The role of transcription-independent damage signals in the initiation of epithelial wound healing.

Wound healing is an essential biological process that comprises sequential steps aimed at restoring the architecture and function of damaged cells and tissues. This process begins with conserved damage signals, such as Ca(2+), hydrogen peroxide (H2O2) and ATP, that diffuse through epithelial tissues and initiate immediate gene transcription-independent cellular effects, including cell shape changes, the formation of functional actomyosin structures and the recruitment of immune cells. These events integrate the ensuing transcription of specific wound response genes that further advance the wound healing response. The immediate importance of transcription-independent damage signals illustrates that healing a wound begins as soon as damage occurs.

The role of transcription-independent damage signals in the initiation of epithelial wound healing.

Wound healing is an essential biological process that comprises sequential steps aimed at restoring the architecture and function of damaged cells and tissues. This process begins with conserved damage signals, such as Ca2+, hydrogen peroxide (H2O2) and ATP, that diffuse through epithelial tissues and initiate immediate gene transcription-independent cellular effects, including cell shape changes, the formation of functional actomyosin structures and the recruitment of immune cells. These events integrate the ensuing transcription of specific wound response genes that further advance the wound healing response. The immediate importance of transcription-independent damage signals illustrates that healing a wound begins as soon as damage occurs.

Fin ray branching is defined by TRAP+ osteolytic tubules in zebrafish.

The shaping of bone structures relies on various cell types and signaling pathways. Here, we use the zebrafish bifurcating fin rays during regeneration to investigate bone patterning. We found that the regenerating fin rays form via two mineralization fronts that undergo an osteoblast-dependent fusion/stitching until the branchpoint, and that bifurcation is not simply the splitting of one unit into two. We identified tartrate-resistant acid phosphatase-positive osteolytic tubular structures at the branchpoints, hereafter named osteolytic tubules (OLTs). Chemical inhibition of their bone-resorbing activity strongly impairs ray bifurcation, indicating that OLTs counteract the stitching process. Furthermore, by testing different osteoactive compounds, we show that the position of the branchpoint depends on the balance between bone mineralization and resorption activities. Overall, these findings provide a unique perspective on fin ray formation and bifurcation, and reveal a key role for OLTs in defining the proximo-distal position of the branchpoint.

Cholesteryl hemiazelate identified in CVD patients causes in vitro and in vivo inflammation.

Oxidation of PUFAs in LDLs trapped in the arterial intima plays a critical role in atherosclerosis. Though there have been many studies on the atherogenicity of oxidized derivatives of PUFA-esters of cholesterol, the effects of cholesteryl hemiesters (ChEs), the oxidation end products of these esters, have not been studied. Through lipidomics analyses, we identified and quantified two ChE types in the plasma of CVD patients and identified four ChE types in human endarterectomy specimens. Cholesteryl hemiazelate (ChA), the ChE of azelaic acid (n-nonane-1,9-dioic acid), was the most prevalent ChE identified in both cases. Importantly, human monocytes, monocyte-derived macrophages, and neutrophils exhibit inflammatory features when exposed to subtoxic concentrations of ChA in vitro. ChA increases the secretion of proinflammatory cytokines such as interleukin-1ß and interleukin-6 and modulates the surface-marker profile of monocytes and monocyte-derived macrophage. In vivo, when zebrafish larvae were fed with a ChA-enriched diet, they exhibited neutrophil and macrophage accumulation in the vasculature in a caspase 1- and cathepsin B-dependent manner. ChA also triggered lipid accumulation at the bifurcation sites of the vasculature of the zebrafish larvae and negatively impacted their life expectancy. We conclude that ChA behaves as an endogenous damage-associated molecular pattern with inflammatory and proatherogenic properties.

Yap induces osteoblast differentiation by modulating Bmp signalling during zebrafish caudal fin regeneration.

