129 research outputs found
Flightless I over-expression impairs skin barrier development, function and recovery following skin blistering
No full textAbstract not availableZlatko Kopecki, Gink N Yang, Ruth M Arkell, Jessica E Jackson, Elizabeth Melville, Hiroaki Iwata, Ralf J Ludwig, Detlef Zillikens, Dedee F Murrell and Allison J Cowi
Regulation of focal adhesions by Flightless I involves inhibition of paxillin phosphorylation via a Rac1-dependent pathway
No full textFlightless I (Flii) is an actin-remodeling protein that influences diverse processes including cell migration and gene transcription and links signal transduction with cytoskeletal regulation. Here, we show that Flii modulation of focal adhesions and filamentous actin stress fibers is Rac1-dependent. Using primary skin fibroblasts from Flii overexpressing (FliiTg/Tg), wild-type, and Flii deficient (Flii+/−) mice, we show that elevated expression of Flii increases stress fiber formation by impaired focal adhesion turnover and enhanced formation of fibrillar adhesions. Conversely, Flii knockdown increases the percentage of focal complex positive cells. We further show that a functional effect of Flii at both the cellular level and in in vivo mouse wounds is through inhibiting paxillin tyrosine phosphorylation and suppression of signaling proteins Src and p130Cas, both of which regulate adhesion signaling pathways. Flii is upregulated in response to wounding, and overexpression of Flii inhibits paxillin activity and reduces adhesion signaling by modulating the activity of the Rho family GTPases. Overexpression of constitutively active Rac1 GTPase restores the spreading ability of FliiTg/Tg fibroblasts and may explain the reduced adhesion, migration, and proliferation observed in FliiTg/Tg mice and their impaired wound healing, a process dependent on effective cellular motility and adhesion.Zlatko Kopecki, Geraldine M. O'Neill, Ruth M. Arkell and Allison J. Cowi
The role of the Zic genes in mouse neural crest development
No full textThe neural crest is an embryonic population of migratory, multipotent cells that are formed at the boundary of the neural and non-neural ectoderm around the time of neural tube closure. After migrating to locations throughout the embryo the neural crest cells differentiate into many different cell types including osteoblasts and chondrocytes of the cranio-facial skeleton and neurones and glia of the peripheral nervous system. Gain of function experiments demonstrate that many genes, including the Zic gene family, are involved in neural crest formation. In order to determine whether they have a role in endogenous neural crest development, loss of function studies are required. The ability to perform genetic analysis in the mouse makes it an ideal organism in which to do this. When this study commenced Zic1, Zic2 and Zic3 were assigned as members of the mammalian Zic gene family. A comparison of their expression patterns during mouse neural crest development reveals Zic2 and Zic3 to be the most likely to have roles in neural crest formation. Analysis of neural crest induction and migration in loss of function alleles of Zic2 and Zic3 reveals that loss of either gene function alone causes both a delay in the onset of trunk neural crest production and a reduction in the number of neural crest progenitor cells. Additionally, loss of Zic2 gene function results in a heightened response to BMP signalling by the neural tube. A more severe neural crest depletion occurs in embryos lacking Zic3 and heterozygous for a mutation in Zic2. These results indicate that Zic2 and Zic3 cooperate in the formation of the neural crest by mediating the competence of the dorsal neural tube to respond to inductive signals. This work provides the first genetic evidence that Zic2 and Zic3 are involved in neural crest development and that they function together during mouse development
Flightless I Regulates Hemidesmosome Formation and Integrin-Mediated Cellular Adhesion and Migration during Wound Repair
