PAX7 Balances the Cell Cycle Progression via Regulating Expression of Dnmt3b and Apobec2 in Differentiating PSCs.

PAX7 transcription factor plays a crucial role in embryonic myogenesis and in adult muscles in which it secures proper function of satellite cells, including regulation of their self renewal. PAX7 downregulation is necessary for the myogenic differentiation of satellite cells induced after muscle damage, what is prerequisite step for regeneration. Using differentiating pluripotent stem cells we documented that the absence of functional PAX7 facilitates proliferation. Such action is executed by the modulation of the expression of two proteins involved in the DNA methylation, i.e., Dnmt3b and Apobec2. Increase in Dnmt3b expression led to the downregulation of the CDK inhibitors and facilitated cell cycle progression. Changes in Apobec2 expression, on the other hand, differently impacted proliferation/differentiation balance, depending on the experimental model used.

MECHANISMS IN ENDOCRINOLOGY: Pioneer transcription factors in pituitary development and tumorigenesis.

Pioneer transcription factors have key roles in development as master regulators of cell fate specification. Only a small fraction of all transcription factors have the pioneer ability that confers access to target genomic DNA sites embedded in so-called 'closed' heterochromatin. This ability to seek and bind target sites within the silenced portion of the epigenome is the basis for their role in changing cell fate. Upon binding heterochromatin sites, pioneer factors trigger remodeling of chromatin from a repressed into an active organization. This action is typically exerted at enhancer regulatory sequences, thus allowing activation of new gene subsets. During pituitary development, the only pioneer with a well-documented role is Pax7 that specifies the intermediate lobe melanotrope cell fate. In this review, a particular focus is placed on this Pax7 function but its properties are also considered within the general context of pioneer factor action. Given their potent activity to reprogram gene expression, it is not surprising that many pioneers are associated with tumor development. Overexpression or chromosomal translocations leading to the production of chimeric pioneers have been implicated in different cancers. We review here the current knowledge on the mechanism of pioneer factor action.

Lineage tracing reveals evidence of a popliteal lymphatic muscle progenitor cell that is distinct from skeletal and vascular muscle progenitors.

Loss of popliteal lymphatic vessel (PLV) contractions, which is associated with damage to lymphatic muscle cells (LMCs), is a biomarker of disease progression in mice with inflammatory arthritis. Currently, the nature of LMC progenitors has yet to be formally described. Thus, we aimed to characterize the progenitors of PLV-LMCs during murine development, towards rational therapies that target their proliferation, recruitment, and differentiation onto PLVs. Since LMCs have been described as a hybrid phenotype of striated and vascular smooth muscle cells (VSMCs), we performed lineage tracing studies in mice to further clarify this enigma by investigating LMC progenitor contribution to PLVs in neonatal mice. PLVs from Cre-tdTomato reporter mice specific for progenitors of skeletal myocytes (Pax7+ and MyoD+) and VSMCs (Prrx1+ and NG2+) were analyzed via whole mount immunofluorescent microscopy. The results showed that PLV-LMCs do not derive from skeletal muscle progenitors. Rather, PLV-LMCs originate from Pax7-/MyoD-/Prrx1+/NG2+ progenitors similar to VSMCs prior to postnatal day 10 (P10), and from a previously unknown Pax7-/MyoD-/Prrx1+/NG2- muscle progenitor pathway during development after P10. Future studies of these LMC progenitors during maintenance and repair of PLVs, along with their function in other lymphatic beds, are warranted.

Blastula stage specification of avian neural crest.

Cell fate specification defines the earliest steps towards a distinct cell lineage. Neural crest, a multipotent stem cell population, is thought to be specified from the ectoderm, but its varied contributions defy canons of segregation potential and challenges its embryonic origin. Aiming to resolve this conflict, we have assayed the earliest specification of neural crest using blastula stage chick embryos. Specification assays on isolated chick epiblast explants identify an intermediate region specified towards the neural crest cell fate. Furthermore, low density culture suggests that the specification of intermediate cells towards the neural crest lineage is independent of contact mediated induction and Wnt-ligand induced signaling, but is, however, dependent on transcriptional activity of ß-catenin. Finally, we have validated the regional identity of the intermediate region towards the neural crest cell fate using fate map studies. Our results suggest a model of neural crest specification within a restricted epiblast region in blastula stage chick embryos.

