Elastogenesis Correlates With Pigment Production in Murine Aortic Valve Leaflets

Objective: Aortic valve (AV) leaflets rely on a precise extracellular matrix (ECM) microarchitecture for appropriate biomechanical performance. The ECM structure is maintained by valvular interstitial cells (VICs), which reside within the leaflets. The presence of pigment produced by a melanocytic population of VICs in mice with dark coats has been generally regarded as a nuisance, as it interferes with histological analysis of the AV leaflets. However, our previous studies have shown that the presence of pigment correlates with increased mechanical stiffness within the leaflets as measured by nanoindentation analyses. In the current study, we seek to better characterize the phenotype of understudied melanocytic VICs, explore the role of these VICs in ECM patterning, and assess the presence of these VICs in human aortic valve tissues. Approach and Results: Immunofluorescence and immunohistochemistry revealed that melanocytes within murine AV leaflets express phenotypic markers of either neuronal or glial cells. These VIC subpopulations exhibited regional patterns that corresponded to the distribution of elastin and glycosaminoglycan ECM proteins, respectively. VICs with neuronal and glial phenotypes were also found in human AV leaflets and showed ECM associations similar to those observed in murine leaflets. A subset of VICs within human AV leaflets also expressed dopachrome tautomerase, a common melanocyte marker. A spontaneous mouse mutant with no aortic valve pigmentation lacked elastic fibers and had reduced elastin gene expression within AV leaflets. A hyperpigmented transgenic mouse exhibited increased AV leaflet elastic fibers and elastin gene expression. Conclusions: Melanocytic VIC subpopulations appear critical for appropriate elastogenesis in mouse AVs, providing new insight into the regulation of AV ECM homeostasis. The identification of a similar VIC population in human AVs suggests conservation across species

The transcription factors Ets1 and Sox10 interact during murine melanocyte development

Melanocytes, the pigment-producing cells, arise from multipotent neural crest (NC) cells during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The variable spotting mouse pigmentation mutant arose spontaneously at the Jackson Laboratory. We identified a G-to-A nucleotide transition in exon 3 of the Ets1 gene in variable spotting, which results in a missense G102E mutation. Homozygous variable spotting mice exhibit sporadic white spotting. Similarly, mice carrying a targeted deletion of Ets1 exhibit hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The transcription factor Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of various NC derivatives, including melanocytes. We show that Ets1 is required early for murine NC cell and melanocyte precursor survival in vivo. Given the importance of Ets1 for Sox10 expression in the chick, we investigated a potential genetic interaction between these genes by comparing the hypopigmentation phenotypes of single and double heterozygous mice. The incidence of hypopigmentation in double heterozygotes was significantly greater than in single heterozygotes. The area of hypopigmentation in double heterozygotes was significantly larger than would be expected from the addition of the areas of hypopigmentation of single heterozygotes, suggesting that Ets1 and Sox10 interact synergistically in melanocyte development. Since Sox10 is also essential for enteric ganglia development, we examined the distal colons of Ets1 null mutants and found a significant decrease in enteric innervation, which was exacerbated by Sox10 heterozygosity. At the molecular level, Ets1 was found to activate an enhancer critical for Sox10 expression in NC-derived structures. Furthermore, enhancer activation was significantly inhibited by the variable spotting mutation. Together, these results suggest that Ets1 and Sox10 interact to promote proper melanocyte and enteric ganglia development from the NC

