In amniotes, it is widely accepted that WNTs secreted by the dorsal neural tube form a concentration gradient that regulates early somite patterning and myotome organization. Here we demonstrate in ...the chicken embryo that WNT protein is not secreted to act at a distance, but rather loaded onto migrating neural crest cells that deliver it to somites. Inhibiting neural crest migration or ablating their population has a profound impact on the WNT response in somites. Furthermore, we show that a central player in the efficient delivery of WNT to somites is the heparan sulfate proteoglycan GPC4, expressed by neural crest. Together, our data describe a novel mode of signaling whereby WNT proteins hitch a ride on migratory neural crest cells to pattern the somites at a distance from its source.
Fusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this ...process. Here, we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGFβ (SMAD2/3-dependent) signalling acts specifically and uniquely as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGFβ signalling in the embryonic muscle relies on a receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is likely restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion relies on TGFβ signalling to regulate its pace.
How cells in the embryo coordinate epithelial plasticity with cell fate decision in a fast changing cellular environment is largely unknown. In chick embryos, skeletal muscle formation is initiated ...by migrating Delta1-expressing neural crest cells that trigger NOTCH signaling and myogenesis in selected epithelial somite progenitor cells, which rapidly translocate into the nascent muscle to differentiate. Here, we uncovered at the heart of this response a signaling module encompassing NOTCH, GSK-3β, SNAI1 and β-catenin. Independent of its transcriptional function, NOTCH profoundly inhibits GSK-3β activity. As a result SNAI1 is stabilized, triggering an epithelial to mesenchymal transition. This allows the recruitment of β-catenin from the membrane, which acts as a transcriptional co-factor to activate myogenesis, independently of WNT ligand. Our results intimately associate the initiation of myogenesis to a change in cell adhesion and may reveal a general principle for coupling cell fate changes to EMT in many developmental and pathological processes.
In the embryo and in the adult, skeletal muscle growth is dependent on the proliferation and the differentiation of muscle progenitors present within muscle masses. Despite the importance of these ...progenitors, their embryonic origin is unclear. Here we use electroporation of green fluorescent protein in chick somites, video confocal microscopy analysis of cell movements, and quail-chick grafting experiments to show that the dorsal compartment of the somite, the dermomyotome, is the origin of a population of muscle progenitors that contribute to the growth of trunk muscles during embryonic and fetal life. Furthermore, long-term lineage analyses indicate that satellite cells, which are known progenitors of adult skeletal muscles, derive from the same dermomyotome cell population. We conclude that embryonic muscle progenitors and satellite cells share a common origin that can be traced back to the dermomyotome.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gene-targeted knockout technologies are invaluable tools for understanding the functions of genes in vivo. CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a ...wide spectrum of organisms. Here, we combined CRISPR with in vivo electroporation in the chicken embryo to efficiently target the transcription factor PAX7 in tissues of the developing embryo. This approach generated mosaic genetic mutations within a wild-type cellular background. This series of proof-of-principle experiments indicate that in vivo CRISPR-mediated cell genome engineering is an effective method to achieve gene loss-of-function in the tissues of the chicken embryo and it completes the growing genetic toolbox to study the molecular mechanisms regulating development in this important animal model.
•We combined CRISPR and electroporation in the chicken embryo.•This led to efficient somatic genetic mutations in its tissues.•This completes the genetic toolbox to study development in amniotes.
Due to its amenability to manipulations, to live observation and its striking similarities to mammals, the chicken embryo has been one of the major animal models in biomedical research. Although it ...is technically possible to genome-edit the chicken, its long generation time (6 months to sexual maturity) makes it an impractical lab model and has prevented it widespread use in research. The Japanese quail (
) is an attractive alternative, very similar to the chicken, but with the decisive asset of a much shorter generation time (1.5 months). In recent years, transgenic quail lines have been described. Most of them were generated using replication-deficient lentiviruses, a technique that presents diverse limitations. Here, we introduce a novel technology to perform transgenesis in quail, based on the in vivo transfection of plasmids in circulating Primordial Germ Cells (PGCs). This technique is simple, efficient and allows using the infinite variety of genome engineering approaches developed in other models. Furthermore, we present a website centralizing quail genomic and technological information to facilitate the design of genome-editing strategies, showcase the past and future transgenic quail lines and foster collaborative work within the avian community.
