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Keywords:

  • AER;
  • β-catenin;
  • chick;
  • expression pattern;
  • limb;
  • Wnt10a

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The apical ectodermal ridge (AER) is indispensable for vertebrate limb development and requires Wnt/β-catenin signaling for induction and maintenance. We report identification and involvement of Wnt10a in AER formation during chick limb development. Chicken Wnt10a has 82% identity with mouse Wnt10a in the amino acid sequence. The Wnt10a gene was expressed broadly in the surface ectoderm from as early as stage 10. By stage 15, the expression was restricted to the surface ectoderm overlying the lateral plate mesoderm. Wnt10a expression became intensified in the presumptive limb ectoderm during AER formation, and subsequently intense expression signals persisted in the AER. Wnt10a misexpression led to ectopic Fgf8 expression in the developing limb ectoderm and induced translocation of β-catenin in chick embryo fibroblasts. These results suggest that Wnt10a is involved in AER formation in the chick limb bud through the Wnt/β-catenin signaling pathway. Developmental Dynamics 233:282–287, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Wnt/β-catenin signaling plays key roles in formation and maintenance of the apical ectodermal ridge (AER) during limb development. Misexpression of the constitutively active form of β-catenin leads to induction of ectopic Fgf8 expression in the developing chick limb bud (Kengaku et al., 1998; Soshnikova et al., 2003). Conditional mutation of β-catenin, on the other hand, results in lack of Fgf8 expression (Barrow et al., 2003; Soshnikova et al., 2003). Compound mutation of Lef1/Tcf1 also impairs AER formation (Galceran et al., 1999). Therefore, Wnt/β-catenin signaling in the AER is required for Fgf8 expression to induce and maintain this activity.

Wnt3a and Wnt3 are known to trigger Wnt/β-catenin signaling during AER formation in chick and mouse embryos, respectively. In chick embryos, Wnt3a is expressed in the presumptive limb ectoderm before initial expression of Fgf8 in the limb ectoderm (Kengaku et al., 1997). Misexpression of mouse Wnt3a leads to induction of ectopic Fgf8 expression in the chicken limb bud (Kengaku et al., 1998). In mouse embryos, Wnt3 but not Wnt3a is expressed ubiquitously in the limb ectoderm (Parr et al., 1993). Conditional Wnt3 mutation results in the lack of Fgf8 expression in the mouse limb bud (Barrow et al., 2003).

In attempting to clarify which Wnt members are implicated in AER formation and maintenance, we found that Wnt10a was expressed in the AER of chick embryos. Here, we report on the expression patterns of chicken and mouse Wnt10a. We show the phenotypic consequence of Wnt10a misexpression in the developing chick limb bud, and the distribution of nuclear β-catenin in Wnt10a-expressing fibroblasts.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Expression Pattern of Wnt10a in Chicken and Mouse Embryos

We screened chicken genomic libraries with mouse Wnt10a as a probe and obtained a full-length chicken Wnt10a cDNA by 5′ rapid amplification of cDNA ends (RACE). The chicken Wnt10a was, respectively, 82.0% and 61.2% identical to the mouse Wnt10a and Wnt10b amino acid sequence (Fig. 1A,B).

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Figure 1. Sequence analysis of chick Wnt10a. A: Comparison of the amino acid sequence of chick Wnt10a and mouse Wnt10a. The amino acid sequence was 82% identical between them. Asterisks indicate the identical amino acid residues. c, chicken; m, mouse. B: A phylogenetic tree predicting the evolutionary relationship among chicken Wnt10a and other known mouse Wnts (mWnt), generated by using the UPGMA method (Genetyx, Tokyo). The chicken Wnt10a sequence is grouped with mouse Wnt10a (arrowhead).

