X-Ray synchronotron study of phase transforms in illite clays to extract information on sigillata manufacturing processes.

The technique of sigillata really began in central Italy during the first century B. C. with the development of red vitrified slips obtained through vitrification of a clay preparation. These ceramics, usually decorated with raised motifs and standardised shapes, quickly took over as semi luxury crockery. Given this success, this technique quickly extended to the entire Italian peninsula and then to the Mediterranean coast. From the very start of our era, great centres of production were set up in the south of Gaul. The aspect of sigillata comes from the nature and the texture of its slip. Studies have shown that sigillata slips of quality were obtained from a non calcareous clay while the local calcareous clay was used for the bodies. During firing the slips are vitrified and get a specific microstructure containing hematite and nanometric corundum crystals [1]. An investigation of the clays surrounding La Graufesenque site started and it seems that only the Trias levels are chemically compatible with the composition of antique slips. Apart from the in depth study of the mineralogical nature of these clays realized at a geological Laboratory, we have studied the structural transformations as a function of temperature of two of these clays, chosen for the quality of vitrification in the firing temperature range of sigillata [1030-1080°C]. The main difference between the chemical composition of these two clays is the amount of Mg (2.4 % and 4.5 % in oxide weight). Time-resolved measurements were made at Daresbury (station 2.3) up to 1100oC in oxidizing conditions. An abrupt increase of the hematite cell was observed around 850°C. Above 1000°C, the hematite peaks get sharper which indicate an increase of coherence length (Fig. 1). A spinel phase with cell parameter close to MgAl2O3 was detected from this temperature. As for the hematite, its coherence length increases with the temperature but also during the beginning of the cooling. For the clay sample with the smaller amount of Mg, a corundum phase with very small coherence length was detected above 1000°C. Slips were prepared from the last clay by modern potters and firing at 1050°C in oxidizing atmosphere. A mineral quantitative analysis performed using the Rietveld method revealed that the amount of spinel phase is very high while the corundum contributes to a small part of crystal phases. It is the inverse in the antique slip where the amount of Mg in oxide weight is around 1%. It is clear that the amount of Mg plays a key role in the corundum/spinel competition and that the present slips contain too much Mg. Two questions arise: (i) As the Trias levels are quite heterogeneous is it possible to find clay with less Mg? and (ii) Did the gallo-roman potters eliminated a great part of Mg during the slip preparation process? We discuss the merits of these two alternative hypotheses

Integrated Functions of Pax3 and Pax7 in the Regulation of Proliferation, Cell Size and Myogenic Differentiation

Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed

Gene Expression Profiling of Muscle Stem Cells Identifies Novel Regulators of Postnatal Myogenesis

International audienceSkeletal muscle growth and regeneration require a population of muscle stem cells, the satellite cells, located in close contact to the myofiber. These cells are specified during fetal and early postnatal development in mice from a Pax3/7 population of embryonic progenitor cells. As little is known about the genetic control of their formation and maintenance, we performed a genome-wide chronological expression profile identifying the dynamic transcriptomic changes involved in establishment of muscle stem cells through life, and acquisition of muscle stem cell properties. We have identified multiple genes and pathways associated with satellite cell formation, including set of genes specifically induced (EphA1, EphA2, EfnA1, EphB1, Zbtb4, Zbtb20) or inhibited (EphA3, EphA4, EphA7, EfnA2, EfnA3, EfnA4, EfnA5, EphB2, EphB3, EphB4, EfnBs, Zfp354c, Zcchc5, Hmga2) in adult stem cells. Ephrin receptors and ephrins ligands have been implicated in cell migration and guidance in many tissues including skeletal muscle. Here we show that Ephrin receptors and ephrins ligands are also involved in regulating the adult myogenic program. Strikingly, impairment of EPHB1 function in satellite cells leads to increased differentiation at the expense of self-renewal in isolated myofiber cultures. In addition, we identified new transcription factors, including several zinc finger proteins. ZFP354C and ZCCHC5 decreased self-renewal capacity when overexpressed, whereas ZBTB4 increased it, and ZBTB20 induced myogenic progression. The architectural and transcriptional regulator HMGA2 was involved in satellite cell activation. Together, our study shows that transcriptome profiling coupled with myofiber culture analysis, provides an efficient system to identify and validate candidate genes implicated in establishment/maintenance of muscle stem cells. Furthermore, tour de force transcriptomic profiling provides a wealth of data to inform for future stem cell-based muscle therapies