No full textFlightless I (Flii), a highly conserved member of the gelsolin family of actin-remodelling proteins associates with actin structures and is involved in cellular motility and adhesion. Our previous studies have shown that Flii is an important negative regulator of wound repair. Here, we show that Flii affects hemidesmosome formation and integrin-mediated keratinocyte adhesion and migration. Impaired hemidesmosome formation and sparse arrangements of keratin cytoskeleton tonofilaments and actin cytoskeleton anchoring fibrils were observed in FliiTg/+ and FliiTg/Tg mice with their skin being significantly more fragile than Flii+/− and WT mice. Flii+/− primary keratinocytes showed increased adhesion on laminin and collagen I than WT and FliiTg/Tg primary keratinocytes. Decreased expression of CD151 and laminin-binding integrins α3, β1, α6 and β4 were observed in Flii overexpressing wounds, which could contribute to the impaired wound re-epithelialization observed in these mice. Flii interacts with proteins directly linked to the cytoplasmic domain of integrin receptors suggesting that it may be a mechanical link between ligand-bound integrin receptors and the actin cytoskeleton driving adhesion-signaling pathways. Therefore Flii may regulate wound repair through its effect on hemidesmosome formation and integrin-mediated cellular adhesion and migration.Zlatko Kopecki, Ruth Arkell, Barry C. Powell and Allison J. Cowi
Decreased expression of Flightless I, a gelsolin family member and developmental regulator, in early-gestation fetal wounds improves healing
No full textUp until late in the third trimester of gestation and through to adulthood, the healing response acts more to regenerate than to repair a wound. The mechanisms underlying this "scar-free" healing remain unknown although the actin cytoskeleton has a major role. Flightless I (Flii), an actin-remodelling protein and essential developmental regulator, negatively affects wound repair but its effect on scar-free fetal healing is unknown. Using fetal skin explants from E17 (regenerate) and E19 (repair) rats, the function of Flii in fetal wound repair was determined. Expression of Flii increased between E17 and E19 days of gestation and wounding transiently increased Flii expression in E17 but not E19 wounds. However, both confocal and immunofluorescent analysis showed E17 keratinocytes immediately adjacent to the wounds downregulated Flii. As a nuclear coactivator and inhibitor of proliferation and migration, the absence of Flii in cells at the edge of the wound could be instrumental in allowing these cells to proliferate and migrate into the wound deficit. In contrast, Flii was strongly expressed within the cytoplasm and nucleus of keratinocytes within epidermal cells at the leading edge of E19 wounded fetal skin explants. This increase in Flii expression in E19 wounds could affect the way these cells migrate into the wound space and contribute to impaired wound healing. Neutralising Flii protein improved healing of early- but not late-gestation wounds. Flii did not colocalise with actin cables formed around E17 wounds suggesting an independent mechanism of action distinct from its actin-binding function in scar-free wound repair.Cheng-Hung Lin, James M. Waters, Barry C. Powell, Ruth M. Arkell, Allison J. Cowi
Fibroblast-specific upregulation of Flightless I impairs wound healing
No full textThe cytoskeletal protein Flightless (Flii) is a negative regulator of wound healing. Upregulation of Flii is associated with impaired migration, proliferation and adhesion of both fibroblasts and keratinocytes. Importantly, Flii translocates from the cytoplasm to the nucleus in response to wounding in fibroblasts but not keratinocytes. This cell-specific nuclear translocation of Flii suggests that Flii may directly regulate gene expression in fibroblasts, providing one potential mechanism of action for Flii in the wound healing response. To determine whether the tissue-specific upregulation of Flii in fibroblasts was important for the observed inhibitory effects of Flii on wound healing, an inducible fibroblast-specific Flii overexpressing mouse model was generated. The inducible ROSA26 system allowed the overexpression of Flii in a temporal and tissue-specific manner in response to tamoxifen treatment. Wound healing in the inducible mice was impaired, with wounds at day 7 postwounding significantly larger than those from non-inducible controls. There was also reduced collagen maturation, increased myofibroblast infiltration and elevated inflammation. The impaired healing response was similar in magnitude to that observed in mice with non-tissue-specific upregulation of Flii suggesting that fibroblast-derived Flii may have an important role in the wound healing response.Christopher T. Turner, James M. Waters, Jessica E. Jackson, Ruth M. Arkell, and Allison J. Cowi
