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DEVELOPMENTAL DYNAMICS 208:139–148 (1997)
Reactivation and Graded Axial Expression Pattern of
Wnt-10a Gene During Early Regeneration Stages of Adult
Tail in Amphibian Urodele Pleurodeles waltl
XAVIER CAUBIT,1* STEPHANE NICOLAS,1 DE-LI SHI,2 AND YANNICK LE PARCO1
de Biologie du Développement de Marseille, Laboratoire de Génétique et Physiologie du Développement UMR C 9943,
Faculté des Sciences de Luminy, 13288 Marseille cedex 9, France
2Laboratoire de Biologie Moléculaire et Cellulaire du Développement, URA 1135, Université Pierre et Marie Curie,
Paris, France
1Institut
ABSTRACT
Adult urodele amphibians such
as Pleurodeles waltl are able to regenerate their
amputated limbs or tail. The mechanisms implicated in growth control and formation of the
blastema are unknown but it has been proposed
that regeneration in newts may proceed through
reactivation of genes involved in embryonic development. Knowing the role of Wnt genes in the
patterning of the primary and secondary axes of
the vertebrate embryo, we suspected that some of
these genes could be involved in axial pattern
during newt tail regeneration. Pwnt-10a gene,
cloned from a newt tail regenerate cDNA library,
showed an expression pattern compatible with
such a role in tail regenerates. Pwnt-10a, which is
highly expressed during embryonic development
(from gastrula to tailbud-stage) and weakly expressed in the adult tail, is strongly re-expressed
during tail regeneration. In the blastemal mesenchyme Pwnt-10a transcripts exhibited a graded
distribution along the antero-posterior axis, the
mRNA accumulation being maximal in the caudal
most part corresponding to the growing zone.
These findings strongly support the view that
Pwnt-10a may act in cooperation with other factors to control growth and patterning in newt tail
regeneration. Until now Wnt-10a was only known
to be involved in central nervous system development; our results suggest that this gene may also
play a role in other developmental processes.
Dev. Dyn. 208:139–148, 1997. r 1997 Wiley-Liss, Inc.
Key words: Wnt-10a; tail regeneration; development; amphibian urodeles
INTRODUCTION
Adult urodele amphibians such as Pleurodeles waltl
(Pw) are characterized by their epimorphic regeneration capacity to replace their amputated appendages
(limbs and tail). Undifferentiated and dividing mesenchymal cells originating from the tissues of the stump
accumulate under the wound epithelium and form the
blastema. The molecular mechanisms that underlie
transition between stump tissue and blastemal cells
are not fully understood. The outgrowth of newt limb
r 1997 WILEY-LISS, INC.
blastemal cells requires neurotrophic factors. The wound
epithelium could contribute to the process of regeneration by influencing or initiating cellular de-differentiation and by stimulating cell proliferation (Stocum and
Dearlove, 1972; Tassava et al., 1987). During tail
regeneration, proliferation of ependymoglial cells of the
spinal cord of the stump gives rise to an ependymal
tube extending into the blastema (Egar and Singer,
1972). As tail regeneration progresses, blastemal cells
condense following a precise pattern to form the cartilage rod and, later, the skeletal muscle masses (Holtzer,
1956, Kiortsis and Droin, 1961). It is now well established that the regenerating tail of urodele amphibian
shows a rostrocaudal gradient of differentiation (Iten
and Bryant, 1976; Geraudie et al., 1988; Arsanto et al.,
1992; Thouveny et al., 1991). Little is known about the
factors which control cell dedifferentiation, cell division
and morphogenetic events during tail regeneration. It
has been proposed that regeneration may proceed
through reactivation of genes involved in tissue patterning during development (Muneoka and Sassoon, 1992).
The possibility that, during regeneration, adult newt
tissues maintain or reactivate expression of homeobox
genes to regulate pattern formation is under investigation (Savard et al., 1988; Brown and Brockes, 1991;
Simon and Tabin, 1993; Beauchemin and Savard, 1992).
