Single Epidermal Cells Research Articles

Epidermal and dermal cells from adult rat eccrine sweat gland-containing skin can reconstruct the three-dimensional structure of eccrine sweat glands

BackgroundPreviously, we have demonstrated that eccrine sweat gland cells (ESGCs) can reconstruct the three-dimensional (3D) structure of eccrine sweat glands (ESGs). However, there is still a need to explore source cells capable of regenerating ESG to address the issue of ESG regeneration in ESGC-deficient conditions, such as severe burns. MethodsThe epidermal cells and dermal cells in adult rat ventral foot skin (ESG-bearing) were isolated. The isolated single epidermal cells and dermal cells were mixed with Matrigel, and then the mixture was implanted into the axillary/inguinal fat pads of nude mice. Five weeks after implantation, the Matrigel plugs were harvested and the morphology and differentiation of the cells were examined by H&E staining and fluorescent immunohistochemical staining for ESG markers, such as Na+ -K+ -2Cl- cotransporter 1 (NKCC1), Na+ -K+ -ATPase (NKA), Foxa1 and K14. ResultsThe epidermal cells and dermal cells of adult rat ventral foot skin can reconstruct 3D structure and express specific markers of ESGs in skin, such as NKCC1, NKA and Foxa1, indicating the ESG-phenotypic differentiation of the 3D structures. Double immunofluorescence staining showed that some 3D structures expressed both the myoepithelial cell marker alpha-SMA and the common marker K14 of duct cells and myoepithelial cells, while some 3D structures expressed only K14, indicating that ESG-like 3D structures differentiated into duct-like and secretory coiled cells. ConclusionEpidermal and dermal cells from adult ESG-bearing skin can be used as a cell source for ESG regeneration.

Alternative Splicing during Fiber Development in G. hirsutum.

Cotton is a valuable cash crop in many countries. Cotton fiber is a trichome that develops from a single epidermal cell and serves as an excellent model for understanding cell differentiation and other life processes. Alternative splicing (AS) of genes is a common post-transcriptional regulatory process in plants that is essential for plant growth and development. The process of AS during cotton fiber formation, on the other hand, is mainly unknown. A substantial number of multi-exon genes were discovered to be alternatively spliced during cotton fiber formation in this study, accounting for 23.31% of the total number of genes in Gossypium hirsutum. Retention intron (RI) is not necessarily the most common AS type, indicating that AS genes and processes during fiber development are very temporal and tissue-specific. When compared to fiber samples, AS is more prevalent at the fiber initiation stages and in the ovule, indicating that development stages and tissues use different AS strategies. Genes involved in fiber development have gone through stage-specific AS, demonstrating that AS regulates cotton fiber development. Furthermore, AS can be regulated by trans-regulation elements such as splicing factor and cis-regulation elements such as gene length, exon numbers, and GC content, particularly at exon-intron junction sites. Our findings also suggest that increased DNA methylation may aid in the efficiency of AS, and that gene body methylation is key in AS control. Finally, our research will provide useful information about the roles of AS during the cotton fiber development process.

Co-option of epidermal cells enables touch sensing

The epidermis is equipped with specialized mechanosensory organs that enable the detection of tactile stimuli. Here, by examining the differentiation of the tactile bristles, mechanosensory organs decorating the Drosophila adult epidermis, we show that neighbouring epidermal cells are essential for touch perception. Each mechanosensory bristle signals to the surrounding epidermis to co-opt a single epidermal cell, which we named the F-Cell. Once specified, the F-Cell adopts a specialized morphology to ensheath each bristle. Functional assays reveal that adult mechanosensory bristles require association with the epidermal F-Cell for touch sensing. Our findings underscore the importance of resident epidermal cells in the assembly of functional touch-sensitive organs.

Observation of the development of leaf vein and stomata and identification candidate transcription factors related to vein/stoma development in grapevine leaf (Vitis vinifera L.)

