1. Genetics and Genomics
  2. Immunology and Inflammation

Combined transient ablation and single cell RNA sequencing reveals the development of medullary thymic epithelial cells

  1. Kristen L Wells
  2. Corey N Miller
  3. Andreas R Gschwind
  4. Wu Wei
  5. Jonah D Phipps
  6. Mark S Anderson  Is a corresponding author
  7. Lars M Steinmetz  Is a corresponding author
  1. Stanford University, United States
  2. University of California, San Francisco, United States
  3. European Molecular Biology Laboratory, Germany
https://doi.org/10.7554/eLife.60188
Share this article
Cite this article
  1. Kristen L Wells
  2. Corey N Miller
  3. Andreas R Gschwind
  4. Wu Wei
  5. Jonah D Phipps
  6. Mark S Anderson
  7. Lars M Steinmetz
(2020)
Combined transient ablation and single cell RNA sequencing reveals the development of medullary thymic epithelial cells
eLife 9:e60188.
https://doi.org/10.7554/eLife.60188
  2. Download BibTeX
  3. Download .RIS

Abstract

Medullary thymic epithelial cells (mTECs) play a critical role in central immune tolerance by mediating negative selection of autoreactive T cells through the collective expression of the peripheral self-antigen compartment, including tissue-specific antigens (TSAs). Recent work has shown that gene expression patterns within the mTEC compartment are remarkably heterogenous and include multiple differentiated cell states. To further define mTEC development and medullary epithelial lineage relationships, we combined lineage tracing and recovery from transient in vivo mTEC ablation with single cell RNA-sequencing in Mus musculus. The combination of bioinformatic and experimental approaches revealed a non-stem transit-amplifying population of cycling mTECs that preceded Aire expression. Based on our findings, we propose a branching model of mTEC development wherein a heterogeneous pool of transit-amplifying cells gives rise to Aire- and Ccl21a-expressing mTEC subsets. We further use experimental techniques to show that within the Aire-expressing developmental branch, TSA expression peaked as Aire expression decreased, implying Aire expression must be established before TSA expression can occur. Collectively, these data provide a higher order roadmap of mTEC development and demonstrate the power of combinatorial approaches leveraging both in vivo models and high-dimensional datasets.

Data availability

RNA-seq data that support the findings of this study have been deposited in the GEO database under accession numbers GSE137699

The following data sets were generated

Article and author information

Author details

  1. Kristen L Wells

    Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7466-8164

  2. Corey N Miller

    Diabetes Center, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Andreas R Gschwind

    Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0769-6907

  4. Wu Wei

    Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Jonah D Phipps

    Diabetes Center, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Mark S Anderson

    Diabetes Center, University of California, San Francisco, San Francisco, United States
    For correspondence
    Mark.Anderson@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3093-4758

  7. Lars M Steinmetz

    Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
    For correspondence
    lars.steinmetz@embl.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3962-2865

Funding

National Science Foundation (DGE- 1656518)

  • Kristen L Wells

National Institutes of Health (P01 HG000205)

  • Lars M Steinmetz

National Institutes of Health (R01 AI097457)

  • Mark S Anderson

National Institutes of Health (R01 AI097457)

  • Mark S Anderson

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

Reviewing Editor

  1. Ellen A Robey, University of California, Berkeley, United States

Ethics

Animal experimentation: Mice were maintained in the University of California San Francisco (UCSF) specific pathogen- free animal facility in accordance with the guidelines established by the Institutional Animal Care and Use Committee (IACUC) and Laboratory Animal Resource Center and all experimental procedures were approved by the Laboratory Animal Resource Center at UCSF. The animal protocol number associated with the study is AN180637-02B.

