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Shiso production will continue in cold greenhouses until late November "We have both dried leaves and pre-developed plants so leaves can be harvested autonomously Each plant produces over a hundred leaves per cycle and our market is expanding not only to Italy but abroad as well." more tannic and pungent and ideal for fermenting Cultivating shiso in the Po Valley immediately seemed like a difficult undertaking but the stubbornness of D'Angelo and his collaborators prevailed: "It certainly wasn't easy organic aromatic herb with the organoleptic characteristics required by the market." For more information:L'Orto di MimìVia Macina, 25030Castel Mella (Brescia)[email protected]www.lortodimimi.com Frontpage photo: © L'ORTO DI MIMI' FreshPublishers © 2005-2025 FreshPlaza.com The cultivation of wasabi (Eutrema japonicum) a brassica native to Japan that is widely used in the kitchen is proving successful in Italy for the first time the grated rhizome is used to accompany raw fish but the leaves are also fried in tempura and the stems are used for making the traditional kizami which has always been closely linked to Japan is now also taking place at the L'Orto di Mimì farm in the Brescia province set up by chef Antonio D'Angelo and recently awarded as the best certified organic 'Japanese Farm' in Europe Talking about this successful project is D'Angelo himself who supported from a technical-scientific point of view by his collaborator Andrea Tessadrelli wasabi is one of the most difficult crops in the world Over 15 months pass between planting and the first harvest which greatly increases the risks involved in production the first harvest partially took place in 2022 with a taste identical to the Japanese original yet initially with some differences in colour and epidermis due to the excessive heat." Producing wasabi in Italy seemed an impossible task at first but Tessadrelli took up D'Angelo's challenge "We recreated an environment similar to the Japanese one from scratch including constantly flowing water at a controlled temperature and trees to reduce solar radiation creating wind currents with a thermal cooling function." The added value of Orto di Mimì was to focus on know-how and scientific research right from the start focusing on the plants that demonstrated the best characteristics from an agronomic and nutritional point of view Tessadrelli adds that "from my first day in this company I have always been convinced that you cannot replicate an exotic crop without investing in research and breeding a variety that can adapt and thrive even at our latitudes we have crossed and improved several varieties over the years We have now reached the stage of worldwide registration of Fant1 a unique hybrid that is resistant up to 40° Celsius and boasts abundant opaque foliage and a rhizome with a good specific weight He concludes: "The company already has four years of intensive research and development behind it with a lot of investments made to try and get the first results but now we are proud to have our own variety to grow and propagate with constant attention to varietal improvement We are only at the beginning and we are very confident knowing that we have reached a goal that was previously unimaginable." For more information L'Orto di MimìVia Macina, 25030Castel Mella (Brescia)[email protected]www.lortodimimi.com Metrics details Skeletal muscle satellite cells are quiescent adult resident stem cells that activate proliferate and differentiate to generate myofibres following injury They harbour a robust proliferation potential and self-renewing capacity enabling lifelong muscle regeneration Although several classes of microRNAs were shown to regulate adult myogenesis systematic examination of stage-specific microRNAs during lineage progression from the quiescent state is lacking Here we provide a genome-wide assessment of the expression of small RNAs during the quiescence/activation transition and differentiation by RNA-sequencing We show that the majority of small RNAs present in quiescent activated and differentiated muscle cells belong to the microRNA class by comparing expression in distinct cell states we report a massive and dynamic regulation of microRNAs highlighting their pivotal role in regulation of quiescence We also identify a number of microRNAs with reliable and specific expression in