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To investigate the possible roles for Wnt10a in chick development, we examined the expression pattern of Wnt10a in chicken embryos from stage 10 to stage 34 using in situ hybridization. At stage 10, Wnt10a transcripts were detected weakly but significantly in the surface ectoderm posterior to the otic vesicle (Fig. 2A). Specifically, intense expression signals were observed in the surface ectoderm along the neural fold of the closing neural tube (Fig. 2A, arrowheads). At stage 12, strong expression of Wnt10a began to be detected in surface ectoderm around the tail bud (Fig. 2B, arrowhead). By stage 15, the expression that had been detected broadly in the surface ectoderm was restricted to the ectoderm overlying the lateral plate mesoderm but not the neural tube and somites (Fig. 2C).

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Figure 2. Wnt10a is localized in the apical ectodermal ridge (AER) in chick and mouse embryos. A–I: Chicken embryo. J–L: Mouse embryo. A,B: Expression of Wnt10a at stage (St.) 10 (A) and 12 (B), respectively. Dorsal view of the whole embryo. The expression is observed broadly in the surface ectoderm and strongly in the surface ectoderm along the neural folds of the closing neural tube (A, arrowheads) and around the tail bud (B, arrowhead). C: Expression of Wnt10a at stage 15. A cross-section at a prospective forelimb level, showing dorsal on the top. The expression is observed in the surface ectoderm overlying the lateral plate mesoderm. D,E: Expression of Wnt10a and Wnt3a, respectively, at stage 16. E: Wnt3a is also expressed in the dorsal neural tube. Lateral view of the prospective forelimb region, showing dorsal on the left. F,G: Expression of Wnt10a and Fgf8, respectively, at stage 18. Wnt10a expression becomes strong at the region between arrowheads. Lateral view of the forelimb and the trunk regions. H,I: Expression of Wnt10a and Fgf8, respectively, at stage 21. Lateral view of the forelimb. H, window: A cross-section through the AER. H: Wnt10a expression is observed in the AER and weakly in the nonridge ectoderm (arrowheads) but is not detected in the limb mesenchyme (window). J,K: Expression of Wnt10a at 10.5 days post coitum. Wnt10a expression is localized in the forelimb and hindlimb AER (arrowheads). J: Lateral view of the whole embryo. Ventral view of the forelimb bud. K: Dorsal on the right. L: Expression of Wnt10a at 11.5 days post coitum. Ventral view of the forelimb bud. Dorsal on the right.

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During limb development, strong expression of Wnt10a was restricted in the ectoderm of the presumptive forelimb and hindlimb fields and then restricted in the developing AER. At stage 16, the expression signals detected uniformly in the surface became intensified in the prospective forelimb field (Fig. 2D). Wnt10a expression was observed uniformly in the limb fields at the dorsal–ventral levels. The expression domain of Wnt10a overlapped that of Wnt3a (Fig. 2D,E). At stage 18, strong expression of Wnt10a became detectable in the ectodermal domain located at the dorsal–ventral border on the developing AER (Fig. 2F, arrowheads). The Wnt10a expression domain was narrower at the dorsal–ventral levels and more extensive at the anterior–posterior levels than the Fgf8 expression domain (Fig. 2G). At stage 21, strong expression of Wnt10a was observed in the AER expressing Fgf8 (Fig. 2H,I). In contrast, in nonridge ectoderm, Wnt10a expression became very weak (Fig. 2H, arrowheads). During later stages, Wnt10a expression in the AER continued, but at stage 29, the expression disappeared in the tip of the forelimb AER (data not shown). By stage 32, Wnt10a expression was undetectable in the forelimb AER (data not shown). At stage 34, Wnt10a expression was observed in the hindlimb AER (data not shown). No expression signals were detected in the limb mesenchyme throughout all stages (Fig. 2H, window).

We also detected Wnt10a expression in mouse embryos. Expression of Wnt10a was first observed at 10.5 days post coitum (d.p.c.) in the AER of the limb buds (Fig. 2J,K) and maintained until 11.5 d.p.c. (Fig. 2L). Wnt10a was expressed in the AER of both chicken and mouse embryos.