It is also highly probable that other patterning genes,
such as those encoding cell signal molecules, are involved in regeneration as they are in development.
Three superfamilies of cell signaling molecules, the
transforming growth factor (TGF-b), fibroblast growth
factor (FGF) and Wnt gene families, play a major role in
the foundation and organization of embryonic tissues.
In particular, the Wnt gene family has been implicated
in multiple developmental processes and more specifically in patterning and growth control during vertebrate embryogenesis (review by McMahon, 1992; Nusse
*Correspondence to: Xavier Caubit, Institut de Biologie du Développement de Marseille, Laboratoire de Génétique et Physiologie du
Développement UMR C 9943, Faculté des Sciences de Luminy, 13288
Marseille cedex 9, France.
Received 21 June 1996; Accepted 9 September 1996
140
CAUBIT ET AL.
and Varmus, 1992; Parr and McMahon, 1994). The Wnt
genes, related to the Drosophila segment polarity gene
wingless, encode secreted cysteine-rich signalling glycoproteins which appear tightly associated with either
the cell surface (Papkoff and Schryver, 1990; Parkin et
al., 1993) or the extracellular matrix (Bradley and
Brown, 1990). A variety of experimental approaches on
vertebrates have shown that Wnt genes are implicated
in many aspects of embryogenesis such as mesoderm
formation, gastrulation, neurogenesis and organogenesis (see for review, Parr and McMahon, 1994). During
development, spatial and temporal expression patterns
have been reported for several Wnt genes in mouse
(Parr et al., 1993; Takada et al., 1994), Xenopus (Moon,
1993, for review), zebrafish (Krauss et al., 1992; Kelly
et al., 1993) and chicken (Dealy et al., 1993; Hollyday et
al., 1995). These studies have demonstrated that Wnt
genes are, in most cases, expressed in specific domains
during central nervous system (CNS), limb and tail
development. Recent studies have shown that Wnt-7a
is important in regulating the dorso-ventral polarity in
the developing limb of mouse (Parr and McMahon,
1995) and chicken (Yang and Niswander, 1995). Among
studies regarding Wnt genes in tail development
(Krauss et al., 1992; Takada et al., 1994), a null
mutation in Wnt-3a resulted in a severe truncation of
the body axis, missing caudal somites and tailbud.
We report here the expression pattern of a Pleurodeles waltl wnt gene (Pwnt-10a) during embryonic development and tail regeneration in adult newts. This gene
is the ortholog of the Xenopus and zebrafish wnt-10a
genes, which have only been detected to date in the
CNS (Wolda and Moon, 1992; Kelly et al., 1993).
Pwnt-10a is highly re-expressed in the blastema mesenchyme at the early regeneration stages of the adult
newt tail. During the growth of the regenerate, the
transcripts exhibit a rostrocaudal graded distribution
with a higher level in its mostcaudal part. These
findings strongly support the view that Pwnt-10a plays
a role in the growth and patterning during newt tail
regeneration.
RESULTS
Cloning of Pwnt-10a
Degenerated oligonucleotide primers matching conserved regions of the Wnt gene sequences were used to
amplify genomic DNA and adult newt tail cDNA.
Amplified fragments were isolated and used to screen a
cDNA library constructed with mRNA isolated from
young tail regenerates. This strategy allows us to
isolate a clone encoding a full-length polypeptide. This
cDNA contains an open reading frame of 1,170-bp
encoding 389 amino acids. The methionin residue encoded by the first ATG in the frame is followed by a
putative hydrophobic signal peptide sequence and a
polypeptide containing 23 of the invariant cysteine
residues characteristic of Wnt (Gavin et al., 1990;
Nusse and Varmus, 1992; Wolda and Moon, 1992). The
38 untranslated region consisting of 1,300 nt, contains a
polyadenylation signal (AAUAAA) 16 nt upstream from
a poly(A) tail (Fig. 1).
