Search

Translational control of gene expression is an important regulator of adult stem cell quiescence, activation and self-renewal. In skeletal muscle, quiescent satellite cells maintain low levels of protein synthesis, mediated in part through the phosphorylation of eIF2α (P-eIF2α). Pharmacological inhibition of the eIF2α phosphatase with the small molecule sal003 maintains P-eIF2α and permits the expansion of satellite cells

An important strategy for establishing mechanisms of gene function during development is through mutation of individual genes and analysis of subsequent effects on cell behavior. Here, we present a single-plasmid approach for genome editing in chick embryos to study experimentally perturbed cells in an otherwise normal embryonic environment. To achieve this, we have engineered a plasmid that encodes Cas9 protein, gene-specific guide RNA (gRNA), and a fluorescent marker within the same construct. Using transfection- and electroporation-based approaches, we show that this construct can be used to perturb gene function in early embryos as well as human cell lines. Importantly, insertion of this cistronic construct into replication-incompetent avian retroviruses allowed us to couple gene knockouts with long-term lineage analysis. We demonstrate the application of our newly engineered constructs and viruses by perturbing β-catenin in vitro and Sox10, Pax6 and Pax7 in the neural crest, retina, and neural tube and segmental plate in vivo, respectively. Together, this approach enables genes of interest to be knocked out in identifiable cells in living embryos and can be broadly applied to numerous genes in different embryonic tissues., Summary: A single-plasmid approach using self-cleaving ribozymes enables delivery of CRISPR components via electroporation and can be integrated into viral delivery systems for long-term tracking of mutant cells.

Sox2 mutations are associated with pituitary hormone deficiencies and the protein is required for pituitary progenitor proliferation, but its function has not been well characterized in this context. SOX2 is known to activate expression of Six6, encoding a homeodomain transcription factor, in the ventral diencephalon. Here, we find that the same relationship likely exists in the pituitary. Moreover, because Six6 deletion is associated with a similar phenotype as described here for loss of Sox2, Six6 appears to be an essential downstream target of SOX2 in the gland. We also uncover a second role for SOX2. Whereas cell differentiation is reduced in Sox2 mutants, some endocrine cells are generated, such as POMC-positive cells in the intermediate lobe. However, loss of SOX2 here results in complete downregulation of the melanotroph pioneer factor PAX7, and subsequently a switch of identity from melanotrophs to ectopic corticotrophs. Rescuing proliferation by ablating the cell cycle negative regulator p27 (also known as Cdkn1b) in Sox2 mutants does not restore melanotroph emergence. Therefore, SOX2 has two independent roles during pituitary morphogenesis; firstly, promotion of progenitor proliferation, and subsequently, acquisition of melanotroph identity., Summary: SOX2 has two independent roles during pituitary morphogenesis: promoting progenitor proliferation via SIX6 and determining melanotroph identity via PAX7.