Regeneration of hair follicles is modulated by flightless I (Flii) in a rodent Vibrissa model
No full textRegeneration of cells, tissues, and organs has long captured the attention of researchers for its obvious potential benefits in biomedical applications. Although mammals are notoriously poor at regeneration compared with many lower-order species, the hair follicle, paradoxically a defining characteristic of mammals, is capable of regeneration following partial amputation. To investigate the role of a negative regulator of wound healing, flightless I (Flii), on hair follicle regeneration, the bulbar region of vibrissae from rats as well as strains of mice expressing low (Flii+/−), normal (Flii+/+), and high (FLIITg/Tg) levels of Flii were surgically amputated, and then allowed to regenerate in vivo. Macroscopic and histological assessment of the regeneration process revealed impaired or delayed regenerative potential in Flii+/− follicles. Regenerated follicles expressing high levels of Flii (FLIITg/Tg) produced significantly longer terminal hair fibers. Immunohistochemical analysis was used to characterize the pattern of expression of Flii, as well as markers of hair follicle development and wound healing-associated factors during hair follicle regeneration. These studies confirmed that Flii appears to have a positive role in the regeneration of hair follicles, contrary to its negative influence on wound healing in skin.James M. Waters, Jessica E. Lindo, Ruth M. Arkell and Allison J. Cowi
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Functional analysis of transforming growth factor-beta related molecules during early mouse development
No full textSIGLEAvailable from British Library Document Supply Centre-DSC:DXN007070 / BLDSC - British Library Document Supply CentreGBUnited Kingdo
The influence of Flightless I on toll-like-receptor-mediated inflammation in a murine model of diabetic wound healing
No full textImpaired wound healing and ulceration represent a serious complication of both type 1 and type 2 diabetes. Cytoskeletal protein Flightless I (Flii) is an important inhibitor of wound repair, and reduced Flii gene expression in fibroblasts increased migration, proliferation, and adhesion. As such it has the ability to influence all phases of wound healing including inflammation, remodelling and angiogenesis. Flii has the potential to modulate inflammation through its interaction with MyD88 which it an adaptor protein for TLR4. To assess the effect of Flii on the inflammatory response of diabetic wounds, we used a murine model of streptozocin-induced diabetes and Flii genetic mice. Increased levels of Flii were detected in Flii transgenic murine wounds resulting in impaired healing which was exacerbated when diabetes was induced. When Flii levels were reduced in diabetic wounds of Flii-deficient mice, healing was improved and decreased levels of TLR4 were observed. In contrast, increasing the level of Flii in diabetic mouse wounds led to increased TLR4 and NF-κB production. Treatment of murine diabetic wounds with neutralising antibodies to Flii led to an improvement in healing with decreased expression of TLR4. Decreasing the level of Flii in diabetic wounds may therefore reduce the inflammatory response and improve healing.Nadira Ruzehaji, Stuart J. Mills, Elizabeth Melville, Ruth Arkell, Robert Fitridge and Allison J. Cowi
Production of Digoxigenin-Labeled Riboprobes for In Situ Hybridization Experiments
No full textExperiments that visualize gene expression in intact tissues or organisms are fundamental to studies of gene function. These experiments, called in situ hybridization, require the production of a riboprobe, which is a labeled antisense RNA corresponding to a particular gene. The most commonly used system for visualizing gene expression via in situ hybridization is the incorporation of a digoxigenin label into an in vitro−transcribed RNA probe. After hybridization of the riboprobe to a target mRNA, its location can be detected via a high‐affinity α‐digoxigenin antibody conjugated to an alkaline‐phosphatase enzyme. The article describes the design and production of digoxigenin‐labeled riboprobes transcribed in vitro from template DNA (either plasmid or PCR amplicon). These riboprobes are suitable for use in tissue and whole‐mount in situ hybridization protocols
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