Wnt10a Induces Ectopic Fgf8 Expression in the Chick Limb Bud

During AER formation, Wnt10a signals were first detected intensely in the surface ectoderm of the presumptive limb field, and subsequently in the AER, suggesting that Wnt10a is involved in AER formation during chick limb development. To test this possibility, we carried out Wnt10a misexpression studies in the chick limb bud using the replication-competent retroviral vector (RCAS). RCAS-Wnt10a viruses were injected into the presumptive limb field of stage 11–13 embryos. Exogenous Wnt10a was misexpressed in the developing limb bud and led to ectopic Fgf8 expression in the limb ectoderm adjacent or distant to the AER (Fig. 3A–C, 8/13). Similar results were obtained by misexpressing mouse Wnt3a in the chick limb bud (Fig. 3D–F).

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Figure 3. Wnt10a can induce ectopic Fgf8 expression and activate the Wnt/β-catenin signaling. AF: Misexpression of chick Wnt10a (cWnt10a, A–C) or mouse Wnt3a (mWnt3a, D–F) using replication-competent retroviral vector in the developing chick limb bud. Expression of Fgf8 in the injected limb bud. Dorsal is on the left. In cWnt10a misexpression, ectopic Fgf8 expression is observed in the ectoderm lateral to the apical ectodermal ridge (AER,A,B) or distant to the AER (C, 8/13), as well as in mWnt3a misexpression (D–F). G: Western blot analysis of β-catenin in the cytoplasmic and membrane fractions of chicken embryonic fibroblasts (CEFs), expressing excess Wnt proteins. Cytoplasmic β-catenin levels are elevated in CEFs overexpressing chicken Wnt10a and mouse Wnt3a, compared with in those overexpressing chicken Wnt5a and control CEFs. Left, position of a molecular weight marker protein. c10a, chicken Wnt10a; m3a, mouse Wnt3a; c5a, chicken Wnt5a; (−), control CEFs alone. H–J: Detection of nuclear β-catenin on CEFs expressing Wnts. H,I: In Wnt10a (H) or mouse Wnt3a (I) misexpression, β-catenin (green) is observed in the nucleus (red) in addition to accumulated cytoplasmic β-catenin. The arrowheads indicate the areas of β-catenin and nucleus colocalization (yellow). J: In contrast, β-catenin is undetectable in the nucleus in control CEFs.

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Wnt10a Activates the Wnt/β-Catenin Signaling Pathway

The β-catenin signaling is known to be critical for AER formation during vertebrate limb development. To test whether Wnt10a can activate the β-catenin signaling pathway, we examined accumulation of cytoplasmic β-catenin in chicken embryonic fibroblasts (CEFs). Overexpression of Wnt10a triggered an increase in cytoplasmic β-catenin levels, compared with the control (Fig. 3G). The increasing levels of β-catenin induced by Wnt10a were approximately equal to those induced by mouse Wnt3a (Fig. 3G). Wnt5a, which does not activate β-catenin signaling, did not accumulate β-catenin in CEFs, as previously reported (Shimizu et al., 1997; Kawakami et al., 2001).

Cytoplasmic β-catenin is translocated into the nucleus and in association with LEF/TCF transcriptional factors regulates transcription of the target genes. We examined whether Wnt10a promoted translocation of β-catenin into the nucleus. RCAS constructs were transfected into CEFs, and then anti–β-catenin antibody and propidium iodide were added to detect β-catenin protein and the nucleus, respectively. Distributions of β-catenin protein (green) and the nucleus (red) into the cells were analyzed using a confocal laser-scanning microscope. In CEFs expressing Wnt10a or mouse Wnt3a, β-catenin protein was detected within the nucleus (yellow, Fig. 3H,I, arrowheads). In contrast, no β-catenin protein was detected within the nucleus in control CEFs (Fig. 3J). These results indicated that Wnt10a activated the β-catenin signaling pathway in AER formation.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In this study, we isolated the Wnt10a gene in chicken embryo and identified Wnt10a expression pattern in both chicken and mouse embryos (Figs. 1, 2). We also found that Wnt10a promotes cytoplasmic accumulation and nuclear distribution of β-catenin (Fig. 3G–J), indicating that it triggers the β-catenin signaling pathway. We showed that Wnt10a misexpression leads to ectopic Fgf8 expression (Fig. 3A–C). We also discussed the roles of Wnt10a in AER formation in the chick and mouse.

