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.
RNA-seq data that support the findings of this study have been deposited in the GEO database under accession numbers GSE137699
Combined transient ablation and single cell RNA sequencing reveals the development of medullary thymic epithelial cellsNCBI Gene Expression Omnibus, GSE137699.
- Kristen L Wells
- Lars M Steinmetz
- Mark S Anderson
- Mark S Anderson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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.
- Ellen A Robey, University of California, Berkeley, United States
© 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.
Homeostatic and circadian processes collaborate to appropriately time and consolidate sleep and wake. To understand how these processes are integrated, we scheduled brief sleep deprivation at different times of day in Drosophila and find elevated morning rebound compared to evening. These effects depend on discrete morning and evening clock neurons, independent of their roles in circadian locomotor activity. In the R5 ellipsoid body sleep homeostat, we identified elevated morning expression of activity dependent and presynaptic gene expression as well as the presynaptic protein BRUCHPILOT consistent with regulation by clock circuits. These neurons also display elevated calcium levels in response to sleep loss in the morning, but not the evening consistent with the observed time-dependent sleep rebound. These studies reveal the circuit and molecular mechanisms by which discrete circadian clock neurons program a homeostatic sleep center.
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