Paired box 7 inhibits differentiation in 3T3-L1 preadipocytes.

Myogenesis is precisely proceeded by myogenic regulatory factors. Myogenic stem cells are activated, proliferated and fused into a multinuclear myofiber. Pax7, paired box 7, one of the earliest markers during myogenesis. It has been reported that Pax7 regulates the muscle marker genes, Myf5 and MyoD toward differentiation. The possible roles of Pax7 in myogenic cells have been well researched. However, it has not yet been clarified if Pax7 itself is able to induce myogenic fate in nonmyogenic lineage cells. In this study, we performed experiments using stably expressed Pax7 in 3T3-L1 preadipocytes to elucidate if Pax7 inhibits adipogenesis. We found that Pax7 represses adipogenic markers and prevents differentiation. These cells showed decreased expression of PDGFRα, PPARγ and Fabp4 and inhibited forming lipid droplets.

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.

Limitations of the Pax7-creERT2 transgene for driving deletion of Nf1 in adult mouse muscle.

Neurofibromatosis Type 1 (NF1) is an autosomal dominant genetic disorder that results in a variety of characteristic manifestations. Prior studies have shown reduced muscle size and global skeletal muscle weakness in children with NF1. This associated weakness can lead to significant challenges impacting on quality of life. Pre-clinical studies using a muscle-specific NF1 knockout mouse have linked this weakness to an underlying primary metabolic deficiency in the muscle. However, the neonatal lethality of this strain prevents analysis of the role of NF1 in adult muscle. In this study, we present the characterization of an inducible muscle-specific NF1 knockout strain (Nf1Pax7i f/f ) produced by cross breeding the Pax7-CreERT2 strain with the conditional Nf1flox/flox line. Tamoxifen dosing of 8-week old Nf1Pax7i f/f mice led to recombination of the floxed allele in muscle, as detected by PCR. Detailed phenotypic analysis of treated adult mice over 8 weeks revealed no changes in bodyweight or muscle weight, no histological signs of myopathy, and no functional evidence of distress or impairment. Subsequent analysis using the Ai9 Cre-dependent tdTomato reporter strain was used to analyse labelling in embryos and in adult mice. Cell tracking studies identified a lower than expected rate of integration of recombined satellite cells into adult muscle. In contrast, a high persistent contribution of embryonic cells that were Pax7+ were found in adult muscle. These findings indicate important caveats with the use of the Pax7-CreER T2 strain and highlight a need to develop new tools for investigating the function of NF1 in mature muscle.

Pax3- and Pax7-mediated Dbx1 regulation orchestrates the patterning of intermediate spinal interneurons.

Transcription factors are key orchestrators of the emergence of neuronal diversity within the developing spinal cord. As such, the two paralogous proteins Pax3 and Pax7 regulate the specification of progenitor cells within the intermediate neural tube, by defining a neat segregation between those fated to form motor circuits and those involved in the integration of sensory inputs. To attain insights into the molecular means by which they control this process, we have performed detailed phenotypic analyses of the intermediate spinal interneurons (IN), namely the dI6, V0D, V0VCG and V1 populations in compound null mutants for Pax3 and Pax7. This has revealed that the levels of Pax3/7 proteins determine both the dorso-ventral extent and the number of cells produced in each subpopulation; with increasing levels leading to the dorsalisation of their fate. Furthermore, thanks to the examination of mutants in which Pax3 transcriptional activity is skewed either towards repression or activation, we demonstrate that this cell diversification process is mainly dictated by Pax3/7 ability to repress gene expression. Consistently, we show that Pax3 and Pax7 inhibit the expression of Dbx1 and of its repressor Prdm12, fate determinants of the V0 and V1 interneurons, respectively. Notably, we provide evidence for the activity of several cis-regulatory modules of Dbx1 to be sensitive to Pax3 and Pax7 transcriptional activity levels. Altogether, our study provides insights into how the redundancy within a TF family, together with discrete dynamics of expression profiles of each member, are exploited to generate cellular diversity. Furthermore, our data supports the model whereby cell fate choices in the neural tube do not rely on binary decisions but rather on inhibition of multiple alternative fates.