quiescence including several maternally-expressed miRNAs generated at the imprinted Dlk1-Dio3 locus the majority of class-switching miRNAs are associated with the quiescence/activation transition suggesting a poised program that is actively repressed These data constitute a key resource for functional analyses of miRNAs in skeletal myogenesis in the regulation of stem cell self-renewal and tissue homeostasis a subset of proliferating satellite cells self-renew in their niche by reversibly exiting the cell cycle skeletal myogenesis is a tractable model to study the regulation of quiescence As previous quantitative and differential data obtained using RT-qPCR or miRNA-microarrays were limited to the quantification of known molecules we performed an unbiased analysis of small-RNA profiles from stem to differentiated cells in adult myogenesis Our data provide a key resource for functional studies of the involvement of small-RNAs - including miRNAs and more broadly in the regulation of stem cell self-renewal and tissue homeostasis Unbiased identification of stage specific small RNAs during lineage progression from muscle stem cells (A) Quiescent satellite cells were isolated after digestion of resting limb muscles and diaphragm from adult Tg:Pax7-nGFP mice by FACS using GFP fluorescence An aliquot was cultured in vitro for 60 h or 7 days and the remainder was lysed directly for RNA extraction After size selecting 15–35 nucleotides small RNAs on a polyacrylamide gel sequencing libraries were prepared and analysed (B) Schematic representation of lineage progression in adult skeletal muscle activated and differentiated samples are represented Immuno-fluorescence images confirmed the cellular identity of the 3 populations (i) quiescent satellite cells: Pax7(+) Myod(−); Activated satellite cells/myoblasts: Pax7(+) MyoD(+); Differentiated muscle cells: Pax7(−) Myog(+) Note the presence of rare self-renewing “reserve cells” expressing Pax7 in the differentiated sample (C) Sequenced small RNA corresponded overwhelmingly to miRNAs in all 3 samples and showed low contamination by degraded tRNA Despite the inclusion of the 25–32 nt size range in the analysis whereas reads mapping to intronic regions were identified in particular in the quiescent samples (>5% reads) (D) 412 and 231 miRNAs were detected in at least one sample type more than 10 or 100 times (E) Frequency histogram displaying the miRNAs distribution according to their expression levels in all 3 samples highlight their large dynamic range in expression Other classes of small RNAs and in particular piRNAs were not detected in our samples We subsequently focused on the expression profiles of miRNAs This observation underscored the importance of robust normalization of the datasets to avoid skewing of the expression profiles as a result of the high expression of a limited number of miRNAs Identification of differentially expressed miRNAs during myogenic lineage progression (A) Scatter plot of miRNA expression level in Quiescent vs Results are presented as the median of log transformed normalized counts for each miRNA 249 showed a modulation that reached statistical significance in the 3 pairwise comparisons (corrected p-value ≤ 0.001) Statistically significant up- or down-regulated miRNAs were coloured in yellow and blue (D) Heatmap presenting 4 classes of differentially expressed miRNAs identified by K-means clustering MicroRNAs are involved in the regulation of all processes – quiescence activation and self-renewal and differentiation and a large number of miRNAs with expression specific of one particular state were identified whereas low expression is blue as in previous panels We then grouped the differentially expressed miRNAs according to their expression profiles using K-means clustering which reveals 4 classes (Fig. 2D and Supplementary Table S2) The first consisted of 59 miRNAs whose expression was found to be associated specifically with quiescence The second and third clusters comprised miRNAs either expressed during activation or conversely silenced in this cell state; they represented 70 and 64 miRNAs the last cluster was composed of miRNAs showing an increase in expression during commitment and differentiation the most important transition was between quiescence and activation where more than half of the differentially expressed miRNAs identified were specific to these states This finding highlights