Eya2 promotes cell cycle progression by regulating DNA damage response during vertebrate limb regeneration

  1. Konstantinos Sousounis
  2. Donald M Bryant
  3. Jose Martinez Fernandez
  4. Samuel S Eddy
  5. Stephanie L Tsai
  6. Gregory C Gundberg
  7. Jihee Han
  8. Katharine Courtemanche
  9. Michael Levin
  10. Jessica L Whited  Is a corresponding author
  1. Harvard University, United States
  2. Brigham & Women's Hospital, United States
  3. Tufts University, United States
  4. Harvard Medical School, United States

Abstract

How salamanders accomplish progenitor cell proliferation while faithfully maintaining genomic integrity and regenerative potential remains elusive. Here we found an innate DNA damage response mechanism that is evident during blastema proliferation (early- to late-bud) and studied its role during tissue regeneration by ablating the function of one of its components, Eyes absent 2. In eya2 mutant axolotls, we found that DNA damage signaling through the H2AX histone variant was deregulated, especially within the proliferating progenitors during limb regeneration. Ultimately, cell cycle progression was impaired at the G1/S and G2/M transitions and regeneration rate was reduced. Similar data were acquired using acute pharmacological inhibition of the Eya2 phosphatase activity and the DNA damage checkpoint kinases Chk1 and Chk2 in wild-type axolotls. Together, our data indicate that highly-regenerative animals employ a robust DNA damage response pathway which involves regulation of H2AX phosphorylation via Eya2 to facilitate proper cell cycle progression upon injury.

Data availability

Raw data can be accessed in the NIH Sequence Read Archive: SUB6297224.

The following previously published data sets were used

Article and author information

Author details

  1. Konstantinos Sousounis

    Stem Cell and Regenerative Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Donald M Bryant

    Stem Cell and Regenerative Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Jose Martinez Fernandez

    Stem Cell and Regenerative Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Samuel S Eddy

    Orthopedic Surgery, Brigham & Women's Hospital, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Stephanie L Tsai

    Stem Cell and Regenerative Biology, Molecular and Cellular Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7549-3418
  6. Gregory C Gundberg

    Stem Cell and Regenerative Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Jihee Han

    Stem Cell and Regenerative Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Katharine Courtemanche

    Stem Cell and Regenerative Biology, Harvard University, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Michael Levin

    The Allen Discovery Center, Tufts University, Medford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7292-8084
  10. Jessica L Whited

    Department of Orthopedic Surgery, Harvard Medical School, Boston, United States
    For correspondence
    jwhited@partners.org
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3709-6515

Funding

Sara Elizabeth O'Brien Trust (Postdoctoral fellowship)

  • Konstantinos Sousounis

National Institutes of Health (K99EY029361)

  • Konstantinos Sousounis

Paul G. Allen Family Foundation (Allen Discovery Center at Tufts)

  • Michael Levin
  • Jessica L Whited

National Institutes of Health (1DP2HD087953)

  • Jessica L Whited

National Institutes of Health (1R01HD095494)

  • Jessica L Whited

Harvard Stem Cell Institute (HIP)

  • Jose Martinez Fernandez

Howard Hughes Medical Institute (Gilliam Fellowship)

  • Donald M Bryant

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 experiments involving animals were performed according to IACUC protocol #2016N000369 at Brigham and Women's Hospital. All surgeries were performed while animals were anesthetized in tricaine. All experiments were planned and executed in manners that minimized animal suffering.

Copyright

© 2020, Sousounis 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

  • 3,657
    views
  • 477
    downloads
  • 30
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. Konstantinos Sousounis
  2. Donald M Bryant
  3. Jose Martinez Fernandez
  4. Samuel S Eddy
  5. Stephanie L Tsai
  6. Gregory C Gundberg
  7. Jihee Han
  8. Katharine Courtemanche
  9. Michael Levin
  10. Jessica L Whited
(2020)
Eya2 promotes cell cycle progression by regulating DNA damage response during vertebrate limb regeneration
eLife 9:e51217.
https://doi.org/10.7554/eLife.51217

Share this article

https://doi.org/10.7554/eLife.51217

Further reading

    1. Cell Biology
    2. Developmental Biology
    Sarah Rubin, Ankit Agrawal ... Elazar Zelzer
    Research Article

    Chondrocyte columns, which are a hallmark of growth plate architecture, play a central role in bone elongation. Columns are formed by clonal expansion following rotation of the division plane, resulting in a stack of cells oriented parallel to the growth direction. In this work, we analyzed hundreds of Confetti multicolor clones in growth plates of mouse embryos using a pipeline comprising 3D imaging and algorithms for morphometric analysis. Surprisingly, analysis of the elevation angles between neighboring pairs of cells revealed that most cells did not display the typical stacking pattern associated with column formation, implying incomplete rotation of the division plane. Morphological analysis revealed that although embryonic clones were elongated, they formed clusters oriented perpendicular to the growth direction. Analysis of growth plates of postnatal mice revealed both complex columns, composed of ordered and disordered cell stacks, and small, disorganized clusters located in the outer edges. Finally, correlation between the temporal dynamics of the ratios between clusters and columns and between bone elongation and expansion suggests that clusters may promote expansion, whereas columns support elongation. Overall, our findings support the idea that modulations of division plane rotation of proliferating chondrocytes determines the formation of either clusters or columns, a multifunctional design that regulates morphogenesis throughout pre- and postnatal bone growth. Broadly, this work provides a new understanding of the cellular mechanisms underlying growth plate activity and bone elongation during development.

    1. Developmental Biology
    2. Neuroscience
    Yuqi Cai, Zhirong Zhao ... Miao He
    Short Report

    Multiple embryonic origins give rise to forebrain oligodendrocytes (OLs), yet controversies and uncertainty exist regarding their differential contributions. We established intersectional and subtractional strategies to genetically fate map OLs produced by medial ganglionic eminence/preoptic area (MGE/POA), lateral/caudal ganglionic eminences (LGE/CGE), and dorsal pallium in the mouse brain. We found that, contrary to the canonical view, LGE/CGE-derived OLs make minimum contributions to the neocortex and corpus callosum, but dominate piriform cortex and anterior commissure. Additionally, MGE/POA-derived OLs, instead of being entirely eliminated, make small but sustained contribution to cortex with a distribution pattern distinctive from those derived from the dorsal origin. Our study provides a revised and more comprehensive view of cortical and white matter OL origins, and established valuable new tools and strategies for future OL studies.