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During myogenesis, muscle precursors become divided into either fast- or slow-twitch fibres, which in the zebrafish occupy distinct domains in the embryo. Genes encoding sarcomeric proteins specific for fast or slow fibres are frequently used as lineage markers. In an attempt to identify and evaluate early definitive markers for cells in the fast-twitch pathway, we analysed genes encoding proteins contributing to the fast sarcomeric structures. The previously uncharacterized zebrafish alkali-like myosin light chain gene (myl1) was found to be expressed exclusively in cells in the fast-twitch pathway initiated at an early stage of fast fibre differentiation. Myl1 was expressed earlier, and in a more fibre type restricted manner, than any of the previously described and frequently used fast myosin light and heavy chain and troponin muscle markers mylz2, mylz3, tnni2, tnnt3a, fMyHC1.3. In summary, this study introduces a novel marker for early differentiating fast muscle cells.

In the embryonic zebrafish, skeletal muscle fibres are formed from muscle progenitors in the paraxial mesoderm. The embryonic myotome is mostly constituted of fast-twitch-specific fibres, which are formed from a fast-specific progenitor cell pool. The most lateral fraction of the fast domain in the myotome of zebrafish embryos derives from the Pax7-positive dermomyotome-like cells. In this study, we show that two genes, belonging to the sine oculus class 1 (six1) genes (six1a and six1b), are both essential for the regulation of Pax7(+) cell proliferation and, consequently, in their differentiation during the establishment of the zebrafish dermomyotome. In both six1a and six1b morphant embryos, Pax7(+) cells are initially formed but fail to proliferate, as detected by reduced levels of the proliferation marker phosphohistone3 and reduced brdU incorporation. In congruence, overexpression of six1a or six1b leads to increased Pax7(+) cell number and reduced or alternatively delayed fibre cell differentiation. Bone morphogenetic protein signalling has previously been suggested to inhibit differentiation of Pax7(+) cells in the dermomyotome. Here we show that the remaining Pax7(+) cells in six1a and six1b morphant embryos also have significantly reduced pSmad1/5/8 levels and propose that this leads to a reduced proliferative activity, which may result in a premature differentiation of Pax7(+) cells in the zebrafish dermomyotome. In summary, we show a mechanism for Six1a and Six1b in establishing the Pax7(+) cell derived part of the fast muscle and suggest new important roles for Six1 in the regulation of the Pax7(+) muscle cell population through pSmad1/5/8 signalling.

Numerous muscle lineages are formed during myogenesis within both slow-and fast-specific cell groups. In this study, we show that six fast muscle-specific myosin heavy chain genes have unique expression patterns in the zebrafish embryo. The expression of tail-specific myosin heavy chain (fmyhc2.1) requires wnt signaling and is essential for fast muscle organization within the tail. Retinoic acid treatment results in reduced wnt signaling, which leads to loss of the fmyhc2.1 domain. Retinoic acid treatment also results in a shift of muscle identity within two trunk domains defined by expression of fmyhc1.2 and fmyhc1.3 in favor of the anteriormost myosin isoform, fmyhc1.2. In summary, we identify new muscle domains along the anteroposterior axis in the zebrafish that are defined by individual nonoverlapping, differentially regulated expression of myosin heavy chain isoforms.