Bullseye Analysis: A Fluorescence Microscopy Technique to Detect Local Changes in Intracellular Reactive Oxygen Species (ROS) Production.

Reactive oxygen species (ROS) are naturally produced compounds that play important roles in cell signaling, gene regulation, and biological defense, including involvement in the oxidative burst that is central to the anti-microbial actions of macrophages. However, these highly reactive, short-lived radical species also stimulate cells to undergo programmed cell death at high concentrations, as well as causing detrimental effects such as oxidation of macromolecules at more moderate levels. Imaging ROS is highly challenging, with many researchers working on the challenge over the past 10-15 years without producing a definitive method. We report a new fluorescence microscopy-based technique, Bullseye Analysis. This methodology is based on concepts provided by the FRAP (Fluorescence Recovery after Photobleaching) technique and refined to evidence the spatiotemporal production of ROS, and the subsequent consequences, on a subcellular scale. To exemplify the technique, we have used the ROS-reporter dye, CellROX, and the ROS-inducing photosensitizer, LightOx58, a potent source of ROS compared with UV irradiation alone. Further validation of the technique was carried out using differing co-stains, notably Mitotracker and JC-1.

Inducible ablation of CD11c+ cells to determine their role in skin wound repair.

Whether resident and recruited myeloid cells may impair or aid healing of acute skin wounds remains a debated question. To begin to address this, we examined the importance of CD11c+ myeloid cells in the early activation of skin wound repair. We find that an absence of CD11c+ cells delays wound closure and epidermal proliferation, likely due to defects in the activation of the IL-23-IL-22 axis that is required for wound healing.

Detection and time-tracking activation of a photosensitiser on live single colorectal cancer cells using Raman spectroscopy.

Raman spectroscopy has been used to observe uptake, metabolism and response of single-cells to drugs. Photodynamic therapy is based on the use of light, a photosensitiser and oxygen to destroy tumour tissue. Here, we used single-cell Raman spectroscopy to study the uptake and intracellular degradation of a novel photosensitiser with a diphenylacetylene structure, DC473, in live single-cells from colorectal adenocarcinoma cell lines SW480, HT29 and SW620. DC473 was seen to predominantly accumulate in lipid droplets, showing higher accumulation in HT29 and SW620 cells than in SW480 cells, with a broader DC473 peak shifted to higher wavenumbers. DC473 activation and effects were tracked on live single-cells for 5 minutes. Upon exposure to UV light, the DC473 signal intensity dropped, with remaining DC473 shifting towards higher wavenumbers and widening, with a lifetime of approximately 50 seconds. Morphologically, SW480 and SW620 cells showed changes upon photodynamic therapy, whereas HT29 cells showed no changes. Morphological changes correlated with higher remaining DC473 signal after UV exposure. Our research suggests that DC473 forms aggregates within the cells that disaggregate following activation, showing the potential of Raman spectroscopy for the study of time-dependent single-cell pharmacodynamics.

Cellular localisation of structurally diverse diphenylacetylene fluorophores.

Fluorescent probes are increasingly used as reporter molecules in a wide variety of biophysical experiments, but when designing new compounds it can often be difficult to anticipate the effect that changing chemical structure can have on cellular localisation and fluorescence behaviour. To provide further chemical rationale for probe design, a series of donor-acceptor diphenylacetylene fluorophores with varying lipophilicities and structures were synthesised and analysed in human epidermal cells using a range of cellular imaging techniques. These experiments showed that, within this family, the greatest determinants of cellular localisation were overall lipophilicity and the presence of ionisable groups. Indeed, compounds with high log D values (>5) were found to localise in lipid droplets, but conversion of their ester acceptor groups to the corresponding carboxylic acids caused a pronounced shift to localisation in the endoplasmic reticulum. Mildly lipophilic compounds (log D = 2-3) with strongly basic amine groups were shown to be confined to lysosomes i.e. an acidic cellular compartment, but sequestering this positively charged motif as an amide resulted in a significant change to cytoplasmic and membrane localisation. Finally, specific organelles including the mitochondria could be targeted by incorporating groups such as a triphenylphosphonium moiety. Taken together, this account illustrates a range of guiding principles that can inform the design of other fluorescent molecules but, moreover, has demonstrated that many of these diphenylacetylenes have significant utility as probes in a range of cellular imaging studies.

Tandem fluorescence and Raman (fluoRaman) characterisation of a novel photosensitiser in colorectal cancer cell line SW480.