The leaf functions through photosynthesis and determines the yield and quality of the grape (Vitis vinifera L.). Understanding the morphology, anatomy, and molecular regulation of leaf development can provide insight into the function of the leaf. In the present study, the microstructure of the vein and stomata was observed using an improved tissue clearing method, and differently expressed transcription factors (TFs) underlying leaf development were determined by transcriptome analysis in the ‘Pinot Noir’ grape. The process of stomata development from a single epidermal cell to a mature stomata cell was observed clearly under light microscopy by improving the method of tissue clearing. The onset of stomata formation in the leaf tip occurred prior to that in the central leaf, and developing stomata were observed in the center of the leaves at different development stages except for L5, when only mature stomata were present. The major vein was initiated before leaf unfolding (L1) and numbers of vessels increased underlying leaf growth. The shape of the vessel presented tapering. Freely ending veins were not observed until stages L4 and L5, and the vessels were different from other order veins in shape and ways of connection. Development-dependent differences between stomata and vein suggested differences in function during leaf development. A total of 445 TFs involved in leaf morphology and anatomy were identified. The members of the bHLH family (SCRM) and MYB family (MYB88) can contribute to stomata patterning during leaf development. Members of the TF families of ARF (ARF5), Dof (TMO6 and HCA2), HD-Zip III (HB14), and GRAS (SCR, SCL14, and SCL28) were potential regulators involved in vein development. The grape orthologs of WOX9, TCP4, and GRF1-9 can participate in leaf outgrowth and expansion. These results will be helpful for our further study of the molecular regulation of leaf development and the relation between leaf structure and function, which can be applied to improve grape production.

Microscope studies of symptomless growth of Botrytis cinerea in Lactuca sativa and Arabidopsis thaliana.

The grey mould pathogen Botrytis cinerea forms systemic associations in some hosts, spreading into plant organs produced a considerable time after initial infection. These infections may have no macroscopic symptoms during much of the hosts' lifetime and are at least partially within the host tissue. The aim of the studies reported here was to locate and visualize these infections at a cellular level in Lactuca sativa (lettuce) and Arabidopsis thaliana. Symptomless but infected plants were produced by dry spore inoculation of plants growing in conditions previously shown to result in fungal spread from the initial inoculation site to newly developing plant organs. Tissue taken from inoculated plants was examined using confocal laser scanning microscopy. Two B. cinerea isolates were used: B05.10 and its GFP-labelled derivative Bcgfp1-3. Spore germination on leaf surfaces was followed by development of subcuticular inclusions and plant cell damage in single infected epidermal cells and sometimes a few nearby cells. Sparsely branched long hyphae arose and spread from the inclusions, mostly on the outer surface of the epidermal layer but occasionally below the cuticle or epidermal cells, where further inclusions formed. This was consistent with the pattern in time of recovery of B. cinerea from surface-sterilized leaf tissue. In the late symptomless phase, mycelium arising from internal fungal inclusions formed mycelial networks on the surface of leaves. Symptomless exterior mycelium grew on the roots in A. thaliana.

Three root hair defective genes, GhRHD3-1, GhRHD4-1, and GhRSL4-1, regulate fiber cell elongation in cotton

Cotton fibers, single epidermal cells, are most prevalent natural product in textile industries. Root hairs, similar to cotton fibers, are also single cell trichomes. Root hair defective gene (RHD) has been found to greatly modulate root hair development. However, the function of RHD remains unexplored in cotton. We identified 19 RHDs in Gossypium hirsutum. GhRHD3–1, GhRHD4–1, and GhRSL4–1 were highly expressed in fiber elongation stage and promoter-driven GUS of these three genes were mainly expressed in root hair. GhRHD3–1, GhRHD4–1, and GhRSL4–1 localized to the endoplasmic reticulum, cytomembrane, and nucleus, respectively. Overexpression of these three genes each in Arabidopsis increased root length. Silencing these three genes each in cotton reduced plant height and fiber length. Gene Ontology (GO) analysis showed that cell wall, photosynthesis, and oxidoreductase activity related processes were enriched in down-regulated genes after silencing GhRHD3–1, GhRHD4–1, and GhRSL4–1, respectively. Transcripts of the genes identified from enriched terms were also reduced after silencing GhRHD3–1, GhRHD4–1, and GhRSL4–1. Our results suggest that GhRHD3–1, GhRHD4–1, and GhRSL4–1 promote fiber elongation through regulating cell wall, photosynthesis, and oxidoreductase activity pathways, respectively. This study deepens our understanding of the GhRHDs in cotton and will help us breed better cotton varieties in future.

