Chromatin accessibility dynamics and single cell RNA-Seq reveal new regulators of regeneration in neural progenitors

Abstract

Vertebrate appendage regeneration requires precisely coordinated remodeling of the transcriptional landscape to enable the growth and differentiation of new tissue, a process executed over multiple days and across dozens of cell types. The heterogeneity of tissues and temporally-sensitive fate decisions involved has made it difficult to articulate the gene regulatory programs enabling regeneration of individual cell types. To better understand how a regenerative program is fulfilled by neural progenitor cells (NPCs) of the spinal cord, we analyzed pax6-expressing NPCs isolated from regenerating Xenopus tropicalis tails. By intersecting chromatin accessibility data with single-cell transcriptomics, we find that NPCs place an early priority on neuronal differentiation. Late in regeneration, the priority returns to proliferation. Our analyses identify Pbx3 and Meis1 as critical regulators of tail regeneration and axon organization. Overall, we use transcriptional regulatory dynamics to present a new model for cell fate decisions and their regulators in NPCs during regeneration.

Data availability

Sequencing data has been deposited in GEO under accession code GSE146837 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE146837)

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Anneke Dixie Kakebeen

    Biochemistry, University of Washington, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Alex D Chitsazan

    Biochemistry, University of Washington, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Madison Corinne Williams

    Biochemistry, University of Washington, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Lauren M Saunders

    Department of Genome Sciences, University of Washington, Seattle, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4377-4252
  5. Andrea Elizabeth Wills

    Biochemistry, University of Washington, Seattle, United States
    For correspondence
    aewills@uw.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3647-8105

Funding

National Institute of Neurological Disorders and Stroke (R01NS099124)

  • Andrea Elizabeth Wills

National Institute of General Medical Sciences (T32GM007270)

  • Anneke Dixie Kakebeen

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

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#4374) of the University of Washington, an AALAC-accredited institution. All surgeries were carried out under MS222 or Benzocaine anesthesia, and every effort was made to minimize suffering.

Copyright

© 2020, Kakebeen 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

  • 7,134
    views
  • 879
    downloads
  • 49
    citations

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

Download links

Share this article

https://doi.org/10.7554/eLife.52648

Further reading

    1. Developmental Biology
    Igor Kondrychyn, Liqun He ... Li-Kun Phng
    Research Article

    Cell migration is a key process in the shaping and formation of tissues. During sprouting angiogenesis, endothelial tip cells invade avascular tissues by generating actomyosin-dependent forces that drive cell migration and vascular expansion. Surprisingly, endothelial cells (ECs) can still invade if actin polymerization is inhibited. In this study, we show that endothelial tip cells employ an alternative mechanism of cell migration that is dependent on Aquaporin (Aqp)-mediated water inflow and increase in hydrostatic pressure. In the zebrafish, ECs express aqp1a.1 and aqp8a.1 in newly formed vascular sprouts in a VEGFR2-dependent manner. Aqp1a.1 and Aqp8a.1 loss-of-function studies show an impairment in intersegmental vessels formation because of a decreased capacity of tip cells to increase their cytoplasmic volume and generate membrane protrusions, leading to delayed tip cell emergence from the dorsal aorta and slower migration. Further inhibition of actin polymerization resulted in a greater decrease in sprouting angiogenesis, indicating that ECs employ two mechanisms for robust cell migration in vivo. Our study thus highlights an important role of hydrostatic pressure in tissue morphogenesis.

    1. Developmental Biology
    2. Stem Cells and Regenerative Medicine
    Ruben Sebastian-Perez, Shoma Nakagawa ... Maria Pia Cosma
    Research Article

    Chromocenters are established after the 2-cell (2C) stage during mouse embryonic development, but the factors that mediate chromocenter formation remain largely unknown. To identify regulators of 2C heterochromatin establishment in mice, we generated an inducible system to convert embryonic stem cells (ESCs) to 2C-like cells. This conversion is marked by a global reorganization and dispersion of H3K9me3-heterochromatin foci, which are then reversibly formed upon re-entry into pluripotency. By profiling the chromatin-bound proteome (chromatome) through genome capture of ESCs transitioning to 2C-like cells, we uncover chromatin regulators involved in de novo heterochromatin formation. We identified TOPBP1 and investigated its binding partner SMARCAD1. SMARCAD1 and TOPBP1 associate with H3K9me3-heterochromatin in ESCs. Interestingly, the nuclear localization of SMARCAD1 is lost in 2C-like cells. SMARCAD1 or TOPBP1 depletion in mouse embryos leads to developmental arrest, reduction of H3K9me3, and remodeling of heterochromatin foci. Collectively, our findings contribute to comprehending the maintenance of chromocenters during early development.