Version history

  1. Received: June 18, 2020
  2. Accepted: November 21, 2020
  3. Accepted Manuscript published: November 23, 2020 (version 1)
  4. Version of Record published: December 29, 2020 (version 2)

Copyright

© 2020, Wells 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

  • 3,618
    views
  • 571
    downloads
  • 52
    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. Kristen L Wells
  2. Corey N Miller
  3. Andreas R Gschwind
  4. Wu Wei
  5. Jonah D Phipps
  6. Mark S Anderson
  7. Lars M Steinmetz
(2020)
Combined transient ablation and single cell RNA sequencing reveals the development of medullary thymic epithelial cells
eLife 9:e60188.
https://doi.org/10.7554/eLife.60188

Share this article

https://doi.org/10.7554/eLife.60188

Categories and tags

Research organism

Further reading

    1. Computational and Systems Biology
    2. Genetics and Genomics

    Imputation of 3D genome structure by genetic–epigenetic interaction modeling in mice

    Lauren Kuffler, Daniel A Skelly ... Gregory W Carter
    Research Article

    Gene expression is known to be affected by interactions between local genetic variation and DNA accessibility, with the latter organized into three-dimensional chromatin structures. Analyses of these interactions have previously been limited, obscuring their regulatory context, and the extent to which they occur throughout the genome. Here, we undertake a genome-scale analysis of these interactions in a genetically diverse population to systematically identify global genetic–epigenetic interaction, and reveal constraints imposed by chromatin structure. We establish the extent and structure of genotype-by-epigenotype interaction using embryonic stem cells derived from Diversity Outbred mice. This mouse population segregates millions of variants from eight inbred founders, enabling precision genetic mapping with extensive genotypic and phenotypic diversity. With 176 samples profiled for genotype, gene expression, and open chromatin, we used regression modeling to infer genetic–epigenetic interactions on a genome-wide scale. Our results demonstrate that statistical interactions between genetic variants and chromatin accessibility are common throughout the genome. We found that these interactions occur within the local area of the affected gene, and that this locality corresponds to topologically associated domains (TADs). The likelihood of interaction was most strongly defined by the three-dimensional (3D) domain structure rather than linear DNA sequence. We show that stable 3D genome structure is an effective tool to guide searches for regulatory elements and, conversely, that regulatory elements in genetically diverse populations provide a means to infer 3D genome structure. We confirmed this finding with CTCF ChIP-seq that revealed strain-specific binding in the inbred founder mice. In stem cells, open chromatin participating in the most significant regression models demonstrated an enrichment for developmental genes and the TAD-forming CTCF-binding complex, providing an opportunity for statistical inference of shifting TAD boundaries operating during early development. These findings provide evidence that genetic and epigenetic factors operate within the context of 3D chromatin structure.

    1. Genetics and Genomics
    2. Neuroscience

    Genetic Chimerism: Marmosets contain multitudes

    Kenneth Chiou, Noah Snyder-Mackler
    Insight

    Single-cell RNA sequencing reveals the extent to which marmosets carry genetically distinct cells from their siblings.

    1. Genetics and Genomics

    Elimination of subtelomeric repeat sequences exerts little effect on telomere essential functions in Saccharomyces cerevisiae

    Can Hu, Xue-Ting Zhu ... Jin-Qiu Zhou
    Research Article

    Telomeres, which are chromosomal end structures, play a crucial role in maintaining genome stability and integrity in eukaryotes. In the baker’s yeast Saccharomyces cerevisiae, the X- and Y’-elements are subtelomeric repetitive sequences found in all 32 and 17 telomeres, respectively. While the Y’-elements serve as a backup for telomere functions in cells lacking telomerase, the function of the X-elements remains unclear. This study utilized the S. cerevisiae strain SY12, which has three chromosomes and six telomeres, to investigate the role of X-elements (as well as Y’-elements) in telomere maintenance. Deletion of Y’-elements (SY12YΔ), X-elements (SY12XYΔ+Y), or both X- and Y’-elements (SY12XYΔ) did not impact the length of the terminal TG1-3 tracks or telomere silencing. However, inactivation of telomerase in SY12YΔ, SY12XYΔ+Y, and SY12XYΔ cells resulted in cellular senescence and the generation of survivors. These survivors either maintained their telomeres through homologous recombination-dependent TG1-3 track elongation or underwent microhomology-mediated intra-chromosomal end-to-end joining. Our findings indicate the non-essential role of subtelomeric X- and Y’-elements in telomere regulation in both telomerase-proficient and telomerase-null cells and suggest that these elements may represent remnants of S. cerevisiae genome evolution. Furthermore, strains with fewer or no subtelomeric elements exhibit more concise telomere structures and offer potential models for future studies in telomere biology.