Cellular origins and lineage relationships of the intestinal epithelium.

Knowledge of the development and hierarchical organization of tissues is key to understanding how they are perturbed in injury and disease, as well as how they may be therapeutically manipulated to restore homeostasis. The rapidly regenerating intestinal epithelium harbors diverse cell types and their lineage relationships have been studied using numerous approaches, from classical label-retaining and genetic lineage tracing methods to novel transcriptome-based annotations. Here, we describe the developmental trajectories that dictate differentiation and lineage specification in the intestinal epithelium. We focus on the most recent single-cell RNA-sequencing (scRNA-seq)-based strategies for understanding intestinal epithelial cell lineage relationships, underscoring how they have refined our view of the development of this tissue and highlighting their advantages and limitations. We emphasize how these technologies have been applied to understand the dynamics of intestinal epithelial cells in homeostatic and injury-induced regeneration models.

Single-Cell Studies of Intestinal Stem Cell Heterogeneity During Homeostasis and Regeneration.

Single-cell RNA-sequencing (scRNA-seq) provides a unique opportunity to study heterogeneous cell populations within tissues, including the intestinal epithelium, to gain detailed molecular insights into their biology. Many new putative markers of intestinal stem cells and their progeny have been described using single-cell transcriptomics, which has contributed to the identification of novel subpopulations of mature cell types and insight into their developmental trajectories. This approach has revealed tremendous cellular heterogeneity within the intestinal epithelium that is concordant with its diverse and multifaceted functions. We discuss the function of these subpopulations during tissue homeostasis, as well as putative subpopulations with inducible regenerative potential following tissue injury.

"Catch and Release": A Variation of the Archetypal Nucleotidyl Transfer Reaction.

Nucleotidyl transfer is an archetypal enzyme reaction central to DNA replication and repair. Here we describe a variation of the nucleotidylation reaction termed "catch and release" that is used by an antibiotic modifying enzyme. The aminoglycoside nucleotidyl transferase 4' (ANT4') inactivates antibiotics such as kanamycin and neomycin through nucleotidylation within an active site that shares significant structural, and inferred underlying catalytic similarity, with human DNA polymerase beta. Here we follow the entire nucleotidyl transfer reaction coordinate of ANT4' covalently inactivating neomycin using X-ray crystallography. These studies show that although the underlying reaction mechanism is conserved with polymerases, a short 2.35 A hydrogen bond is initially formed to facilitate tight binding of the aminoglycoside substrate and is subsequently disrupted by the assembly of the catalytically active ternary complex. This enables the release of products post catalysis due to a lower free energy of the product state compared to the starting substrate complex. We propose that this "catch and release" mechanism of antibiotic turnover observed in ANT4' is a variation of nucleotidyl transfer that has been adapted for the inactivation of antibiotics.