- Histological, transcriptomic and in vitro analysis reveal an intrinsic activated state of myogenic precursors in hyperplasic muscle of trout. - For this purpose, we compared in situ proliferation, in vitro cell behavior and transcriptomic profile of myogenic precursors originating from hyperplasic muscle of juvenile trout (JT) and from non-hyperplasic muscle of fasted juvenile trout (FJT) and adult trout (AT).. - Results: For the first time, we showed that myogenic precursors proliferate in hyperplasic muscle from JT as shown by in vivo BrdU labeling. - In line with our transcriptomic data, in vitro JT myogenic precursors displayed higher proliferation and differentiation capacities than FJT and AT myogenic precursors.. - Muscle hyperplasia requires muscle stem cells, also called satellite cells [10] which are localized between. - Once activated during de- velopment, growth or after muscle injury, myogenic precursors proliferate and differentiate to eventually form nascent myofibres [11, 12]. - Myogenic differenti- ation is associated with the expression of the myogenic regulatory factor MyoD [13]. - Then, the expression of myogenin at the onset of the satellite cell differentiation triggers the expression of fusogenic and structural protein such as myomaker and myosin heavy chain [14, 15].. - Satellite cells have been clearly identified in situ in muscle of carp [16] and zebrafish [17]. - Myogenic precursors proliferate in hyperplasic muscle during post-larval growth. - In order to quantify the number of proliferative satellite cells in trout of 2 g, 500 g and fasted trout of 5 g, we devel- oped immunofluorescence analysis to locate proliferative nuclei in satellite cell position, i.e. - 1 Quantification of satellite cells proliferation in hyperplasic and non-hyperplasic muscle of trout (a) Muscle cross sections stained with anti-laminin (red) and anti-BrdU (green) in trout of 2 g, 500 g and of 3-weeks fasted trout (5 g). - SD) in satellite cells position, (under the basal lamina), in white muscle of trout weighing 2 g, 500 g and of 3-weeks fasted trout weighing 5 g. - Myogenic precursors extracted from hyperplasic and non- hyperplasic trout muscles exhibit distinct transcriptome To better known the intrinsic molecular properties of myo- genic precursors from hyperplasic muscle, we compared the transcriptome of myogenic precursors extracted from juvenile trout (JT) displaying hyperplasic muscle growth with that of myogenic precursors extracted from non-hyperplasic muscle resulting from fasted juven- ile trout (FJT) and adult trout (AT). - For this purpose, we first compared gene expression profiles between myogenic precursors from FJT and AT in order to identify genes whose differential expression would be specifically related to age or fasting. - The cluster 1 comprised 1865 genes up regulated in JT myogenic precursors and cluster 2 comprised 758 genes down regulated in JT myogenic precursors.. - JT myogenic precursors exhibit transcriptomic signature of activated state cell. - DAVID analysis of the 1206 eligible genes from cluster 1 revealed significant enrichment (Table 1) in genes involved in translation (p = 2.8E − 26. - to the formation of the myofibrils (i.e, 8 genes encoding myosins, 5 genes encoding troponins, 2 genes encoding capping protein muscle Z-line and 3 genes encoding tropomyosins). - 2 Hierarchical clustering of differentially expressed genes between JT myogenic precursors and FJT and AT myogenic precursors. - Each row represents the expression pattern of a single gene and each column corresponds to a single sample: columns 1 to 5: JT myogenic precursors sampled. - columns 6 to 10: FJT myogenic precursors sampled. - and columns 11 to 15: AT myogenic precursors sampled.. - Genes associated with the quiescent state are down regulated in JT myogenic precursors. - Cluster 2 comprised genes that were down regulated in JT myogenic precursors compared to both FJT and AT myogenic precursors. - In this cluster, we identified genes of the Notch pathway, suggesting a repression of quies- cent state. - Moreover, a marker of quiescent satellite cells [32], nestin was down regulation in JT myogenic precursors. - We also observed a global repression of the TGFβ pathway in JT myogenic precursors. - Indeed, 7 genes involved in TGFβ pathway were down regulated in JT myogenic precursors (tgfb2, tgfbr1, bmpr2b, bmpr1bb, smad3b, smad6a and acvrrl1) whereas 5 in- hibitors of TGFβ pathway were up regulated in JT myo- genic precursors (Bmp7a, gremlin2, dcn, fstl1b and fsta).. - JT myogenic precursors have an enhanced intrinsic capacity for in vitro proliferation. - To know more about the intrinsic molecular properties of myogenic precursors of hyperplasic muscle compared to myogenic precursors of non-hyperplasic muscle, we car- ried out a primary cell culture of myogenic progenitors extracted from JT, FJT and AT conditions. - Cell prolifer- ation assays using BrdU showed a higher proliferation rate of JT myogenic precursors (40.1%) after two days of culture compared to FJT (0.8%) and AT (10.3%) myogenic precursors (Fig. - In JT myogenic precursors the prolifera- tion rate increased from D2 to reach a maximum rate at D5 with 61.4% of BrdU positive nuclei, then the pro- liferation rate decreased from D8 to 42.4% to D11 to 31.4%. - For AT myogenic precursors, the proliferation rate increased at D5 to 48.3% to almost Table 1 Functional categories inferred from up regulated genes in JT myogenic precursors. - Table of the most significant Gene Ontology terms in Biological Process and Cellular Component that were found following functional enrichment analysis (DAVID Software 6.7) among genes up regulated in JT myogenic precursors. - 3 Hierarchical clustering of differentially expressed cell cycle genes between JT myogenic precursors and FJT and AT myogenic precursors. - and columns 11 to 15: AT myogenic precursors sampled. - 4 Hierarchical clustering of differentially expressed myogenic genes between JT myogenic precursors and FJT and AT myogenic precursors. - reach the proliferation rate in JT myogenic precursors and decreased from D8 to 31.6% and at D11 to 19.1%.. - Thus, the kinetic of proliferation of the AT precursors was close to that one of JT but with a lower rate from D5 to D11. - Overall, myogenic precursors of JT exhibit a global enhanced proliferation capacity under in vitro conditions compared to FJT and AT.. - JT myogenic precursors have an enhanced capacity for in vitro myogenic differentiation. - To go further on the characterization of the intrinsic molecular properties of myogenic precursors of hyper- plastic muscle, we quantified the in vitro differentiation capacities of JT, FJT and AT myogenic precursors. - In JT myogenic precursors the differentiation rate increased at D5 to 11.6%, D8 to 24.4% and reach a maximum rate at D11 with 28% of nuclei contained in myosin positive cells. - For AT myogenic precursors, no significant increase of the differ- entiation rate was observed even after 11 days of culture.. - Overall, JT myogenic precursors exhibited a global en- hanced differentiation capacity under in vitro conditions.. - Evaluation of the expression level by qPCR of myo- genin and myomaker after 2 days in cell culture validated. - Indeed, the expression of myogenin and myomaker were higher in JT myogenic precursors compared to AT and FJT myogenic precursors. - In addition, the expression level of myogenin and myomaker after 8 days in culture increased in FJT myogenic precursors. - Overall, qPCR data validated our previous results with JT myo- genic precursors as more engaged in differentiation pro- gram than AT and FJT myogenic precursors.. - For this purpose, we examined proliferation of trout satellite cells in vivo and compared gene expres- sion profiling and in vitro myogenic potential of satellite cells extracted from juvenile trout muscle displaying intense hyperplastic growth (JT), with satellite cells ex- tracted from trout muscle that no longer exhibited muscle hyperplasia, namely fasted juvenile trout (FJT) and adult trout (AT).. - Many studies on mammalian isolated satellite cells were carried out on cells directly isolated from muscle and purified by FACS using fluorescent reporters or cell surface marker [33]. - As these technologies cannot yet be used in trout fish, we took advantage of the specific. - 5 Proliferation rate of JT, FJT and AT myogenic precursors after days of plating (D2, D5, D8 and D11). - 6 Differentiation rate of JT, FJT and AT myogenic precursors after days in culture (D2, D5, D8 and D11). - SD) of the percentage of nuclei contained in MyHC positive cells for each condition at D2, D5, D8 and D11.. - adhesion of satellite cells on laminin substrate to enrich them in culture [19, 34]. - Although it has been reported that isolation procedures alter gene expression of myo- genic precursors [35, 36], we assumed in this study that the differential ex vivo properties of trout satellite cells originating either from hyperplastic or non-hyperplastic muscle, somehow reflect intrinsic differences preexisting before their extraction from muscle.. - First, we sought to identify and quantify proliferative satellite cells in muscle of growing versus non-growing trout using in vivo BrdU injection followed by double immuno-labeling of laminin and BrdU. - This shows that fish hyperplastic muscle contains proliferative satellite cells well after hatching, what sharply contrasts with the mi- totic quiescence of satellite cells located in mature mouse muscle [38].. - Relative to satellite cells from non-hyperplastic muscle, satellite cells from juvenile trout were found to exhibit. - Mitochondrial biogenesis has been associated with the shift from quiescence to proliferation of satellite cells [39, 40]. - In keeping with this, our result that matches meta-analyses of multiple transcriptomes revealing low expression of genes associated with oxidative phosphor- ylation in mouse quiescent satellite cells [41], supports the view that JT cells are intrinsically activated com- pared to satellite cells from non-hyperplastic muscle.. - Other major functional categories inferred from genes up-regulated in