Abstract
BackgroundComponents of the limb musculoskeletal system have distinct mesoderm origins. Limb skeletal muscles originate from somites, while the skeleton and attachments (tendons and connective tissues) derive from limb lateral plate. Despite distinct mesoderm origins, the development of muscle, skeleton and attachments is highly coordinated both spatially and temporally to ensure complete function of the musculoskeletal system. A system to study molecular interactions between somitic-derived tissues (muscles) and lateral-plate-derived tissues (skeletal components and attachments) during limb development is missing.ResultsWe designed a gene delivery system in chick embryos with the ultimate aim to study the interactions between the components of the musculoskeletal system during limb development. We combined the Tol2 genomic integration system with the viral T2A system and developed new vectors that lead to stable and bicistronic expression of two proteins at comparable levels in chick cells. Combined with limb somite and lateral plate electroporation techniques, two fluorescent reporter proteins were co-expressed in stoichiometric proportion in the muscle lineage (somitic-derived) or in skeleton and their attachments (lateral-plate-derived). In addition, we designed three vectors with different promoters to target muscle cells at different steps of the differentiation process.ConclusionLimb somite electroporation technique using vectors containing these different promoters allowed us to target all myogenic cells, myoblasts or differentiated muscle cells. These stable and promoter-specific vectors lead to bicistronic expression either in somitic-derived myogenic cells or lateral plate-derived cells, depending on the electroporation sites and open new avenues to study the interactions between myogenic cells and tendon or connective tissue cells during limb development.
Highlights
Components of the limb musculoskeletal system have distinct mesoderm origins
Stable and bicistronic expression of TdTomato and enhanced Green Fluorescent Protein (EGFP) fluorescent reporter proteins using the Tol2 transposon and the viral 2A peptide systems We previously designed stable vectors based on the Tol2 transposon, which allowed us to stably misexpress genesof-interest in chick embryos [44]
In order to stably integrate this cassette into the chick genome, we inserted the CMV/βactin promoter and the bicistronic cassette into the stable vector based on the Tol2 transposon system [44]
Summary
Components of the limb musculoskeletal system have distinct mesoderm origins. Limb skeletal muscles originate from somites, while the skeleton and attachments (tendons and connective tissues) derive from limb lateral plate. The development of muscle, skeleton and attachments is highly coordinated both spatially and temporally to ensure complete function of the musculoskeletal system. A system to study molecular interactions between somitic-derived tissues (muscles) and lateral-plate-derived tissues (skeletal components and attachments) during limb development is missing. Myogenic cells originate from somites, while components of the skeletal system originate from limb lateral plate mesoderm [1,2,3,4]. Embryonic myogenesis establishes the scaffold of muscles, while foetal myogenesis ensures muscle growth and maturation [5, 6] Both embryonic and foetal myogenesis rely on muscle progenitors that express the Paired homeobox transcription factors Pax and Pax7 [7]. Once the muscle differentiation process has started, muscle masses split progressively to give rise to individualised limb skeletal muscles [12]
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