We compared the predicted amino-acid sequence of
this Pleurodeles waltl clone with Wnt sequences from
mouse (Gavin et al., 1990), Xenopus (Christian et al.,
1991a) and zebrafish (Krauss et al., 1992; Kelly et al.,
1993). The strongest homology was observed with zebrafish wnt-10A. Figure 2 shows the alignment of the
deduced amino acid sequence of the Pleurodeles clone
with that of zebrafish wnt-10a. The two proteins are
71% identical. Furthermore, the Pleurodeles clone shows
considerable amino-acid identity with three wnt-10a
partial sequences from Xenopus, salamander (Plethodon Jordani) and thresler shark (Alopius vulpinus)
(see Sidow, 1992; Wolda and Moon, 1992). The homology score with other Wnt sequences is about 30% (not
shown). It appears therefore that the wnt polypeptide
we isolated from Pleurodeles represents a true ortholog
of wnt-10a proteins.
Pleurodeles wnt-10a contains an insertion of 35 aminoacids between residues 153 and 189 which is not
present in any mouse (Gavin et al., 1990) or Xenopus
Wnt proteins (data not shown). Interestingly, the zebrafish wnt-10a contains the same conserved insertion.
Developmental Expression of PWnt-10a
RNAase protection analysis was performed to investigate the expression of Pwnt-10a mRNA during embryonic development of Pleurodeles. Total cellular RNA
was extracted from five embryos at different stages of
development, from fertilized eggs until late larval stage.
The Pwnt-10a antisense probe is 190 bp in length,
complementary to nucleotides 1,634–1,824 in 38 untranslated sequence (Fig. 1). A 117 bp GAPDH probe
was included in each hybridization to control for RNA
equivalence. As shown in Figure 3A, Pwnt-10a transcripts were first detected at mid-gastrula stage (stage
10) and transcription was maintained until tail-bud
stage. A very low expression level was detected at larval
stage. We examined Pwnt-10a mRNA distribution along
the antero-posterior axis of the tail-bud embryo. For
this purpose, embryos were dissected into head, trunk
and tail regions. Total RNA was extracted from these
regions and each fraction was analyzed by RNAase
protection. Pwnt-10a transcripts were detected in these
three regions of the embryo (Fig. 3A).
Expression of Pwnt-10A mRNA in Adult and
Regenerating Tissues
RNAase protection assay was used to analyze the
distribution of Pwnt-10a in various adult tissues. A low
level of expression was found in lung and kidney, and a
very faint signal was observed in adult brain after a
long exposure (Fig. 3B). During regeneration, expression pattern of Pwnt-10a was analyzed using mRNA
extracted from regenerates removed 3 days to 8 weeks
after the amputation. During the early stages of regeneration, the level of expression was significantly higher
(four- to fivefold) in regenerating tissues than in adult
Wnt-10a EXPRESSION IN NEWT TAIL REGENERATION
Fig. 1. Nucleotide sequence and deduced amino acid sequence of the
Pwnt-10a cDNA clone. A putative hydrophobic leader sequence, two
possible sites of N-linked glycosylation (N-X-S/T) and a polyadenylation
141
signal (AATAAA) in the 38 untranslated region are underlined. The
nucleotide sequence has been deposited in the GenBank data base with
accession number U65428.
142
CAUBIT ET AL.
Fig. 2. Comparison of amino acid sequences of Pleurodeles waltl wnt10a [Pwnt-10a and Zebrafish wnt-10a
(Zwnt-10a)]. Conserved cysteine residues are marked with arrowheads.
newt tail (Fig. 4). Interestingly, a sharp increase of
Pwnt-10a mRNA was detected as soon as 3 days after
tail amputation. This high level of transcription was
maintained during the first 2 weeks of regeneration.
The Pwnt-10a expression then progressively declined.
revealed a proximal decrease of Pwnt-10a expression,
at positions where, in the regenerate, extensive morphogenesis and differentiation took place.