TRAF6 regulates satellite stem cell self-renewal and function during regenerative myogenesis.

Satellite cells are a stem cell population within adult muscle and are responsible for myofiber regeneration upon injury. Satellite cell dysfunction has been shown to underlie the loss of skeletal muscle mass in many acquired and genetic muscle disorders. The transcription factor paired box-protein-7 (PAX7) is indispensable for supplementing the reservoir of satellite cells and driving regeneration in normal and diseased muscle. TNF receptor-associated factor 6 (TRAF6) is an adaptor protein and an E3 ubiquitin ligase that mediates the activation of multiple cell signaling pathways in a context-dependent manner. Here, we demonstrated that TRAF6-mediated signaling is critical for homeostasis of satellite cells and their function during regenerative myogenesis. Selective deletion of Traf6 in satellite cells of adult mice led to profound muscle regeneration defects and dramatically reduced levels of PAX7 and late myogenesis markers. TRAF6 was required for the activation of MAPKs ERK1/2 and JNK1/2, which in turn activated the transcription factor c-JUN, which binds the Pax7 promoter and augments Pax7 expression. Moreover, TRAF6/c-JUN signaling repressed the levels of the microRNAs miR-1 and miR-206, which promote differentiation, to maintain PAX7 levels in satellite cells. We also determined that satellite cell-specific deletion of Traf6 exaggerates the dystrophic phenotype in the mdx (a mouse model of Duchenne muscular dystrophy) mouse by blunting the regeneration of injured myofibers. Collectively, our study reveals an essential role for TRAF6 in satellite stem cell function.

PAX3 and PAX7 as upstream regulators of myogenesis.

Like other subclasses within the PAX transcription factor family, PAX3 and PAX7 play important roles in the emergence of a number of different tissues during development. PAX3 regulates neural crest and, together with its orthologue PAX7, is also expressed in parts of the central nervous system. In this chapter we will focus on their role in skeletal muscle. Both factors are key regulators of myogenesis where Pax3 plays a major role during early skeletal muscle formation in the embryo while Pax7 predominates during post-natal growth and muscle regeneration in the adult. We review the expression and functions of these factors in the myogenic context. We also discuss mechanistic aspects of PAX3/7 function and modulation of their activity by interaction with other proteins, as well as the post-transcriptional and transcriptional regulation of their expression.

CRISPR mediated somatic cell genome engineering in the chicken.

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.

Pax3 and Pax7 play essential safeguard functions against environmental stress-induced birth defects.

Exposure to environmental teratogenic pollutant leads to severe birth defects. However, the biological events underlying these developmental abnormalities remain undefined. Here, we report a molecular link between an environmental stress response pathway and key developmental genes during craniofacial development. Strikingly, mutant mice with impaired Pax3/7 function display severe craniofacial defects. We show that these are associated with an upregulation of the signaling pathway mediated by the Aryl hydrocarbon receptor (AHR), the receptor to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), revealing a genetic interaction between Pax3 and AHR signaling. Activation of AHR signaling in Pax3-deficient embryos drives facial mesenchymal cells out of the cell cycle through the upregulation of p21 expression. Accordingly, inhibiting AHR activity rescues the cycling status of these cells and the facial closure of Pax3/7 mutants. Together, our findings demonstrate that the regulation of AHR signaling by Pax3/7 is required to protect against TCDD/AHR-mediated teratogenesis during craniofacial development.

The quest for male germline stem cell markers: PAX7 gets ID'd.