Osteoblast differentiation is a key process for bone homeostasis and repair. Multiple signalling pathways have been associated with osteoblast differentiation, yet much remains unknown on how this process is regulated in vivo Previous studies have proposed that the Hippo pathway transcriptional co-activators YAP and TAZ (also known as YAP1 and WWTR1, respectively) maintain progenitor stemness and inhibit terminal differentiation of osteoblasts, whereas others suggest they potentiate osteoblast differentiation and bone formation. Here, we use zebrafish caudal fin regeneration as a model to clarify how the Hippo pathway regulates de novo bone formation and osteoblast differentiation. We demonstrate that Yap inhibition leads to accumulation of osteoprogenitors and prevents osteoblast differentiation in a cell non-autonomous manner. This effect correlates with a severe impairment of Bmp signalling in osteoblasts, likely by suppressing the expression of the ligand bmp2a in the surrounding mesenchymal cells. Overall, our findings provide a new mechanism of bone formation through the Hippo-Yap pathway, integrating Yap in the signalling cascade that governs osteoprogenitor maintenance and subsequent differentiation during zebrafish caudal fin regeneration.

Novel role for Grainy head in the regulation of cytoskeletal and junctional dynamics during epithelial repair.

Tissue repair is critical for the maintenance of epithelial integrity and permeability. Simple epithelial repair relies on a combination of collective cell movements and the action of a contractile actomyosin cable at the wound edge that together promote the fast and efficient closure of tissue discontinuities. The Grainy head family of transcription factors (Grh in flies; GRHL1-GRHL3 in mammals) are essential proteins that have been implicated both in the development and repair of epithelia. However, the genes and the molecular mechanisms that it controls remain poorly understood. Here, we show that Grh knockdown disrupts actomyosin dynamics upon injury of the Drosophila pupa epithelial tissue. This leads to the formation of an ectopic actomyosin cable away from the wound edge and impaired wound closure. We also uncovered that E-Cadherin is downregulated in the Grh-depleted tissue around the wound, likely as a consequence of Dorsal (an NF-κB protein) misregulation, which also affects actomyosin cable formation. Our work highlights the importance of Grh as a stress response factor and its central role in the maintenance of epithelial characteristics necessary for tissue repair through regulating cytoskeleton and E-Cadherin dynamics.

Trends in tissue repair and regeneration.

The 6th EMBO conference on the Molecular and Cellular Basis of Regeneration and Tissue Repair took place in Paestum (Italy) on the 17th-21st September, 2016. The 160 scientists who attended discussed the importance of cellular and tissue plasticity, biophysical aspects of regeneration, the diverse roles of injury-induced immune responses, strategies to reactivate regeneration in mammals, links between regeneration and ageing, and the impact of non-mammalian models on regenerative medicine.

Correction: Telomerase Is Required for Zebrafish Lifespan.

[This corrects the article DOI: 10.1371/journal.pgen.1003214.].

Control of tissue growth by Yap relies on cell density and F-actin in zebrafish fin regeneration.

Caudal fin regeneration is characterized by a proliferation boost in the mesenchymal blastema that is controlled precisely in time and space. This allows a gradual and robust restoration of original fin size. However, how this is established and regulated is not well understood. Here, we report that Yap, the Hippo pathway effector, is a chief player in this process: functionally manipulating Yap during regeneration dramatically affects cell proliferation and expression of key signaling pathways, impacting regenerative growth. The intracellular location of Yap is tightly associated with different cell densities along the blastema proximal-distal axis, which correlate with alterations in cell morphology, cytoskeleton and cell-cell contacts in a gradient-like manner. Importantly, Yap inactivation occurs in high cell density areas, conditional to F-actin distribution and polymerization. We propose that Yap is essential for fin regeneration and that its function is dependent on mechanical tension, conferred by a balancing act of cell density and cytoskeleton activity.

Renal regeneration after acute kidney injury.

