Sox9 marks limbal stem cells and is required for asymmetric cell fate switch in the corneal epithelium.

Adult tissues with high cellular turnover require a balance between stem cell renewal and differentiation, yet the mechanisms underlying this equilibrium are unclear. The cornea exhibits a polarized lateral flow of progenitors from the peripheral stem cell niche to the center; attributed to differences in cellular fate. To identify genes that are critical for regulating the asymmetric fates of limbal stem cells and their transient amplified progeny in the central cornea, we utilized an in vivo cell cycle reporter to isolate proliferating basal cells across the anterior ocular surface epithelium and performed single-cell transcriptional analysis. This strategy greatly increased the resolution and revealed distinct basal cell identities with unique expression profiles of structural genes and transcription factors. We focused on Sox9; a transcription factor implicated in stem cell regulation across various organs. Sox9 was found to be differentially expressed between limbal stem cells and their progeny in the central corneal. Lineage tracing analysis confirmed that Sox9 marks long-lived limbal stem cells and conditional deletion led to abnormal differentiation and squamous metaplasia in the central cornea. These data suggest a requirement for Sox9 for the switch to asymmetric fate and commitment toward differentiation, as transient cells exit the limbal niche. By inhibiting terminal differentiation of corneal progenitors and forcing them into perpetual symmetric divisions, we replicated the Sox9 loss-of-function phenotype. Our findings reveal an essential role for Sox9 for the spatial regulation of asymmetric fate in the corneal epithelium that is required to sustain tissue homeostasis.

A PDX1 cistrome and single-cell transcriptome resource of the developing pancreas.

Pancreatic and duodenal homeobox 1 (PDX1) is crucial for pancreas organogenesis, yet the dynamic changes in PDX1 binding in human or mouse developing pancreas have not been examined. To address this knowledge gap, we performed PDX1 ChIP-seq and single-cell RNA-seq using fetal human pancreata. We integrated our datasets with published datasets and revealed the dynamics of PDX1 binding and potential cell lineage-specific PDX1-bound genes in the pancreas from fetal to adult stages. We identified a core set of developmentally conserved PDX1-bound genes that reveal the broad multifaceted role of PDX1 in pancreas development. Despite the well-known dramatic changes in PDX1 function and expression, we found that PDX1-bound genes are largely conserved from embryonic to adult stages. This points towards a dual role of PDX1 in regulating the expression of its targets at different ages, dependent on other functionally congruent or directly interacting partners. We also showed that PDX1 binding is largely conserved in mouse pancreas. Together, our study reveals PDX1 targets in the developing pancreas in vivo and provides an essential resource for future studies on pancreas development.

Lgr6 marks epidermal stem cells with a nerve-dependent role in wound re-epithelialization.

Stem cells support lifelong maintenance of adult organs, but their specific roles during injury are poorly understood. Here we demonstrate that Lgr6 marks a regionally restricted population of epidermal stem cells that interact with nerves and specialize in wound re-epithelialization. Diphtheria toxin-mediated ablation of Lgr6 stem cells delays wound healing, and skin denervation phenocopies this effect. Using intravital imaging to capture stem cell dynamics after injury, we show that wound re-epithelialization by Lgr6 stem cells is diminished following loss of nerves. This induces recruitment of other stem cell populations, including hair follicle stem cells, which partially compensate to mediate wound closure. Single-cell lineage tracing and gene expression analysis reveal that the fate of Lgr6 stem cells is shifted toward differentiation following loss of their niche. We conclude that Lgr6 epidermal stem cells are primed for injury response and interact with nerves to regulate their fate.

Two-photon live imaging of single corneal stem cells reveals compartmentalized organization of the limbal niche.

The functional heterogeneity of resident stem cells that support adult organs is incompletely understood. Here, we directly visualize the corneal limbus in the eyes of live mice and identify discrete stem cell niche compartments. By recording the life cycle of individual stem cells and their progeny, we directly analyze their fates and show that their location within the tissue can predict their differentiation status. Stem cells in the inner limbus undergo mostly symmetric divisions and are required to sustain the population of transient progenitors that support corneal homeostasis. Using in situ photolabeling, we captured their progeny exiting the niche before moving centripetally in unison. The long-implicated slow-cycling stem cells are functionally distinct and display local clonal dynamics during homeostasis but can contribute to corneal regeneration after injury. This study demonstrates how the compartmentalized organization of functionally diverse stem cell populations supports the maintenance and regeneration of an adult organ.

Advances in resolving the heterogeneity and dynamics of keratinocyte differentiation.

The mammalian skin is equipped with a highly dynamic stratified epithelium. The maintenance and regeneration of this epithelium is supported by basally located keratinocytes, which display stem cell properties, including lifelong proliferative potential and the ability to undergo diverse differentiation trajectories. Keratinocytes support not just the surface of the skin, called the epidermis, but also a range of ectodermal structures including hair follicles, sebaceous glands, and sweat glands. Recent studies have shed light on the hitherto underappreciated heterogeneity of keratinocytes by employing state-of-the-art imaging technologies and single-cell genomic approaches. In this mini review, we highlight major recent discoveries that illuminate the dynamics and cellular mechanisms that govern keratinocyte differentiation in the live mammalian skin and discuss the broader implications of these findings for our understanding of epithelial and stem cell biology in general.