Christophe Marcelle

A common somitic origin for embryonic muscle progenitors and satellite cells

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.

Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration

BACKGROUND In the trunk of avian embryos, neural crest migration through the somites is segmental, with neural crest cells entering the rostral half of each somitic sclerotome but avoiding the caudal half. Little is known about the molecular nature of the cues-intrinsic to the somites-that are responsible for this segmental migration of neural crest cells. RESULTS We demonstrate that Eph-related receptor tyrosine kinases and their ligands are essential for the segmental migration of avian trunk neural crest cells through the somites. EphB3 localizes to the rostral half-sclerotome, including the neural crest, and the ligand ephrin-B1 has a complementary pattern of expression in the caudal half-sclerotome. To test the functional significance of this striking asymmetry, soluble ligand ephrin-B1 was added to interfere with receptor function in either whole trunk explants or neural crest cells cultured on alternating stripes of ephrin-B1 versus fibronection. Neural crest cells in vitro avoided migrating on lanes of immobilized ephrin-B1; the addition of soluble ephrin-B1 blocked this inhibition. Similarly, in whole trunk explants, the metameric pattern of neural crest migration was disrupted by addition of soluble ephrin-B1, allowing entry of neural crest cells into caudal portions of the sclerotome. CONCLUSIONS Both in vivo and in vitro, the addition of soluble ephrin-B1 results in a loss of the metameric migratory pattern and a disorganization of neural crest cell movement. These results demonstrate that Eph-family receptor tyrosine kinases and their transmembrane ligands are involved in interactions between neural crest and sclerotomal cells, mediating an inhibitory activity necessary to constrain neural precursors to specific territories in the developing nervous system.

Wnt regulation of chondrocyte differentiation

The Wnt family of growth factors are important regulators of several developmental processes including skeletogenesis. To further investigate the role of Wnts we analysed their expression in the developing chick limb and performed functional analyses in vivo and in vitro. We found that Wnt5b and Wnt11 are restricted within the prehypertrophic chondrocytes of the cartilage elements, Wnt5a is found in the joints and perichondrium, while Wnt4 is expressed in the developing joints and, in some bones, a subset of the hypertrophic chondrocytes. These Wnts mediate distinct effects on the initiation of chondrogenesis and differentiation of chondrocytes in vitro and in vivo. Wnt4 blocks the initiation of chondrogenesis and accelerates terminal chondrocyte differentiation in vitro. In contrast, Wnt5a and Wnt5b promote early chondrogenesis in vitro while inhibiting terminal differentiation in vivo. As Wnt5b and Wnt11 expression overlaps with and appears after Indian hedgehog (Ihh), we also compared their effects with Ihh to see if they mediate aspects of Ihh signalling. This showed that Ihh and Wnt5b and Wnt11 control chondrogenesis in parallel pathways.

WNT11 acts as a directional cue to organize the elongation of early muscle fibres

The early vertebrate skeletal muscle is a well-organized tissue in which the primitive muscle fibres, the myocytes, are all parallel and aligned along the antero-posterior axis of the embryo. How myofibres acquire their orientation during development is unknown. Here we show that during early chick myogenesis WNT11 has an essential role in the oriented elongation of the myocytes. We find that the neural tube, known to drive WNT11 expression in the medial border of somites, is necessary and sufficient to orient myocyte elongation. We then show that the specific inhibition of WNT11 function in somites leads to the disorganization of myocytes. We establish that WNT11 mediates this effect through the evolutionary conserved planar cell polarity (PCP) pathway, downstream of the WNT/β-catenin-dependent pathway, required to initiate the myogenic program of myocytes and WNT11 expression. Finally, we demonstrate that a localized ectopic source of WNT11 can markedly change the orientation of myocytes, indicating that WNT11 acts as a directional cue in this process. All together, these data show that the sequential action of the WNT/PCP and the WNT/β-catenin pathways is necessary for the formation of fully functional embryonic muscle fibres. This study also provides evidence that WNTs can act as instructive cues to regulate the PCP pathway in vertebrates.

Myostatin promotes the terminal differentiation of embryonic muscle progenitors

Myostatin, a TGF-beta family member, is an important regulator of adult muscle size. While extensively studied in vitro, the mechanisms by which this molecule mediates its effect in vivo are poorly understood. We addressed this question using chick and mouse embryos. We show that while myostatin overexpression in chick leads to an exhaustion of the muscle progenitor population that ultimately results in muscle hypotrophy, myostatin loss of function in chick and mouse provokes an expansion of this population. Our data demonstrate that myostatin acts in vivo to regulate the balance between proliferation and differentiation of embryonic muscle progenitors by promoting their terminal differentiation through the activation of p21 and MyoD. Previous studies have suggested that myostatin imposes quiescence on muscle progenitors. Our data suggest that myostatins effect on muscle progenitors is more complex than previously realized and is likely to be context-dependent. We propose a novel model for myostatin mode of action in vivo, in which myostatin affects the balance between proliferation and differentiation of embryonic muscle progenitors by enhancing their differentiation.

