Erratum: Canonical Wnt signalling regulates nuclear export of Setdb1 during skeletal muscle terminal differentiation.

[This corrects the article DOI: 10.1038/celldisc.2016.37.].

Canonical Wnt signalling regulates nuclear export of Setdb1 during skeletal muscle terminal differentiation.

The histone 3 lysine 9 methyltransferase Setdb1 is essential for both stem cell pluripotency and terminal differentiation of different cell types. To shed light on the roles of Setdb1 in these mutually exclusive processes, we used mouse skeletal myoblasts as a model of terminal differentiation. Ex vivo studies on isolated single myofibres showed that Setdb1 is required for adult muscle stem cells expansion following activation. In vitro studies in skeletal myoblasts confirmed that Setdb1 suppresses terminal differentiation. Genomic binding analyses showed a release of Setdb1 from selected target genes upon myoblast terminal differentiation, concomitant to a nuclear export of Setdb1 to the cytoplasm. Both genomic release and cytoplasmic Setdb1 relocalisation during differentiation were dependent on canonical Wnt signalling. Transcriptomic assays in myoblasts unravelled a significant overlap between Setdb1 and Wnt3a regulated genetic programmes. Together, our findings revealed Wnt-dependent subcellular relocalisation of Setdb1 as a novel mechanism regulating Setdb1 functions and myogenesis.

ß-Catenin Activation in Muscle Progenitor Cells Regulates Tissue Repair.

Skeletal muscle regeneration relies on a pool of resident muscle stem cells called satellite cells (MuSCs). Following injury-induced destruction of the myofibers, quiescent MuSCs are activated and generate transient amplifying progenitors (myoblasts) that will fuse to form new myofibers. Here, we focus on the canonical Wnt signaling pathway and find that either conditional ß-catenin disruption or activation in adult MuSCs results in perturbation of muscle regeneration. Using both in vivo and in vitro approaches, we observed that myoblasts lacking ß-catenin show delayed differentiation, whereas myoblasts with constitutively active ß-catenin undergo precocious growth arrest and differentiation. Transcriptome analysis further demonstrated that Wnt/ß-catenin signaling interacts with multiple pathways and, more specifically, TGF-ß signaling. Indeed, exogenous TGF-ß2 stimulation restores the regenerative potential of muscles with targeted ß-catenin disruption in MuSCs. We conclude that a precise level of ß-catenin activity is essential for regulating the amplification and differentiation of MuSC descendants during adult myogenesis.

Distinct genetic programs guide Drosophila circular and longitudinal visceral myoblast fusion.

BACKGROUND: The visceral musculature of Drosophila larvae comprises circular visceral muscles tightly interwoven with longitudinal visceral muscles. During myogenesis, the circular muscles arise by one-to-one fusion of a circular visceral founder cell (FC) with a visceral fusion-competent myoblast (FCM) from the trunk visceral mesoderm, and longitudinal muscles arise from FCs of the caudal visceral mesoderm. Longitudinal FCs migrate anteriorly under guidance of fibroblast growth factors during embryogenesis; it is proposed that they fuse with FCMs from the trunk visceral mesoderm to give rise to syncytia containing up to six nuclei. RESULTS: Using fluorescence in situ hybridization and immunochemical analyses, we investigated whether these fusion events during migration use the same molecular repertoire and cellular components as fusion-restricted myogenic adhesive structure (FuRMAS), the adhesive signaling center that mediates myoblast fusion in the somatic mesoderm. Longitudinal muscles were formed by the fusion of one FC with Sns-positive FCMs, and defects in FCM specification led to defects in longitudinal muscle formation. At the fusion sites, Duf/Kirre and the adaptor protein Rols7 accumulated in longitudinal FCs, and Blow and F-actin accumulated in FCMs. The accumulation of these four proteins at the fusion sites argues for FuRMAS-like adhesion and signaling centers. Longitudinal fusion was disturbed in rols and blow single, and scar wip double mutants. Mutants of wasp or its interaction partner wip had no defects in longitudinal fusion. CONCLUSIONS: Our results indicated that all embryonic fusion events depend on the same cell-adhesion molecules, but that the need for Rols7 and regulators of F-actin distinctly differs. Rols7 was required for longitudinal visceral and somatic myoblast fusion but not for circular visceral fusion. Importantly, longitudinal fusion depended on Kette and SCAR/Wave but was independent of WASp-dependent Arp2/3 activation. Thus, the complexity of the players involved in muscle formation increases from binucleated circular muscles to longitudinal visceral muscles to somatic muscles.

The Hydra FGFR, Kringelchen, partially replaces the Drosophila Heartless FGFR.

Fibroblast growth factor receptors (FGFR) are highly conserved receptor tyrosine kinases, and evolved early in metazoan evolution. In order to investigate their functional conservation, we asked whether the Kringelchen FGFR in the freshwater polyp Hydra vulgaris, is able to functionally replace FGFR in fly embryos. In Drosophila, two endogenous FGFR, Breathless (Btl) and Heartless (Htl), ensure formation of the tracheal system and mesodermal cell migration as well as formation of the heart. Using UAS-kringelchen-5xmyc transgenic flies and targeted expression, we show that Kringelchen is integrated correctly into the cell membrane of mesodermal and tracheal cells in Drosophila. Nevertheless, Kringelchen expression driven in tracheal cells failed to rescue the btl (LG19) mutant. The Hydra FGFR was able to substitute for Heartless in the htl (AB42) null mutant; however, this occurred only during early mesodermal cell migration. Our data provide evidence for functional conservation of this early-diverged FGFR across these distantly related phyla, but also selectivity for the Htl FGFR in the Drosophila system.

Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7.

Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell-cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles.

The syncytial visceral and somatic musculature develops independently of ß3-Tubulin during Drosophila embryogenesis, while maternally supplied ß1-Tubulin is stable until the early steps of myoblast fusion.

Microtubules are necessary for fusion and elongation of vertebrate muscle cells. In Drosophila, several isoforms of ß-Tubulin, the functional subunit of microtubules, are expressed in different tissues of the developing embryo, while solely the ß3-Tubulin isoform is detected in large amounts during differentiation of the somatic and visceral musculature. Here we show the unexpected result that all mesodermal tissues develop correctly in ß3-Tubulin loss of function mutants. Furthermore, we show that ß2-Tubulin transcripts are not detectable in embryos and an exceptional zygotic ß1-Tubulin expression in ß3-Tubulin mutants cannot be observed. Nevertheless, a maternally contributed ß1-Tubulin-GFP fusion protein (from protein trap collection, Buszczak et al., 2007, Genetics 175, 1505-1531) acts in a dominant negative way, disturbing embryonic development from early stages on. This effect can be observed to the same extent in a zygotic ß3-Tubulin mutant situation. Our results indicate that the maternally supplied ß1-Tubulin based microtubule network is sufficient for myoblast fusion, myotube elongation and sarcomere formation both during visceral and somatic muscle development in Drosophila embryogenesis.