the concerted role that miRNAs play during the regulation in this transition Validation of miRNA regulation on in vivo activated satellite cells Histogram presenting parallel expression measured by small-RNAseq following in vitro culture compared to in vivo activated satellite cells and isolated single muscle fibres (A–F) The trend in expression was confirmed for 6 out 6 tested miRNAs and only miR-26a did not show the same amplitude of deregulation on in vivo activated samples (G–J) identical results were obtained for activation specific miRNAs thus validating the miRNA-sequencing data using an in vitro activation paradigm Normalization based on cell number allowed to confirm the higher expression level of many miRNAs during quiescence given the per-cell normalization we used in our RT-qPCR assay our analysis leads us to propose that tens of miRNAs have higher levels of expression in quiescent vs these findings suggest that the miRNAs over-expressed during quiescence are potent regulators that exert their effect in satellite cells we also identified functions related to skeletal muscle tissue development (p-value < 5.9E-9) regulation of ubiquitin-protein transferase activity (p-value < 6.6E-4) and somatic stem cell division (p-value < 1.1E-4) Functional analysis of miRNA from the Dlk1-Dio3 locus expressed in quiescent satellite cells (A) Schematic representation of the maternally expressed miR-127/miR-136 and miR-379/miR-410 gene cluster in the imprinted Dlk1-Dio3 locus located on mouse chromosome 12 Dlk1 and Dio3 are expressed from paternally inherited chromosome (B) Heatmap representing the expression level of miRNAs from the Dlk1-Dio3 locus during lineage progression from quiescent to activated and differentiated satellite cells The two miRNAs studied in more detail are highlighted with orange arrows (C) Expression of coding and non-coding genes from the Dlk1-Dio3 locus in control and mutant mice (D) Counting of satellite cell numbers on EDL muscle fibres from mutant and WT mice; n = 6–7 mice/genotype; ≥15 fibres/mice The bar in the violin plot represents the median (E) H&E staining of TA muscle sections from WT and miR-379/410 mutants 6 and 9 weeks after Notexin injury of muscle (F) Anti-Myog staining (top) and EdU reaction (bottom) in satellite cells isolated from Dll1-Dio3 Control (WT) and mutant (KO) mice at 72 h and 24 h Quantification of Myog+ and EdU+ cells at each time point revealed no difference between WT and KO satellite cells anti-Myog stainings (top) and EdU reaction (bottom) in satellite cells isolated from Tg:Pax7-nGFP mice 72 h after Mimic-127 Mimic-379 or Scramble control transfection Myogenin-positive and EdU positive cells at 72 h following Mimic-127 Expression levels of the corresponding genes in the dataset of Garcia-Prat et al ITGB6 and CD44) are among candidates that could mediate the effect of miR-127 and miR-379 We observed an overall downregulation in quiescence of the several thousand mRNAs putatively targeted by quiescence miRNAs These observations point to a collective control by miRNAs on the expression of specific mRNAs during these cell transitions The number of miRNAs expressed in quiescence and thus the number of potential targets identified complicated the global analysis of their function using a restricted list of potential targets generated by combining 3 prediction tools we found Gene Ontologies associated with regulation of muscle development Having identified the overexpression of tens of miRNAs from two clusters located in the Dlk1-Dio3 locus in quiescent satellite cells we investigated the impact of the deletion of the miR-379/410 cluster in vivo Our observation that this resulted in no overt regeneration phenotype or modulation of cell fates of isolated satellite cells leads us to speculate that other miRNAs have overlapping functions with this cluster functional analysis by sustained expression of two miRNAs from the miR-127 and miR-379 loci resulted in a robust phenotype corresponding to an increase in Pax7 expression and reduced commitment towards differentiation thereby indicating that these miRNAs promote the stem cell state These findings suggest that these miRNAs play key roles in regulating satellite cell quiescence in adult resting muscles but instead increased self-renewal and decreased differentiation of satellite cells The absence of a regeneration phenotype in the miR-379/410 mutant