Arabidopsis thaliana CYCLIC NUCLEOTIDE-GATED CHANNEL2 mediates extracellular ATP signal transduction in root epidermis.

Summary Damage can be signalled by extracellular ATP (eATP) using plasma membrane (PM) receptors to effect cytosolic free calcium ion ([Ca2+]cyt) increase as a second messenger. The downstream PM Ca2+ channels remain enigmatic. Here, the Arabidopsis thaliana Ca2+ channel subunit CYCLIC NUCLEOTIDE‐GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca2+]cyt signalling in roots.Extracellular ATP‐induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root [Ca2+]cyt were measured with aequorin, and root transcriptional changes were determined by quantitative real‐time PCR. Two cngc2 loss‐of‐function mutants were used: cngc2‐3 and defence not death1 (which expresses cytosolic aequorin).Extracellular ATP‐induced transient depolarization of Arabidopsis root elongation zone epidermal PM voltage was Ca2+ dependent, requiring CNGC2 but not CNGC4 (its channel co‐subunit in immunity signalling). Activation of PM Ca2+ influx currents also required CNGC2. The eATP‐induced [Ca2+]cyt increase and transcriptional response in cngc2 roots were significantly impaired.CYCLIC NUCLEOTIDE‐GATED CHANNEL2 is required for eATP‐induced epidermal Ca2+ influx, causing depolarization leading to [Ca2+]cyt increase and damage‐related transcriptional response.

Imaging of I, Re and Tc plant uptake on the single-cell scale using SIMS and rL-SNMS

In radioecological studies, there is a significant need for understanding the plant uptake of radionuclides on a cellular level. The present work applies mass spectrometry to image the radionuclide distribution within the cellular structures of plants at varying concentrations. In a first step, plants of Daucus carota and Pisum sativum labelled with iodine and rhenium were examined, at concentrations in the range of 10 mM. Cross sections of several plant parts were imaged by secondary ion mass spectrometry (SIMS) after cryogenation in order to preserve cell structure. In a second step, the distribution of 99Tc in the two plant species was determined. For radiological reasons, a concentration three orders of magnitude lower was used, rendering measurements with SIMS impossible. Therefore, resonant laser secondary neutral mass spectrometry (rL-SNMS) was used for the first time to image 99Tc with suppression of molecular isobaric interferences. The measurement of only about 1010 atoms of 99Tc atoms is demonstrated and the distribution of 99Tc within a single epidermal cell is imaged.

Different potato virus Y strains frequently co-localize in single epidermal leaf cells and in the aphid stylet.

Single aphids can simultaneously or sequentially acquire and transmit multiple potato virus Y (PVY) strains. Multiple PVY strains are often found in the same field and occasionally within the same plant, but little is known about how PVY strains interact in plants or in aphid stylets. Immuno-staining and confocal microscopy were used to examine the spatial and temporal dynamics of PVY strain mixtures (PVYO and PVYNTN or PVYO and PVYN) in epidermal leaf cells of 'Samsun NN' tobacco and 'Goldrush' potato. Virus binding and localization was also examined in aphid stylets following acquisition. Both strains systemically infected tobacco and co-localized in cells of all leaves examined; however, the relative amounts of each virus changed over time. Early in the tobacco infection, when mosaic symptoms were observed, PVYO dominated the infection although PVYNTN was detected in some cells. As the infection progressed and vein necrosis developed, PVYNTN was prevalent. Co-localization of PVYO and PVYN was also observed in epidermal cells of potato leaves with most cells infected with both viruses. Furthermore, two strains could be detected binding to the distal end of aphid stylets following virus acquisition from a plant infected with a strain mixture. These data are in contrast with the traditional belief of spatial separation of two closely related potyviruses and suggest apparent non-antagonistic interaction between PVY strains that could help explain the multitude of emerging recombinant PVY strains discovered in potato in recent years.

