Alessandro Magli

Nfix Regulates Fetal-Specific Transcription in Developing Skeletal Muscle

Skeletal myogenesis, like hematopoiesis, occurs in successive developmental stages that involve different cell populations and expression of different genes. We show here that the transcription factor nuclear factor one X (Nfix), whose expression is activated by Pax7 in fetal muscle, in turn activates the transcription of fetal specific genes such as MCK and beta-enolase while repressing embryonic genes such as slow myosin. In the case of the MCK promoter, Nfix forms a complex with PKC theta that binds, phosphorylates, and activates MEF2A. Premature expression of Nfix activates fetal and suppresses embryonic genes in embryonic muscle, whereas muscle-specific ablation of Nfix prevents fetal and maintains embryonic gene expression in the fetus. Therefore, Nfix acts as a transcriptional switch from embryonic to fetal myogenesis.

Modulation of TGF-β signaling by endoglin in murine hemangioblast development and primitive hematopoiesis.

Endoglin (Eng), an accessory receptor for the transforming growth factor β (TGF-β) superfamily, is required for proper hemangioblast and primitive hematopoietic development. However the mechanism by which endoglin functions at this early developmental stage is currently unknown. Transcriptional analyses of differentiating eng(-/-) and eng(+/+) ES cells revealed that lack of endoglin leads to profound reductions in the levels of key hematopoietic regulators, including Scl, Lmo2, and Gata2. We also detected lower levels of phosphorylated Smad1 (pSmad1), a downstream target signaling molecule associated with the TGF-β pathway. Using doxycycline-inducible ES cell lines, we interrogated the TGF-β signaling pathway by expressing activated forms of ALK-1 and ALK-5, type I receptors for TGF-β. Our results indicate that ALK-1 signaling promotes hemangioblast development and hematopoiesis, as evidenced by colony assays, gene expression and FACS analyses, whereas signaling by ALK-5 leads to the opposite effect, inhibition of hemangioblast and hematopoietic development. In Eng(-/-) ES cells, ALK-1 rescued both the defective hemangioblast development, and primitive erythropoiesis, indicating that ALK-1 signaling can compensate for the absence of endoglin. We propose that endoglin regulates primitive hematopoiesis by modulating the activity of the Smad1/5 signaling pathway in early stages of development.

Differentiation-dependent lysine 4 acetylation enhances MEF2C binding to DNA in skeletal muscle cells

Myocyte enhancer factor 2 (MEF2) proteins play a key role in promoting the expression of muscle-specific genes in differentiated muscle cells. MEF2 activity is regulated by the association with several transcriptional co-factors and by post-translational modifications. In the present report, we provide evidence for a novel regulatory mechanism of MEF2C activity, which occurs at the onset of skeletal muscle differentiation and is based on Lys4 acetylation. This covalent modification results in the enhancement of MEF2C binding to DNA and chromatin. In particular, we report that the kinetic parameters of MEF2/DNA association change substantially upon induction of differentiation to give a more stable complex and that this effect is mediated by Lys4 acetylation. We also show that Lys4 acetylation plays a prominent role in the p300-dependent activation of MEF2C.

Proline Isomerase Pin1 represses terminal differentiation and Myocyte Enhancer Factor 2C function in Skeletal Muscle Cells

Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser98 and Ser110, which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.

Functional Dissection of Pax3 in Paraxial Mesoderm Development and Myogenesis

The paired box transcription factor Pax3 is well‐known as a major regulator of embryonic myogenesis. Before Pax3 expression becomes restricted to the dermomyotome, this transcription factor is also expressed in the developing somites. The role of Pax3 at this early stage is unclear, in particular because of the scarce frequency of Pax3‐positive cells in the early mouse embryo. Inducible gene expression in embryonic stem cells (ESCs) represents an excellent tool to overcome this limitation, since it can provide large quantities of otherwise rare embryonic populations expressing a factor of interest. Here we used engineered mouse ESCs to perform a functional analysis of Pax3 with the aim to identify the molecular determinants involved in the early functions of this transcription factor. We find that Pax3 induction during embryoid body differentiation results in the upregulation of genes expressed in the presomitic and somitic mesoderm. Moreover, we show that paraxial mesoderm induced by transient expression of Pax3 is not irreversibly committed to myogenesis rather requires sustained Pax3 expression. Using a series of deletion mutants of Pax3, which differentially affect its transcriptional activity, we map protein domains necessary for induction of paraxial mesoderm and induction of the myogenic program. The paired, homeo‐, and transcriptional activation domains were each required for both processes, however, the paired‐c‐terminal RED domain showed a paraxial mesoderm‐specific activity that was dispensable for myogenesis. These findings demonstrate and provide mechanistic insight into an early role for Pax3 in the generation of paraxial mesoderm. STEM Cells2013;31:59–70

Nuclear localization of cationic solid lipid nanoparticles containing Protamine as transfection promoter.