PAX7 target genes are globally repressed in facioscapulohumeral muscular dystrophy skeletal muscle

Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy, linked to hypomethylation of D4Z4 repeats on chromosome 4q causing expression of the DUX4 transcription factor. However, DUX4 is difficult to detect in FSHD muscle biopsies and it is debatable how robust changes in DUX4 target gene expression are as an FSHD biomarker. PAX7 is a master regulator of myogenesis that rescues DUX4-mediated apoptosis. Here, we show that suppression of PAX7 target genes is a hallmark of FSHD, and that it is as major a signature of FSHD muscle as DUX4 target gene expression. This is shown using meta-analysis of over six FSHD muscle biopsy gene expression studies, and validated by RNA-sequencing on FSHD patient-derived myoblasts. DUX4 also inhibits PAX7 from activating its transcriptional target genes and vice versa. Furthermore, PAX7 target gene repression can explain oxidative stress sensitivity and epigenetic changes in FSHD. Thus, PAX7 target gene repression is a hallmark of FSHD that should be considered in the investigation of FSHD pathology and therapy

HACD1, a regulator of membrane composition and fluidity, promotes myoblast fusion and skeletal muscle growth

The reduced diameter of skeletal myofibres is a hallmark of several congenital myopathies, yet the underlying cellular and molecular mechanisms remain elusive. In this study, we investigate the role of HACD1/PTPLA, which is involved in the elongation of the very long chain fatty acids, in muscle fibre formation. In humans and dogs, HACD1 deficiency leads to a congenital myopathy with fibre size disproportion associated with a generalized muscle weakness. Through analysis of HACD1-deficient Labradors, Hacd1-knockout mice, and Hacd1-deficient myoblasts, we provide evidence that HACD1 promotes myoblast fusion during muscle development and regeneration. We further demonstrate that in normal differentiating myoblasts, expression of the catalytically active HACD1 isoform, which is encoded by a muscle-enriched splice variant, yields decreased lysophosphatidylcholine content, a potent inhibitor of myoblast fusion, and increased concentrations of ≥C18 and monounsaturated fatty acids of phospholipids. These lipid modifications correlate with a reduction in plasma membrane rigidity. In conclusion, we propose that fusion impairment constitutes a novel, non-exclusive pathological mechanism operating in congenital myopathies and reveal that HACD1 is a key regulator of a lipid-dependent muscle fibre growth mechanism

The myogenic transcriptional network

Myogenesis has been a leading model for elucidating the molecular mechanisms that underlie tissue differentiation and development since the discovery of MyoD. During myogenesis, the fate of myogenic precursor cells is first determined by Pax3/Pax7. This is followed by regulation of the myogenic differentiation program by muscle regulatory factors (Myf5, MyoD, Myog, and Mrf4) to form muscle tissues. Recent studies have uncovered a detailed myogenic program that involves the RP58 (Zfp238)-dependent regulatory network, which is critical for repressing the expression of inhibitor of DNA binding (Id) proteins. These novel findings contribute to a comprehensive understanding of the muscle differentiation transcriptional program

Transcriptome analyses based on genetic screens for Pax3 myogenic targets in the mouse embryo

