The Secreted Metalloprotease ADAMTS20 Is Required for Melanoblast Survival

ADAMTS20 (A disintegrin-like and metalloprotease domain with thrombospondin type-1 motifs) is a member of a family of secreted metalloproteases that can process a variety of extracellular matrix (ECM) components and secreted molecules. Adamts20 mutations in belted (bt) mice cause white spotting of the dorsal and ventral torso, indicative of defective neural crest (NC)-derived melanoblast development. The expression pattern of Adamts20 in dermal mesenchymal cells adjacent to migrating melanoblasts led us to initially propose that Adamts20 regulated melanoblast migration. However, using a Dct-LacZ transgene to track melanoblast development, we determined that melanoblasts were distributed normally in whole mount E12.5 bt/bt embryos, but were specifically reduced in the trunk of E13.5 bt/bt embryos due to a seven-fold higher rate of apoptosis. The melanoblast defect was exacerbated in newborn skin and embryos from bt/bt animals that were also haploinsufficient for Adamts9, a close homolog of Adamts20, indicating that these metalloproteases functionally overlap in melanoblast development. We identified two potential mechanisms by which Adamts20 may regulate melanoblast survival. First, skin explant cultures demonstrated that Adamts20 was required for melanoblasts to respond to soluble Kit ligand (sKitl). In support of this requirement, bt/bt;Kittm1Alf/+ and bt/bt;KitlSl/+ mice exhibited synergistically increased spotting. Second, ADAMTS20 cleaved the aggregating proteoglycan versican in vitro and was necessary for versican processing in vivo, raising the possibility that versican can participate in melanoblast development. These findings reveal previously unrecognized roles for Adamts proteases in cell survival and in mediating Kit signaling during melanoblast colonization of the skin. Our results have implications not only for understanding mechanisms of NC-derived melanoblast development but also provide insights on novel biological functions of secreted metalloproteases

A change in the optical polarization associated with a gamma-ray flare in the blazar 3C 279

It is widely accepted that strong and variable radiation detected over all accessible energy bands in a number of active galaxies arises from a relativistic, Doppler-boosted jet pointing close to our line of sight. The size of the emitting zone and the location of this region relative to the central supermassive black hole are, however, poorly known, with estimates ranging from light-hours to a light-year or more. Here we report the coincidence of a gamma-ray flare with a dramatic change of optical polarization angle. This provides evidence for co-spatiality of optical and gamma-ray emission regions and indicates a highly ordered jet magnetic field. The results also require a non-axisymmetric structure of the emission zone, implying a curved trajectory for the emitting material within the jet, with the dissipation region located at a considerable distance from the black hole, at about 10^5 gravitational radii.Comment: Published in Nature issued on 18 February 2010. Corresponding authors: Masaaki Hayashida and Greg Madejsk

In Vitro Dedifferentiation of Melanocytes from Adult Epidermis

In previous work we described a novel culture technique using a cholera toxin and PMA-free medium (Mel-mix) for obtaining pure melanocyte cultures from human adult epidermis. In Mel-mix medium the cultured melanocytes are bipolar, unpigmented and highly proliferative. Further characterization of the cultured melanocytes revealed the disappearance of c-Kit and TRP-1 and induction of nestin expression, indicating that melanocytes dedifferentiated in this in vitro culture. Cholera toxin and PMA were able to induce c-Kit and TRP-1 protein expressions in the cells, reversing dedifferentiation. TRP-1 mRNA expression was induced in dedifferentiated melanocytes by UV-B irradiated keratinocyte supernatants, however direct UV-B irradiation of the cells resulted in further decrease of TRP-1 mRNA expression. These dedifferentiated, easily accessible cultured melanocytes provide a good model for studying melanocyte differentiation and possibly transdifferentiation. Because melanocytes in Mel-mix medium can be cultured with human serum as the only supplement, this culture system is also suitable for autologous cell transplantation

Zebrafish Endzone Regulates Neural Crest-Derived Chromatophore Differentiation and Morphology

The development of neural crest-derived pigment cells has been studied extensively as a model for cellular differentiation, disease and environmental adaptation. Neural crest-derived chromatophores in the zebrafish (Danio rerio) consist of three types: melanophores, xanthophores and iridiphores. We have identified the zebrafish mutant endzone (enz), that was isolated in a screen for mutants with neural crest development phenotypes, based on an abnormal melanophore pattern. We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced. Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size. We demonstrate that enz function is required cell autonomously by melanophores and that the enz locus is located on chromosome 7. In addition, zebrafish enz appears to selectively regulate chromatophore development within the neural crest lineage since all other major derivatives develop normally. Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology. Thus, although developmental regulation of different chromatophore sublineages in zebrafish is in part genetically distinct, enz provides an example of a common regulator of neural crest-derived chromatophore differentiation and morphology