The functional role of Pax7-expressing satellite cells (SCs) in postnatal skeletal muscle development beyond weaning remains obscure. Therefore, the relevance of SCs during prepubertal growth, a period after weaning but prior to the onset of puberty, has not been examined. Here, we have characterized mouse skeletal muscle growth during prepuberty and found significant increases in myofiber cross-sectional area that correlated with SC-derived myonuclear number. Remarkably, genome-wide RNA-sequencing analysis established that post-weaning juvenile and early adolescent skeletal muscle have markedly different gene expression signatures. These distinctions are consistent with extensive skeletal muscle maturation during this essential, albeit brief, developmental phase. Indelible labeling of SCs with Pax7CreERT2/+; Rosa26nTnG/+ mice demonstrated SC-derived myonuclear contribution during prepuberty, with a substantial reduction at puberty onset. Prepubertal depletion of SCs in Pax7CreERT2/+; Rosa26DTA/+ mice reduced myofiber size and myonuclear number, and caused force generation deficits to a similar extent in both fast and slow-contracting muscles. Collectively, these data demonstrate SC-derived myonuclear accretion as a cellular mechanism that contributes to prepubertal hypertrophic skeletal muscle growth., Summary: Examination of gene expression and morphological changes in mouse skeletal muscle during prepuberty demonstrates that satellite cell-derived myonuclear accretion contributes to prepubertal hypertrophic skeletal muscle growth.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder characterized by muscle wasting and premature death. The defective gene is dystrophin, a structural protein, absence of which causes membrane fragility and myofiber necrosis. Several lines of evidence showed that in adult DMD patients dystrophin is involved in signaling pathways that regulate calcium homeostasis and differentiation programs. However, secondary aspects of the disease, such as inflammation and fibrosis development, might represent a bias in the analysis. Because fetal muscle is not influenced by gravity and does not suffer from mechanical load and/or inflammation, we investigated 12-week-old fetal DMD skeletal muscles, highlighting for the first time early alterations in signaling pathways mediated by the absence of dystrophin itself. We found that PLC/IP3/IP3R/Ryr1/Ca2+ signaling is widely active in fetal DMD skeletal muscles and, through the calcium-dependent PKCα protein, exerts a fundamental regulatory role in delaying myogenesis and in myofiber commitment. These data provide new insights into the origin of DMD pathology during muscle development.

International audience; A central question in development is to define how the equilibrium between cell proliferation and differentiation is temporally and spatially regulated during tissue formation. Here, we address how interactions between cyclin-dependent kinase inhibitors essential for myogenic growth arrest (p21 cip1 and p57 kip2), the Notch pathway and myogenic regulatory factors (MRFs) orchestrate the proliferation, specification and differentiation of muscle progenitor cells. We first show that cell cycle exit and myogenic differentiation can be uncoupled. In addition, we establish that skeletal muscle progenitor cells require Notch signaling to maintain their cycling status. Using several mouse models combined with ex vivo studies, we demonstrate that Notch signaling is required to repress p21 cip1 and p57 kip2 expression in muscle progenitor cells. Finally, we identify a muscle-specific regulatory element of p57 kip2 directly activated by MRFs in myoblasts but repressed by the Notch targets Hes1/Hey1 in progenitor cells. We propose a molecular mechanism whereby information provided by Hes/Hey downstream of Notch as well as MRF activities are integrated at the level of the p57 kip2 enhancer to regulate the decision between progenitor cell maintenance and muscle differentiation.

The neural crest (NC) is a migratory population of cells unique to vertebrates that generates many diverse derivatives. NC cells arise during gastrulation at the neural plate border (NPB), which is later elevated as the neural folds (NFs) form and fuse in the dorsal region of the closed neural tube, from where NC cells emigrate. In chick embryos, Pax7 is an early marker, and necessary component of NC development. Unlike other early NPB markers, which are co-expressed in lateral ectoderm, medial neural plate or posterior-lateral mesoderm, Pax7 early expression seems more restricted to the NPB. However, the molecular mechanisms controlling early Pax7 expression remain poorly understood. Here, we identify a novel enhancer of Pax7 in avian embryos that replicates the expression of Pax7 associated with early NC development. Expression from this enhancer is found in early NPB, NFs and early emigrating NC, but unlike Pax7, which is also expressed in mesodermal derivatives, this enhancer is not active in somites. Further analysis demonstrates that cMyb is able to interact with this enhancer and modulates reporter and endogenous early Pax7 expression; thus, cMyb is identified as a novel regulator of Pax7 in early NC development.

