A dynamic analysis of muscle fusion in the chick embryo

Abstract

Skeletal muscle development, growth and regeneration depend upon the ability of muscle cells to fuse into multinucleated fibers. Surprisingly little is known about the cellular events that underlie fusion during amniote development. Here, we have developed novel molecular tools to characterize muscle cell fusion during chick embryo development. We show that all cell populations arising from somites fuse, but each with unique characteristics. Fusion in the trunk is slow and independent of fiber length. By contrast, the addition of nuclei in limb muscles is three times more rapid than in trunk and is tightly associated with fiber growth. A complex interaction takes place in the trunk, where primary myotome cells from the medial somite border rarely fuse to one another, but readily do so with anterior and posterior border cells. Conversely, resident muscle progenitors actively fuse with one another, but poorly with the primary myotome. In summary, this study unveils an unexpected variety of fusion behaviors in distinct embryonic domains that is likely to reflect a tight molecular control of muscle fusion in vertebrates.

Making muscle: skeletal myogenesisin vivoandin vitro

“…Denetclaw et al, 1997;Gros et al, 2009). More cells are progressively added to the myotome by the other dermomyotomal lips (Gros et al, 2004) and these cells fuse to existing myocytes leading to the formation of slow MyHC + myofibers (Sieiro-Mosti et al, 2014) (Fig. 3A).…”

CRISPR mediated somatic cell genome engineering in the chicken

“…Also we required a system that was amenable to long-term analyses and be inducible, so that we could activate the CRISPR-mediated deletion at different stages of embryogenesis. We therefore designed an inducible CRISPR vector strategy, which combines features from the Tet-On Advanced system (Clontech), Cas9 and gRNA vectors (Mali et al, 2013a) with the Tol2 transposable elements (Sato et al, 2007;Serralbo et al, 2013;Sieiro-Mosti et al, 2014;Yokota et al, 2011).…”

The chicken embryo as an efficient model to test the function of muscle fusion genes in amniotes

“…In order to create the fusion-targeting doxycycline inducible system used here, new vectors were created (see Fig 1): i) The “Tol2 CAGGS-NLSmCherry IRES rtTA advanced” plasmid (Fig 1A) was made by replacing the mEGFP from “Tol2-CAGGS-NLSCherry-IRES-mEGFP” [25]with an rtTA (reverse tetracycline-controlled transactivator protein) Advanced sequence (Clontech) used in[28] and ii) the “Tol2 pBI Rac1 T17N EGFPcaax” and “Tol2 pBI Cdc42 T17N EGFPcaax” plasmids (Fig 1B) were created by cloning Rac1 T17N and Cdc42 T17N (see below) in the bidirectional “pBI-EGFP” used in [28]. The EGFP from pBI-EGFP was replaced by a membranal EGFPcaax and this was flanked by two sequences for the Tol2 transposable elements.…”

“…A single human influenza hemagglutinin (HA) tag was added in-frame at the c-terminus of each coding sequence. Finally, a “CAGGS Transposase” plasmid (Fig 1C) [25] was co-electroporated to ensure genomic integration of sequences described above.…”

“…Using the technique of in vivo electroporation in the chicken embryo, we have previously characterized muscle cell fusion during early embryonic development [25]. Here, we have tested whether it is a suitable and advantageous model to assay the function of candidate genes during muscle cell fusion.…”