Acute kidney injury is common and associated with negative renal and patient outcomes. The human kidney has a real but limited regeneration capacity. Understanding renal regeneration may allow us to manipulate this process and thus develop therapeutic weapons to improve patients' outcome. In the first part of this paper we discuss the clinical factors associated with renal recovery: baseline patient particularities, acute kidney injury characteristics and the medical approach taken in the short and long-term. In the second part, the cellular and molecular mechanisms underlying renal regeneration are explored. The immune system seems to have an important role, first promoting inflammation and then tissue healing. Other players, such as cellular senescence, mitochondrial dysfunction, renal haemodynamics and metabolic reprogramming also have a role in renal regeneration. We aim to develop a short review of renal regeneration, offering a holistic view of this process.

Cholesteryl hemiesters alter lysosome structure and function and induce proinflammatory cytokine production in macrophages.

RATIONALE: Cholesteryl hemiesters are oxidation products of polyunsaturated fatty acid esters of cholesterol. Their oxo-ester precursors have been identified as important components of the "core aldehydes" of human atheromata and in oxidized lipoproteins (Ox-LDL). We had previously shown, for the first time, that a single compound of this family, cholesteryl hemisuccinate (ChS), is sufficient to cause irreversible lysosomal lipid accumulation (lipidosis), and is toxic to macrophages. These features, coupled to others such as inflammation, are typically seen in atherosclerosis. OBJECTIVE: To obtain insights into the mechanism of cholesteryl hemiester-induced pathological changes in lysosome function and induction of inflammation in vitro and assess their impact in vivo. METHODS AND RESULTS: We have examined the effects of ChS on macrophages (murine cell lines and primary cultures) in detail. Specifically, lysosomal morphology, pH, and proteolytic capacity were examined. Exposure of macrophages to sub-toxic ChS concentrations caused enlargement of the lysosomes, changes in their luminal pH, and accumulation of cargo in them. In primary mouse bone marrow-derived macrophages (BMDM), ChS-exposure increased the secretion of IL-1ß, TNF-α and IL-6. In zebrafish larvae (wild-type AB and PU.1:EGFP), fed with a ChS-enriched diet, we observed lipid accumulation, myeloid cell-infiltration in their vasculature and decrease in larval survival. Under the same conditions the effects of ChS were more profound than the effects of free cholesterol (FC). CONCLUSIONS: Our data strongly suggest that cholesteryl hemiesters are pro-atherogenic lipids able to mimic features of Ox-LDL both in vitro and in vivo.

The Toll/NF-κB signaling pathway is required for epidermal wound repair in Drosophila.

The Toll/NF-κB pathway, first identified in studies of dorsal-ventral polarity in the early Drosophila embryo, is well known for its role in the innate immune response. Here, we reveal that the Toll/NF-κB pathway is essential for wound closure in late Drosophila embryos. Toll mutants and Dif dorsal (NF-κB) double mutants are unable to repair epidermal gaps. Dorsal is activated on wounding, and Dif and Dorsal are required for the sustained down-regulation of E-cadherin, an obligatory component of the adherens junctions (AJs), at the wound edge. This remodeling of the AJs promotes the assembly of an actin-myosin cable at the wound margin; contraction of the actin cable, in turn, closes the wound. In the absence of Toll or Dif and dorsal (dl), both E-cadherin down-regulation and actin-cable formation fail, thus resulting in open epidermal gaps. Given the conservation of the Toll/NF-κB pathway in mammals and the epithelial expression of many components of the pathway, this function in wound healing is likely to be conserved in vertebrates.

Telomerase is required for zebrafish lifespan.

Telomerase activity is restricted in humans. Consequentially, telomeres shorten in most cells throughout our lives. Telomere dysfunction in vertebrates has been primarily studied in inbred mice strains with very long telomeres that fail to deplete telomeric repeats during their lifetime. It is, therefore, unclear how telomere shortening regulates tissue homeostasis in vertebrates with naturally short telomeres. Zebrafish have restricted telomerase expression and human-like telomere length. Here we show that first-generation tert(-/-) zebrafish die prematurely with shorter telomeres. tert(-/-) fish develop degenerative phenotypes, including premature infertility, gastrointestinal atrophy, and sarcopaenia. tert(-/-) mutants have impaired cell proliferation, accumulation of DNA damage markers, and a p53 response leading to early apoptosis, followed by accumulation of senescent cells. Apoptosis is primarily observed in the proliferative niche and germ cells. Cell proliferation, but not apoptosis, is rescued in tp53(-/-)tert(-/-) mutants, underscoring p53 as mediator of telomerase deficiency and consequent telomere instability. Thus, telomerase is limiting for zebrafish lifespan, enabling the study of telomere shortening in naturally ageing individuals.

Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila.

Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in Drosophila, and paves the way for genetic dissection of the mechanisms at work behind steroid regulation of innate immune cells.

Denervation impairs regeneration of amputated zebrafish fins.

BACKGROUND: Zebrafish are able to regenerate many of its tissues and organs after damage. In amphibians this process is regulated by nerve fibres present at the site of injury, which have been proposed to release factors into the amputated limbs/fins, promoting and sustaining the proliferation of blastemal cells. Although some candidate factors have been proposed to mediate the nerve dependency of regeneration, the molecular mechanisms involved in this process remain unclear. RESULTS: We have used zebrafish as a model system to address the role of nerve fibres in fin regeneration. We have developed a protocol for pectoral fin denervation followed by amputation and analysed the regenerative process under this experimental conditions. Upon denervation fins were able to close the wound and form a wound epidermis, but could not establish a functional apical epithelial cap, with a posterior failure of blastema formation and outgrowth, and the accumulation of several defects. The expression patterns of genes known to be key players during fin regeneration were altered upon denervation, suggesting that nerves can contribute to the regulation of the Fgf, Wnt and Shh pathways during zebrafish fin regeneration. CONCLUSIONS: Our results demonstrate that proper innervation of the zebrafish pectoral fin is essential for a successful regenerative process, and establish this organism as a useful model to understand the molecular and cellular mechanisms of nerve dependence, during vertebrate regeneration.

Differentiated skeletal cells contribute to blastema formation during zebrafish fin regeneration.

The origin of cells that generate the blastema following appendage amputation has been a long-standing question in epimorphic regeneration studies. The blastema is thought to originate from either stem (or progenitor) cells or differentiated cells of various tissues that undergo dedifferentiation. Here, we investigate the origin of cells that contribute to the regeneration of zebrafish caudal fin skeletal elements. We provide evidence that the process of lepidotrichia (bony rays) regeneration is initiated as early as 24 hours post-amputation and that differentiated scleroblasts acquire a proliferative state, detach from the lepidotrichia surface, migrate distally, integrate into the blastema and dedifferentiate. These findings provide novel insights into the origin of cells in epimorphic appendage regeneration in zebrafish and suggest conservation of regeneration mechanisms between fish and amphibians.

Video force microscopy reveals the mechanics of ventral furrow invagination in Drosophila.

The absence of tools for mapping the forces that drive morphogenetic movements in embryos has impeded our understanding of animal development. Here we describe a unique approach, video force microscopy (VFM), that allows detailed, dynamic force maps to be produced from time-lapse images. The forces at work in an embryo are considered to be decomposed into active and passive elements, where active forces originate from contributions (e.g., actomyosin contraction) that do mechanical work to the system and passive ones (e.g., viscous cytoplasm) that dissipate energy. In the present analysis, the effects of all passive components are considered to be subsumed by an effective cytoplasmic viscosity, and the driving forces are resolved into equivalent forces along the edges of the polygonal boundaries into which the region of interest is divided. Advanced mathematical inverse methods are used to determine these driving forces. When applied to multiphoton sections of wild-type and mutant Drosophila melanogaster embryos, VFM is able to calculate the equivalent driving forces acting along individual cell edges and to do so with subminute temporal resolution. In the wild type, forces along the apical surface of the presumptive mesoderm are found to be large and to vary parabolically with time and angular position, whereas forces along the basal surface of the ectoderm, for example, are found to be smaller and nearly uniform with position. VFM shows that in mutants with reduced junction integrity and myosin II activity, the driving forces are reduced, thus accounting for ventral furrow failure.

An amputation resets positional information to a proximal identity in the regenerating zebrafish caudal fin.