Interplay of opposing fate choices stalls oncogenic growth in murine skin epithelium.

Skin epithelium can accumulate a high burden of oncogenic mutations without morphological or functional consequences. To investigate the mechanism of oncogenic tolerance, we induced HrasG12V in single murine epidermal cells and followed them long term. We observed that HrasG12V promotes an early and transient clonal expansion driven by increased progenitor renewal that is replaced with an increase in progenitor differentiation leading to reduced growth. We attribute this dynamic effect to emergence of two populations within oncogenic clones: renewing progenitors along the edge and differentiating ones within the central core. As clone expansion is accompanied by progressive enlargement of the core and diminishment of the edge compartment, the intraclonal competition between the two populations results in stabilized oncogenic growth. To identify the molecular mechanism of HrasG12V-driven differentiation, we screened known Ras-effector in vivo and identified Rassf5 as a novel regulator of progenitor fate choice that is necessary and sufficient for oncogene-specific differentiation.

Genetic mapping and transcriptomic characterization of a new fuzzless-tufted cottonseed mutant.

Fiber mutants are unique and valuable resources for understanding the genetic and molecular mechanisms controlling initiation and development of cotton fibers that are extremely elongated single epidermal cells protruding from the seed coat of cottonseeds. In this study, we reported a new fuzzless-tufted cotton mutant (Gossypium hirsutum) and showed that fuzzless-tufted near-isogenic lines (NILs) had similar agronomic traits and a higher ginning efficiency compared to their recurrent parents with normal fuzzy seeds. Genetic analysis revealed that the mutant phenotype is determined by a single incomplete dominant locus, designated N5. The mutation was fine mapped to an approximately 250-kb interval containing 33 annotated genes using a combination of bulked segregant sequencing, SNP chip genotyping, and fine mapping. Comparative transcriptomic analysis using 0–6 days post-anthesis (dpa) ovules from NILs segregating for the phenotypes of fuzzless-tufted (mutant) and normal fuzzy cottonseeds (wild-type) uncovered candidate genes responsible for the mutant phenotype. It also revealed that the flanking region of the N5 locus is enriched with differentially expressed genes (DEGs) between the mutant and wild-type. Several of those DEGs are members of the gene families with demonstrated roles in cell initiation and elongation, such as calcium-dependent protein kinase and expansin. The transcriptome landscape of the mutant was significantly reprogrammed in the 6 dpa ovules and, to a less extent, in the 0 dpa ovules, but not in the 2 and 4 dpa ovules. At both 0 and 6 dpa, the reprogrammed mutant transcriptome was mainly associated with cell wall modifications and transmembrane transportation, while transcription factor activity was significantly altered in the 6 dpa mutant ovules. These results imply a similar molecular basis for initiation of lint and fuzz fibers despite certain differences.

Symplasmic Isolation Contributes to Somatic Embryo Induction and Development in the Tree Fern Cyathea delgadii Sternb.

In this report, we describe studies on symplasmic communication and cellular rearrangement during direct somatic embryogenesis (SE) in the tree fern Cyathea delgadii. We analyzed changes in the symplasmic transport of low-molecular-weight fluorochromes, such as 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) and fluorescein (delivered to cells as fluorescein diacetate, FDA), within stipe explants and somatic embryos originating from single epidermal cells and developing during 16-d long culture. Induction of SE is preceded by a restriction in fluorochrome distribution between certain explant cells. Microscopic analysis showed a series of cellular changes like a decrease in vacuole size, increase in vacuole numbers, and increased density of cytoplasm and deposition of electron-dense material in cell walls that may be related with embryogenic transition. In somatic embryos, the limited symplasmic communication between cells was observed first in linear tri-cellular embryos. Further development of the fern embryo was associated with the formation of symplasmic domains corresponding to the four segments of the plant body. Using symplasmic tracers, we provided evidence that the changes in plasmodesmata permeability are corelated with somatic-to-embryogenic transition and somatic embryo development.