myogenic precursors derived from hy- perplastic muscle were related to DNA replication and cell cycle. - In keeping with this, it is interesting to note that mito- chondrial activity, which is higher in JT satellite cells relative to FJT and AT cells, has been reported to posi- tively regulate myogenesis [43]. - These results are in agreement with data obtained in mouse showing an up regulation of notch and Hey genes in quiescent satellite cells [45]. - In addition, the up regulation of several genes involved in TGFbeta pathway was in line with a repression of differentiation of myogenic precursors [46]. - Indeed, we notably observed an up-regulation of BMP receptor type 1 which knock-down in mouse satellite cells caused prema- ture myogenic differentiation [47]. - All these data support the view that satellite cells extracted from muscle of fasted trout or adult trout are close to a quiescent state com- pared to satellite cells from juvenile trout.. - Another major result of our study was that behavior of satellite cells from hyperplastic muscle quite differs from that of satellite cells extracted from non-hyperplastic muscle. - Specifically, we found that cultured JT myogenic precursors exhibited higher proliferation rate and dif- ferentiation capacities than FJT and AT myogenic pre- cursors. - 7 Quantification of the expression of myogenin and myomaker in JT, FJT and AT myogenic precursors. - hyperplastic muscle of juvenile trout are intrinsically more potent to form myofibres than satellite cells from non-hyperplastic muscle. - However, differentiation of FJT myogenic precursors was only delayed because qPCR and immunofluores- cence analysis showed resumption of differentiation after 8 days of culture. - In contrast, AT myogenic pre- cursors exhibited a strong proliferation capacity in vitro suggesting that AT myogenic precursors were not de- layed or close to senescent state that could explain their low myogenic capacities as observed in old mouse [48].. - The satellite cells from muscle of trout juveniles exhibit in vivo and ex vivo features of activation that are not found in satellite cells isolated from non-hyperplastic muscle. - Thus, muscle hyperplastic growth in fish likely relates to the fact that satellite cells in these animals are intrinsically potent to form myofibres well after hatching.. - was used following the recommenda- tions of manufacturer to measure the proliferation of the cells. - For all studies, myogenic precursors were isolated from dorsal part of the white muscle of juvenile trout (5 g, JT), of 3–4 weeks fasted juvenile rainbow trout (5 g, FJT) and of adult rainbow trout (1.5-2 kg, AT) as previ- ously described [19]. - Isolated myogenic precursors were plated on poly-L-lysine and laminin-coated plates at 80,000 cells per cm 2 for every analysis except to prolif- eration measurement which were 60,000 cells per cm 2. - Relative quantification of the target gene transcripts was made using 18S gene expression as reference. - The efficiency of the qPCR was about 80–100%. - Data are pre- sented as the ratio between the relative expression of the target gene and 18S.. - A BrdU labeling and detection kit Sigma) was used following the recommendations of manufacturer to measure the proliferation of the cells. - A Kruskal-Wallis test with a Dunn ’ s post hoc multiple comparisons test was performed on in situ satellite cells proliferation data. - The funders did not intervene in the design, analysis and interpretation of the data.. - Coordinated regulation of the GH/IGF system genes during refeeding in rainbow trout (Oncorhynchus mykiss). - Temporal expression of regulatory and structural muscle proteins during Myogenesis of satellite cells on isolated adult rat fibers. - In vitro characterization of proliferation and differentiation of trout satellite cells. - compensatory growth of the trout (Oncorhynchus mykiss). - STAT3 regulates self- renewal of adult muscle satellite cells during injury-induced muscle regeneration. - An adult tissue-specific stem cell in its niche: a gene profiling analysis of in vivo quiescent and activated muscle satellite cells. - Expression of the dermatomyositis autoantigen TIF1 γ in regenerating muscle. - Nestin- GFP reporter expression defines the quiescent state of skeletal muscle satellite cells. - Satellite cells are mitotically quiescent in mature mouse muscle: an EM and radioautographic study. - A critical requirement for notch signaling in maintenance of the quiescent skeletal muscle stem cell state. - Molecular signature of quiescent satellite cells in adult skeletal muscle. - BMP signalling permits population expansion by preventing premature myogenic differentiation in muscle satellite cells. - Gene expression profiling of the hyperplastic growth zones of the late trout embryo myotome using laser capture microdissection and microarray analysis. - Gene expression profiling of trout regenerating muscle reveals common transcriptional signatures with hyperplastic growth zones of the post-embryonic myotome
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