Catherine P. Lu

Identification of Stem Cell Populations in Sweat Glands and Ducts Reveals Roles in Homeostasis and Wound Repair

Sweat glands are abundant in the body and essential for thermoregulation. Like mammary glands, they originate from epidermal progenitors. However, they display few signs of cellular turnover, and whether they have stem cells and tissue-regenerative capacity remains largely unexplored. Using lineage tracing, we here identify in sweat ducts multipotent progenitors that transition to unipotency after developing the sweat gland. In characterizing four adult stem cell populations of glandular skin, we show that they display distinct regenerative capabilities and remain unipotent when healing epidermal, myoepithelial-specific, and lumenal-specific injuries. We devise purification schemes and isolate and transcriptionally profile progenitors. Exploiting molecular differences between sweat and mammary glands, we show that only some progenitors regain multipotency to produce de novo ductal and glandular structures, but that these can retain their identity even within certain foreign microenvironments. Our findings provide insight into glandular stem cells and a framework for the further study of sweat gland biology.

Skin Stem Cells Orchestrate Directional Migration by Regulating Microtubule-ACF7 Connections through GSK3β

Homeostasis and wound healing rely on stem cells (SCs) whose activity and directed migration are often governed by Wnt signaling. In dissecting how this pathway integrates with the necessary downstream cytoskeletal dynamics, we discovered that GSK3β, a kinase inhibited by Wnt signaling, directly phosphorylates ACF7, a > 500 kDa microtubule-actin crosslinking protein abundant in hair follicle stem cells (HF-SCs). We map ACF7s GSK3β sites to the microtubule-binding domain and show that phosphorylation uncouples ACF7 from microtubules. Phosphorylation-refractile ACF7 rescues overall microtubule architecture, but phosphorylation-constitutive mutants do not. Neither mutant rescues polarized movement, revealing that phospho-regulation must be dynamic. This circuitry is physiologically relevant and depends upon polarized GSK3β inhibition at the migrating front of SCs/progeny streaming from HFs during wound repair. Moreover, only ACF7 and not GSKβ-refractile-ACF7 restore polarized microtubule-growth and SC-migration to ACF7 null skin. Our findings provide insights into how this conserved spectraplakin integrates signaling, cytoskeletal dynamics, and polarized locomotion of somatic SCs.

Sweat Gland Progenitors in Development, Homeostasis, and Wound Repair

The human body is covered with several million sweat glands. These tiny coiled tubular skin appendages produce the sweat that is our primary source of cooling and hydration of the skin. Numerous studies have been published on their morphology and physiology. Until recently, however, little was known about how glandular skin maintains homeostasis and repairs itself after tissue injury. Here, we provide a brief overview of sweat gland biology, including newly identified reservoirs of stem cells in glandular skin and their activation in response to different types of injuries. Finally, we discuss how the genetics and biology of glandular skin has advanced our knowledge of human disorders associated with altered sweat gland activity.

Amino acid residues in Rag1 crucial for DNA hairpin formation

The Rag proteins carry out V(D)J recombination through a process mechanistically similar to cut-and-paste transposition. Specifically, Rag complexes form DNA hairpins through direct transesterification, using a catalytic Asp-Asp-Glu (DDE) triad in Rag1. How is sufficient DNA distortion introduced to allow hairpin formation? We hypothesized that, like certain transposases, the Rag proteins might use aromatic amino acid residues to stabilize a flipped-out base. Through in vivo and in vitro experiments and structural predictions, we identified residues in Rag1 crucial for hairpin formation. One of these, a conserved tryptophan (Trp893), probably participates in base-stacking interactions near the cleavage site, as do Trp298, Trp265 and Trp319 in the Tn5, Tn10 and Hermes transposases, respectively. Other residues surrounding the catalytic glutamate (YKEFRK) may share functional similarities with the YREK motif in IS4 family transposases.

Stem Cell Lineage Infidelity Drives Wound Repair and Cancer

Tissue stem cells contribute to tissue regeneration and wound repair through cellular programs that can be hijacked by cancer cells. Here, we investigate such a phenomenon in skin, where during homeostasis, stem cells of the epidermis and hair follicle fuel their respective tissues. We find that breakdown of stem cell lineage confinement-granting privileges associated with both fates-is not only hallmark but also functional in cancer development. We show that lineage plasticity is critical in wound repair, where it operates transiently to redirect fates. Investigating mechanism, we discover that irrespective of cellular origin, lineage infidelity occurs in wounding when stress-responsive enhancers become activated and override homeostatic enhancers that govern lineage specificity. In cancer, stress-responsive transcription factor levels rise, causing lineage commanders to reach excess. When lineage and stress factors collaborate, they activate oncogenic enhancers that distinguish cancers from wounds.