Wnt10a and Wnt3a in Chicken Embryo

Wnts and Fgfs play a key role in the genetic cascade that controls limb development. First, Wnt2b/8c is expressed in the lateral plate mesoderm of the presumptive limb fields and induces Fgf10 expression in the lateral mesoderm (Kawakami et al., 2001). Fgf10 induces Wnt3a expression in the overlying ectoderm (Kawakami et al., 2001). Then, Wnt3a promotes Fgf8 expression in the surface ectoderm, which maintains Fgf10 expression in the underlying mesenchyme (Ohuchi et al., 1997a; Kengaku et al., 1998).

We found that Wnt10a expression started with the same timing as Wnt3a. Although we tested whether Wnt10a expression is induced by the implantation of cells expressing Fgf10 in the flank region, Wnt10a was not detected after 24 hr (data not shown). Wnt3a induction, on the other hand, was observed after 24 hr (data not shown). Therefore, Wnt10a expression may be regulated directly by a more-upstream factor such as Wnt2b/8c or Tbx5/4, which is involved in induction of the forelimb/hindlimb (Kawakami et al., 2001; Takeuchi et al., 2003).

On the basis of an analysis of its overexpression and expression patterns, Wnt3a is considered a candidate of AER inducers, because mouse Wnt3a was able to induce ectopic Fgf8 expression when misexpressed in the chick limb bud (Kengaku et al., 1998). Wnt10a as well as Wnt3a leads to ectopic Fgf8 expression. During AER formation, Wnt10a expression overlaps that of Wnt3a. Wnt10a expression becomes strong in the presumptive limb ectoderm at stage 16 before Fgf8 expression. Wnt3a expression is first detected in the region at stage 16 (Kengaku et al., 1997). The expression of Wnt10a and Wnt3a is maintained until stage 27. However, there is a difference in the duration of Wnt10a and Wnt3a expression in the AER. Although Wnt10a expression continues until stage 32, Wnt3a expression becomes undetectable at stage 29. In addition, Wnt10a is not expressed in the dorsal neural tube, proximal otic vesicle, and feather buds, whereas Wnt3a is detected in these regions (Hollyday et al., 1995; Chang et al., 2004). These observations suggest that Wnt10a function is redundant with that of Wnt3a in induction of Fgf8 expression.

Wnt10a and Wnt3 in Mouse Embryo

Mouse Wnt3, an AER inducer, is expressed ubiquitously in the limb ectoderm (Parr et al., 1993; Barrow et al., 2003). Because the AER is localized at the dorsal–ventral border, this means that other Wnt members and/or more factors, such as bone morphogenetic protein signaling molecules, are also involved in AER formation (Barrow et al., 2003; Soshnikova et al., 2003). Wnt10a expression is localized in developing AER in both mice and chickens. In addition, overexpression of mouse Wnt10a led to ectopic Fgf8 expression in chicken embryos (data not shown). These data suggest that Wnt10a is also involved in AER formation. To elucidate the role of Wnt10a in AER formation, analysis of the targeted disruption of the Wnt10a gene in mice is needed.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Chicken Embryos

Fertilized chicken eggs were incubated at 38°C. The embryos were staged according to Hamburger and Hamilton (1951). Chicken embryo fibroblasts were cultured in Dulbecco's modified Eagle medium (D-MEM) containing 2% fetal bovine serum (FBS) and 1% chicken serum.

Cloning of Chicken Wnt10a

To isolate chicken Wnt10a cDNA, we first screened genomic libraries using mouse Wnt10a as a probe. To isolate full-length cDNA, we performed 5′-RACE (Invitrogen). The sequence was determined and deposited to the GenBank/DDBJ with the accession number AB177400.

