Mechanical Forces in the Skin : Roles in Tissue Architecture, Stability, and Function

Tissue shape emerges from the collective mechanical properties and behavior of individual cells and the ways by which they integrate into the surrounding tissue. Tissue architecture and its dynamic changes subsequently feed back to guide cell behavior. The skin is a dynamic, self-renewing barrier that is subjected to large-scale extrinsic mechanical forces throughout its lifetime. The ability to withstand this constant mechanical stress without compromising its integrity as a barrier requires compartment-specific structural specialization and the capability to sense and adapt to mechanical cues. This review discusses the unique mechanical properties of the skin and the importance of signals that arise from mechanical communication between cells and their environment.Peer reviewe

Novel strategies for expansion of tooth epithelial stem cells and ameloblast generation

Enamel is secreted by ameloblasts derived from tooth epithelial stem cells (SCs). Humans cannot repair or regenerate enamel, due to early loss of tooth epithelial SCs. Contrarily in the mouse incisors, epithelial SCs are maintained throughout life and endlessly generate ameloblasts, and thus enamel. Here we isolated Sox2-GFP+ tooth epithelial SCs which generated highly cellular spheres following a novel in vitro strategy. This system enabled analysis of SC regulation by various signaling molecules, and supported the stimulatory and inhibitory roles of Shh and Bmp, respectively; providing better insight into the heterogeneity of the SCs. Further, we generated a novel mouse reporter, Enamelin-tdTomato for identification of ameloblasts in live tissues and cells, and used it to demonstrate presence of ameloblasts in the new 3D co-culture system of dental SCs. Collectively, our results provide means of generating 3D tooth epithelium from adult SCs which can be utilized toward future generation of enamel.Peer reviewe

Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage

Summary Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.Peer reviewe

Functionally Distinctive Ptch Receptors Establish Multimodal Hedgehog Signaling in the Tooth Epithelial Stem Cell Niche

Continuous growth of the mouse incisor teeth is due to the life-long maintenance of epithelial stem cells (SCs) in their niche called cervical loop (CL). Several signaling factors regulate SC maintenance and/or their differentiation to achieve organ homeostasis. Previous studies indicated that Hedgehog signaling is crucial for both the maintenance of the SCs in the niche, as well as for their differentiation. How Hedgehog signaling regulates these two opposing cellular behaviors within the confinement of the CL remains elusive. In this study, we used in vitro organ and cell cultures to pharmacologically attenuate Hedgehog signaling. We analyzed expression of various genes expressed in the SC niche to determine the effect of altered Hedgehog signaling on the cellular hierarchy within the niche. These genes include markers of SCs (Sox2 and Lgr5) and transit-amplifying cells (P-cadherin, Sonic Hedgehog, and Yap). Our results show that Hedgehog signaling is a critical survival factor for SCs in the niche, and that the architecture and the diversity of the SC niche are regulated by multiple Hedgehog ligands. We demonstrated the presence of an additional Hedgehog ligand, nerve-derived Desert Hedgehog, secreted in the proximity of the CL. In addition, we provide evidence that Hedgehog receptors Ptch1 and Ptch2 elicit independent responses, which enable multimodal Hedgehog signaling to simultaneously regulate SC maintenance and differentiation. Our study indicates that the cellular hierarchy in the continuously growing incisor is a result of complex interplay of two Hedgehog ligands with functionally distinct Ptch receptors. Stem Cells 2019;37:1238-1248Peer reviewe

Hair follicle dermal condensation forms via Fgf20 primed cell cycle exit, cell motility, and aggregation

Mesenchymal condensation is a critical step in organogenesis, yet the underlying molecular and cellular mechanisms remain poorly understood. The hair follicle dermal condensate is the precursor to the permanent mesenchymal unit of the hair follicle, the dermal papilla, which regulates hair cycling throughout life and bears hair inductive potential. Dermal condensate morphogenesis depends on epithelial Fibroblast Growth Factor 20 (Fgf20). Here, we combine mouse models with 3D and 4D microscopy to demonstrate that dermal condensates form de novo and via directional migration. We identify cell cycle exit and cell shape changes as early hallmarks of dermal condensate morphogenesis and find that Fgf20 primes these cellular behaviors and enhances cell motility and condensation. RNAseq profiling of immediate Fgf20 targets revealed induction of a subset of dermal condensate marker genes. Collectively, these data indicate that dermal condensation occurs via directed cell movement and that Fgf20 orchestrates the early cellular and molecular events.Peer reviewe

Transforming growth factor Beta 3 is required for excisional wound repair in vivo.