The development of new imaging tools, molecules and modalities is crucial to understanding biological processes and the localised cellular impact of bioactive compounds. A small molecule photosensitiser, DC473, has been designed to be both highly fluorescent and to exhibit a strong Raman signal in the cell-silent region of the Raman spectrum due to a diphenylacetylene structure. DC473 has been utilised to perform a range of novel tandem fluorescence and Raman (fluoRaman) imaging experiments, enabling a thorough examination of the compound's cellular localisation, exemplified in colorectal cancer cells (SW480). This multifunctional fluoRaman imaging modality revealed the presence of the compound in lipid droplets and only a weak signal in the cytosol, by both Raman and fluorescence imaging. In addition, Raman microscopy detected the compound in a cell compartment we labelled as the nucleolus, whereas fluorescence microscopy did not detect the fluoRaman probe due to solvatochromatic effects in a local polar environment. This last finding was only possible with the use of tandem confocal Raman and fluorescence methods. By following the approach detailed herein, incorporation of strong Raman functional groups into fluorophores can enable a plethora of fluoRaman experiments, shedding further light on potential drug compound's cellular behaviour and biological activity.

Epidermal Notch signalling: differentiation, cancer and adhesion.

The Notch pathway plays an important role in regulating epidermal differentiation. Notch ligands, receptors and effectors are expressed in a complex and dynamic pattern in embryonic and adult skin. Genetic ablation or activation of the pathway reveals that Notch signalling promotes differentiation of the hair follicle, sebaceous gland and interfollicular epidermal lineages and that Notch acts as an epidermal tumour suppressor. Notch signalling interacts with a range of other pathways to fulfil these functions and acts via RBP-Jkappa dependent and independent mechanisms. The effects on differentiation can be cell autonomous and non-autonomous, and Notch contributes to stem cell clustering via modulation of cell adhesion.

Adult epidermal Notch activity induces dermal accumulation of T cells and neural crest derivatives through upregulation of jagged 1.

Notch signalling regulates epidermal differentiation and tumour formation via non-cell autonomous mechanisms that are incompletely understood. This study shows that epidermal Notch activation via a 4-hydroxy-tamoxifen-inducible transgene caused epidermal thickening, focal detachment from the underlying dermis and hair clumping. In addition, there was dermal accumulation of T lymphocytes and stromal cells, some of which localised to the blisters at the epidermal-dermal boundary. The T cell infiltrate was responsible for hair clumping but not for other Notch phenotypes. Notch-induced stromal cells were heterogeneous, expressing markers of neural crest, melanocytes, smooth muscle and peripheral nerve. Although Slug1 expression was expanded in the epidermis, the stromal cells did not arise through epithelial-mesenchymal transition. Epidermal Notch activation resulted in upregulation of jagged 1 in both epidermis and dermis. When Notch was activated in the absence of epidermal jagged 1, jagged 1 was not upregulated in the dermis, and epidermal thickening, blister formation, accumulation of T cells and stromal cells were inhibited. Gene expression profiling revealed that epidermal Notch activation resulted in upregulation of several growth factors and cytokines, including TNFα, the expression of which was dependent on epidermal jagged 1. We conclude that jagged 1 is a key mediator of non-cell autonomous Notch signalling in skin.

Regulation of epidermal proliferation and hair follicle cycling by synthetic photostable retinoid EC23.

BACKGROUND: Retinoid signaling is an important regulator of the epidermis and skin appendages. Therefore, synthetic retinoids have been developed for therapeutic use for skin disorders such as psoriasis and acne. AIMS: In previous studies, we showed how the photostable retinoid EC23 induces neuronal differentiation in stem cell-like cell populations, and here, we aim to investigate its ability to influence epidermal and hair follicle growth. METHODS: EC23 influence on skin biology was investigated initially in cultures of monolayer keratinocytes and three-dimentional in vitro models of skin, and finally in in vivo studies of mice back skin. RESULTS: EC23 induces keratinocyte hyperproliferation in vitro and in vivo, and when applied to mouse skin increases the number of involucrin-positive suprabasal cell layers. These phenotypic changes are similar in skin treated with the natural retinoid all-trans retinoic acid (ATRA); however, EC23 is more potent; a tenfold lower dose of EC23 is sufficient to induce epidermal thickening, and resulting hyperproliferation is sustained for a longer time period after first dose. EC23 treatment resulted in a disorganized stratum corneum, reduced cell surface lipids and compromised barrier, similar to ATRA treatment. However, EC23 induces a rapid telogen to anagen transition and hair re-growth in 6-week-old mice with synchronously resting back skin follicles. The impact of EC23 on the hair cycle was surprising as similar results have not been seen with ATRA. CONCLUSIONS: These data suggest that synthetic retinoid EC23 is a useful tool in exploring the turnover and differentiation of cells and has a potent effect on skin physiology.

The antibacterial activity of a photoactivatable diarylacetylene against Gram-positive bacteria.