Elastogenesis Correlates With Pigment Production in Murine Aortic Valve Leaflets

Objective: Aortic valve (AV) leaflets rely on a precise extracellular matrix (ECM) microarchitecture for appropriate biomechanical performance. The ECM structure is maintained by valvular interstitial cells (VICs), which reside within the leaflets. The presence of pigment produced by a melanocytic population of VICs in mice with dark coats has been generally regarded as a nuisance, as it interferes with histological analysis of the AV leaflets. However, our previous studies have shown that the presence of pigment correlates with increased mechanical stiffness within the leaflets as measured by nanoindentation analyses. In the current study, we seek to better characterize the phenotype of understudied melanocytic VICs, explore the role of these VICs in ECM patterning, and assess the presence of these VICs in human aortic valve tissues.Approach and Results: Immunofluorescence and immunohistochemistry revealed that melanocytes within murine AV leaflets express phenotypic markers of either neuronal or glial cells. These VIC subpopulations exhibited regional patterns that corresponded to the distribution of elastin and glycosaminoglycan ECM proteins, respectively. VICs with neuronal and glial phenotypes were also found in human AV leaflets and showed ECM associations similar to those observed in murine leaflets. A subset of VICs within human AV leaflets also expressed dopachrome tautomerase, a common melanocyte marker. A spontaneous mouse mutant with no aortic valve pigmentation lacked elastic fibers and had reduced elastin gene expression within AV leaflets. A hyperpigmented transgenic mouse exhibited increased AV leaflet elastic fibers and elastin gene expression.Conclusions: Melanocytic VIC subpopulations appear critical for appropriate elastogenesis in mouse AVs, providing new insight into the regulation of AV ECM homeostasis. The identification of a similar VIC population in human AVs suggests conservation across species

A Complex Genomic Rearrangement Involving the Endothelin 3 Locus Causes Dermal Hyperpigmentation in the Chicken

Dermal hyperpigmentation or Fibromelanosis (FM) is one of the few examples of skin pigmentation phenotypes in the chicken, where most other pigmentation variants influence feather color and patterning. The Silkie chicken is the most widespread and well-studied breed displaying this phenotype. The presence of the dominant FM allele results in extensive pigmentation of the dermal layer of skin and the majority of internal connective tissue. Here we identify the causal mutation of FM as an inverted duplication and junction of two genomic regions separated by more than 400 kb in wild-type individuals. One of these duplicated regions contains endothelin 3 (EDN3), a gene with a known role in promoting melanoblast proliferation. We show that EDN3 expression is increased in the developing Silkie embryo during the time in which melanoblasts are migrating, and elevated levels of expression are maintained in the adult skin tissue. We have examined four different chicken breeds from both Asia and Europe displaying dermal hyperpigmentation and conclude that the same structural variant underlies this phenotype in all chicken breeds. This complex genomic rearrangement causing a specific monogenic trait in the chicken illustrates how novel mutations with major phenotypic effects have been reused during breed formation in domestic animals

Effect of the relative shift between the electron density and temperature pedestal position on the pedestal stability in JET-ILW and comparison with JET-C

The electron temperature and density pedestals tend to vary in their relative radial positions, as observed in DIII-D (Beurskens et al 2011 Phys. Plasmas 18 056120) and ASDEX Upgrade (Dunne et al 2017 Plasma Phys. Control. Fusion 59 14017). This so-called relative shift has an impact on the pedestal magnetohydrodynamic (MHD) stability and hence on the pedestal height (Osborne et al 2015 Nucl. Fusion 55 063018). The present work studies the effect of the relative shift on pedestal stability of JET ITER-like wall (JET-ILW) baseline low triangularity (\u3b4) unseeded plasmas, and similar JET-C discharges. As shown in this paper, the increase of the pedestal relative shift is correlated with the reduction of the normalized pressure gradient, therefore playing a strong role in pedestal stability. Furthermore, JET-ILW tends to have a larger relative shift compared to JET carbon wall (JET-C), suggesting a possible role of the plasma facing materials in affecting the density profile location. Experimental results are then compared with stability analysis performed in terms of the peeling-ballooning model and with pedestal predictive model EUROPED (Saarelma et al 2017 Plasma Phys. Control. Fusion). Stability analysis is consistent with the experimental findings, showing an improvement of the pedestal stability, when the relative shift is reduced. This has been ascribed mainly to the increase of the edge bootstrap current, and to minor effects related to the increase of the pedestal pressure gradient and narrowing of the pedestal pressure width. Pedestal predictive model EUROPED shows a qualitative agreement with experiment, especially for low values of the relative shift

The untranslated side of hair and skin mammalian pigmentation : beyond coding sequences