231 Novel protocol for the invitro maintenance and expansion of adult epidermal LGR5+ stem cells

Currently, the standard for treatment of full-thickness skin wounds is skin grafts or bioengineered skin substitutes; however, this method is limited by the amount of intact donor skin and lack of follicles and architecture. Thus, a protocol is needed for the expansion and differentiation of adult epidermal and hair follicle stem cells for use in scaffold mediated tissue engineering. Recently, we developed a transgenic porcine model in which H2B-GFP is under the control of the LGR5 promoter. LGR5 is an established marker of stem cells, meaning this model can be used to track the development and behaviour of these cells. The focus of this project was to create a novel culture method for the maintenance and expansion of LGR5+ epidermal adult stem cells utilising the green fluorescent protein (GFP) tag. Single cell epidermal stem cells were isolated from porcine skin using dispase II (10mgmL−1; Sigma) and trypsin (0.05%; Corning). Porcine fetal fibroblasts (PFF) or mouse embryonic fibroblasts (MEF) were grown to 95% confluence in a 6-well plate. Feeder layer cells were mitotically inactivated by incubation with mitomycin C (Sigma Aldrich, 10μgmL−1). Three different media were tested: basal medium [Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, Corning; Ham's F12, ThermoFisher; fetal bovine serum, Gemini Bio-Products], basal media + 5-azacytidine (Sigma Aldrich) and CHIR99021 (Tocris), or basal media + keratinocyte growth supplements (transferrin, hydrocortisone, T3, adenine, insulin, cholera toxin; Sigma Aldrich, epidermal growth factor; R&D Systems). Epidermal cells were plated in each medium for both PFF and MEF feeder layers. Experiments were performed in technical duplicates and replicated 3 times. On Day 9, total numbers of colonies in each well were counted and number of GFP-positive cells were quantified using ImageJ (National Institutes of Health). Results in Table 1 show that overall, the MEF feeder layer was able to support a higher rate of growth (P<0.05) and maintain the LGR5+ lineage at a higher proportion under all of the experimental conditions (P<0.05). In the growth-supplemented media, MEFs had fewer colonies than PFFs, but MEF colonies were, on average, 2.5 times larger (P<0.05). Conditions containing 5-aza and CHIR were the only conditions to maintain the LGR5+ lineage on the feeder layer. Statistically significant differences (P<0.05) were determined using two-way ANOVA, followed by Tukey's HSD test. Next, LGR5+ cells will be plated in media containing additional growth factors to stimulate expansion, while using CHIR and 5-aza to maintain the LGR5+ lineage. This protocol could be used in scaffolds to create three-dimensional growth of skin invitro and lead to better grafts for burn victims. Table 1.Growth of LGR5+ cells in different media including 5-azacytidine (5-aza), CHIR 99021 (CHIR), and keratinocyte growth supplements Group1 Basal medium (BM) BM + 5-aza+ CHIR BM + growth supplements No. of colonies/well MEF 127.7±40.8AB 189.3±16.9A 87.3±14.6B PFF 65.0±14.1A 83.3±17.0AB 148±33.7B Average no. of GFP+ cells per frame MEF 0.5±0.8B 65.7±18.4A 1.8±1.7B PFF 0.9±1.0B 22.6±4.5A 0.3±0.6B A,BValues within rows with different superscripts differ (P ≤ 0.05). 1MEF=mouse embryonic fibroblasts; PFF=porcine fetal fibroblasts.

Anchoring of greenhouse whitefly eggs on different rose cultivars

Whiteflies attach their eggs to plants by implanting the egg pedicel into the epidermis of the underside of leaves. This pedicel works like a wall plug embedded in sealing cement, presenting a smart interconnection, which was exemplarily studied in Trialeurodes vaporariorum eggs on two stages of abaxial leaflets of four cut rose cultivars using a combination of microscopic and biomechanical techniques. The penetration force obtained by piercing single epidermal cells with an insect minuten pin did not significantly correlate with the force which was applied to pull off the greenhouse whitefly eggs from abaxial leaves. A maximum pull-off force of 5.4 mN was measured on young leaves of the rose cultivar ‘Schloss Ippenburg®’, corresponding to maximum 941 times the egg weight. Egg pull-off force significantly differed between cut rose cultivars and leaf ages. On greenhouse whitefly-susceptible cultivars ‘Poesie®’ and ‘Reggae®’, eggs detached, applying less force compared to that on resistant cultivars. Leaf structural features had no significant impact on greenhouse whitefly egg pull-off forces. A major effect of leaf turgor pressure and swelling of colleterial gland secretion (cement) surrounding the whitefly egg is assumed to facilitate the firm interconnection between egg and plant epidermis by a combination of form closure, friction locking, and adhesive bond forming a composite material in the contact region. This bond exhibits a maximum adhesive strength of 12.2 MPa, which is much higher than those in beetle and moth eggs glued to oviposition substrates.