The emergence of antibiotic resistance is a growing threat to human health, and therefore, alternatives to existing compounds are urgently needed. In this context, a novel fluorescent photoactivatable diarylacetylene has been identified and characterised for its antibacterial activity, which preferentially eliminates Gram-positive over Gram-negative bacteria. Experiments confirmed that the Gram-negative lipopolysaccharide-rich outer surface is responsible for tolerance, as strains with reduced outer membrane integrity showed increased susceptibility. Additionally, bacteria deficient in oxidative damage repair pathways also displayed enhanced sensitivity, confirming that reactive oxygen species production is the mechanism of antibacterial activity. This new diarylacetylene shows promise as an antibacterial agent against Gram-positive bacteria that can be activated in situ, potentially for the treatment of skin infections.

Fluorescent Retinoic Acid Analogues as Probes for Biochemical and Intracellular Characterization of Retinoid Signaling Pathways.

Retinoids, such as all- trans-retinoic acid (ATRA), are endogenous signaling molecules derived from vitamin A that influence a variety of cellular processes through mediation of transcription events in the cell nucleus. Because of these wide-ranging and powerful biological activities, retinoids have emerged as therapeutic candidates of enormous potential. However, their use has been limited, to date, due to a lack of understanding of the complex and intricate signaling pathways that they control. We have designed and synthesized a family of synthetic retinoids that exhibit strong, intrinsic, solvatochromatic fluorescence as multifunctional tools to interrogate these important biological activities. We utilized the unique photophysical characteristics of these fluorescent retinoids to develop a novel in vitro fluorometric binding assay to characterize and quantify their binding to their cellular targets, including cellular retinoid binding protein II (CRABPII). The dihydroquinoline retinoid, DC360, exhibited particularly strong binding ( Kd = 34.0 ± 2.5 nM), and we further used X-ray crystallography to determine the structure of the DC360-CRABPII complex to 1.8 Å, which showed that DC360 occupies the known hydrophobic retinoid binding pocket. Finally, we used confocal fluorescence microscopy to image the cellular behavior of the compounds in cultured human epithelial cells, highlighting a fascinating nuclear localization, and used RNA sequencing to confirm that the compounds regulate cellular processes similar to those of ATRA. We anticipate that the unique properties of these fluorescent retinoids can now be used to cast new light on the vital and highly complex retinoid signaling pathway.

Photoactivated cell-killing involving a low molecular weight, donor-acceptor diphenylacetylene.

Photoactivation of photosensitisers can be utilised to elicit the production of ROS, for potential therapeutic applications, including the destruction of diseased tissues and tumours. A novel class of photosensitiser, exemplified by DC324, has been designed possessing a modular, low molecular weight and 'drug-like' structure which is bioavailable and can be photoactivated by UV-A/405 nm or corresponding two-photon absorption of near-IR (800 nm) light, resulting in powerful cytotoxic activity, ostensibly through the production of ROS in a cellular environment. A variety of in vitro cellular assays confirmed ROS formation and in vivo cytotoxic activity was exemplified via irradiation and subsequent targeted destruction of specific areas of a zebrafish embryo.

Quantitative Methods for Measuring Repair Rates and Innate-Immune Cell Responses in Wounded Mouse Skin.

In skin wounds, innate-immune cells clear up tissue debris and microbial contamination, and also secrete cytokines and other growth factors that impact repair process such as re-epithelialization and wound closure. After injury, there is a rapid influx and efflux of immune cells at wound sites, yet the function of each innate cell population in skin repair is still under investigation. Flow cytometry is a valuable research tool for detecting and quantifying immune cells; however, in mouse back skin, the difficulty in extracting immune cells from small area of skin due to tissue complexity has made cytometric analysis an underutilized tool. In this paper, we provide detailed methods on the digestion of lesion-specific skin without disrupting antigen expression followed by multiplex cell staining that allows for identification of seven innate-immune populations, including rare subsets such as group-3 innate lymphoid cells (ILC3s), by flow-cytometry analysis. Furthermore, when studying the functions of immune cells to tissue repair an important metric to monitor is size of the wound opening. Normal wounds close steadily albeit at non-linear rates, while slow or stalled wound closure can indicate an underlying problem with the repair process. Calliper measurements are difficult and time-consuming to obtain and can require repeated sedation of experimental animals. We provide advanced methods for measuring of wound openness; digital 3D image capture and semi-automated image processing that allows for unbiased, reliable measurements that can be taken repeatedly over time.

Epidermal Notch1 recruits RORγ(+) group 3 innate lymphoid cells to orchestrate normal skin repair.

Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ(+) ILC3s into wounded dermis; RORγ(+) ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ(+) ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair.

Assembly and patterning of vertebrate blood vessels.