The development of a functional tissue requires coordination of the amplification of progenitors and their differentiation into specific cell types. The molecular basis for this coordination during myotome ontogeny is not well understood. Dermomytome progenitors that colonize the myotome first acquire myocyte identity and subsequently proliferate as Pax7-expressing progenitors before undergoing terminal differentiation. We show that the dynamics of sonic hedgehog (Shh) signaling is crucial for this transition in both avian and mouse embryos. Initially, Shh ligand emanating from notochord/floor plate reaches the dermomyotome, where it both maintains the proliferation of dermomyotome cells and promotes myogenic differentiation of progenitors that colonized the myotome. Interfering with Shh signaling at this stage produces small myotomes and accumulation of Pax7-expressing progenitors. An in vivo reporter of Shh activity combined with mouse genetics revealed the existence of both activator and repressor Shh activities operating on distinct subsets of cells during the epaxial myotomal maturation. In contrast to observations in mice, in avians Shh promotes the differentiation of both epaxial and hypaxial myotome domains. Subsequently, myogenic progenitors become refractory to Shh; this is likely to occur at the level of, or upstream of, smoothened signaling. The end of responsiveness to Shh coincides with, and is thus likely to enable, the transition into the growth phase of the myotome.

During salamander limb regeneration, only the structures distal to the amputation plane are regenerated, a property known as the rule of distal transformation. Multiple cell types are involved in limb regeneration; therefore, determining which cell types participate in distal transformation is important for understanding how the proximo-distal outcome of regeneration is achieved. We show that connective tissue-derived blastema cells obey the rule of distal transformation. They also have nuclear MEIS, which can act as an upper arm identity regulator, only upon upper arm amputation. By contrast, myogenic cells do not obey the rule of distal transformation and display nuclear MEIS upon amputation at any proximo-distal level. These results indicate that connective tissue cells, but not myogenic cells, are involved in establishing the proximo-distal outcome of regeneration and are likely to guide muscle patterning. Moreover, we show that, similarly to limb development, muscle patterning in regeneration is influenced by β-catenin signalling.

Skeletal muscle tissue provides mechanical force for locomotion of all vertebrate animals. It is prone to damage from acute physical trauma and physiological stress. To cope with this, it possesses a tremendous capacity for rapid and effective repair that is widely held to be accomplished by the satellite cells lying between the muscle fiber plasmalemma and the basement membrane. Cell transplantation and lineage-tracing studies have demonstrated that Pax7-expressing (Pax7+) satellite cells can repair damaged muscle tissue repeatedly after several bouts of acute injury. These findings provided evidence that Pax7+ cells are muscle stem cells. However, stem cells from a variety of other origins are also reported to contribute to myofibers upon engraftment into muscles, questioning whether satellite cells are the only stem cell source for muscle regeneration. Here, we have engineered genetic ablation of Pax7+ cells to test whether there is any significant contribution to muscle regeneration after acute injury from cells other than this source. We find that such elimination of Pax7+ cells completely blocks regenerative myogenesis either following injury to the tibialis anterior (TA) muscle or after transplantation of extensor digitorum longus (EDL) muscles into nude mice. As Pax7 is specifically expressed in satellite cells, we conclude that they are essential for acute injury-induced muscle regeneration. It remains to be established whether there is any significant role for stem cells of other origins. The implications of our results for muscle stem cell-based therapy are discussed.

International audience; Distinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches.

The position of the olfactory placodes suggests that these epithelial thickenings might provide morphogenetic information to the adjacent facial mesenchyme. To test this, we performed in ovo manipulations of the nasal placode in the avian embryo. Extirpation of placodal epithelium or placement of barriers on the lateral side of the placode revealed that the main influence is on the lateral nasal, not the frontonasal, mesenchyme. These early effects were consistent with the subsequent deletion of lateral nasal skeletal derivatives. We then showed in rescue experiments that FGFs are required for nasal capsule morphogenesis. The instructive capacity of the nasal pit epithelium was tested in a series of grafts to the face and trunk. Here, we showed for the first time that nasal pits are capable of inducing bone, cartilage and ectopic PAX7 expression, but these effects were only observed in the facial grafts. Facial mesenchyme also supported the initial projection of the olfactory nerve and differentiation of the olfactory epithelium. Thus, the nasal placode has two roles: as a signaling center for the lateral nasal skeleton and as a source of olfactory neurons and sensory epithelium.

