Abstract Skeletal muscle formation involves tight interactions between muscle cells and associated connective tissue fibroblasts. Every muscle displays the same type of organisation, they are innervated in the middle and attached at both extremities to tendons. Myonuclei are heterogeneous along myotubes and regionalised according to these middle and tip domains. During development, as soon as myotubes are formed, myonuclei at muscle tips facing developing tendons display their own molecular program. In addition to molecular heterogeneity, a subset of tip myonuclei has a fibroblastic origin different to the classical somitic origin, highlighting a cellular heterogeneity of myonuclei in foetal myotubes. To gain insights on the functional relevance of myonucleus heterogeneity during limb development, we used 2D culture and co-culture systems to dissociate autonomous processes (occurring in 2D-cultures) from 3D-spatial environment of tissue development. We also assessed the role of mechanical parameters in myonucleus heterogeneity in paralysed limb muscles. The regionalisation of cellular heterogeneity was lost in 2D cell culture systems and paralyzed muscles. The molecular signature of MTJ myonuclei was also lost a dish and paralysed muscles indicating a requirement of spatial and mechanical input for MTJ formation. Tip genes that maintain their expression in a dish are involved in myofibrillogenesis and myotube attachments. The behaviour of regionalized markers in cultured myotubes and paralyzed muscles allows us to deduce whether the genes intervene in myogenesis, myotube attachment or MTJ formation. Highlights The regionalisation of cellular heterogeneity of myonuclei is lost in cultured myotubes BMP signalling regulates fibroblast nucleus incorporation into cultured myotubes The molecular signature of MTJ myonuclei is lost in cultured myotubes and paralysed muscles Tip genes involved in myofibrillogenesis maintain their regionalised expression in cultured myotubes