<p>Abstract</p> <p>Background</p> <p>Pax3 is a key upstream regulator of the onset of myogenesis, controlling progenitor cell survival and behaviour as well as entry into the myogenic programme. It functions in the dermomyotome of the somite from which skeletal muscle derives and in progenitor cell populations that migrate from the somite such as those of the limbs. Few Pax3 target genes have been identified. Identifying genes that lie genetically downstream of <it>Pax3 </it>is therefore an important endeavour in elucidating the myogenic gene regulatory network.</p> <p>Results</p> <p>We have undertaken a screen in the mouse embryo which employs a <it>Pax3^GFP</it>allele that permits isolation of Pax3 expressing cells by flow cytometry and a <it>Pax3^PAX3-FKHR</it>allele that encodes PAX3-FKHR in which the DNA binding domain of Pax3 is fused to the strong transcriptional activation domain of FKHR. This constitutes a gain of function allele that rescues the <it>Pax3 </it>mutant phenotype. Microarray comparisons were carried out between <it>Pax3^GFP/+</it>and <it>Pax3^{GFP/PAX3-FKHR}</it>preparations from the hypaxial dermomyotome of somites at E9.5 and forelimb buds at E10.5. A further transcriptome comparison between Pax3-GFP positive and negative cells identified sequences specific to myogenic progenitors in the forelimb buds. Potential Pax3 targets, based on changes in transcript levels on the gain of function genetic background, were validated by analysis on loss or partial loss of function <it>Pax3 </it>mutant backgrounds. Sequences that are up- or down-regulated in the presence of PAX3-FKHR are classified as somite only, somite and limb or limb only. The latter should not contain sequences from Pax3 positive neural crest cells which do not invade the limbs. Verification by whole mount <it>in situ </it>hybridisation distinguishes myogenic markers. Presentation of potential Pax3 target genes focuses on signalling pathways and on transcriptional regulation.</p> <p>Conclusions</p> <p>Pax3 orchestrates many of the signalling pathways implicated in the activation or repression of myogenesis by regulating effectors and also, notably, inhibitors of these pathways. Important transcriptional regulators of myogenesis are candidate Pax3 targets. Myogenic determination genes, such as <it>Myf5 </it>are controlled positively, whereas the effect of <it>Pax3 </it>on genes encoding inhibitors of myogenesis provides a potential brake on differentiation. In the progenitor cell population, <it>Pax7 </it>and also <it>Hdac5 </it>which is a potential repressor of <it>Foxc2</it>, are subject to positive control by <it>Pax3</it>.</p

Mapping and identification of candidate loci responsible for Peromyscus hybrid overgrowth

Crosses between two recently diverged rodent species of the genus Peromyscus result in dramatic parent-of-origin effects on growth and development. P. maniculatus females crossed with P. polionotus males yield growth-retarded conceptuses, whereas the reciprocal cross results in overgrowth and lethality. These hybrid effects are particularly pronounced in the placenta. We previously detected linkage to two regions of the genome involved in the overgrowth effects. One locus, termed Peal, is a paternally expressed autosomal locus mapping to a domain whose house mouse equivalent contains several clusters of imprinted genes. The other locus, termed Mexl, maps to a gene-poor region of the X chromosome. Here we use an advanced intercross line to verify and narrow the regions of linkage and identify candidate genes for Mexl and Peal. While we have previously shown that Mexl affects both pre-and postnatal growth, we show here that Peal affects only prenatal growth. Utilizing criteria such as mutant phenotypes and allelic expression, we identify the loci encoding the homeobox protein Esx1 and the zinc-finger protein Pw1/Peg3 as candidates. Both loci exhibit expression changes in the hybrids

Further Characterisation of the Molecular Signature of Quiescent and Activated Mouse Muscle Satellite Cells

Satellite cells are the resident stem cells of adult skeletal muscle. To date though, there is a paucity of native markers that can be used to easily identify quiescent satellite cells, with Pax7 probably being the best that is currently available. Here we have further characterized a number of recently described satellite cell markers, and also describe novel ones. Caveolin-1, integrin α7 and the calcitonin receptor proved reliable markers for quiescent satellite cells, being expressed by all satellite cells identified with Pax7. These three markers remained expressed as satellite cells were activated and underwent proliferation. The nuclear envelope proteins lamin A/C and emerin, mutations in which underlie Emery-Dreifuss muscular dystrophy, were also expressed in both quiescent and proliferating satellite cells. Conversely, Jagged-1, a Notch ligand, was not expressed in quiescent satellite cells but was induced upon activation. These findings further contribute to defining the molecular signature of muscle satellite cells

Clinicopathological significance of homeoprotein Six1 in hepatocellular carcinoma

Tumour recurrence and metastases of hepatocellular carcinoma (HCC) after hepatectomy are the major obstacles of long-term survival. The present study investigated the clinicopathological significance of a possible metastasis regulator Six1 in HCC patients who were undergone hepatectomy. Seventy-two pairs of RNA and 103 pairs of protein from tumour and adjacent nontumour liver tissues of HCC patients were examined. About 85 and 60% of HCC tumour tissues were found to overexpress Six1 mRNA and protein, respectively, compared with nontumour liver tissues. No Six1 protein was detected in HCC nontumour liver tissues and normal liver tissues. Increased Six1 protein expression in HCC patients was significantly correlated with pathologic tumour-node-metastasis (pTNM) stage (P=0.002), venous infiltration (P=0.004) and poor overall survival (P=0.0423). We concluded that Six1 is frequently overexpressed in HCC patients and elevated Six1 protein in HCC patients may be an indication of advanced stage and poor overall survival after hepatectomy