More Than the Sum of Its Parts: Single-Cell Transcriptomics Reveals Epidermal Cell States

Compared to mouse models, less is known about human epidermal cell states and differentiation. In this issue of Cell Reports, Cheng etal. (2018) dissect the cell states and heterogeneity in human epidermis with large-scale transcriptomics of 92,889 single epidermal cells from normal and inflamed skin.

Development, structure and evolutionary significance of seed appendages in Salix matsudana (Salicaceae).

The seeds of Salix and Populus (Salicaceae) are characterized by having numerous long hairs which loosely accompanying the seeds and a small annular appendage which surrounding the base of the seed along with tufted hairs. In this study, the complete development and detailed structure of the hairs and annular appendage in Salix matsudana were investigated using standard techniques for plant anatomy and histochemistry. The results show that the hairs originate successively from the single epidermal cells of the placenta (in megaspore mother cell phase) and funiculus (in eight-nucleate phase), and that their development consists of a progressive increase in cell size and an absence of cell division. The annular appendage is initiated from four to five rows of cells at the distal end of the funiculus in octant proembryo phase and its development is characterized by reactivated meristematic activity and a size increase of these cells. The initiation and development of the hairs are irrelevant to ovule development but fertilization and a developed embryo is necessary for the annular appendage to occur. Considering the reliable fossils, we inferred that the feature of seeds surrounded by long hairs is an ancestral character, and that the detachment of hairs from the funiculus and the occurrence of an annular appendage with tufts of hairs may be the more derived states for seed dispersal in Salix and Populus.

1441 3D cell culture system as a model to assess clonal growth and spontaneous differentiation of human epidermal keratinocytes

Regeneration of human epidermis after injury is a key function of progenitor cells. Understanding of decision-making mechanisms for self-renewal and differentiation relies on the development of experimental approaches, including surrogate cell culture-based epidermal regeneration models for assessment of progenitor cell activation, autonomous growth and spontaneous differentiation at the clonal level. Here we propose a 3D epidermal cell culture system which satisfies aforementioned criteria. Growth from single epidermal cells in thick basal membrane-derived gel results in the formation of multilayered spheroids with an inward differentiation. Unlike classical human epidermal culture systems in which differentiation is induced experimentally by stress stimuli, differentiation in 3D is spontaneous, implying in vivo-like context. As such, 3D culture conditions allowed us to evaluate the behavior of primary human keratinocytes and commercially available keratinocyte-derived cell line N/TERT1 (in which telomerase is overexpressed), as well as lentiviral-infected N/TERT1-derivatives, produced in the lab. It is generally assumed that immortalization by telomerase does not interfere with differentiation and hence N/TERT1 is considered a reliable tool for epidermal studies. However, N/TERT1-derived spheroids displayed marked changes in their shape and in the content of Involucrin+ cells, suggesting that telomerase delayed differentiation. This was accompanied by an increased Ki67 index and expansion of K14+/p63+ cell subset. Furthermore, we observed modest disorganization of epidermal tight and adherence junctions, while epithelial polarity and stratification were preserved. Strikingly, secondary infection of N/TERT1 cells with “empty” lentivirus resulted in disruption of epithelial polarity, loss of ability for stratification and absence of Involucrin+ cells. Overall, we suggest a novel experimental approach for assessment of epidermal cell fate regulation and polarity. This approach might be also feasible for the elucidation of mechanisms driving epidermal cell transformation and oncogenesis.

Non-invasive metabolic imaging of melanoma progression.