Spatiotemporal antagonism in mesenchymal-epithelial signaling in sweat versus hair fate decision.

How to grow hair or sweat glands Unlike other mammals that must pant or seek shade or water when overheated, humans are able to self-cool to tolerate extreme heat. Sweat glands, which enable humans to run in marathons, are instrumental for this remarkable feat. Lu et al. investigated skin appendage diversity during development of the furry backs and sweaty paws of mice (see the Perspective by Lai and Chuong). They also examined human skin, which is capable of making both hairs and sweat glands in the same area of the body. Epithelialmesenchymal interactions, with varied signaling pathways that act at specific times in development, are key to producing different skin appendages for adaptation to the environment. Science, this issue p. 10.1126/science.aah6102; see also p. 1533 Mice and humans exploit the epithelial-mesenchymal circuitry in different ways to direct sweat gland or hair follicle generation. INTRODUCTION Across the vertebrate kingdom, epithelial appendages—including mammary, sweat, and salivary glands; hair follicles (HFs); teeth; scales; and feathers—begin to form during embryogenesis when WNT signaling instructs progenitors within the epithelial sheet to organize into morphologically similar placodes. Most animals restrict these epidermal appendages to distinct body regions. This paradigm shifted late in mammalian evolution; the dual presence of HFs and eccrine sweat glands (SwGs) in the skin is a recent acquisition of primates. Classical tissue recombination experiments from the 1960s revealed that mesenchyme directs the divergent downstream events that determine appendage specification and patterning. Relatively little is known about the specific spatiotemporal cross-talk and molecular mechanisms that underlie epithelial fate specification in response to mesenchymal signals. Elucidating the epithelial-mesenchymal cross-talk involved in regional skin appendage specification is integral to understanding how humans have adjusted these mechanisms to endow them with a greater capacity than that of their hairy cousins to live in diverse environments. RATIONALE The acquisition of SwGs and their importance in thermoregulation and water balance are underscored by human patients who suffer from the life-threatening condition of lacking SwGs, either from loss in severe burns or from genetic disorders. Conversely, a gain-of-function variant that elevates SwG numbers has been expanding among the Southeast Asian population, where excessive SwGs are desirable. By elucidating the underlying mechanisms that distinguish humans from other mammals in their ability to make both glands and follicles over their body skin, our findings could pave the way for future therapeutic advances in skin regeneration with dual appendages. RESULTS Using mouse as a model, we explored the differences between the back skin mesenchyme, which is only competent to specify HFs, and the foot skin mesenchyme, which can only make SwGs. Using genome-wide analyses and functional studies, we discovered that just after the formation of morphologically similar epidermal buds, appendage choice is determined through regional skin differences in mesenchymal expression of bone morphogenetic proteins (BMPs). Probing into mechanisms, we showed that when BMPs are elevated in foot skin mesenchyme, BMP signaling is activated in both dermis and epidermis. This triggers a cascade of downstream signaling events. WNT signaling is elevated in the dermis and reduced in the epidermis. Mesenchymal fibroblast growth factors (FGFs) appear and affect the overlying epithelium. These converging pathways lead to suppression of sonic hedgehog (SHH) in the epithelium. This BMP:SHH antagonism within the epithelial bud specifies SwG fate and prevents HF fate. Moreover, by manipulating gene expression in vivo at specific developmental stages, we demonstrated that this signaling circuitry acts only within a narrow window of time during mouse embryogenesis. Thus, when SHH is ectopically expressed in foot skin epithelium during the permissive phase, HF-specific gene expression is up-regulated in the epithelial bud, whereas SHH signaling in the mesenchyme stimulates expression of BMP antagonists, further suppressing local BMP signaling and blocking SwG fate. In human skins, this antagonistic interplay of BMP:SHH signaling occurs temporally, in addition to spatially. The first bud waves are specified as HFs, and then a tipping in the balance of BMP:SHH signaling results in the last waves of buds becoming SwGs. CONCLUSION Our findings revealed a differential impact of BMP signaling on appendage fate specification that has ancient roots and occurs repeatedly throughout vertebrate evolution. In the evolutionary developmental biology view of BMP signaling and fate specification of integument, chicken scales and mammalian SwGs require BMP signaling to specify their fate, whereas feathers and HFs must suppress it. Our discovery of BMP-SHH antagonism in bud fate choice uncovered additional evolutionary parallels, this time between two even more distantly related epidermal appendages, the mammalian SwG and the fly wing. Our studies provide new insights into how elevated mesenchymal BMP signaling triggers a self-perpetuating molecular cascade that culminates in silencing of SHH signaling to suppress one appendage fate and specify another. In most mammals, the BMP:SHH antagonism is regulated spatially. Humans, however, have evolved to regulate it temporally, endowing them with greater ability to run marathons and survive in extreme climates. BMP-SHH antagonism specifies SwG versus HF fate. To specify SwGs, mesenchymal-derived BMPs and FGFs signal to epithelial buds and suppress epithelially derived SHH production. Conversely, hair follicles are specified when mesenchymal BMP signaling is inhibited, permitting SHH production. This antagonism is spatially restricted in most mammals but temporally regulated in humans, permitting the presence of HFs (pink), SwGs (purple), or both HFs and SwGs (pink with purple droplet) throughout our body skin. The gain of eccrine sweat glands in hairy body skin has empowered humans to run marathons and tolerate temperature extremes. Epithelial-mesenchymal cross-talk is integral to the diverse patterning of skin appendages, but the molecular events underlying their specification remain largely unknown. Using genome-wide analyses and functional studies, we show that sweat glands are specified by mesenchymal-derived bone morphogenetic proteins (BMPs) and fibroblast growth factors that signal to epithelial buds and suppress epithelial-derived sonic hedgehog (SHH) production. Conversely, hair follicles are specified when mesenchymal BMP signaling is blocked, permitting SHH production. Fate determination is confined to a critical developmental window and is regionally specified in mice. In contrast, a shift from hair to gland fates is achieved in humans when a spike in BMP silences SHH during the final embryonic wave(s) of bud morphogenesis.

