Canonical Wnt signaling and the regulation of divergent mesenchymal Fgf8 expression in Axolotl limb development and regeneration

  1. Giacomo Leon Glotzer
  2. Pietro Tardivo  Is a corresponding author
  3. Elly M Tanaka  Is a corresponding author
  1. Research Institute of Molecular Pathology, Austria


The expression of Fibroblast growth factors (Fgf) ligands in a specialized epithelial compartment, the Apical Ectodermal Ridge (AER), is a conserved feature of limb development across vertebrate species. In vertebrates, Fgf 4, 8, 9, and 17 are all expressed in the AER. An exception to this paradigm is the salamander (axolotl) developing and regenerating limb, where key Fgf ligands are expressed in the mesenchyme. The mesenchymal expression of Amex.Fgf8 in axolotl has been suggested to be critical for regeneration. To date, there is little knowledge regarding what controls Amex.Fgf8 expression in the axolotl limb mesenchyme. A large body of mouse and chick studies have defined a set of transcription factors and canonical Wnt signaling as the main regulators of epidermal Fgf8 expression in these organisms. In this study, we address the hypothesis that alterations to one or more of these components during evolution has resulted in mesenchymal Amex.Fgf8 expression in the axolotl. To sensitively quantify gene expression with spatial precision, we combined optical clearing of whole-mount axolotl limb tissue with single molecule fluorescen in situ hybridization and a semi-automated quantification pipeline. Several candidate upstream components were found expressed in the axolotl ectoderm, indicating that they are not direct regulators of Amex.Fgf8 expression. We found that Amex.Wnt3a is expressed in axolotl limb epidermis, similarly to chicken and mouse. However, unlike in amniotes, Wnt target genes are activated preferentially in limb mesenchyme rather than in epidermis. Inhibition and activation of Wnt signaling results in downregulation and upregulation of mesenchymal Amex.Fgf8 expression respectively. These results implicate a shift in tissue responsiveness to canonical Wnt signaling from epidermis to mesenchyme as one step contributing to the unique mesenchymal Amex.Fgf8 expression seen in the axolotl.

Data availability

Figure 5- source data contains the numerical data used to generate the figureFiji macros used for image analysis have been uploaded to Github:

The following previously published data sets were used

Article and author information

Author details

  1. Giacomo Leon Glotzer

    Vienna BioCenter, Research Institute of Molecular Pathology, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  2. Pietro Tardivo

    Vienna BioCenter, Research Institute of Molecular Pathology, Vienna, Austria
    For correspondence
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7878-5272
  3. Elly M Tanaka

    Vienna BioCenter, Research Institute of Molecular Pathology, Vienna, Austria
    For correspondence
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4240-2158


Marshallplan-Jubiläumsstiftung (1062 1304 14 24 2020)

  • Giacomo Leon Glotzer

H2020 European Research Council (AdG 7420346)

  • Elly M Tanaka

Austrian Science Fund (I4846)

  • Elly M Tanaka

Research Institute of Molecular Pathology

  • Pietro Tardivo

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.


Animal experimentation: All axolotl handling and surgical procedures were performed in accordance with local ethics committee guidelines. Animal experiments were performed as approved by the Magistrate of Vienna (animal license number GZ: 9418/2017/12).

Reviewing Editor

  1. Marianne E Bronner, California Institute of Technology, United States

Publication history

  1. Preprint posted: April 25, 2022 (view preprint)
  2. Received: April 29, 2022
  3. Accepted: May 6, 2022
  4. Accepted Manuscript published: May 19, 2022 (version 1)
  5. Version of Record published: May 31, 2022 (version 2)


© 2022, Glotzer et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.


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  1. Giacomo Leon Glotzer
  2. Pietro Tardivo
  3. Elly M Tanaka
Canonical Wnt signaling and the regulation of divergent mesenchymal Fgf8 expression in Axolotl limb development and regeneration
eLife 11:e79762.

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