Regional Distribution of Pwnt-10a mRNA in
Regenerates
In order to compare the expression levels of Pwnt-10a
in several tissue types of normal and regenerating
adult newt tails, reverse transcription polymerase chain
reaction (RT-PCR) assays were performed (Fig. 6). RNA
was extracted from spinal cord, skin and muscle of
normal tails. In addition, RNA was extracted from
wound epithelium, ependymal tubes, presumptive
muscle and cartilage regions collected with biopsy
needles from 800 µm thick cryostat sections of 3-weekold tail regenerates in which histological structures are
well defined (Nicolas et al., 1996). cDNAs from these
fractions were prepared and calibrated using GAPDH
amplification on serial dilutions of the samples (data
not shown). These normalized cDNA samples were then
used to PCR-amplify Pwnt-10a. Our results indicated
that Pwnt-10a transcripts were abundantly present in
the skin of adult newt tail and in the wound epithelium
of regenerating tail as well. No significant Pwnt-10a
expression was found in the muscle of adult newt tail
whereas high expression was detected in the mesenchymal fraction of the regenerating tail. Pwnt-10a showed
a significantly higher expression in the mesenchymal
fraction than in the epithelial one. It is noteworthy that
a sustained high expression was detected in tissue
To investigate for putative variations of Pwnt-10a
expression level along the antero-posterior and dorsoventral axes of the regenerating tail, 2–3-week-old
regenerates were dissected according to the two schemes
presented in Figure 5A. RNA preparations originating
from these fractions were normalized by optical density
and each fraction (10 µg of total RNA) was analyzed by
RNAase protection. As shown in Figure 5B, a protected
band was obtained with the preparations derived from
all parts of a regenerate. When the intensity of the
protected fragment from Pwnt-10a mRNA was normalized to the protected fragment of GAPDH mRNA, we
found that the level of Pwnt-10a mRNA varied along
the antero-posterior axis of the regenerate, the highest
transcript level being detected in its most posterior
part. Densitometry analysis (Fig. 5C) indicated that
Pwnt-10a mRNA exhibited a graded distribution along
the antero-posterior axis of the regenerate, with a
threefold higher abundance in the posterior part (apex)
of the regenerate compared to the anterior part (transition zone). The analysis did not reveal significant
variations along the dorso-ventral axis. This result
Tissue Distribution of Pwnt-10a Transcripts in
Adult and Regenerating Tails
Wnt-10a EXPRESSION IN NEWT TAIL REGENERATION
143
Fig. 3. RNAase protection analysis of the expression of Pwnt-10a
mRNA. A: Developmental expression of Pwnt-10a. Total RNA was
isolated from embryos of each stage and from sectioned regions corresponding to head, trunk and tail of tailbud stage embryos. Samples were
hybridized with 5 3 105 cts/min of Pwnt-10a probe; 5 3 104 cts/min of the
GAPDH probe was included in each sample to control the relative amount
of total RNA loaded for each sample. Note that Pwnt-10a mRNA is
observed from mid-gastrula stage to tailbud stage. B: Analysis of
expression of Pwnt-10a in various tissues of the adult newt.
regions corresponding to differentiating vertebral cartilage. Whereas we don’t detected wnt-10a expression in
the caudal spinal cord of adult animals by RNAase
protection (Fig. 3B), our RT-PCR assays demonstrated
that the gene is expressed in this tissus. We detected
also a slightly higher expression in the regenerating
spinal cord of blastema.
detected at late tail-bud stage (Wolda and Moon, 1992).
In addition, in situ hybridization data have shown that
wnt-10a transcripts have a precise distribution in the
developing brain. In the Pleurodeles embryo, wnt-10a
transcripts were detected by RNAase protection assays
at an earlier embryonic stage (mid-gastrula). Analyses
on RNA extracted from various parts (head, trunk and
tail) of the embryo demonstrated that these transcripts
were expressed along the embryonic axis. Our results
showed that, during development, wnt-10a was expressed in the tail bud, at least in urodeles. The
apparent discrepancies between Wnt-10a expression in
Pleurodeles, Xenopus and zebrafish could result from
the different sensitivity of the techniques used. It is
noteworthy that, until now, Wnt genes (especially wnt5a, wnt-5b, wnt-3a) other than Wnt-10a were shown to
be expressed in the tail bud in zebrafish (Krauss et al.,
1992), Xenopus (Moon et al., 1993) and mouse (Takada
et al., 1994).