Base Flipping in V(D)J Recombination: Insights into the Mechanism of Hairpin Formation, the 12/23 Rule, and the Coordination of Double-Strand Breaks

ABSTRACT Tn5 transposase cleaves the transposon end using a hairpin intermediate on the transposon end. This involves a flipped base that is stacked against a tryptophan residue in the protein. However, many other members of the cut-and-paste transposase family, including the RAG1 protein, produce a hairpin on the flanking DNA. We have investigated the reversed polarity of the reaction for RAG recombination. Although the RAG proteins appear to employ a base-flipping mechanism using aromatic residues, the putatively flipped base is not at the expected location and does not appear to stack against any of the said aromatic residues. We propose an alternative model in which a flipped base is accommodated in a nonspecific pocket or cleft within the recombinase. This is consistent with the location of the flipped base at position −1 in the coding flank, which can be occupied by purine or pyrimidine bases that would be difficult to stabilize using a single, highly specific, interaction. Finally, during this work we noticed that the putative base-flipping events on either side of the 12/23 recombination signal sequence paired complex are coupled to the nicking steps and serve to coordinate the double-strand breaks on either side of the complex.

Understanding how the V(D)J recombinase catalyzes transesterification: distinctions between DNA cleavage and transposition

The Rag1 and Rag2 proteins initiate V(D)J recombination by introducing site-specific DNA double-strand breaks. Cleavage occurs by nicking one DNA strand, followed by a one-step transesterification reaction that forms a DNA hairpin structure. A similar reaction allows Rag transposition, in which the 3′-OH groups produced by Rag cleavage are joined to target DNA. The Rag1 active site DDE triad clearly plays a catalytic role in both cleavage and transposition, but no other residues in Rag1 responsible for transesterification have been identified. Furthermore, although Rag2 is essential for both cleavage and transposition, the nature of its involvement is unknown. Here, we identify basic amino acids in the catalytic core of Rag1 specifically important for transesterification. We also show that some Rag1 mutants with severe defects in hairpin formation nonetheless catalyze substantial levels of transposition. Lastly, we show that a catalytically defective Rag2 mutant is impaired in target capture and displays a novel form of coding flank sensitivity. These findings provide the first identification of components of Rag1 that are specifically required for transesterification and suggest an unexpected role for Rag2 in DNA cleavage and transposition.

A RAG1 Mutation Found in Omenn Syndrome Causes Coding Flank Hypersensitivity: A Novel Mechanism for Antigen Receptor Repertoire Restriction

Hypomorphic RAG mutants with severely reduced V(D)J recombination activity cause Omenn Syndrome (OS), an immunodeficiency with features of immune dysregulation and a restricted TCR repertoire. Precisely how RAG mutants produce autoimmune and allergic symptoms has been unclear. Current models posit that the severe recombination defect restricts the number of lymphocyte clones, a few of which are selected upon Ag exposure. We show that murine RAG1 R972Q, corresponding to an OS mutation, renders the recombinase hypersensitive to selected coding sequences at the hairpin formation step. Other RAG1 OS mutants tested do not manifest this sequence sensitivity. These new data support a novel mechanism for OS: by selectively impairing recombination at certain coding flanks, a RAG mutant can cause primary repertoire restriction, as opposed to a more random, limited repertoire that develops secondary to severely diminished recombination activity.