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A myogenic factor from sea urchin embryos capable of programming muscle differentiation in mammalian cells.

ABSTRACT: Using the basic helix-loop-helix domain of the myogenic factor myogenin as a probe, we identified a clone from a sea urchin cDNA library with considerable sequence similarity to the vertebrate myogenic factors. This cDNA, sea urchin myogenic factor 1 (SUM-1), transactivated a muscle creatine kinase-chloramphenicol acetyltransferase reporter gene in 10T1/2 fibroblasts to a level comparable to that of the vertebrate myogenic factors. In addition, bacterially expressed beta-galactosidase-SUM-1 fusion protein interacted directly with the kappa E-2 site in the muscle creatine kinase enhancer core as assayed by electrophoretic mobility shift assays. Stably transfected SUM-1 activated the muscle differentiation program and converted 10T1/2 cells from fibroblasts to myotubes. In sea urchin embryos, SUM-1 RNA was not detected before gastrulation. It accumulated to its highest levels during the prism stage when myoblasts were first detected by myosin immunostaining and then diminished as myocytes differentiated. SUM-1 protein was localized in secondary mesenchyme cells when they could first be identified as muscle cells by myosin immunostaining. These results implicate SUM-1 as a regulatory factor involved in the early decision of a pluripotent secondary mesenchyme cell to convert to a myogenic fate. SUM-1 is an example of an invertebrate myogenic factor that is capable of functioning in mammalian cells.

SUBMITTER: Venuti JM

PROVIDER: S-EPMC52054 | biostudies-other | 1991 Jul

REPOSITORIES: biostudies-other

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A myogenic factor from sea urchin embryos capable of programming muscle differentiation in mammalian cells.

Venuti J M JM Goldberg L L Chakraborty T T Olson E N EN Klein W H WH

Proceedings of the National Academy of Sciences of the United States of America 19910701 14

Using the basic helix-loop-helix domain of the myogenic factor myogenin as a probe, we identified a clone from a sea urchin cDNA library with considerable sequence similarity to the vertebrate myogenic factors. This cDNA, sea urchin myogenic factor 1 (SUM-1), transactivated a muscle creatine kinase-chloramphenicol acetyltransferase reporter gene in 10T1/2 fibroblasts to a level comparable to that of the vertebrate myogenic factors. In addition, bacterially expressed beta-galactosidase-SUM-1 fusi ...[more]

PMID: 2068103

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Project description:The sea urchin Paracentrotus lividus (Lamarck, 1816) is a keystone herbivore in the Mediterranean Sea due to its ability to transform macroalgal-dominated communities into barren areas characterized by increased cover of bare substrates and encrusting coralline algae, reduced biodiversity and altered ecosystem functions. P. lividus is also an excellent animal model for toxicology, physiology and biology investigations having been used for more than a century as a model for embryological studies with synchronously developing embryos which are easy to manipulate and analyze for morphological aberrations. Despite its importance for the scientific community, the complete genome is still not fully annotated. To date, only a few molecular tools are available and a few Next Generation Sequencing (NGS) studies have been performed. Here we aimed at setting-up an RNA extraction method to obtain high quality and sufficient quantity of RNA for NGS from P. lividus embryos at the pluteus stage. We compared five different RNA extraction protocols from four different pools of plutei (500, 1000, 2500 and 5000 embryos): TRIzol®, and four widely-used Silica Membrane kits, GenElute™ Mammalian Total RNA Miniprep Kit, RNAqueous® Micro Kit, RNeasy® Micro Kit and Aurum™ Total RNA Mini Kit. The quantity of RNA isolated was evaluated using NanoDrop. The quality, considering the purity, was measured as A260/A280 and A260/230 ratios. The integrity was measured by RNA Integrity Number (RIN). Our results demonstrated that the most efficient procedures were GenElute, RNeasy and Aurum, producing a sufficient quantity of RNA for NGS. The Bioanalyzer profiles and RIN values revealed that the most efficient methods guaranteeing for RNA integrity were RNeasy and Aurum combined with an initial preservation in RNAlater. This research represents the first attempt to standardize a method for high-quality RNA extraction from sea urchin embryos at the pluteus stage, providing a new resource for this established model marine organism.

Project description:BackgroundSarcopenia and osteoporosis frequently co-occur in the elderly and have common pathophysiological determinants. Slit guidance ligand 3 (SLIT3) has been recently discovered as a novel therapeutic factor against osteoporosis, and a SLIT3 fragment containing the second leucine-rich repeat domain (LRRD2) had a therapeutic efficacy against osteoporosis. However, a role of SLIT3 in the skeletal muscle is unknown.MethodsSkeletal muscle mass, strength, and/or physical activity were evaluated in Slit3-/- , ovariectomized, and aged mice, based on the measurements of muscle weight and grip strength, Kondziella's inverted hanging test, and/or wheel-running test. Skeletal muscles were also histologically evaluated by haematoxylin and eosin staining and/or immunofluorescence. The ovariectomized and aged mice were intravenously injected with recombinant SLIT3 LRRD2 for 4 weeks. C2C12 cells were used to know cellular effects of SLIT3, such as in vitro myogenesis, fusion, cell viability, and proliferation, and also used to evaluate its molecular mechanisms by immunocytochemistry, immunoprecipitation, western blotting, real-time PCR, siRNA transfection, and receptor-ligand binding ELISA.ResultsSlit3-deficient mice exhibited decreased skeletal muscle mass, muscle strength, and physical activity. The relative masses of gastrocnemius and soleus were lower in the Slit3-/- mice (0.580 ± 0.039% and 0.033 ± 0.003%, respectively) than those in the WT littermates (0.622 ± 0.043% and 0.038 ± 0.003%, respectively) (all, P < 0.05). Gastrocnemius of Slit3-/- mice showed the reduced number of Type I and Type IIa fibres (all, P < 0.05), but not of Type IIb and Type IIx fibres. SLIT3 activated β-catenin signalling by promoting its release from M-cadherin, thereby increasing myogenin expression to stimulate myoblast differentiation. In vitro experiments involving ROBO2 expression, knockdown, and interaction with SLIT3 indicated that ROBO2 functions as a SLIT3 receptor to aid myoblast differentiation. SLIT3 LRRD2 dissociated M-cadherin-bound β-catenin and up-regulated myogenin expression to increase myoblast differentiation, in a manner similar to full-length SLIT3. Systemic treatment with SLIT3 LRRD2 increased skeletal muscle mass in both ovariectomized and aged mice (all, P < 0.05). The relative masses of gastrocnemius and soleus were higher in the treated aged mice (0.548 ± 0.045% and 0.033 ± 0.005%, respectively) than in the untreated aged mice (0.508 ± 0.016% and 0.028 ± 0.003%, respectively) (all, P < 0.05). SLIT3 LRRD2 treatment increased the hanging duration of the aged mice by approximately 1.7-fold (P < 0.05).ConclusionsSLIT3 plays a sarcoprotective role by activating β-catenin signalling. SLIT3 LRRD2 can potentially be used as a therapeutic agent against muscle loss.

Dataset Information

A myogenic factor from sea urchin embryos capable of programming muscle differentiation in mammalian cells.

Publications

A myogenic factor from sea urchin embryos capable of programming muscle differentiation in mammalian cells.

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