DISCUSSION
We report here a Pleurodeles cDNA that encodes a
Wnt protein. Expression patterns of this gene, which
we referred to as Pwnt-10a—given that it is the ortholog of Zebrafish wnt-10a—were analyzed during embryonic development and tail regeneration in the adult
newt. Our main findings regarding this gene are discussed as follows.
Pwnt-10a Is Expressed in the Tail Bud During
Newt Development
During embryonic development in newts, Pwnt-10a
transcripts were detected from mid-gastrula to tail-bud
stages. The spatio-temporal expression pattern of Wnt10a gene was previously examined during zebrafish
and Xenopus development (Kelly et al., 1993; Wolda
and Moon, 1992). Zwnt-10a transcripts were first detected during the late stages of CNS development
(Kelly et al., 1993). Xwnt-10a transcripts were first
Pwnt-10a Is Re-expressed in a Rostrocaudal
Gradient in Tail Regenerates
In the adult newt, Pwnt-10a was detected in internal
organs (lung, kidney and brain) but also in the tail.
RT-PCR allowed us to show that Pwnt-10a was constitutively expressed in the adult normal tail skin. As
recently suggested for the homeodomain containing
144
CAUBIT ET AL.
Fig. 4. RNAase protection analysis of Pwnt-10a expression in adult
and in regenerating tail. Ten micrograms of total RNA was hybridized with
5 3 105 cts/min PWnt-10a probe and 5 3 104 cts/min GAPDH probe.
Lanes 3–6, tail regenerates of 3 days, 2, 4 and 8 weeks. Lanes 2 and 7,
normal tail of adult newt. Pwnt-10a is expressed at low level in adult tail.
Notice that Pwnt-10a mRNA are always more transcribed in regenerating
tissues than in adult tail tissues. Note the strong accumulation of
Pwnt-10a mRNA during the first 2 weeks of the regenerating process.
gene Pw-dll (Nicolas et al., 1996), the maintenance of
Pwnt-10a expression in the adult skin is probably due
to the periodical renewal of the epidermis in adulthood.
We also detected, by RT-PCR, a Pwnt-10a expression in
the caudal spinal cord. No significant expression was
observed in the adult tail muscle whereas strong expression was induced in the mesenchyme of tail regenerates. As soon as 3 days after tail amputation, Pwnt-10a
expression in regenerating tissues increased to a high
level which persisted for 2 weeks. In 2-week-old regenerates, Pwnt-10a expression was not confined to any
particular tissue. Indeed, analysis of epithelial and
mesenchymal fractions revealed that mRNA was present in the both. As Pwnt-10a was expressed at a same
level in adult skin and wound epithelium, the increased
amount of Pwnt-10a transcripts in tail regenerate was
most probably due to their accumulation in the blastemal mesenchyme. Different possibilities may account
for the high Pwnt-10a mRNA accumulation detected 3
days after amputation and in young (2-week-old) regenerates. For instance, this increase could be explained by
1) a gene regulation by transcriptional or post-transcriptional mechanisms in response to the amputation
trauma and 2) a selective recruitment and proliferation
of Pwnt-10a expressing mesenchymal cells from adult
normal tail to form the blastema. Further investigations are needed to identify the cells which express
Pwnt-10a in normal and regenerating tissues of adult
newt tail. In spite of repeated attempts, we have been
unable to detect Pwnt-10a transcripts using in situ
hybridization experiments, even with the most sensitive radioactive probes. Pwnt-10a transcripts display a
graded distribution along the antero-posterior axis
with maximal accumulation in the most posterior part
of 3-week-old regenerates, where mitotic activity is
