Hepatocytes are replenished gradually during homeostasis and robustly after liver injury^{1, 2}. In adults, new hepatocytes originate from the existing hepatocyte pool^3-8, but the cellular source of renewing hepatocytes remains unclear. Telomerase is expressed in many stem cell populations, and mutations in telomerase pathway genes have been linked to liver diseases^9-11. Here we identify a subset of hepatocytes that expresses high levels of telomerase and show that this hepatocyte subset repopulates the liver during homeostasis and injury. Using lineage tracing from the telomerase reverse transcriptase (Tert) locus in mice, we demonstrate that rare hepatocytes with high telomerase expression (TERT^High hepatocytes) are distributed throughout the liver lobule. During homeostasis, these cells regenerate hepatocytes in all lobular zones, and both self-renew and differentiate to yield expanding hepatocyte clones that eventually dominate the liver. In response to injury, the repopulating activity of TERT^High hepatocytes is accelerated and their progeny cross zonal boundaries. RNA sequencing shows that metabolic genes are downregulated in TERT^High hepatocytes, indicating that metabolic activity and repopulating activity may be segregated within the hepatocyte lineage. Genetic ablation of TERT^High hepatocytes combined with chemical injury causes a marked increase in stellate cell activation and fibrosis. These results provide support for a 'distributed model' of hepatocyte renewal in which a subset of hepatocytes dispersed throughout the lobule clonally expands to maintain liver mass.

Squamous cell carcinoma (SCC) occurs frequently in the human Xeroderma Pigmentosum (XP) syndrome and is characterized by deficient UV-damage repair. SCC is the most common equine ocular cancer and the only associated genetic risk factor is a UV-damage repair protein. Specifically, a missense mutation in horse DDB2 (T338M) was strongly associated with both limbal SCC and third eyelid SCC in three breeds of horses (Halflinger, Belgian, and Rocky Mountain Horses) and was hypothesized to impair binding to UV-damaged DNA. Here, we investigate DDB2-T338M mutant's capacity to recognize UV lesions in vitro and in vivo, together with human XP mutants DDB2-R273H and -K244E. We show that the recombinant DDB2-T338M assembles with DDB1, but fails to show any detectable binding to DNA substrates with or without UV lesions, due to a potential structural disruption of the rigid DNA recognition β-loop. Consistently, we demonstrate that the cellular DDB2-T338M is defective in its recruitment to focally radiated DNA damages, and in its access to chromatin. Thus, we provide direct functional evidence indicating the DDB2-T338M recapitulates molecular defects of human XP mutants, and is the causal loss-of-function allele that gives rise to equine ocular SCCs. Our findings shed new light on the mechanism of DNA recognition by UV-DDB and on the initiation of ocular malignancy.

Video abstract

Aging is a complex process that affects multiple organs. Modeling aging and age-related diseases in the lab is challenging because classical vertebrate models have relatively long lifespans. Here, we develop the first platform for rapid exploration of age-dependent traits and diseases in vertebrates, using the naturally short-lived African turquoise killifish. We provide an integrative genomic and genome-editing toolkit in this organism using our de-novo-assembled genome and the CRISPR/Cas9 technology. We mutate many genes encompassing the hallmarks of aging, and for a subset, we produce stable lines within 2-3 months. As a proof of principle, we show that fish deficient for the protein subunit of telomerase exhibit the fastest onset of telomere-related pathologies among vertebrates. We further demonstrate the feasibility of creating specific genetic variants. This genome-to-phenotype platform represents a unique resource for studying vertebrate aging and disease in a high-throughput manner and for investigating candidates arising from human genome-wide studies.

Cell-free RNA from liquid biopsies can be analyzed to determine disease tissue of origin. We extend this concept to identify cell types of origin using the Tabula Sapiens transcriptomic cell atlas as well as individual tissue transcriptomic cell atlases in combination with the Human Protein Atlas RNA consensus dataset. We define cell type signature scores, which allow the inference of cell types that contribute to cell-free RNA for a variety of diseases.