Male germline or spermatogonial stem cells (SSCs) are conserved across many species and essential for uninterrupted production of sperm over long periods of reproductive life span. A better understanding of SSC biology provides limitless opportunities in male reproductive health, fertility preservation, and regenerative medicine. Although several potential markers define SSCs, not many definitive markers exist that are specific for a rare subset of SSCs that self-renew and have the ability to give rise to other progenitors, eventually contributing to all stages of spermatogenesis. In the September 2014 issue of the JCI, Aloisio and colleagues report that PAX7 is a new marker expressed uniquely in a rare subset of SSCs in mouse testes. PAX7+ cells fulfill all the criteria required for bona fide SSCs. Surprisingly, male germline-specific deletion of Pax7 indicates that it is dispensable for spermatogenesis.

PAX7 expression defines germline stem cells in the adult testis.

Spermatogenesis is a complex, multistep process that maintains male fertility and is sustained by rare germline stem cells. Spermatogenic progression begins with spermatogonia, populations of which express distinct markers. The identity of the spermatogonial stem cell population in the undisturbed testis is controversial due to a lack of reliable and specific markers. Here we identified the transcription factor PAX7 as a specific marker of a rare subpopulation of A(single) spermatogonia in mice. PAX7+ cells were present in the testis at birth. Compared with the adult testis, PAX7+ cells constituted a much higher percentage of neonatal germ cells. Lineage tracing in healthy adult mice revealed that PAX7+ spermatogonia self-maintained and produced expanding clones that gave rise to mature spermatozoa. Interestingly, in mice subjected to chemotherapy and radiotherapy, both of which damage the vast majority of germ cells and can result in sterility, PAX7+ spermatogonia selectively survived, and their subsequent expansion contributed to the recovery of spermatogenesis. Finally, PAX7+ spermatogonia were present in the testes of a diverse set of mammals. Our data indicate that the PAX7+ subset of A(single) spermatogonia functions as robust testis stem cells that maintain fertility in normal spermatogenesis in healthy mice and mediate recovery after severe germline injury, such as occurs after cancer therapy.

Chronic binge alcohol consumption alters myogenic gene expression and reduces in vitro myogenic differentiation potential of myoblasts from rhesus macaques.

Chronic alcohol abuse is associated with skeletal muscle myopathy. Previously, we demonstrated that chronic binge alcohol (CBA) consumption by rhesus macaques accentuates skeletal muscle wasting at end-stage of simian immunodeficiency virus (SIV) infection. A proinflammatory, prooxidative milieu and enhanced ubiquitin proteasome activity were identified as possible mechanisms leading to loss of skeletal muscle. The possibility that impaired regenerative capacity, as reflected by the ability of myoblasts derived from satellite cell (SCs) to differentiate into myotubes has not been examined. We hypothesized that the inflammation and oxidative stress in skeletal muscle from CBA animals impair the differentiation capacity of myoblasts to form new myofibers in in vitro assays. We isolated primary myoblasts from the quadriceps femoris of rhesus macaques that were administered CBA or isocaloric sucrose (SUC) for 19 mo. Proliferation and differentiation potential of cultured myoblasts were examined in vitro. Myoblasts from the CBA group had significantly reduced PAX7, MYOD1, MYOG, MYF5, and MEF2C expression. This was associated with decreased myotube formation as evidenced by Jenner-Giemsa staining and myonuclei fusion index. No significant difference in the proliferative ability, cell cycle distribution, or autophagy was detected between myoblasts isolated from CBA and SUC groups. Together, these results reflect marked dysregulation of myoblast myogenic gene expression and myotube formation, which we interpret as evidence of impaired skeletal muscle regenerative capacity in CBA-administered macaques. The contribution of this mechanism to alcoholic myopathy warrants further investigation.

Foxc2 induces Wnt4 and Bmp4 expression during muscle regeneration and osteogenesis.