Neural crest regulates myogenesis through the transient activation of NOTCH

How dynamic signalling and extensive tissue rearrangements interact to generate complex patterns and shapes during embryogenesis is poorly understood. Here we characterize the signalling events taking place during early morphogenesis of chick skeletal muscles. We show that muscle progenitors present in somites require the transient activation of NOTCH signalling to undergo terminal differentiation. The NOTCH ligand Delta1 is expressed in a mosaic pattern in neural crest cells that migrate past the somites. Gain and loss of Delta1 function in neural crest modifies NOTCH signalling in somites, which results in delayed or premature myogenesis. Our results indicate that the neural crest regulates early muscle formation by a unique mechanism that relies on the migration of Delta1-expressing neural crest cells to trigger the transient activation of NOTCH signalling in selected muscle progenitors. This dynamic signalling guarantees a balanced and progressive differentiation of the muscle progenitor pool.

In Ovo Electroporation of Avian Somites

In ovo electroporation is a well‐established method of gene transfer into neural and mesenchymal tissue in chick embryos. Electroporation of somites, however, has been hampered by low efficiency due to technical difficulties. Here, we present a powerful technique to electroporate avian somites and subpopulations of somitic cells at high efficiency in ovo. We demonstrate specific targeting of distinct somitic compartments and their derivatives using single or combinations of plasmid expression vectors. This technique opens new perspectives to investigate the morphologic and genetic basis of somite development. Developmental Dynamics 229:643–650, 2004.

Intrinsic signals regulate the initial steps of myogenesis in vertebrates

In vertebrates, despite the evidence that extrinsic factors induce myogenesis in naive mesoderm, other experiments argue that the initiation of the myogenic program may take place independent of these factors. To resolve this discrepancy, we have re-addressed this issue, using short-term in vivo microsurgery and culture experiments in chick. Our results show that the initial expression of the muscle-specific markers Myf5 and MyoD is regulated in a mesoderm-autonomous fashion. The reception of a Wnt signal is required for MyoD, but not Myf5 expression; however, we show that the source of the Wnt signal is intrinsic to the mesoderm. Gain- and loss-of-function experiments indicate that Wnt5b, which is expressed in the presomitic mesoderm, represents the MyoD-activating cue. Despite Wnt5b expression in the presomitic mesoderm, MyoD is not expressed in this tissue: our experiments demonstrate that this is due to a Bmp inhibitory signal that prevents the premature expression of MyoD before somites form. Our results indicate that myogenesis is a multistep process which is initiated prior to somite formation in a mesoderm-autonomous fashion; as somites form, influences from adjacent tissues are likely to be required for maintenance and patterning of early muscles.

Molecular cloning of Quek 1 and 2, two quail vascular endothelial growth factor (VEGF) receptor-like molecules

We have previously reported the cloning of two partial cDNAs corresponding to two quail (Coturnix coturnix japonica) receptor tyrosine kinases (RTKs), named Quek 1 and Quek 2, and their expression in endothelial cells of the early avian embryo. We here report the cloning of the full-size cDNAs for both molecules. Sequence comparison shows that Quek 1 and 2 share an overall amino acid (aa) identity of 49%. They both comprise seven extracellular immunoglobulin-like (Ig-like) domains, a single transmembrane domain, and an intracellular kinase domain split into two by a 70 aa insertion. These structural characteristics are shared by the members of the recently discovered VEGF receptor (VEGFR) family. We have compared the sequences of Quek 1 and 2 to the other VEGFRs. At the aa level, Quek 1 is most closely related to KDR/flk-1 (VEGFR 2) (aa identity of 69% and 71%, respectively). Quek 2 shows a similar degree of aa identity to fit-4 (VEGFR 3). Quek 1 and 2 display a lower homology to fit-1 (VEGFR 1) (about 45% aa identity). These data suggest that Quek 1 and 2 are the avian homologues of VEGFRs 2 and 3, respectively.

Muscle stem cells

Despite being mainly composed of highly differentiated contractile fibers, the adult skeletal muscle possesses the remarkable ability to regenerate, following injury. The cells that are responsible for this capacity are the satellite cells, a small population of adult stem cells positioned under the basal lamina of muscle fibers and that can give rise to both differentiated myogenic cells while maintaining a stem cell pool by a self-renewal mechanism. We will discuss here recent publications on the developmental origin of muscle stem cells, on the signaling pathways that affect their proliferation and differentiation, with reference to works on skeletal muscle formation in the embryo as well as the adult, using the mouse and chick as reference models.