mice could be explained by compensations by other miRNAs expressed in quiescence targeting common targets or other compensatory mechanisms during development following the germline deletion The implication of a re-expression of the paternal allele of the miRNA-cluster is however unlikely a maternally expressed ncRNAs that overlaps with the miR-379/411 cluster was detected in satellite cells in mutant mice the size of the locus to be deleted precludes the use of a cell type specific inducible Cre-recombinase under the control of Pax7 to study the role of these miRNAs in more detail Future work will be required in gain or loss of function experiments to uncover the molecular function of additional differentially expressed miRNAs and to identify their relevant targets in the context of induction and maintenance of quiescence beyond the pivotal role of miR-489 and miR-195/497 already noted in Pax7-positive cells identifying the signalling pathways upstream of these miRNAs will shed light on this tightly regulated biological process our findings that a relatively significant variety of miRNAs are dedicated to negotiate the quiescence to activation states of muscle stem cells suggests that quiescence is actively repressed by this class of regulators These results can impact on our views of genetic and epigenetic regulation of quiescence and how this critical cell state is regulated in homeostasis and trauma All experiments with animals were performed under conditions established by the European Community and approved by the Ethics Committee at Institut Pasteur EDL muscles were dissected and incubated in 0.1% w/v collagenase (Sigma C0130)/DMEM for 1 h in a 37 °C shaking water bath individual myofibres were either processed for RNA extraction to validate the RNA sequencing in differentiation conditions or directly fixed in 4% paraformaldehyde (PFA Freshly isolated satellite cells were seeded at 3,000 cells/cm2 in growth medium containing 1:1 DMEM:MCDB (Gibco and Sigma-Aldrich respectively) supplemented with 20% serum FBS (Gibco) and 1% Ultroser G (Pall) on Matrigel coated flasks (BD Biosciences) and cultured in an incubator under physiological oxygen pressure (37 °C cells were pulsed with the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU 2 h prior to fixation (ThermoFisher Click-iT Plus EdU kit and cells were cultured for a total of 7 days to reach early differentiation Freshly isolated satellite cells from Tg:Pax7-nGFP were transfected in suspension immediately after FACS with miRIDIAN microRNA hsa-miR-126-3p mimic (UCGUACCGUGAGUAAUAAUGCG C310397) and Scramble Control#1 (UCACAACCUCCUAGAAAGAGUAGA; Dharmacon CN-001000) at 200 nM final concentration using Lipofectamine 2000 (ThermoFisher) in Opti-MEM (Gibco) 3 volumes of fresh growth medium were added and cells were cultured for the indicated time Cells and myofibers were fixed in 4% PFA for 5 min permeabilised 5 min in 0.5% Triton-X100 (Sigma-Aldrich) and blocked in 10% normal goat-serum (Gibco) for 30 min at RT Cells and fibres were then incubated with primary antibodies (Chick GFP Samples were washed with 1X PBS three times and incubated with Alexa-conjugated secondary antibodies (Life Technologies EdU staining was chemically revealed using the Click-iT Plus kit according to manufacturer’s recommendations (Life Technologies Images were acquired using an upright fluorescent microscope (Zeiss) Isolated TA muscles were fixed in PFA 2%/0.2% Triton/PBS 1X for 2 h at 4 °C washed in PBS 1X overnight at 4 °C and incubated in 20% sucrose/PBS 1X for an additional 12 h TA muscles were embedded in OCT and frozen in liquid nitrogen prior to cryosectioning (10 µm) and stained with Hematoxylin/Eosin Images were acquired with Zeiss Axioscan microscope quiescent cells were directly sorted into Trizol-LS reagent (Invitrogen) and in-vitro cultured cells (activated at 60 hours and differentiated at 7 days) collected in Qiazol reagent (QIagen) Total RNA was subsequently purified using the miRNeasy Mini Kit following the manufacturer's instructions (Qiagen) Ten micrograms of total RNA obtained from several animals for the quiescent samples were used for each biological replicate prepared for deep sequencing (i.e 2 replicates for the quiescent and differentiated samples and 3 replicates for the in vitro activated sample) For RT-qPCR validations all samples were extracted using the same methods (Trizol LS after FACS for