Plasmid Constructions

To carry out misexpression in CEFs and in the developing limb bud, the full coding regions of chicken Wnt10a and mouse Wnt3a were subcloned into RCAS L14 or L44 (Kawakami et al., 1996). The RCAS construct for chicken Wnt5a has been reported previously (Kawakami et al., 1999).

In Situ Hybridization

A plasmid containing the entire coding region of chicken Wnt10a was digested with NsiI followed by blunting and was transcribed with T3 RNA polymerase to prepare the antisense probe. Antisense RNA probes for chicken Fgf8 (Ohuchi et al., 1997b) and chicken Wnt3a (Kawakami et al., 2000) had been synthesized previously. Embryos were fixed in 4% paraformaldehyde (PFA) in PBS at 4°C overnight and dehydrated in ethanol. Whole-mount in situ hybridization was performed, as described previously (Kawakami et al., 1996). Stained embryos were embedded in 0.5% gelatin/30% albumin and sectioned with a Vibratome at 30–50 μm.

Western Analysis

Cytoplasmic β-catenin accumulation assay was carried out using a method previously described (Shimizu et al., 1997; Kawakami et al., 2001). CEFs were transfected with the RCAS constructs. Samples containing 50 μg of total protein were subjected to electrophoresis through sodium dodecyl sulfate-polyacrylamide (7.5%) gels. The primary antibody against β-catenin (BD Transduction Lab.) was used at a dilution of 1:2,000. The protein was detected using the ECL Plus System (Amersham Pharmacia Biotech) with a 1/10 dilution of the reagent mixture.

Detection of Nuclear β-Catenin

CEFs were transfected with the RCAS constructs and cultured on cover glasses until the cultures became confluent. After fixation in 4% PFA in PBS at room temperature for 15 min, cells were treated with 50% methanol in PBS at 4°C for 5 min. After incubation with 20 μg/ml RNase A in PBS at 37°C for 30 min, cells were treated with 1% bovine serum albumin in PBS at room temperature for 60 min. The primary antibody against β-catenin (BD Transduction Lab.) was used at a dilution of 1:2,000 at 4°C overnight. The secondary antibody (fluorescein isothiocyanate conjugate) against mouse-IgG was used at a dilution of 1:1,000 at room temperature for 60 min. The nuclei were stained with 50 μg/ml propidium iodide at room temperature for 2 min. The cells were observed using a confocal laser-scanning microscope.

Misexpression in Chick Limb Buds

Preparation of the retroviruses bearing the Wnt genes was performed according to the method of Fekete and Cepko (1993). The RCAS construct was transfected into DF-1 cells (ATCC; #CRL-12203) using Lipofectamine 2000 (Invitrogen). Cells were grown in the culture medium (5% FBS and 1% chicken serum in D-MEM). After confluence in a 15-cm culture dish, the medium was replaced with 20 ml of fresh medium (2% FBS and 0.5% chicken serum in D-MEM). On the next day, the medium was harvested and replaced. The medium was pooled after four replacements. To concentrate the virus particles in the culture medium, the medium was centrifuged at 4°C for 10 min at 3,000 × g. The supernatant was filtered through a membrane with a pore size of 0.45 μm (Whatmann; 6896-2504). After the medium was centrifuged again at 4°C for 3 hr at 70,000 × g, the major part of the medium was discarded by decantation and the virus particles in the bottom pellet were resuspended in a small volume of the remaining medium (approximately 200 μl). The final titer of the virus was usually 180- to 240-fold concentration.

Virus infection was carried out using Line M, retrovirus-free, fertilized chicken eggs (Nisseiken). The retrovirus was injected into the presumptive limb fields on the right side of stage 10–13 embryos. After 1.5–2 days of reincubation, the embryos were fixed in 4% PFA in PBS and the gene expression pattern was determined using in situ hybridization.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Ms. C. Komaguchi-Wada and A. Shiga-Oda for technical assistance. We also thank D.H. Waterbury for critical reading of the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES