino-benzidine-4HCl (DAB) and 0.006%
H2O2. Although the immunostaining with the monoclonal
antibody was somewhat weaker than that with the polyclonal antibody, their
expression profiles were spatio-temporally consistent (data not shown).
To distinguish between the adult progenitor cells and the remaining larval
cells during stages 60-62, some sections close to the sections used for Msi-1
immunohistochemistry were stained with methyl green-pyronin Y (Muto, Tokyo,
Japan) for 5 minutes. The adult progenitor cells were stained intensely red
because of their RNA-rich cytoplasm, whereas the larval epithelial cells
undergoing apoptosis were stained much weaker
(Ishizuya-Oka and Ueda, 1996).
In addition, other sections were incubated at room temperature for 1 hour with
the following antibodies: the mouse anti-PCNA monoclonal antibody (diluted
1:100; Novacastra, Newcastle, UK) for proliferating cells; the rabbit
anti-Xenopus IFABP antibody (diluted 1:1000) (generous gift of Dr
Y.-B. Shi) (Ishizuya-Oka et al.,
1997
) for differentiated intestinal absorptive cells; and the
rabbit anti-bullfrog Pg antibody (diluted 1:10,000) (generous gift of Dr T.
Inokuchi) (Ishizuya-Oka et al.,
1998
) for differentiated glandular cells. They were then
visualized by sequential incubation with streptavidinbiotin-peroxidase complex
(Nichirei, Tokyo, Japan) and DAB/H2O2 as described
above. There was no positive staining when the same concentration of
pre-immune or normal serum was applied for each antiserum as the specificity
control (not shown). At least three specimens were examined for each
developmental stage or culture day.
Finally, to examine the relationship between Msi-1 immunoreactivity and cell proliferation more directly, some sections were double immunostained with a mixture containing the rabbit anti-Msi-1 antibody and the mouse anti-PCNA antibody at 4°C overnight. They were then incubated with a mixture containing Alexa Fluor488-conjugated anti-rabbit IgG (1:500; Molecular Probes, Eugene, OR) and Alexa Fluor568-conjugated anti-mouse IgG (1:500; Molecular Probes) and observed by fluorescence microscopy.
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Results |
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Msi-1 mRNA expression is specific for progenitor cells of adult
epithelium
To identify which type of cells express Msi-1 mRNA, we performed in situ
hybridization analysis. Consistent with the RT-PCR data described above
(Fig. 1A), Msi-1 mRNA was
neither detected in the intestine (Fig.
2A) nor the stomach (Fig.
2F) throughout pre- and prometamorphosis. In the intestine, around
the onset of metamorphic climax (stages 60-61), when progenitor cells of the
adult epithelium were identified as islets stained strongly with pyronin-Y
(Fig. 2B), Msi-1 mRNA became
detectable only in these adult progenitor cells
(Fig. 2C). By contrast, all the
control hybridizations with the sense RNA probe gave only background levels of
signals (Fig. 2D). Thereafter,
as morphogenesis of intestinal folds proceeded, the level of Msi-1 mRNA
rapidly decreased from the crest to trough of the folds
(Fig. 2E). By the end of
metamorphosis (stage 66), Msi-1 mRNA became hardly detectable, if any.
Similarly, in the stomach, Msi-1 mRNA was transiently expressed only in
pyronin-Y-positive adult progenitor cells during stages 60-61
(Fig. 2G,H). Then, as the adult
progenitor cells differentiated into the surface and glandular epithelia, the
level of Msi-1 mRNA rapidly decreased. By stage 66, Msi-1 mRNA became hardly
detectable, if any, except for nonspecific staining in the neck region of
glands (Fig. 2I).
|
Transient expression of Msi-1 protein correlates with active
proliferation of adult progenitor cells
To investigate the relationship between Msi-1 expression and development of
the adult epithelium more precisely, we analyzed the expression profile of
Msi-1 proteins by immunohistochemistry.
Small intestine
Throughout pre- and prometamorphosis, the larval epithelium remained simple
columnar and negative for Msi-1 (Fig.
3A). At stage 60, when progenitor cells of the adult epithelium
were first identified as pyronin-Y-positive small islets
(Fig. 3B), Msi-1
immunoreactivity became weakly detectable only in these islets
(Fig. 3C). Then, at stage 61,
when the adult progenitor cells rapidly replaced the larval epithelium
(Fig. 3D) by active
proliferation (Fig. 3E), almost
all of the progenitor cells were positive for Msi-1
(Fig. 3F). Double
immunofluorescence labeling with Msi-1 and PCNA antibodies indicated that
Msi-1-positive cells have a high activity of cell proliferation
(Fig. 4A). By contrast, the
degenerating larval epithelial cells remained negative
(Fig. 3D,F). After the
completion of larval-to-adult epithelial cell replacement (stage 63)
(Fig. 3G), Msi-1
immunoreactivity rapidly decreased and became weakly detectable only in the
trough of newly formed folds (Fig.
3H), where proliferating cells became localized
(Fig. 3I). By contrast, the
immunoreactivity of IFABP, a marker for differentiated absorptive cells,
increased from the crest to trough of the folds
(Fig. 3J). At the end of
metamorphosis, Msi-1 expression was hardly detectable, if any.
|
|
Stomach
As in the intestine, Msi-1 became detectable at stage 60 only in the adult
progenitor cells, which appeared in the basal region of larval glands
(Fig. 5A-C) and were actively
proliferating (Fig. 5D). Then,
at stage 61, the proliferating adult progenitor cells rapidly replaced the
degenerating larval epithelium (Fig.
5E,F). Almost all of the adult progenitor cells were strongly
positive for Msi-1, whereas all of the larval epithelial cells remained
negative (Fig. 5G).
Msi-1-positive cells at this stage have a high activity of cell proliferation
(Fig. 4B) just like in the
intestine. After stage 62, when the adult epithelium completely replaced the
larval epithelium and began to differentiate into the surface and glandular
epithelia (Fig. 5H) expressing
Pg, a marker for differentiated glands
(Fig. 5I), Msi-1 expression
rapidly decreased (Fig. 5J).
Towards the end of metamorphosis, Msi-1 immunoreactivity became hardly
detectable except for a very weak one in the neck region of adult glands
(Fig. 5L), where proliferating
cells were localized (Fig.
5K).
|
TH organ-autonomously upregulates Msi-1 expression in vitro
To clarify whether TH upregulates Msi-1 expression in vitro, we cultured
larval intestines isolated from stage 57 tadpoles in the presence or absence
of TH. Around day 5 in the presence of TH, progenitor cells of the adult
epithelium were identified as pyronin-Y-positive islets
(Fig. 6A) that were actively
proliferating (Fig. 6B).
Although the intensity of Msi-1 immunoreactivity was lower than in vivo, it
was localized in the adult progenitor cells but not in the remaining larval
cells as in vivo (Fig. 6C). Towards day 7, when the adult epithelium differentiated into the absorptive
epithelium expressing IFABP (Fig.
6E), Msi-1-immunoreactive cells became undetectable again
(Fig. 6F). By contrast, in the
absence of TH, the differentiated larval epithelium remained negative for
Msi-1 throughout the cultivation (Fig.
6D).
|
Furthermore, to determine whether the connective tissue surrounding the epithelium is involved in TH upregulation of Msi-1 expression, we separated the epithelium from the connective tissue and cultured it alone in the presence of TH. Around day 5, some epithelial cells became negative for pyronin-Y staining, just like degenerating larval cells in the presence of the connective tissue (Fig. 6G). However, the adult progenitor cells could be hardly distinguished by pyronin-Y staining because the other cells were stained at various intensities. In addition, the epithelial cells remained as a single layer and did not form islet structures. Thereafter, the epithelium gradually decreased in cell number and never differentiated into the adult absorptive epithelium expressing IFABP. In this condition, no Msi-1-immunoreactive cells could be detected throughout the cultivation (Fig. 6H).