quiescent and in-vivo activated satellite cells; Qiazol for isolated single fibres) were isolated from quiescent and 48 h in vitro activated satellite cells using Direct-zol RNA Microprep (Zymo Research) according to manufacturer’s recommendations Total RNA and small RNA ratio were quantified using the Agilent 2100 Bioanalyzer PicoChip and SmallChip and analyzed with 2100 Expert software 10 µg of total RNA (in 10 µl) were mixed with 10 µl of 2X TBE-Urea Sample Buffer (Invitrogen) and loaded in a well of a 15% polyacrylamide TBE-urea gel (Biorad) the gel was soaked in a SYBR gold (Invitrogen) solution and imaged on a Dark Reader transilluminator The 18–35 nucleotide region was cut using a scalpel for each sample and the RNA eluted in 300 µl of 0.3 M NaCl solution under rotation for 4 hours at room temperature The eluate was transferred together with gel debris onto a Spin X cellulose acetate filter (VWR) and centrifuged for 2 minutes at 12,000 xg Small RNAs were finally precipitated by addition of 1 μl of glycogen (Invitrogen) and 750 μl of room temperature 100% ethanol followed by an incubation at −80 °C for 30 min and centrifugation for 25 min at 14,000 rpm and + 4 °C The pellet was washed with 750 µl 75% Ethanol dried and resuspended in 5 µl ultrapure water with 0.5 µl of RNAseOUT (Invitrogen) Small RNAs purified on gel were mixed to 1 µl of 10 µM pre-adenylated 3′ Illumina linker V1.5 (5′-rAppATCTCGTATGCCGTCTTCTGCTTG/3ddC/-3′) and further mixed with 1 µl of 10x T4 RNA-Ligase Truncated Reaction buffer 0.5 µl RNaseOut and 1.5 µl of T4 RNA Ligase 2 truncated (New England Biolabs) 0.5 µl of 5′-RNA adapter (5′-r(GUU CAG AGU UCU ACA GUC CGA CGA UC)-3′) 1 µl of 10 mM ATP and 1 µl T4 RNA ligase (Ambion) were added and ligation was performed at 20 °C for 6 h Adaptor ligated RNA in a volume of 4 µl were then mixed with 1 µl of 20 μM Solexa RT primer (5′-CAA GCA GAA GAC GGC ATA CGA-3′) and denatured at 70 °C and cooled on ice Reverse transcription was then performed after addition of 2 µl 5x first strand buffer (Invitrogen) 0.5 µl_ RNase OUT and 1 µl SuperScript III Reverse Transcriptase (Invitrogen) at 50 °C for 1 h The obtained cDNA was PCR-amplified by addition of 27 µl Ultra-pure water 1 µl of 25 µM Forward Primer (5′-AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-3′) 1 µl of 25 µM reverse Primer (5′-CAA GCA GAA GAC GGC ATA CGA-3′) and 0.5 µl Phusion DNA Polymerase (Finnzymes) using 12 cycles 98 °C 10 sec/60 °C 30 sec/72 °C 15 sec The library was finally purified on a 5% TBE PAGE gel by cutting the region corresponding to the 92–106 bp (the ligated linkers corresponding to a 73 bp band visible on the gel) The gel was crushed by centrifugation and eluted in 1X Elution buffer (Illumina) by rotation for 2 h at RT The eluate was cleared using a Spin-X column and precipitated after addition of 1 µl of glycogen 10 µl of 3 M NaOAc and 325 µl of −20 °C 100% ethanol followed by centrifugation for 20 min at 14,000 rpm the sample was diluted to 10 nM and submitted to sequencing on a Solexa GA-IIX at the core sequencing facility K-means clustering of differentially expressed miRNAs was performed using Cluster 3.0 (available at bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) using normalized read count as input. After a step of median centering of expression level for each miRNA, clustering was perform using centered Pearson correlation with 1,000 iterations. The corresponding heatmap was generated using JavaTreeView (http://jtreeview.sourceforge.net) Comparisons of expression level between the groups of transcripts in the different satellite cell states (i.e quiescent or activated in vivo 3 days post injury) were performed using a Mann-Whitney test The small RNA-seq data generated and analysed during the current study have been deposited in the ArrayExpress database at EMBL-EBI under accession number E-MTAB-5955 [https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5955] and Behavioral Heterogeneity of Cells from the Adult Muscle Satellite Cell Niche Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing Inducible deletion of epidermal Dicer and Drosha reveals multiple functions for miRNAs in postnatal skin The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation Myogenic factors that regulate expression of muscle-specific microRNAs MicroRNA-378 targets the myogenic repressor MyoR during myoblast differentiation Essential role for Dicer