Proliferation and fusion of myoblasts is a well-orchestrated process occurring during muscle development and regeneration. Although myoblasts are known to originate from muscle satellite cells, the molecular mechanisms that coordinate their commitment toward differentiation are poorly understood. Here, we present a novel role for the transcription factor Forkhead box protein C2 (Foxc2) in regulating proliferation and preventing premature differentiation of activated muscle satellite cells. We demonstrate that Foxc2 expression is upregulated early in activated mouse muscle satellite cells and then diminishes during myogenesis. In undifferentiated C2C12 myoblasts, downregulation of endogenous Foxc2 expression leads to a decrease in proliferation, whereas forced expression of FOXC2 sustains proliferation and prevents differentiation into myotubes. We also show that FOXC2 induces Wnt signaling by direct interaction with the Wnt4 (wingless-type MMTV integration site family member-4) promoter region. The resulting elevated expression of bone morphogenetic protein-4 (Bmp4) and RhoA-GTP proteins inhibits the proper myoblast alignment and fusion required for myotube formation. Interestingly, continuous forced expression of FOXC2 alters the commitment of C2C12 myoblasts toward osteogenic differentiation, which is consistent with FOXC2 expression observed in patients with myositis ossificans, an abnormal bone growth within muscle tissue. In summary, our results suggest that (a) Foxc2 regulates the proliferation of multipotent muscle satellite cells; (b) downregulation of Foxc2 is critical for myogenesis to progress; and (c) sustained Foxc2 expression in myoblast cells suppresses myogenesis and alters their lineage commitment toward osteogenesis by inducing the Wnt4 and Bmp4 signaling pathways.

A mouse model of rhabdomyosarcoma originating from the adipocyte lineage.

Rhabdomyosarcoma (RMS) is an aggressive skeletal muscle-lineage tumor composed of malignant myoblasts that fail to exit the cell cycle and are blocked from fusing into syncytial muscle. Rhabdomyosarcoma includes two histolopathologic subtypes: alveolar rhabdomyosarcoma, driven by the fusion protein PAX3-FOXO1 or PAX7-FOXO1, and embryonal rhabdomyosarcoma (ERMS), which is genetically heterogeneous. Here, we show that adipocyte-restricted activation of Sonic hedgehog signaling through expression of a constitutively active Smoothened allele in mice gives rise to aggressive skeletal muscle tumors that display the histologic and molecular characteristics of human ERMS with high penetrance. Our findings suggest that adipocyte progenitors can be a cell of origin for Sonic hedgehog-driven ERMS, showing that RMS can originate from nonskeletal muscle precursors.

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.

Endogenous erythropoietin signaling facilitates skeletal muscle repair and recovery following pharmacologically induced damage.

Erythropoietin acts by binding to its cell surface receptor on erythroid progenitor cells to stimulate erythrocyte production. Erythropoietin receptor expression in nonhematopoietic tissue, including skeletal muscle progenitor cells, raises the possibility of a role for erythropoietin beyond erythropoiesis. Mice with erythropoietin receptor restricted to hematopoietic tissue were used to assess contributions of endogenous erythropoietin to promote skeletal myoblast proliferation and survival and wound healing in a mouse model of cardiotoxin induced muscle injury. Compared with wild-type controls, these mice had fewer skeletal muscle Pax-7(+) satellite cells and myoblasts that do not proliferate in culture, were more susceptible to skeletal muscle injury and reduced maximum load tolerated by isolated muscle. In contrast, mice with chronic elevated circulating erythropoietin had more Pax-7(+) satellite cells and myoblasts with increased proliferation and survival in culture, decreased muscle injury, and accelerated recovery of maximum load tolerated by isolated muscle. Skeletal muscle myoblasts also produced endogenous erythropoietin that increased at low O(2). Erythropoietin promoted proliferation, survival, and wound recovery in myoblasts via the phosphoinositide 3-kinase/AKT pathway. Therefore, endogenous and exogenous erythropoietin contribute to increasing satellite cell number following muscle injury, improve myoblast proliferation and survival, and promote repair and regeneration in this mouse induced muscle injury model independent of its effect on erythrocyte production.