Expression of SCF splice variants in human melanocytes and melanoma cell lines: potential prognostic implications

Stem cell factor (SCF), the ligand for c-Kit, is known to regulate developmental and functional processes of haematopoietic stem cells, mast cells and melanocytes. Two different splice variants form predominantly soluble (sSCF or SCF-1) and in addition some membrane-bound SCF (mSCF or SCF-2). In order to explore the prognostic significance of these molecules in melanoma, total SCF, SCF splice variants and c-Kit expression were studied in normal skin melanocytes and in 11 different melanoma cell lines, using reverse transcription polymerase chain reaction, immunocytochemistry and enzyme-linked immunosorbent assay. Nine of the 11 melanoma cell lines expressed SCF-1 mRNA, only two of them SCF-2, and these two also SCF-1. Coexpression of both SCF-1 and c-Kit was noted in five cell lines, and only one cell line as well as normal melanocytes expressed both SCF-1 and SCF-2 as well as c-Kit. Corresponding results were obtained on immunocytochemical staining. Of three exemplary melanoma cell lines studied, two expressing SCF mRNA also released SCF spontaneously and on stimulation, whereas the line lacking SCF and c-kit mRNA (SK-Mel-23) failed to do so. These data demonstrate thus that melanoma cell lines, particularly those known to metastasize in vivo, lose the ability to express SCF-2 mRNA, suggesting that this molecule may serve, next to c-Kit, as a prognostic marker for malignant melanoma. © 2000 Cancer Research Campaig

Skipping of Exons by Premature Termination of Transcription and Alternative Splicing within Intron-5 of the Sheep SCF Gene: A Novel Splice Variant

Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (−) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as ‘soluble’ isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a ‘novel’ mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D175G site which is necessary to produce ‘soluble’ form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6–9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (−) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals

Multiband variability studies and novel broadband SED modeling of Mrk 501 in 2009

Aims. We present an extensive study of the BL Lac object Mrk 501 based on a data set collected during the multi-instrument campaign spanning from 2009 March 15 to 2009 August 1, which includes, among other instruments, MAGIC, VERITAS, Whipple 10 m, and Fermi-LAT to cover the gamma-ray range from 0.1 GeV to 20 TeV; RXTE and Swift to cover wavelengths from UV to hard X-rays; and GASP-WEBT, which provides coverage of radio and optical wavelengths. Optical polarization measurements were provided for a fraction of the campaign by the Steward and St. Petersburg observatories. We evaluate the variability of the source and interband correlations, the gamma-ray flaring activity occurring in May 2009, and interpret the results within two synchrotron self-Compton (SSC) scenarios.Methods. The multiband variability observed during the full campaign is addressed in terms of the fractional variability, and the possible correlations are studied by calculating the discrete correlation function for each pair of energy bands where the significance was evaluated with dedicated Monte Carlo simulations. The space of SSC model parameters is probed following a dedicated grid-scan strategy, allowing for a wide range of models to be tested and offering a study of the degeneracy of model-to-data agreement in the individual model parameters, hence providing a less biased interpretation than the "single-curve SSC model adjustment" typically reported in the literature.Results. We find an increase in the fractional variability with energy, while no significant interband correlations of flux changes are found on the basis of the acquired data set. The SSC model grid-scan shows that the flaring activity around May 22 cannot be modeled adequately with a one-zone SSC scenario (using an electron energy distribution with two breaks), while it can be suitably described within a two (independent) zone SSC scenario. Here, one zone is responsible for the quiescent emission from the averaged 4.5-month observing period, while the other one, which is spatially separated from the first, dominates the flaring emission occurring at X-rays and very-high-energy (> 100 GeV, VHE) gamma-rays. The flaring activity from May 1, which coincides with a rotation of the electric vector polarization angle (EVPA), cannot be satisfactorily reproduced by either a one-zone or a two-independent-zone SSC model, yet this is partially affected by the lack of strictly simultaneous observations and the presence of large flux changes on sub-hour timescales (detected at VHE gamma rays).Conclusions. The higher variability in the VHE emission and lack of correlation with the X-ray emission indicate that, at least during the 4.5-month observing campaign in 2009, the highest energy (and most variable) electrons that are responsible for the VHE gamma rays do not make a dominant contribution to the similar to 1 keV emission. Alternatively, there could be a very variable component contributing to the VHE gamma-ray emission in addition to that coming from the SSC scenario. The studies with our dedicated SSC grid-scan show that there is some degeneracy in both the one-zone and the two-zone SSC scenarios probed, with several combinations of model parameters yielding a similar model-to-data agreement, and some parameters better constrained than others. The observed gamma-ray flaring activity, with the EVPA rotation coincident with the first gamma-ray flare, resembles those reported previously for low frequency peaked blazars, hence suggesting that there are many similarities in the flaring mechanisms of blazars with different jet properties