during skeletal muscle development Maintenance of muscle stem-cell quiescence by microRNA-489 Identification of tissue-specific microRNAs from mouse The phylogenetic distribution of metazoan microRNAs: insights into evolutionary complexity and constraint MicroRNA-1 and microRNA-133a expression are decreased during skeletal muscle hypertrophy A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance Identification of differences in microRNA transcriptomes between porcine oxidative and glycolytic skeletal muscles Profiling of differentially expressed microRNA and the bioinformatic target gene analyses in bovine fast- and slow-type muscles by massively parallel sequencing Muscle stem cell behavior is modified by microRNA-27 regulation of Pax3 expression Muscle Satellite Cells Are Primed for Myogenesis but Maintain Quiescence with Sequestration of Myf5 mRNA Targeted by microRNA-31 in mRNP Granules miR-195/497 induce postnatal quiescence of skeletal muscle stem cells Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates Skeletal muscle as a paradigm for regenerative biology and medicine A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division mTORC1 controls the adaptive transition of quiescent stem cells from G0 to G(Alert) A global downregulation of microRNAs occurs in human quiescent satellite cells during myogenesis Predicting effective microRNA target sites in mammalian mRNAs Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging Autophagy maintains stemness by preventing senescence Comparison of multiple transcriptomes exposes unified and divergent features of quiescent and activated skeletal muscle stem cells Karagkouni, D. et al. DIANA-TarBasev8: a decade-long collection of experimentally supported miRNA-gene interactions. Nucleic Acids Res. https://doi.org/10.1093/nar/gkx1141 (2017) miRDB: an online resource for microRNA target prediction and functional annotations Genomic imprinting at the mammalian Dlk1-Dio3 domain Regulation of DLK1 by the maternally expressed miR-379/miR-544 cluster may underlie callipyge polar overdominance inheritance The miR-379/miR-410 cluster at the imprinted Dlk1-Dio3 domain controls neonatal metabolic adaptation Attenuation of miR-126 activity expands HSC in vivo without exhaustion A Critical Requirement for Notch Signaling in Maintenance of the Quiescent Skeletal Muscle Stem Cell State The Dlk1-Gtl2 Locus Preserves LT-HSC Function by Inhibiting the PI3K-mTOR Pathway to Restrict Mitochondrial Metabolism A novel class of small RNAs bind to MILI protein in mouse testes Transcriptional and Chromatin Dynamics of Muscle Regeneration after Severe Trauma Notch signaling is necessary to maintain quiescence in adult muscle stem cells Dlk1 is necessary for proper skeletal muscle development and regeneration A role for the myogenic determination gene Myf5 in adult regenerative myogenesis Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting Isolation of Muscle Stem Cells from Mouse Skeletal Muscle Myf5 haploinsufficiency reveals distinct cell fate potentials for adult skeletal muscle stem cells Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Naive and primed murine pluripotent stem cells have distinct miRNA expression profiles Download references We acknowledge the Flow Cytometry Platform of the Technology Core-Center for Translational Science (CRT) and the Transcriptome and EpiGenome Platform of the Center for Innovation & Technological Research at Institut Pasteur for support in conducting this study Centre National pour la Recherche Scientifique and the Agence Nationale de la Recherche (Laboratoire d’Excellence Revive the European Research Council (Advanced Research Grant 332893) and the French Muscular Dystrophy Association (AFM-Téléthon) was funded by the Fondation pour la Recherche Médicale (FRM) We thank the ABC Facility of ANEXPLO Toulouse for mouse husbandry Present address: Département de Cancérologie de l’Enfant et de l’Adolescent & UMR8203 “Vectorologie et Thérapeutiques Anticancéreuses” Department of Developmental & Stem Cell Biology Barbara Gayraud-Morel & Shahragim Tajbakhsh Laboratoire de Biologie Moléculaire Eucaryote The authors declare no competing interests Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-018-21991-w Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Journal of Muscle Research and Cell Motility (2024) Sign up for the Nature Briefing newsletter — what matters in science