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Discussion |
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Recently, in the mammalian CNS, it has been shown that Msi-1 protein binds
to (G/A) UnAGU sequences in the 3'-untranslated region (3'-UTR) of
Numb mRNA and represses the expression of Numb protein
(Imai et al., 2001), which is a
membrane-associated antagonist of Notch signaling
(Wakamatsu et al., 1999
).
Meanwhile, in the mammalian intestine, Notch signaling has been suggested to
be involved in the determination and/or maintenance of stem cells
(van den Brink et al., 2001
).
Taken together, one possible role of Msi-1 is to maintain the adult progenitor
cells through activation of Notch signaling. Alternatively, Msi-1 may bind to
(G/A) UnAGU sequences of other target genes that activate proliferation of the
adult progenitor cells. The nature of these target genes awaits further
investigation.
TH upregulates Msi-1 expression in adult progenitor cells in vitro as
in vivo
In the mammalian intestine, it has been reported that Msi-1 expression is
upregulated during development of adenomas and during regeneration after
irradiation (Potten et al.,
2003). However, it remains unknown which molecules upregulate
Msi-1 expression. In the present study, the upregulation of Msi-1 expression
coincides temporally with the peak of circulating TH level during X.
laevis metamorphosis (Leloup and
Buscaglia, 1977
). More importantly, our culture study has shown
that TH upregulates organ-autonomously Msi-1 expression in the larval
intestine. This means that Msi-1 gene is a TH response gene, as recently
reported in the developing rat brain
(Cuadrado et al., 2002
).
Furthermore, we have shown that Msi-1 expression in vitro is closely
associated with adult progenitor cells that are actively proliferating just
like in vivo. This reinforces our proposal that TH-induced Msi-1 is involved
in the maintenance and/or active proliferation of adult progenitor cells.
Until now, little is known about the origin of the adult progenitor cells
in the amphibian gastrointestine. In the intestinal epithelium before
metamorphosis, no undifferentiated cells can be morphologically identified
(Marshall and Dixon, 1978). In
agreement with this, Msi-1 expression was not detected in the intestinal
epithelium before metamorphosis in the present study. This implies that at
least partially differentiated cells can give rise to progenitor and/or stem
cells during amphibian metamorphosis. Similar cases have recently been
reported in the mammalian tissues such as committed oligodendrocyte precursor
cells that become multipotent stem cells
(Kondo and Raff, 2000
) and
multinucleated myotubes that give rise to mononucleated proliferative
myoblasts (Odelberg et al.,
2000
). Although the important roles of microenvironments known as
`niche' in the control of stem cells are generally recognized
(Potten et al., 1997
;
Blau et al., 2001
;
Mills and Gordon, 2001
;
Spradling et al., 2001
), far
less is known about microenvironmental factors that play key roles in
reversing the differentiated state (i.e. de-differentiation into stem cells).
In this study, we have shown that the adult progenitor cells expressing Msi-1
could be detected after 5 days of TH treatment in vitro in the connective
tissue but not in its absence. This predicts that genes whose expression is
upregulated by TH in the connective tissue within 5 days play important roles
in the de-differentiation. To identify such factors, we recently isolated
genes whose expression is upregulated by TH in the connective tissue by
subtractive differential screening
(Shimizu et al., 2002
). It is
worth functionally analyzing these genes as well as other TH response genes
previously isolated from the X. laevis intestine
(Shi and Brown, 1993
;
Amano and Yoshizato, 1998
).
In conclusion, Msi-1 is useful as a marker for adult progenitor cells including stem cells in the amphibian gastrointestinal epithelium like in the mammalian intestinal epithelium. Amphibian gastrointestinal remodeling, where TH can induce stem cells expressing Msi-1 followed by a new epithelial formation, should provide a unique model system to clarify microenvironmental factors playing key roles in the control of stem cells.
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Acknowledgments |
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