Tail regeneration in urodeles requires the coordinated growth and patterning of the regenerating tissues types, including the spinal cord,cartilage and muscle. The dorsoventral (DV) orientation of the spinal cord at the amputation plane determines the DV patterning of the regenerating spinal cord as well as the patterning of surrounding tissues such as cartilage. We investigated this phenomenon on a molecular level. Both the mature and regenerating axolotl spinal cord express molecular markers of DV progenitor cell domains found during embryonic neural tube development, including Pax6, Pax7 and Msx1. Furthermore, the expression of Sonic hedgehog (Shh) is localized to the ventral floor plate domain in both mature and regenerating spinal cord. Patched1 receptor expression indicated that hedgehog signaling occurs not only within the spinal cord but is also transmitted to the surrounding blastema. Cyclopamine treatment revealed that hedgehog signaling is not only required for DV patterning of the regenerating spinal cord but also had profound effects on the regeneration of surrounding, mesodermal tissues. Proliferation of tail blastema cells was severely impaired, resulting in an overall cessation of tail regeneration, and blastema cells no longer expressed the early cartilage marker Sox9. Spinal cord removal experiments revealed that hedgehog signaling, while required for blastema growth is not sufficient for tail regeneration in the absence of the spinal cord. By contrast to the cyclopamine effect on tail regeneration, cyclopamine-treated regenerating limbs achieve a normal length and contain cartilage. This study represents the first molecular localization of DV patterning information in mature tissue that controls regeneration. Interestingly, although tail regeneration does not occur through the formation of somites, the Shh-dependent pathways that control embryonic somite patterning and proliferation may be utilized within the blastema,albeit with a different topography to mediate growth and patterning of tail tissues during regeneration.

Recent studies have demonstrated that a distinct subpopulation with stem cell-like characteristics in myoblast culture is responsible for new muscle fiber formation after intramuscular transplantation. The identification and isolation of stem-like cells would have significant implications for successful myogenic cell transfer therapy in human muscle disorders. Using a clonal culture system for mouse muscle satellite cells, we have identified two cell types, designated `round cells' and `thick cells', in clones derived from single muscle satellite cells that have been taken from either slow or fast muscle. Clonal analysis of satellite cells revealed that the round cells are immediate descendants of quiescent satellite cells in adult muscle. In single-myofiber culture, round cells first formed colonies and then generated progeny, thick cells, that underwent both myogenic and osteogenic terminal differentiation under the appropriate culture conditions. Thick cells, but not round cells, responded to terminal differentiation-inducing signals. Round cells express Pax7, a specific marker of satellite cells, at high levels. Myogenic cell transfer experiments showed that round cells reconstitute myofibers more efficiently than thick cells. Furthermore, round cells restored dystrophin in myofibers of mdx nude mice, even when as few as 5000 cells were transferred into the gastrocnemius muscle. These results suggest that round cells are satellite-cell descendants with stem cell-like characteristics and represent a useful source of donor cells to improve muscle regeneration.

In the spinal neural tube, populations of neuronal precursors that express a unique combination of transcription factors give rise to specific classes of neurons at precise locations along the dorsoventral axis. Understanding the patterning mechanisms that generate restricted gene expression along the dorsoventral axis is therefore crucial to understanding the creation of diverse neural cell types. Bone morphogenetic proteins (BMPs) and other transforming growth factor β (TGFβ) proteins are expressed by the dorsal-most cells of the neural tube (the roofplate) and surrounding tissues, and evidence indicates that they play a role in assigning cell identity. We have manipulated the level of BMP signaling in the chicken neural tube to show that BMPs provide patterning information to both dorsal and intermediate cells. BMP regulation of the expression boundaries of the homeobox proteins Pax6, Dbx2 and Msx1 generates precursor populations with distinct developmental potentials. Within the resulting populations, thresholds of BMP act to set expression domain boundaries of developmental regulators of the homeobox and basic helix-loop-helix (bHLH) families, ultimately leading to the generation of a diversity of differentiated neural cell types. This evidence strongly suggests that BMPs are the key regulators of dorsal cell identity in the spinal neural tube.