Protamine has attracted much attention as DNA condenser and nuclear transfer enhancer although the excess of hydrophilicity and the strong DNA pack restrain its potentialities. In order to overcome this limitation, we added Protamine in the composition of solid lipid nanoparticles (SLN-Protamine) and we compared this carrier with the same kind of SLN containing Esterquat 1 instead of Protamine (SLN-EQ1). Carriers cytotoxicity was assessed on COS-I cells evaluating the cell cycle by propidium iodide test, while the transfection efficiency was studied using pEGFP as plasmid model. The cell penetrating activity of Protamine inside the lipid vectors was evaluated studying cell internalization by confocal microscopy using Red Nile-labeled carriers. SLN-Protamine:pDNA showed a mean diameter five-times smaller than the size of SLN-EQ1:pDNA and a remarkably lesser cytotoxicity. Transfection by SLN-Protamine:pDNA was seven-times more effective compared with the Protamine:pDNA polyplexes while no transfection capacity was observed for SLN-EQ1:pDNA complexes due to their inability to be internalized owing to their larger dimension. Red Nile-SLN-Protamine were localized in endocytic-like vesicles into the nuclear membrane suggesting the inclusion of Protamine in nano-lipophilic systems may enhance the reduction in the complex dimensions, the nuclear pDNA translocation and the pDNA release in the cells.

Pax3 and Tbx5 Specify Whether PDGFRα+ Cells Assume Skeletal or Cardiac Muscle Fate in Differentiating Embryonic Stem Cells

Embryonic stem cells (ESCs) represent an ideal model to study how lineage decisions are established during embryonic development. Using a doxycycline‐inducible mouse ESC line, we have previously shown that expression of the transcriptional activator Pax3 in early mesodermal cells leads to the robust generation of paraxial mesoderm progenitors that ultimately differentiate into skeletal muscle precursors. Here, we show that the ability of this transcription factor to induce the skeletal myogenic cell fate occurs at the expenses of the cardiac lineage. Our results show that the PDGFRα+FLK1− subfraction represents the main population affected by Pax3, through downregulation of several transcripts encoding for proteins involved in cardiac development. We demonstrate that although Nkx2–5, Tbx5, and Gata4 negatively affect Pax3 skeletal myogenic activity, the cardiac potential of embryoid body‐derived cultures is restored solely by forced expression of Tbx5. Taking advantage of this model, we used an unbiased genome‐wide approach to identify genes whose expression is rescued by Tbx5, and which could represent important regulators of cardiac development. These findings elucidate mechanisms regulating the commitment of mesodermal cells in the early embryo and identify the Tbx5 cardiac transcriptome. Stem Cells 2014;32:2072–2083

EFFECT OF ENDOGLIN OVEREXPRESSION DURING EMBRYOID BODY DEVELOPMENT

Increasing evidence points to endoglin (Eng), an accessory receptor for the transforming growth factor-β superfamily commonly associated with the endothelial lineage, as an important regulator of the hematopoietic lineage. We have shown that lack of Eng results in reduced numbers of primitive erythroid colonies as well as downregulation of key hematopoietic genes. To determine the effect of Eng overexpression in hematopoietic development, we generated a doxycycline-inducible embryonic stem cell line. Our results demonstrate that induction of Eng during embryoid body differentiation leads to a significant increase in the frequency of hematopoietic progenitors, in particular, the erythroid lineage, which correlated with upregulation of Scl, Gata1, Runx1, and embryonic globin. Interestingly, activation of the hematopoietic program happened at the expense of endothelial and cardiac cells, as differentiation into these mesoderm lineages was compromised. Eng-induced enhanced erythroid activity was accompanied by high levels of Smad1 phosphorylation. This effect was attenuated by addition of a bone morphogenetic protein (BMP) signaling inhibitor to these cultures. Among the BMPs, BMP4 is well known for its role in hematopoietic specification from mesoderm by promoting expression of several hematopoietic genes, including Scl. Because Scl is considered the master regulator of the hematopoietic program, we investigated whether Scl would be capable of rescuing the defective hematopoietic phenotype observed in Eng(-/-) embryonic stem cells. Scl expression in Eng-deficient embryonic stem cells resulted in increased erythroid colony-forming activity and upregulation of Gata1 and Gata2, positioning Eng upstream of Scl. Taken together, these findings support the premise that Eng modulates the hematopoietic transcriptional network, most likely through regulation of BMP4 signaling.

Expansion and Purification Are Critical for the Therapeutic Application of Pluripotent Stem Cell-Derived Myogenic Progenitors

Summary Recent reports have documented the differentiation of human pluripotent stem cells toward the skeletal myogenic lineage using transgene- and cell purification-free approaches. Although these protocols generate myocytes, they have not demonstrated scalability, safety, and in vivo engraftment, which are key aspects for their future clinical application. Here we recapitulate one prominent protocol, and show that it gives rise to a heterogeneous cell population containing myocytes and other cell types. Upon transplantation, the majority of human donor cells could not contribute to myofiber formation. As a proof-of-principle, we incorporated the inducible PAX7 lentiviral system into this protocol, which then enabled scalable expansion of a homogeneous population of skeletal myogenic progenitors capable of forming myofibers in vivo. Our findings demonstrate the methods for scalable expansion of PAX7+ myogenic progenitors and their purification are critical for practical application to cell replacement treatment of muscle degenerative diseases.

Myogenic progenitor specification from pluripotent stem cells

Pluripotent stem cells represent important tools for both basic and translational science as they enable to study mechanisms of development, model diseases in vitro and provide a potential source of tissue-specific progenitors for cell therapy. Concomitantly with the increasing knowledge of the molecular mechanisms behind activation of the skeletal myogenic program during embryonic development, novel findings in the stem cell field provided the opportunity to begin recapitulating in vitro the events occurring during specification of the myogenic lineage. In this review, we will provide a perspective of the molecular mechanisms responsible for skeletal myogenic commitment in the embryo and how this knowledge was instrumental for specifying this lineage from pluripotent stem cells. In addition, we will discuss the current limitations for properly recapitulating skeletal myogenesis in the petri dish, and we will provide insights about future applications of pluripotent stem cell-derived myogenic cells.