Skin cancer is associated with abnormal cellular metabolism which if identified early introduces the possibility of intervention to prevent its progress to a deadly metastatic stage. This study combines multiphoton microscopy with fluorescence lifetime imaging (FLIM) using a syngeneic melanoma mouse model, to detect changes in metabolic state of single epidermal cells as a metabolic marker to monitor the progress of tumor growth. This method utilizes imaging of the ratio of the amounts of the free and protein-bound forms of the intracellular autofluorescent metabolic co-enzyme nicotinamide adenine dinucleotide (NADH). Here, we investigate the impact of the primary tumor lesion on the epidermal layers at three different growth stages of melanoma lesion compared to normal skin as a control. We showed a significant increase in the free-to-bound NADH ratio with the growth of the solid melanoma tumor, while concurrently the short and the long lifetime components of NADH remained constant. These results demonstrate the ability of FLIM for rapid, non-invasive and sensitive assessment of melanoma progression revealing its potential as a diagnostic tool for melanoma detection and as an aid for melanoma staging.

The content of endogenous hormones and sugars in the process of early somatic embryogenesis in the tree fern Cyathea delgadii Sternb.

Somatic embryogenesis (SE) of Cyathea delgadii presents a model system for investigating the mechanisms associated with the acquisition of embryogenic competence by single epidermal cells of stipe explants cultured on plant growth regulator-free medium. The present work reveals relationship between endogenous hormone and sugar content in the process of early SE in C. delgadii. By comparing two types of initial explants, i.e. incapable (non-etiolated) and capable (etiolated) of SE, it was established that in etiolated explants, the glucose, fructose, sucrose, and abscisic acid (ABA) contents diminished, but indole-3-acetic acid (IAA) and cytokinins (CKs; i.e. cis/trans zeatin, cis/trans-zeatin riboside, kinetin, kinetin riboside, isopentenyladenosine) contents increased. The ratios between phytohormones revealed that a high concentration of ABA is the main factor inhibiting SE induction. Because of explant excision, a dramatic reduction in concentration of all phytohormones studied was observed, but hormonal balance and sugar content remained almost unchanged. During the 14-day-long culture, the ABA/CKs and ABA/IAA ratios remained constant, whereas the greatest differences were detected for the IAA/CKs and Z-type/iPA cytokinin ratios. Excluding day 6 of culture, cytokinins were found to be the predominant phytohormones over IAA. An almost 12-fold increase in soluble sucrose concentration at day 6 of culture might be the switch to the SE expression phase. Frequent cell divisions leading to somatic embryo formation are clearly associated with increase in trans-zeatin riboside content.

RSL Class I Genes Controlled the Development of Epidermal Structures in the Common Ancestor of Land Plants

SummaryThe colonization of the land by plants, sometime before 470 million years ago, was accompanied by the evolution tissue systems [1, 2, 3]. Specialized structures with diverse functions—from nutrient acquisition to reproduction—derived from single cells in the outermost layer (epidermis) were important sources of morphological innovation at this time [2, 4, 5]. In extant plants, these structures may be unicellular extensions, such as root hairs or rhizoids [6, 7, 8, 9], or multicellular structures, such as asexual propagules or secretory hairs (papillae) [10, 11, 12]. Here, we show that a ROOTHAIR DEFECTIVE SIX-LIKE (RSL) class I basic helix-loop-helix transcription factor positively regulates the development of the unicellular and multicellular structures that develop from individual cells that expand out of the epidermal plane of the liverwort Marchantia polymorpha; mutants that lack MpRSL1 function do not develop rhizoids, slime papillae, mucilage papillae, or gemmae. Furthermore, we discovered that RSL class I genes are also required for the development of multicellular axillary hairs on the gametophyte of the moss Physcomitrella patens. Because class I RSL proteins also control the development of rhizoids in mosses and root hairs in angiosperms [13, 14], these data demonstrate that the function of RSL class I genes was to control the development of structures derived from single epidermal cells in the common ancestor of the land plants. Class I RSL genes therefore controlled the generation of adaptive morphological diversity as plants colonized the land from the water.