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. 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