especially high (Holtzer, 1956). Weak Pwnt-10a expression was detected in the anterior parts (including the
transition zone) of the regenerate, where extensive
morphogenetic events take place. This observation
suggests that Pwnt-10a function is required before
differentiation. Moreover, Pwnt-10a is expressed during blastema formation, so that it could participate in
the cell dedifferentiation process. It is then preferentially expressed in the caudal most part of the blastemal mesenchyme which corresponds to a ‘‘growth zone,’’
moving caudally in respect to the rostrocaudal differentiation gradient. Since transcripts are abundant within
this apical subectodermal area of the regenerate, an
attractive hypothesis is that Pwnt-10a could maintain,
in an undifferentiated state, blastemal mesenchymal
cells and/or regulate their proliferation. Several studies
have shown that Wnt proteins, at least some of them,
possess growth factor activity (Zakany and Duboule,
1993; Dickinson et al., 1994). It may be suggested that,
during newt tail regeneration, Wnt products could
cooperate with other growth factors to control cell
proliferation and patterning, as proposed for mesoderm
induction and axial specification in Xenopus embryo
(Christian et al., 1992) or limb development in vertebrates (Yang and Niswander, 1995; Parr and McMahon,
1995). Regarding this point, we have to keep in mind
that FGF and some of its related receptors have been
shown to be present in limb blastema (Boilly et al.,
1991; Poulain et al., 1993).
Therefore, findings reported in this paper led us to
the following conclusions: 1) For the first time in
amphibian urodeles, we cloned a Wnt gene identified as
an ortholog of Zebrafish Wnt-10a. Data on the expression pattern of this gene, which we referred to as
Pwnt-10a, support the view that it may play a role in
newt tail development, but also in tail regeneration, a
process during which it was shown to be re-expressed in
the blastemal mesenchyme in a rostrocaudal gradient.
2) Furthermore, since until now Wnt-10a was only
known to be involved in CNS development, our results
suggest that this gene may also play a role in other
developmental processes.
EXPERIMENTAL PROCEDURES
Animal Surgery Procedure
The urodelean amphibian used in this study were
embryos, larvae and adults of the European newt,
Pleurodeles waltl. Pw were obtained from the C.N.R.S.
Amphibian Farm, ‘‘Centre de Biologie du Développe-
Wnt-10a EXPRESSION IN NEWT TAIL REGENERATION
145
Fig. 5. Regional distribution of Pwnt-10a mRNA in 3-week-old regenerates. A: Schematic illustration of sectioned regions from regenerates.
Three-week-old regenerating tails were cut in three parts along the
antero-posterior axis respectively called: TZ (transition zone), M (median), Ap (apical); and three parts along the dorso-ventral axis: D (dorsal),
A (axial part of the regenerate), V (ventral). B: RNAase protection analysis
of Pwnt-10a mRNA in each region. C: Histogram of the relative distribution of Pwnt-10a transcripts in the different regions of the regenerate.
Autoradiograms were scanned with a densitometer and the relative
intensity of Pwnt-10a protected fragments were normalized to the amount
of GAPDH mRNA.
ment,’’ Université Paul Sabatier, Toulouse, France.
Animals were reared in groups of 10–12 and maintained in circulating tap water at 18–20°C. Before
surgery, adult animals were anesthetized with 1:1,000
MS 222 (tricaine methane sulfonate, Sigma). Amputations were performed in the third rostral part of the
tail. After appropriate periods of regeneration the blastema were harvested by reamputation.
amino acid sequence QECKCHG. The 38 set encompassed the sequence CXFHWCC. First strand cDNA
was generated by oligo(dt)-primed reversed transcription of 2 µg of adult tail poly(A)1 RNA using the
‘‘timeSaver cDNA synthesis kit’’ (Pharmacia). The conditions for PCR were as follows: 33 cycles at 94°C for 1
min, 47°C for 1 min, 72°C for 2 min. The Taq DNA
polymerase (Promega) was used. The reaction products
were separated in a 2.5% agarose (LMP) gel. Specific
bands (about 400–430 pb) were extracted to produce
probes. To obtain a cDNA clone encompassing the
full-length coding sequence of the Wnt genes, a newt
tail regenerate cDNA library constructed in ZAP II
(Stratagene) was screened using the amplification products. PCR generated fragments for Wnt were labeled by
random priming. Hybridizations were performed overnight at 50°C in 25% formamide, 0.2% polyvinylpyrrolidone, 0.2% BSA, 0.2% ficoll, 0.1% SDS, 1 mM EDTA,
Cloning of the Pwnt-10a cDNA
To clone wnt genes from Pleurodeles waltl, a PCR
based strategy (modified from Christian et al., 1991,
and Gavin et al., 1990) in combination with a library
screening were used. Genomic DNA (200 to 500 ng) and
adult tail cDNA were amplified using two sets of
degenerated oligonucleotide primers encoding two
highly conserved amino acid sequences found in all
Wnt-proteins. The 58 set encompassed the conserved
146
CAUBIT ET AL.
Fig. 6. RT-PCR analysis of the expression of Pwnt-10a in tissue
fractions from adult and regenerating tails. From left to right, lanes show
muscle of adult newt tail; mesenchymal fraction from regenerating tail;
skin of normal tail; epithelial fraction from regenerating tail; spinal cord of
adult newt tail; regenerating spinal cord; and tissue region corresponding
to differentiating cartilage. For each sample, the quantity of cDNA used for
Pwnt-10a-specific amplification was determined after GAPDH-specific
amplification on serial dilutions of reverse transcription mix. Note that
Pwnt-10a is not expressed in the muscle of the adult newt tail whereas it is
highly expressed in the mesenchymal fraction of the regenerating tail.
5 3 SSC, 10 mM HEPES (pH 7), 5 µg/ml denatured
fragmented salmon sperm DNA and about 100 µg/ml
yeast RNA. Filters were washed 3 3 10 min at room
temperature in 2 3 SSC, 0.1% SDS, then 3 3 15 min at
65°C in 1 3 SSC, 0.1% SDS. About 4 3 105 recombinant
phages were screened under these conditions and nine
positive clones were obtained. These clones were plaquepurified and converted into pBluescript plasmids. One
of these isolated clones contained a full-length cDNA
encoding the Pleurodeles wnt-10a (Pwnt-10a). The cDNA
was sequenced using subclones derived from convenient restriction sites. Additional sequences were obtained using several oligonucleotide primers to cover
the regions for which suitable subclones were unavailable. Five micrograms of double-stranded DNA was
sequenced using the dideoxynucleotide chain termination method (Sanger et al., 1977).
RNA Isolation and RNAase Protection Analysis
For adult and larval tissues, RNA was isolated from
frozen tissues by guanidium isothiocyanate extraction
followed by CsCl gradient centrifugation (Chirgwin et
al., 1979; Sambrook et al., 1989). For embryos, total
RNA was isolated using a modification of the guanidine
isothiocyanate/acid/phenol method (Chomczynski and
Sacchi, 1987). Embryos were homogenized in 4 M
guanidine isothiocyanate, 25 mM sodium citrate, pH
7.0, and 0.5% (w/v) Sarkosyl. RNA was extracted with
phenol/chloroform and then precipitated with ethanol.
