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

Abstract

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: https://github.com/labtanaka/glotzer_fiji_scripts

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
    pietro.tardivo@imp.ac.at
    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
    elly.tanaka@imp.ac.at
    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

Funding

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.

Ethics

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)

Copyright

© 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.

Metrics

  • 788
    Page views
  • 221
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Giacomo Leon Glotzer
  2. Pietro Tardivo
  3. Elly M Tanaka
(2022)
Canonical Wnt signaling and the regulation of divergent mesenchymal Fgf8 expression in Axolotl limb development and regeneration
eLife 11:e79762.
https://doi.org/10.7554/eLife.79762

Further reading

    1. Developmental Biology
    Qiyan Mao et al.
    Research Article Updated

    Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. More efficient myofiber bundling accelerates the speed of sarcomerogenesis suggesting that tension generated by bundling promotes sarcomerogenesis. We tested this hypothesis by directly probing tension and found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.

    1. Developmental Biology
    2. Stem Cells and Regenerative Medicine
    Marta Perera et al.
    Research Article

    During embryonic development cells acquire identity at the same time as they are proliferating, implying that an intrinsic facet of cell fate choice requires coupling lineage decisions to rates of cell division. How is the cell cycle regulated to promote or suppress heterogeneity and differentiation? We explore this question combining time lapse imaging with single cell RNA-seq in the contexts of self-renewal, priming and differentiation of mouse embryonic stem cells (ESCs) towards the Primitive Endoderm lineage (PrE). Since ESCs are derived from the Inner Cell Mass of the mammalian blastocyst, ESCs in standard culture conditions are transcriptionally heterogeneous containing subfractions that are primed for either of the two ICM lineages, Epiblast and PrE. These subfractions represent dynamic states that can readily interconvert in culture, and the PrE subfraction is functionally primed for endoderm differentiation. Here we find that differential regulation of cell cycle can tip the balance between these primed populations, such that naïve ESC culture conditions promote Epiblast-like expansion and PrE differentiation stimulates the selective survival and proliferation of PrE-primed cells. In endoderm differentiation, we find that this change is accompanied by a counter-intuitive increase in G1 length that also appears replicated in vivo. While FGF/ERK signalling is a known key regulator of ESCs and PrE differentiation, we find it is not just responsible for ESCs heterogeneity, but also cell cycle synchronisation, required for the inheritance of similar cell cycles between sisters and cousins. Taken together, our results point to a tight relationship between transcriptional heterogeneity and cell cycle regulation in the context of lineage priming, with primed cell populations providing a pool of flexible cell types that can be expanded in a lineage-specific fashion while allowing plasticity during early determination.