Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells’ native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens¹. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.     

Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo

In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (−/−) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (−/−) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.     

Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair

Cadherin-mediated cell–cell adhesion is required for epithelial tissue integrity in homeostasis, during development, and in tissue repair. E-cadherin stability depends on F-actin, but the mechanisms regulating actin polymerization at cell–cell junctions remain poorly understood. Here we investigated a role for formin-mediated actin polymerization at cell–cell junctions. We identify mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell–cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. In summary, our results show that formin activity at epithelial cell–cell junctions is important for adhesion and the maintenance of epithelial cohesion during dynamic processes, such as wound repair.     

Genome-wide structural and evolutionary analysis of the P450 monooxygenase genes (P450ome) in the white rot fungus Phanerochaete chrysosporium : Evidence for gene duplications and extensive gene clustering

Background

Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor) that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian progranulin may be represented by two co-orthologues in zebrafish.

Results

The cDNAs encoding two zebrafish granulin precursors, progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of progranulin. Chromosomal localization of the four progranulin (grn) genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial compartments of various organs.

Conclusion

In support of the duplication-degeneration-complementation model of duplicate gene retention, partitioning of expression between grna and grnb was observed in the intermediate cell mass and yolk syncytial layer, respectively. Taken together these expression patterns suggest that the function of an ancestral grn gene has been devolved upon four Paralogues in zebrafish.     

In vivo imaging of epithelial wound healing in the cnidarian <Emphasis Type="Italic">Clytia hemisphaerica</Emphasis> demonstrates early evolution of purse string and cell crawling closure mechanisms

Background

All animals have mechanisms for healing damage to the epithelial sheets that cover the body and line internal cavities. Epithelial wounds heal either by cells crawling over the wound gap, by contraction of a super-cellular actin cable (“purse string”) that surrounds the wound, or some combination of the two mechanisms. Both cell crawling and purse string closure of epithelial wounds are widely observed across vertebrates and invertebrates, suggesting early evolution of these mechanisms. Cnidarians evolved ~600 million years ago and are considered a sister group to the Bilateria. They have been much studied for their tremendous regenerative potential, but epithelial wound healing has not been characterized in detail. Conserved elements of wound healing in bilaterians and cnidarians would suggest an evolutionary origin in a common ancestor. Here we test this idea by characterizing epithelial wound healing in live medusae of Clytia hemisphaerica.

Results

We identified cell crawling and purse string-mediated mechanisms of healing in Clytia epithelium that appear highly analogous of those seen in higher animals, suggesting that these mechanisms may have emerged in a common ancestor. Interestingly, we found that epithelial wound healing in Clytia is 75 to >600 times faster than in cultured cells or embryos of other animals previously studied, suggesting that Clytia may provide valuable clues about optimized healing efficiency. Finally, in Clytia, we show that damage to the basement membrane in a wound gap causes a rapid shift between the cell crawling and purse string mechanisms for wound closure. This is consistent with work in other systems showing that cells marginal to a wound choose between a super-cellular actin cable or lamellipodia formation to close wounds, and suggests a mechanism underlying this decision.

Conclusions

1. Cell crawling and purse string mechanisms of epithelial wound healing likely evolved before the divergence of Cnidaria from the bilaterian lineage ~ 600mya 2. In Clytia, the choice between cell crawling and purse string mechanisms of wound healing depends on interactions between the epithelial cells and the basement membrane.

     

Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure

Background

Epimorphic regeneration results in the restoration of lost tissues and structures from an aggregation of proliferating cells known as a blastema. Among amniotes the most striking example of epimorphic regeneration comes from tail regenerating lizards. Although tail regeneration is often studied in the context of ecological costs and benefits, details of the sequence of tissue-level events are lacking. Here we investigate the anatomical and histological events that characterize tail regeneration in the leopard gecko, Eublepharis macularius.

Results

Tail structure and tissue composition were examined at multiple days following tail loss, revealing a conserved pattern of regeneration. Removal of the tail results in a consistent series of morphological and histological events. Tail loss is followed by a latent period of wound healing with no visible signs of regenerative outgrowth. During this latent period basal cells of the epidermis proliferate and gradually cover the wound. An additional aggregation of proliferating cells accumulates adjacent to the distal tip of the severed spinal cord marking the first appearance of the blastema. Continued growth of the blastema is matched by the initiation of angiogenesis, followed by the re-development of peripheral axons and the ependymal tube of the spinal cord. Skeletal tissue differentiation, corresponding with the expression of Sox9, and muscle re-development are delayed until tail outgrowth is well underway.

Conclusions

We demonstrate that tail regeneration in lizards involves a highly conserved sequence of events permitting the establishment of a staging table. We show that tail loss is followed by a latent period of scar-free healing of the wound site, and that regeneration is blastema-mediated. We conclude that the major events of epimorphic regeneration are highly conserved across vertebrates and that a comparative approach is an invaluable biomedical tool for ongoing regenerative research.     

Early redox,Src family kinase,and calcium signaling integrate wound responses and tissue regeneration in zebrafish

Tissue injury can lead to scar formation or tissue regeneration. How regenerative animals sense initial tissue injury and transform wound signals into regenerative growth is an unresolved question. Previously, we found that the Src family kinase (SFK) Lyn functions as a redox sensor in leukocytes that detects H₂O₂ at wounds in zebrafish larvae. In this paper, using zebrafish larval tail fins as a model, we find that wounding rapidly activated SFK and calcium signaling in epithelia. The immediate SFK and calcium signaling in epithelia was important for late epimorphic regeneration of amputated fins. Wound-induced activation of SFKs in epithelia was dependent on injury-generated H₂O₂. A SFK member, Fynb, was responsible for fin regeneration. This work provides a new link between early wound responses and late regeneration and suggests that redox, SFK, and calcium signaling are immediate “wound signals” that integrate early wound responses and late epimorphic regeneration.     

Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

Background

Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations.

Results

Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres.

Conclusions

Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms.     

PhOTO zebrafish: a transgenic resource for in vivo lineage tracing during development and regeneration

Background

Elucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo.

Methodology

PhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ∼100µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin.

Conclusions/Significance

PhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior.     

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart¹, retina², spinal cord³, optic nerve⁴, sensory hair cells⁵, and fins⁶.The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 °C, this process occurs within six hours post-amputation (hpa, Figure 1B)^6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)⁸. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)⁸. Depending on the level of the amputation, full regeneration is completed in a week to a month.The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration^9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists¹³, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated^7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development^17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.     

Heat Shock Modulates Neutrophil Motility in Zebrafish

Heat shock is a routine method used for inducible gene expression in animal models including zebrafish. Environmental temperature plays an important role in the immune system and infection progression of ectotherms. In this study, we analyzed the impact of short-term heat shock on neutrophil function using zebrafish (Danio rerio) as an animal model. Short-term heat shock decreased neutrophil recruitment to localized Streptococcus iniae infection and tail fin wounding. Heat shock also increased random neutrophil motility transiently and increased the number of circulating neutrophils. With the use of the translating ribosome affinity purification (TRAP) method for RNA isolation from specific cell types such as neutrophils, macrophages and epithelial cells, we found that heat shock induced the immediate expression of heat shock protein 70 (hsp70) and a prolonged expression of heat shock protein 27 (hsp27). Heat shock also induced cell stress as detected by the splicing of X-box binding protein 1 (xbp1) mRNA, a marker for endoplasmic reticulum (ER) stress. Exogenous expression of Hsp70, Hsp27 and spliced Xbp1 in neutrophils or epithelial cells did not reproduce the heat shock induced effects on neutrophil recruitment. The effect of heat shock on neutrophils is likely due to a combination of complex changes, including, but not limited to changes in gene expression. Our results indicate that routine heat shock can alter neutrophil function in zebrafish. The findings suggest that caution should be taken when employing a heat shock-dependent inducible system to study the innate immune response.     

Epithelial Cells Are Active Participants in Vocal Fold Wound Healing: An In Vivo Animal Model of Injury

Vocal fold epithelial cells likely play an important, yet currently poorly defined, role in healing following injury, irritation and inflammation. In the present study, we sought to identify a possible role for growth factors, epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGFβ1), in epithelial regeneration during wound healing as a necessary first step for uncovering potential signaling mechanisms of vocal fold wound repair and remodeling. Using a rat model, we created unilateral vocal fold injuries and examined the timeline for epithelial healing and regeneration during early and late stages of wound healing using immunohistochemistry (IHC). We observed time-dependent secretion of the proliferation marker, ki67, growth factors EGF and TGFβ1, as well as activation of the EGF receptor (EGFR), in regenerating epithelium during the acute phase of injury. Ki67, growth factor, and EGFR expression peaked at day 3 post-injury. Presence of cytoplasmic and intercellular EGF and TGFβ1 staining occurred up to 5 days post-injury, consistent with a role for epithelial cells in synthesizing and secreting these growth factors. To confirm that epithelial cells contributed to the cytokine secretion, we examined epithelial cell growth factor secretion in vitro using polymerase chain reaction (PCR). Cultured pig vocal fold epithelial cells expressed both EGF and TGFβ1. Our in vivo and in vitro findings indicate that epithelial cells are active participants in the wound healing process. The exact mechanisms underlying their roles in autocrine and paracrine signaling guiding wound healing await study in a controlled, in vitro environment.     

A transgenic reporter under control of an es1 promoter/enhancer marks wound epidermis and apical epithelial cap during tail regeneration in Xenopus laevis tadpole

Rapid wound healing and subsequent formation of the apical epithelial cap (AEC) are believed to be required for successful appendage regeneration in amphibians. Despite the significant role of AEC in limb regeneration, its role in tail regeneration and the mechanisms that regulate the wound healing and AEC formation are not well understood. We previously identified Xenopus laevis es1, which is preferentially expressed in wounded regions, including the AEC after tail regeneration. In this study we established and characterized transgenic Xenopus laevis lines harboring the enhanced green fluorescent protein (EGFP) gene under control of an es1 gene regulatory sequence (es1:egfp).The EGFP reporter expression was clearly seen in several regions of the embryo and then declined to an undetectable level in larvae, recapitulating the endogenous es1 expression. After amputation of the tadpole tail, EGFP expression was re-activated at the edge of the stump epidermis and then increased in the wound epidermis (WE) covering the amputation surface. As the stump started to regenerate, the EGFP expression became restricted to the most distal epidermal region, including the AEC. EGFP was preferentially expressed in the basal or deep cells but not in the superficial cells of the WE and AEC.We performed a small-scale pharmacological screening for chemicals that affected the expression of EGFP in the stump epidermis after tail amputation. The EGFP expression was attenuated by treatment with an inhibitor for ERK, TGF-β or reactive oxygen species (ROS) signaling. These treatments also impaired wound closure of the amputation surface, suggesting that the three signaling activities are required for es1 expression in the WE and successful wound healing after tail amputation.These findings showed that es1:egfp Xenopus laevis should be a useful tool to analyze molecular mechanisms regulating wound healing and appendage regeneration.     

Antisense morpholino targeting just upstream from a poly(A) tail junction of maternal mRNA removes the tail and inhibits translation

Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3′-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.