The mechanisms that regulate blood vessel assembly at appropriate sites in the organism are poorly understood, yet understanding this regulation is critical to the ability to design therapeutics around vessel production in vivo. Classic embryologic studies have yielded descriptive analyses of vascular pattern formation, and they show that angioblasts and endothelial cells respond to environmental cues to assemble at precise embryologic sites. The present study incorporated a genetic model, the mouse, into these studies to obtain mechanistic information regarding vessel assembly and patterning. The data show that both embryo-derived and stem-cell-derived mouse angioblasts respond to host cues in the avian embryo and pattern properly, and also highlight a critical role for the vascular endothelial cell growth factor signaling pathway in these patterning events.

Modified methods for growing 3-D skin equivalents: an update.

Artificial epidermis can be reconstituted in vitro by seeding primary epidermal cells (keratinocytes) onto a supportive substrate and then growing the developing skin equivalent at the air-liquid interface. In vitro skin models are widely used to study skin biology and for industrial drug and cosmetic testing. Here, we describe updated methods for growing 3-dimensional skin equivalents using de-vitalized, de-epidermalized dermis (DED) substrates including methods for DED substrate preparation, cell seeding, growth conditions, and fixation procedures.

Keratinocytes propagated in serum-free, feeder-free culture conditions fail to form stratified epidermis in a reconstituted skin model.

Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification.

Development of the mouse dermal adipose layer occurs independently of subcutaneous adipose tissue and is marked by restricted early expression of FABP4.

The laboratory mouse is a key animal model for studies of adipose biology, metabolism and disease, yet the developmental changes that occur in tissues and cells that become the adipose layer in mouse skin have received little attention. Moreover, the terminology around this adipose body is often confusing, as frequently no distinction is made between adipose tissue within the skin, and so called subcutaneous fat. Here adipocyte development in mouse dorsal skin was investigated from before birth to the end of the first hair follicle growth cycle. Using Oil Red O staining, immunohistochemistry, quantitative RT-PCR and TUNEL staining we confirmed previous observations of a close spatio-temporal link between hair follicle development and the process of adipogenesis. However, unlike previous studies, we observed that the skin adipose layer was created from cells within the lower dermis. By day 16 of embryonic development (e16) the lower dermis was demarcated from the upper dermal layer, and commitment to adipogenesis in the lower dermis was signalled by expression of FABP4, a marker of adipocyte differentiation. In mature mice the skin adipose layer is separated from underlying subcutaneous adipose tissue by the panniculus carnosus. We observed that the skin adipose tissue did not combine or intermix with subcutaneous adipose tissue at any developmental time point. By transplanting skin isolated from e14.5 mice (prior to the start of adipogenesis), under the kidney capsule of adult mice, we showed that skin adipose tissue develops independently and without influence from subcutaneous depots. This study has reinforced the developmental link between hair follicles and skin adipocyte biology. We argue that because skin adipocytes develop from cells within the dermis and independently from subcutaneous adipose tissue, that it is accurately termed dermal adipose tissue and that, in laboratory mice at least, it represents a separate adipose depot.

Expression of Notch pathway genes in mammalian epidermis and modulation by beta-catenin.

The Notch pathway is required for hair follicle maintenance and is activated through beta-catenin induced transcription of the Notch ligand Jagged1. We show that hair follicles in the resting phase express low levels of Jagged1 and Hes1, and other Notch target genes are undetectable. In growing (anagen) follicles, Jagged1 and Hes1 expression increases, Hes5 and HeyL are expressed in distinct cell layers, and Hey2 is expressed in the dermal papilla. When beta-catenin is activated by means of an inducible transgene, Jagged1, Hes1, Hes5, HeyL, and Hey2 are up-regulated, the sites of expression being the same in beta-catenin induced ectopic follicles as in anagen follicles. beta-Catenin also induces Hey1 in dermal papilla cells. beta-Catenin-induced up-regulation of Jagged1 precedes induction of other Notch target genes. The different sites of expression of Hes and Hey genes suggest input from additional signaling pathways.

Jagged 1 is a beta-catenin target gene required for ectopic hair follicle formation in adult epidermis.

The Wnt and Notch signalling pathways regulate hair follicle maintenance, but how they intersect is unknown. We show that Notch signalling is active in the hair follicle pre-cortex, a region of high Wnt activity, where commitment to hair lineages occurs. Deletion of jagged 1 (Jag1) results in inhibition of the hair growth cycle and conversion of hair follicles into cysts of cells undergoing interfollicular epidermal differentiation. Conversely, activation of Notch in adult epidermis triggers expansion of the base of the hair follicle, sebaceous gland enlargement and abnormal clumping of the follicles. In adult epidermis, the induction of new hair follicle formation by beta-catenin is prevented by blocking Notch signalling pharmacologically or through Jag1 deletion. Conversely, activation of both pathways accelerates growth and differentiation of ectopic follicles. beta-catenin stimulates Notch signalling by inducing Jag1 transcription. We conclude that the Notch pathway acts downstream of the Wnt/beta-catenin pathway to determine epidermal cell fate.