BACKGROUND: Zebrafish has emerged as a powerful model organism to study the process of regeneration. This teleost fish has the ability to regenerate various tissues and organs like the heart, spinal cord, retina and fins. In this study, we took advantage of the existence of an excellent morphological reference in the zebrafish caudal fin, the bony ray bifurcations, as a model to study positional information upon amputation. We investigated the existence of positional information for bifurcation formation by performing repeated amputations at different proximal-distal places along the fin. RESULTS: We show that, while amputations performed at a long distance from the bifurcation do not change its final proximal-distal position in the regenerated fin, consecutive amputations done at 1 segment proximal to the bifurcation (near the bifurcation) induce a positional reset and progressively shift its position distally. Furthermore, we investigated the potential role of Shh and Fgf signalling pathways in the determination of the bifurcation position and observed that they do not seem to be involved in this process. CONCLUSIONS: Our results reveal that, an amputation near the bifurcation inhibits the formation of the regenerated bifurcation in the pre-amputation position, inducing a distalization of this structure. This shows that the positional memory for bony ray bifurcations depends on the proximal-distal level of the amputation.

Urinary immune cell phenotype of severe AKI in critically ill patients.

PURPOSE: Cellular mechanisms involved in human renal recovery after an episode of acute kidney injury (AKI) are understudied. We aim to characterize the urinary immune cell phenotype of patients with AKI and evaluate its ability to predict renal recovery. METHODS: A prospective study of critically ill patients with stage ≥ 2 AKI by KDIGO and sterile leukocyturia at admission was performed. Urine samples were collected fresh at day 0 and 2 and samples were analyzed by flow cytometry for different leukocytes. Patients were categorized in renal recovery or no-recovery groups. RESULTS: 28 patients were included, all with sepsis, 60.7% of which recovered renal function. The main urinary leukocytes present were neutrophils, followed by mononuclear phagocytic cells and B cells. Patients who recovered renal function had more M2 macrophages at day 2 (p = 0.043) and less B cells at admission (p = 0.006). M2 macrophages had an AUC-ROC of 0.796 (0.601-0.990) for recovery prediction and B cells an AUC-ROC of 0.743 (0.560-0.926) for no recovery. B regulatory cells were found in the urine of AKI patients. CONCLUSIONS: The urinary immune cell phenotype of severe AKI patients was composed essentially of neutrophils, mononuclear phagocytic cells and B cells. Our data suggest that M2 macrophages may promote and B cells preclude renal recovery. More studies are needed to validate our results and further explore the role of immune cells in renal recovery.

A regeneration-triggered metabolic adaptation is necessary for cell identity transitions and cell cycle re-entry to support blastema formation and bone regeneration.

Regeneration depends on the ability of mature cells at the injury site to respond to injury, generating tissue-specific progenitors that incorporate the blastema and proliferate to reconstitute the original organ architecture. The metabolic microenvironment has been tightly connected to cell function and identity during development and tumorigenesis. Yet, the link between metabolism and cell identity at the mechanistic level in a regenerative context remains unclear. The adult zebrafish caudal fin, and bone cells specifically, have been crucial for the understanding of mature cell contribution to tissue regeneration. Here, we use this model to explore the relevance of glucose metabolism for the cell fate transitions preceding new osteoblast formation and blastema assembly. We show that injury triggers a modulation in the metabolic profile at early stages of regeneration to enhance glycolysis at the expense of mitochondrial oxidation. This metabolic adaptation mediates transcriptional changes that make mature osteoblast amenable to be reprogramed into pre-osteoblasts and induces cell cycle re-entry and progression. Manipulation of the metabolic profile led to severe reduction of the pre-osteoblast pool, diminishing their capacity to generate new osteoblasts, and to a complete abrogation of blastema formation. Overall, our data indicate that metabolic alterations have a powerful instructive role in regulating genetic programs that dictate fate decisions and stimulate proliferation, thereby providing a deeper understanding on the mechanisms regulating blastema formation and bone regeneration.