Shape analysis of the basioccipital bone in Pax7-deficient mice.

We compared the cranial base of newborn Pax7-deficient and wildtype mice using a computational shape modeling technology called particle-based modeling (PBM). We found systematic differences in the morphology of the basiooccipital bone, including a broadening of the basioccipital bone and an antero-inferior inflection of its posterior edge in the Pax7-deficient mice. We show that the Pax7 cell lineage contributes to the basioccipital bone and that the location of the Pax7 lineage correlates with the morphology most effected by Pax7 deficiency. Our results suggest that the Pax7-deficient mouse may be a suitable model for investigating the genetic control of the location and orientation of the foramen magnum, and changes in the breadth of the basioccipital.

High-resolution myogenic lineage mapping by single-cell mass cytometry.

Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force-directed layout visualization defined a molecular signature of the activated stem cell state (CD44+/CD98+/MyoD+) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and ageing.

Myogenic potential of mouse embryonic stem cells lacking functional Pax7 tested in vitro by 5-azacitidine treatment and in vivo in regenerating skeletal muscle.

Regeneration of skeletal muscle relies on the presence of satellite cells. Satellite cells deficiency accompanying some degenerative diseases is the reason for the search for the "replacement cells" that can be used in the muscle therapies. Due to their unique properties embryonic stem cells (ESCs), as well as myogenic cells derived from them, are considered as a promising source of therapeutic cells. Among the factors crucial for the specification of myogenic precursor cells is Pax7 that sustains proper function of satellite cells. In our previous studies we showed that ESCs lacking functional Pax7 are able to form myoblasts in vitro when differentiated within embryoid bodies and their outgrowths. In the current study we showed that ESCs lacking functional Pax7, cultured in vitro in monolayer in the medium supplemented with horse serum and 5azaC, expressed higher levels of factors associated with myogenesis, such as Pdgfra, Pax3, Myf5, and MyoD. Importantly, skeletal myosin immunolocalization confirmed that myogenic differentiation of ESCs was more effective in case of cells lacking Pax7. Our in vivo studies showed that ESCs transplanted into regenerating skeletal muscles were detectable at day 7 of regeneration and the number of Pax7-/- ESCs detected was significantly higher than of control cells. Our results support the concept that lack of functional Pax7 promotes proliferation of differentiating ESCs and for this reason more of them can turn into myogenic lineage.

Sdf-1 (CXCL12) induces CD9 expression in stem cells engaged in muscle regeneration.

INTRODUCTION: Understanding the mechanism of stem cell mobilization into injured skeletal muscles is a prerequisite step for the development of muscle disease therapies. Many of the currently studied stem cell types present myogenic potential; however, when introduced either into the blood stream or directly into the tissue, they are not able to efficiently engraft injured muscle. For this reason their use in therapy is still limited. Previously, we have shown that stromal-derived factor-1 (Sdf-1) caused the mobilization of endogenous (not transplanted) stem cells into injured skeletal muscle improving regeneration. Here, we demonstrate that the beneficial effect of Sdf-1 relies on the upregulation of the tetraspanin CD9 expression in stem cells. METHODS: The expression pattern of adhesion proteins, including CD9, was analysed after Sdf-1 treatment during regeneration of rat skeletal muscles and mouse Pax7-/- skeletal muscles, that are characterized by the decreased number of satellite cells. Next, we examined the changes in CD9 level in satellite cells-derived myoblasts, bone marrow-derived mesenchymal stem cells, and embryonic stem cells after Sdf-1 treatment or silencing expression of CXCR4 and CXCR7. Finally, we examined the potential of stem cells to fuse with myoblasts after Sdf-1 treatment. RESULTS: In vivo analyses of Pax7-/- mice strongly suggest that Sdf-1-mediates increase in CD9 levels also in mobilized stem cells. In the absence of CXCR4 receptor the effect of Sdf-1 on CD9 expression is blocked. Next, in vitro studies show that Sdf-1 increases the level of CD9 not only in satellite cell-derived myoblasts but also in bone marrow derived mesenchymal stem cells, as well as embryonic stem cells. Importantly, the Sdf-1 treated cells migrate and fuse with myoblasts more effectively. CONCLUSIONS: We suggest that Sdf-1 binding CXCR4 receptor improves skeletal muscle regeneration by upregulating expression of CD9 and thus, impacting at stem cells mobilization to the injured muscles.

Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage.

Following their discovery in 1961, it was speculated that satellite cells were dormant myoblasts, held in reserve until required for skeletal muscle repair. Evidence for this accumulated over the years, until the link between satellite cells and the myoblasts that appear during muscle regeneration was finally established. Subsequently, it was demonstrated that, when grafted, satellite cells could also self-renew, conferring on them the coveted status of 'stem cell'. The emergence of other cell types with myogenic potential, however, questioned the precise role of satellite cells. Here, we review recent recombination-based studies that have furthered our understanding of satellite cell biology. The clear consensus is that skeletal muscle does not regenerate without satellite cells, confirming their pivotal and non-redundant role.

Bone marrow-derived hematopoietic cells undergo myogenic differentiation following a Pax-7 independent pathway.

Several reports showed that hematopoietic stem cells (HSCs) participate in muscle regeneration, raising hope for their therapeutic potential for degenerative muscle diseases. However, proof that HSCs are able to reprogram their fate and enter a myogenic pathway, remains elusive. We demonstrate that murine bone marrow (BM)-derived hematopoietic cells, carrying reporter genes controlled by muscle-specific regulatory elements from the Myf5, myosin light chain (MLC3F), or MCK genes, are induced by myoblasts to activate muscle-specific genes. This potential resides in the more undifferentiated progenitors, expressing surface markers typical of HSCs. Comparative gene expression profiling of CD45(+)/Sca1(+) cells isolated from muscle or BM shows that hematopoietic cells participate to muscle regeneration, by undergoing a profound although incomplete myogenic reprogramming on interaction with the muscle microenviroment. These cells undergo specification and differentiation independently from Pax7 and MyoD, and lack Pax7-associated properties, such as self-renewal and proliferation, distinguishing from satellite cells. Our findings indicate that hematopoietic cells, on seeding in the muscle, become a distinct cell population endowed with myogenic potential.

Deletion of Pax7 changes the tunica muscularis of the mouse esophagus from an entirely striated into a mixed phenotype.

The mechanisms responsible for the different amounts of striated muscle in mammalian esophagi are still enigmatic. A recent ultrastructural analysis in mouse esophagus pointed to a particular role of satellite cells during postnatal growth of striated muscle. The aim of this study was to investigate satellite cell development and the influence of Pax7 on this process. Developing and adult esophagi of wild-type and mice carrying a targeted mutation in Pax7 were analyzed by electron microscopy. We found a gene dose-dependent delayed development of striated muscle and a severe loss of satellite cells in Pax7(+/-) and Pax7(-/-) esophagi. In contrast to the entirely striated wild-type esophagus, Pax7(-/-) mutants developed a mixed phenotype with predominantly smooth muscle caudally. We conclude that Pax7-dependent myogenic progenitor cells are of prime importance for striated muscle formation and the degree of smooth-to-striated muscle conversion during esophageal ontogeny.

Comparative expression analysis of Pax3 and Pax7 during mouse myogenesis.

Pax3 and Pax7 are closely related transcription factors involved in the commitment of myogenic precursors in the developing trunk. However, it is not yet clear whether these genes are required for myogenic cell specification in the head and for post-somitic myogensis per se. In part, this uncertainty is due to the scarce information about their normal time course and pattern of expression. Here, we present a systematic immunohistochemical in situ analysis of spatiotemporal characteristics of Pax3 and Pax7 protein expression in comparison to that of MyoD and myogenin in the developing trunk and head muscles. The observed patterns of expression suggest that Pax3 is not involved in myogenesis in the head and its post-somitic expression in the trunk and limb muscles is mostly repressed after stage E13.5. In contrast, Pax7 expression is shared among all striated muscles and exhibits a uniform pattern. Pax7 is expressed only in mononucleated cells that either differentiate into myotubes or later form satellite cells. During development of head muscles, expression of Pax7 follows expression of MyoD and myogenin, implying that Pax7 is not required to induce the initial steps of the myogenic program in the head. In Pax7 homozygous mutants, in which muscle development proceeds normally, expression of Pax3 is indistinguishable from its wild-type pattern (i. e. absent), suggesting that after stage E13.5 myogenesis does not require Pax3 and Pax7. These data challenge the concept that Pax3 and Pax7 determine a persistent lineage of myogenic precursors in pre-natal and post-natal muscle development.