Wound healing is a complex process that relies on proper levels of cytokines and growth factors to successfully repair the tissue. Of particular interest are the members of the transforming growth factor family. There are three TGF-ß isoforms-TGF- ß 1, 2, and 3, each isoform showing a unique expression pattern, suggesting that they each play a distinct function during development and repair. Previous studies reported an exclusive role for TGF-ß 3 in orofacial development and a potent anti-scarring effect. However, the role of TGF- ß 3 in excisional wound healing and keratinocyte migration remains poorly understood. We tested the effect of TGF-ß 3 levels on excisional cutaneous wounds in the adult mouse by directly injecting recombinant TGF-ß 3 or neutralizing antibody against TGF-ß 3 (NAB) in the wounds. Our results demonstrate that TGF-ß 3 does not promote epithelialization. However, TGF-ß 3 is necessary for wound closure as wounds injected with neutralizing antibody against TGF-ß 3 showed increased epidermal volume and proliferation in conjunction with a delay in keratinocyte migration. Wild type keratinocytes treated with NAB and Tgfb3-deficient keratinocytes closed an in vitro scratch wound with no delay, suggesting that our in vivo observations likely result from a paracrine effect

Glutamine Metabolism Controls Stem Cell Fate Reversibility and Long-Term Maintenance in the Hair Follicle

Stem cells reside in specialized niches that are critical for their function. Upon activation, hair follicle stem cells (HFSCs) exit their niche to generate the outer root sheath (ORS), but a subset of ORS progeny returns to the niche to resume an SC state. Mechanisms of this fate reversibility are unclear. We show that the ability of ORS cells to return to the SC state requires suppression of a metabolic switch from glycolysis to oxidative phosphorylation and glutamine metabolism that occurs during early HFSC lineage progression. HFSC fate reversibility and glutamine metabolism are regulated by the mammalian target of rapamycin complex 2 (mTORC2)-Akt signaling axis within the niche. Deletion of mTORC2 results in a failure to re-establish the HFSC niche, defective hair follicle regeneration, and compromised long-term maintenance of HFSCs. These findings highlight the importance of spatiotemporal control of SC metabolic states in organ homeostasis.Peer reviewe

Macroscopic photomicrographs of excisional wounds.

<p>Six-mm excisional punch wounds were performed on the back of wild type mice. One day later, wounds were treated with saline (a, e, i), TGF-ß3 and neutralizing antibody (NAB) against TGF-ß3 (b, f, j), TGF-ß3 (c, g, k), and NAB (d, h, l). Wounds were harvested 4 days (a–d), 7 days (e–h) and 11 days (i–l) post-wounding.</p

Schematic of the morphometric analysis.

<p>l = length of the wound (blue solid line); le = length of the epidermis (yellow solid line); ae = area of the epidermis (black dotted line); aw = area of the granulation tissue (green solid line); wd = wound depth (blue dotted line); d = distance between two sections (black dotted line). Note that in case of closed wound, le = l; in case of non-epithelialized wounds (this example), le</p

TGF-ß3 is not required for epidermal homeostasis and in vitro scratch wounds.

<p>Histological sections of skin from E17.5 wild type (a) and <i>Tgfb3</i>-deficient (b) embryos stained with hematoxylin and eosin. Similar sections (c, wild type; d, <i>Tgfb3</i>-deficient) were immunolabeled for PCNA (red) while nuclei were labeled with DAPI (blue). Dotted line indicates the basement membrane. Percentage of basal cells that were PCNA positive was calculated for both genotypes (e). (f) Interferon Regulatory Factor 6 (Irf6) expression in E17.5 cutaneous extracts of wild type and <i>Tgfb3</i>-deficient animals (2 samples are represented out of 6 tested). B-actin was used as loading control and densitometry used to quantify the level of Irf6 expression. (g–j) Keratinocytes were extracted from E17.5 skin and plated in culture. At confluence, in vitro scratch wounds were performed and followed over time (g, h: time point 0 h; I, j: time point 12 h). Percentage of closure was calculated for each time point (k). N = 3 per group. (f) Percentage of cells incorporating BrdU after a 2 h pulse was calculated. Scale bar = 50 µm.</p