methods-for-skin-wounding-and-assays-for-wound-responses-in-c-elegans

Methods for Skin Wounding and Assays for Wound Responses in <em>C. elegans</em>

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

University of California, San Diego

Repair of skin wounds is essential for animals to survive in a harsh environment, yet the signaling pathways initiating wound repair in vivo remain little understood. In Caenorhabditis elegans, a p38 mitogen-activated protein kinase (MAPK) cascade promotes innate immune responses to wounding but is not required for other aspects of wound healing. We therefore set out to identify additional wound response pathways in C. elegans epidermis.

A Gαq-Ca²⁺ signaling pathway promotes actin-mediated epidermal wound closure in C. elegans.

Dorsal-ventral specification in the amphibian embryo is controlled by β-catenin, whose activation in all dorsal cells is dependent on maternal Wnt11. However, it remains unknown whether other maternally secreted factors contribute to β-catenin activation in the dorsal ectoderm. Here, we show that maternal Xenopus Norrin (xNorrin) promotes anterior neural tissue formation in ventralized embryos. Conversely, when xNorrin function is inhibited, early canonical Wnt signaling in the dorsal ectoderm and the early expression of the zygotic neural inducers Chordin, Noggin, and Xnr3 are severely suppressed, causing the loss of anterior structures. In addition, xNorrin potently inhibits BMP- and Nodal/Activin-related functions through direct binding to the ligands. Moreover, a subset of Norrin mutants identified in humans with Norrie disease retain Wnt activation but show defective inhibition of Nodal/Activin-related signaling in mesoderm induction, suggesting that this disinhibition causes Norrie disease. Thus, xNorrin is an unusual molecule that acts on two major signaling pathways, Wnt and TGF-β, in opposite ways and is essential for early neuroectoderm specification.

Maternal xNorrin, a canonical Wnt signaling agonist and TGF-β antagonist, controls early neuroectoderm specification in Xenopus.

The Caenorhabditis elegans epidermis forms one of the principal barrier epithelia of the animal. Differentiation of the epidermis begins in mid embryogenesis and involves apical-basal polarization of the cytoskeletal and secretory systems as well as cellular junction formation. Secretion of the external cuticle layers is one of the major developmental and physiological specializations of the epidermal epithelium. The four post-embryonic larval stages are separated by periodic moults, in which the epidermis generates a new cuticle with stage-specific characteristics. The differentiated epidermis also plays key roles in endocrine signaling, fat storage, and ionic homeostasis. The epidermis is intimately associated with the development and function of the nervous system, and may have glial-like roles in modulating neuronal function. The epidermis provides passive and active defenses against skin-penetrating pathogens and can repair small wounds. Finally, age-dependent deterioration of the epidermis is a prominent feature of aging and may affect organismal aging and lifespan.

The Caenorhabditis elegans epidermis as a model skin. II: differentiation and physiological roles.

The ability to heal wounds is an ancient and conserved function of epidermal epithelial layers. The importance of skin wound healing to human life and biology has long been evident, however many of the molecular mechanisms underlying wound repair remain little understood. In the past several years, analysis of the C. elegans innate immune response to fungal infection of the epidermis has led to investigations of the ability of the C. elegans skin to respond to damage. In a recent paper we used live imaging to investigate the cell biological basis of wound repair in the adult C. elegans epidermis. We found that needle or laser injury of the skin triggers a large and sustained increase in epidermal calcium. Epidermal calcium signals appear to specifically promote actin-dependent processes of wound closure. The innate immune and wound closure responses act in parallel to promote survival after injury. Our findings indicate that wounding triggers multiple signals in the C. elegans skin. C. elegans offers a tractable model to dissect how epidermal epithelia activate coordinated responses to repair damage.

The wounded worm: Using C. elegans to understand the molecular basis of skin wound healing.

Reactive oxygen species (ROS) such as hydrogen peroxide are generated at wound sites and act as long-range signals in wound healing. The roles of other ROS in wound repair are little explored. Here, we reveal a cytoprotective role for mitochondrial ROS (mtROS) in Caenorhabditis elegans skin wound healing. We show that skin wounding causes local production of mtROS superoxide at the wound site. Inhibition of mtROS levels by mitochondrial superoxide-specific antioxidants blocks actin-based wound closure, whereas elevation of mtROS promotes wound closure and enhances survival of mutant animals defective in wound healing. mtROS act downstream of wound-triggered Ca(2+) influx. We find that the mitochondrial calcium uniporter MCU-1 is essential for rapid mitochondrial Ca(2+) uptake and mtROS production after wounding. mtROS can promote wound closure by local inhibition of Rho GTPase activity via a redox-sensitive motif. These findings delineate a pathway acting via mtROS that promotes cytoskeletal responses in wound healing.

Article	Year
A Gαq-Ca²⁺ signaling pathway promotes actin-mediated epidermal wound closure in C. elegans. Current biology : CB\| PubMed ID: 22100061	2011
Maternal xNorrin, a canonical Wnt signaling agonist and TGF-β antagonist, controls early neuroectoderm specification in Xenopus. PLoS biology\| PubMed ID: 22448144	2012
The Caenorhabditis elegans epidermis as a model skin. II: differentiation and physiological roles. Wiley interdisciplinary reviews. Developmental biology\| PubMed ID: 23539358	2012
The wounded worm: Using C. elegans to understand the molecular basis of skin wound healing. Worm\| PubMed ID: 24058838	2012
C. elegans Epidermal Wounding Induces a Mitochondrial ROS Burst that Promotes Wound Repair. Developmental cell\| PubMed ID: 25313960	2014

Suhong Xu

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