For several decades, tremendous advances in studying skin and hair pigmentation of mammals have been made using Mendelian genetics principles. A number of loci and their associated traits have been extensively examined, crossings performed, and phenotypes well documented. Continuously improving PCR techniques allowed the molecular cloning and sequencing of the first pigmentation genes at the end of the 20th century, a period followed by an intense effort to detect and describe polymorphisms in the coding regions and correlate allelic combinations with the observed melanogenic phenotypes. However, a number of phenotypes and biological events could not be elucidated solely by analysis of the coding regions of genes. Messenger RNA isolation, characterization and quantification techniques allowed groups to move ahead and investigate molecular mechanisms whose secrets lay within the noncoding regions of pigmentation genes transcripts such as MC1R, ASIP, or W. The untranslated elements contain specific nucleotidic sequences and structures that dramatically influence the mRNA half-life and processing thus impacting protein translation and melanin production. As we are progressively uncovering the complex processes regulating melanocyte biology, unraveling complete mRNA structures and understanding mechanisms beyond coding regions has become critical

Structural and functional characterization of the mouse tescalcin promoter

Tescalcin, an EF-hand calcium binding protein that regulates the Na(+)/H(+) exchanger 1 (NHE1), is highly expressed in various mouse tissues such as heart and brain. Despite its potentially important role in cell physiology, the mechanisms that regulate tescalcin gene (Tesc) expression are unknown. In this study, we report two new Tesc mRNA variants (V2 and V3) and characterize the mouse Tesc promoter. The V2 and V3 transcripts result from alternative splicing of intron 5. Our results show that Tesc mRNA variants are expressed in various mouse tissues. Primer extension analysis located the transcription start site at 94 nucleotides upstream of the translation start codon. The DNA nucleotide sequence of the 5'-flanking region contained a CpG island spanning the promoter region from nucleotides -372 to +814, a canonical TATA box (-38/-32), and putative transcription factor binding sites for Sp1, EGR1, ZBP-89, KLF3, MZF1, AP2, ZF5, and CDF-1. Transient transfection of the Y1 and msc-1 cell lines with a series of 5'-deleted promoter constructs indicated that the minimal promoter region was between nucleotides -130 and -40. Electrophoresis mobility shift assays, supershift assays, and mutation studies demonstrated that Sp1 and Sp3 bind to the GC-rich motifs, a CACCC box and three GC boxes, located within the Tesc proximal promoter. Nonetheless, mutations that abolished interaction of Sp1 and Sp3 with the GC-rich motifs located within the minimal promoter region did not abrogate promoter activity in Y1 cells. Mithramycin A, an inhibitor of Sp1-DNA interaction, reduced Tesc promoter activity in msc-1 cells in a dose-dependent manner. Sp3 was a weaker transactivator compared to Sp1 in Drosophila D.mel-2 cells. However, when Sp1 and Sp3 were coexpressed, they transactivated the Tesc promoter in a synergistic manner. In Y1 cells, mutation analysis of a putative ZF5 motif located within the Tesc minimal promoter indicated that this motif was critical for activity of Tesc promoter. Taken together, the data demonstrated that Sp1 and Sp3 transcription factors cooperate positively in the regulation of Tesc promoter, and that the putative ZF5 motif is critical for its activation

Endothelin signalling in the development of neural crest-derived melanocytes

In both mice and humans, mutations in the genes encoding the endothelin B receptor and its ligand endothelin 3 lead to deficiencies in neural crest- derived melanocytes and enteric neurons. The discrete steps at which endothelins exert their functions in melanocyte development were examined in mouse neural crest cell cultures. Such cultures, kept in the presence of fetal calf serum, gave rise to cells expressing the early melanoblast marker Dct+ even in the absence of the phorbol ester tetradecanoyl phorbol acetate (TPA) or endothelins. However, these early Dct+ cells did not proliferate and pigmented cells never formed unless TPA or endothelins were added. In fact, endothelin 2 was as potent as TPA in promoting the generation of both Dct+ melanoblasts and pigmented cells, and endothelin 1 or endothelin 3 stimulated the generation of melanoblasts and of pigmented cells to an even greater extent. The inhibition of this stimulation by the selective endothelin B receptor antagonist BQ-788 (N-cis-2,6- dimethylpiperidinocarbonyl-L-α-methylleucyl-D-1-methoxycarbonyltryptophanyl- D-norleucine) suggested that the three endothelins all signal through the endothelin B receptor. This receptor was indeed expressed in Dct+ melanoblasts, in addition to cells lacking Dct expression. The results demonstrate that endothelins are potent stimulators of melanoblast proliferation and differentiation.SCOPUS: ar.jinfo:eu-repo/semantics/publishe