Satellite cells are resident stem cells of skeletal muscle, supplying myoblasts for post-natal muscle growth, hypertrophy and repair. Many regulatory networks control satellite cell function, which ...includes EGF signalling via the ErbB family of receptors. Here we investigated the role of ErbB3 binding protein-1 (Ebp1) in regulation of myogenic stem cell proliferation and differentiation. Ebp1 is a well-conserved DNA/RNA binding protein that is implicated in cell growth, apoptosis and differentiation in many cell types. Of the two main Ebp1 isoforms, only p48 was expressed in satellite cells and C2C12 myoblasts. Although not present in quiescent satellite cells, p48 was strongly induced during activation, remaining at high levels during proliferation and differentiation. While retroviral-mediated over-expression of Ebp1 had only minor effects, siRNA-mediated Ebp1 knockdown inhibited both proliferation and differentiation of satellite cells and C2C12 myoblasts, with a clear failure of myotube formation. Ebp1-knockdown significantly reduced ErbB3 receptor levels, yet over-expression of ErbB3 in Ebp1 knockdown cells did not rescue differentiation. Ebp1 was also expressed by muscle cells during developmental myogenesis in mouse. Since Ebp1 is well-conserved between mouse and chick, we switched to chick to examine its role in muscle formation. In chick embryo, Ebp1 was expressed in the dermomyotome, and myogenic differentiation of muscle progenitors was inhibited by specific Ebp1 down-regulation using shRNA electroporation. These observations demonstrate a conserved function of Ebp1 in the regulation of embryonic muscle progenitors and adult muscle stem cells, which likely operates independently of ErbB3 signaling.
•ErbB3 binding protein-1 (Ebp1) is expressed during myogenesis in satellite cells.•Ebp1 knockdown inhibits both proliferation and differentiation of satellite cells.•ErbB3 is unable to rescue the differentiation defect in Ebp1 knockdown cells.•Ebp1 is also expressed during developmental myogenesis.•Differentiation of muscle progenitors is also inhibited by Ebp1 down-regulation.
The fusion of myoblasts into multinucleated myotubes is a crucial step of muscle growth during development and of muscle repair in the adult. While multiple genes were shown to play a role in this ...process, a vertebrate model where novel candidates can be tested and analyzed at high throughput and relative ease has been lacking. Here, we show that the early chicken embryo is a fast and robust model in which functional testing of muscle fusion candidate genes can be performed. We have used known modulators of muscle fusion, Rac1 and Cdc42, along with the in vivo electroporation of integrated, inducible vectors, to show that the chicken embryo is a suitable model in which their function can be tested and quantified. In addition to nuclei content, specific characteristics of the experimental model allow a fine characterization of additional morphological features that are nearly impossible to assess in other model organisms. This study should establish the chicken embryo as a cheap, reliable and powerful model in which novel vertebrate muscle fusion candidates can be evaluated.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Wnt/β-catenin signaling pathway is highly conserved throughout evolution, playing crucial roles in several developmental and pathological processes. Wnt ligands can act at a considerable distance ...from their sources and it is therefore necessary to examine not only the Wnt-producing but also the Wnt-receiving cells and tissues to fully appreciate the many functions of this pathway. To monitor Wnt activity, multiple tools have been designed which consist of multimerized Wnt signaling response elements (TCF/LEF binding sites) driving the expression of fluorescent reporter proteins (e.g. GFP, RFP) or of LacZ. The high stability of those reporters leads to a considerable accumulation in cells activating the pathway, thereby making them easily detectable. However, this makes them unsuitable to follow temporal changes of the pathway’s activity during dynamic biological events. Even though fluorescent transcriptional reporters can be destabilized to shorten their half-lives, this dramatically reduces signal intensities, particularly when applied in vivo. To alleviate these issues, we developed two transgenic quail lines in which high copy number (12× or 16×) of the TCF/LEF binding sites drive the expression of destabilized GFP variants. Translational enhancer sequences derived from viral mRNAs were used to increase signal intensity and specificity. This resulted in transgenic lines efficient for the characterization of TCF/β-catenin transcriptional dynamic activities during embryogenesis, including using in vivo imaging. Our analyses demonstrate the use of this transcriptional reporter to unveil novel aspects of Wnt signaling, thus opening new routes of investigation into the role of this pathway during amniote embryonic development.
The study of the morphogenetic cell movements underlying myotome formation in the chick embryo has led to the emergence of highly controversial models. Here we report a real-time cell lineage ...analysis of myotome development using electroporation of a GFP reporter in newly formed chick somites. Confocal analysis of cell movements demonstrates that myotome formation involves two sequential steps. In a first phase, incremental myotome growth results from a contribution of myocytes derived solely from the medial border of the dermomyotome. In a second phase, myocytes are produced from all four borders of the dermomyotome. The relative distribution of myocytes demonstrates that the medial and the lateral borders of the somite generate exclusively epaxial and hypaxial muscles. This analysis also identified five myotomal regions, characterized by the origin of the myocytes that constitute them. Together, our results provide a comprehensive model describing the morphogenesis of the early myotome in higher vertebrates.
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