Genomic DNA and polysaccharides were removed by a
further LiCl precipitation. RNAase protection assays
were carried out according to Krieg and Melton (1987)
with minor modifications. To generate Pwnt-10a spe-
cific riboprobe, a 1,004 bp Pst I-Pst I fragment was
subcloned into the Pst I site of pBluescript SK1. To
produce an antisense transcript, this subclone was
digested with AccI and in vitro transcription was
carried out using T3 RNA polymerase in the presence of
(a32P) rUTP (400–800 Ci/mmole, Amersham). A fulllength probe was purified from a 0.4 mm thin polyacrylamide gel by elution at 37°C in 0.3 M Na acetate, 0.5%
SDS, 2 mM EDTA and 20 µg/ml tRNA. Hybridization
was carried out in the presence of 80% formamide, 0.4
M NaCl, 40 mM PIPES and 1 mM EDTA at 50°C for 36
hr. The samples were digested for 1 hr at 37°C using 10
µg/ml RNAase A and 500 units/ml RNase T1 (all from
Boehringer Mannheim), followed by proteinase K at
37°C for 15 min. After phenol/chloroform extraction and
ethanol precipitation, the protected fragments were
resolved by electrophoresis on a 5% polyacrylamide gel,
and exposed to Kodak X-OMAT AR film with intensifying screens at 80°C.
To control for RNA equivalence, a Pleurodeles glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was
included in each sample (Shi et al., 1992). GAPDH is a
typical ‘‘housekeeping gene’’ (Fort et al., 1985). This
transcript is an adequate normalizing signal. It represents the overall amount of RNA isolated from tissues
without spatial restriction. The probes contained a
small portion of pBluescript polylinker sequence as an
aid in the identification of complete RNAase digestion
and the specificity of the protected fragments. The
signals were quantified using a scanner (Pharmacia
LKB) linked to a computer.
RT-PCR Assay
Adult and regenerating tissues were collected as
previously described (Nicolas et al., 1996). Total RNA
were isolated using RNA NOW (Biogentex) according to
the manufacturer’s instructions. Specific primers for
the Pleurodeles waltl GAPDH gene: 58-GCCAGACAGTTTGTAGTCCAAGAGG-38 and the Pwnt-10a
gene: 58-GGACTGCTTGACTTCCGAGAA TCTG-38 position 1,405–1,429, were used to synthesize cDNA with
the First-Strand cDNA Synthesis Kit (Pharmacia) according to the supplier’s instructions. Before amplification, the reaction mixture was denatured for 5 min at
94°C. Amplification of cDNA was made in a DNA
thermal cycler (Perkin Elmer Cetus) in a final volume
of 50 µl and 1.25 U of Taq DNA polymerase (Promega).
GAPDH and Pwnt-10a amplifications were performed
for 22 cycles under the following conditions: denaturation for 1 min at 94°C, primer annealing for 1 min at
57°C, extension for 1 min at 72°C. 40% of the RT-PCR
products were separated in a 2% agarose gel, transferred to nitrocellulose membrane (Schleicher and
Schuell). After transfer, the membranes were hybridized with Pwnt-10a and GAPDH radioactive probes.
Band intensity was measured by densitometry and
values obtained for Wnt-10a in different samples were
normalized relative to the GAPDH signals. Amplifications were performed on serial dilutions of cDNA to be
Wnt-10a EXPRESSION IN NEWT TAIL REGENERATION
sure that the signal obtained was a linear function of
the input cDNA.
The following oligonucleotides were used during the
PCR procedure: Pwnt-10a: Sense primer 58-AATGAGGCTCCACAACAACC-38 (Fig. 1: positions 902–921),
antisense primer 58-CCGAGAATCTGCCTACTTGC-38
(Fig. 1: positions 1,396–1,415). GAPDH: Sense primer
58-GATTCAAAGGCACCGTCAAG-38, antisense primer
58-GCGTTG CTTACTACCAGGGA-38. This primer pair
gives a 277 bp fragment.
ACKNOWLEDGMENTS
The skilful assistance of P. Sauve for oligonucleotides
synthesis, and G. Turini and M. Berthoumieux for
photography, were greatly appreciated. We thank Jean
Pierre Arsanto and Jacques Pradel for critical reading
this manuscript. This work was supported by the
Association Francaise contre les Myopathies (AFM)
and by the Institut pour la Recherche sur la Moelle
Epinière (IRME). X.C. is supported by a fellowship from
the Ligue National Contre le Cancer. S.N. is supported
by a fellowship from AFM.
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