A change in cis-regulatory logic underlying obligate versus facultative muscle multinucleation in chordates.

Vertebrates and tunicates are sister groups that share a common fusogenic factor, Myomaker (Mymk), that drives myoblast fusion and muscle multinucleation. Yet they are divergent in when and where they express Mymk. In vertebrates, all developing skeletal muscles express Mymk and are obligately multinucleated. In tunicates, Mymk is expressed only in post-metamorphic multinucleated muscles, but is absent from mononucleated larval muscles. In this study, we demonstrate that cis-regulatory sequence differences in the promoter region of Mymk underlie the different spatiotemporal patterns of its transcriptional activation in tunicates and vertebrates. Although in vertebrates myogenic regulatory factors (MRFs) such as MyoD1 alone are required and sufficient for Mymk transcription in all skeletal muscles, we show that transcription of Mymk in post-metamorphic muscles of the tunicate Ciona requires the combinatorial activity of MRF, MyoD and Early B-cell Factor (Ebf). This macroevolutionary difference appears to be encoded in cis, likely due to the presence of a putative Ebf-binding site adjacent to predicted MRF binding sites in the Ciona Mymk promoter. We further discuss how Mymk and myoblast fusion might have been regulated in the last common ancestor of tunicates and vertebrates, for which we propose two models.

Cell fusion: Inter-organ tissue communication promotes a union between myoblasts.

Repeated rounds of fusion between apposing myoblasts allow muscles to become multinucleated. New research finds that myoblasts undergoing fusion in the Drosophila embryo respond to hormone signaling from a nearby tissue, resulting in the activation of a myoblast-specific gene necessary for the fusion process.

A change in cis-regulatory logic underlying obligate versus facultative muscle multinucleation in chordates.

Vertebrates and tunicates are sister groups that share a common fusogenic factor, Myomaker (Mymk), that drives myoblast fusion and muscle multinucleation. Yet they are divergent in when and where they express Mymk. In vertebrates, all developing skeletal muscles express Mymk and are obligately multinucleated. In tunicates, Mymk is only expressed in post-metamorphic multinucleated muscles, but is absent from mononucleated larval muscles. In this study, we demonstrate that cis-regulatory sequence differences in the promoter region of Mymk underlie the different spatiotemporal patterns of its transcriptional activation in tunicates and vertebrates. While in vertebrates Myogenic Regulatory Factors (MRFs) like MyoD1 alone are required and sufficient for Mymk transcription in all skeletal muscles, we show that transcription of Mymk in post-metamorphic muscles of the tunicate Ciona requires the combinatorial activity of MRF/MyoD and Early B-Cell Factor (Ebf). This macroevolutionary difference appears to be encoded in cis, likely due to the presence of a putative Ebf binding site adjacent to predicted MRF binding sites in the Ciona Mymk promoter. We further discuss how Mymk and myoblast fusion might have been regulated in the last common ancestor of tunicates and vertebrates, for which we propose two models.

Evolution of a chordate-specific mechanism for myoblast fusion.

Vertebrate myoblast fusion allows for multinucleated muscle fibers to compound the size and strength of mononucleated cells, but the evolution of this important process is unknown. We investigated the evolutionary origins and function of membrane-coalescing agents Myomaker and Myomixer in various groups of chordates. Here, we report that Myomaker likely arose through gene duplication in the last common ancestor of tunicates and vertebrates, while Myomixer appears to have evolved de novo in early vertebrates. Functional tests revealed a complex evolutionary history of myoblast fusion. A prevertebrate phase of muscle multinucleation driven by Myomaker was followed by the later emergence of Myomixer that enables the highly efficient fusion system of vertebrates. Evolutionary comparisons between vertebrate and nonvertebrate Myomaker revealed key structural and mechanistic insights into myoblast fusion. Thus, our findings suggest an evolutionary model of chordate fusogens and illustrate how new genes shape the emergence of novel morphogenetic traits and mechanisms.

Loss of Myomixer Results in Defective Myoblast Fusion, Impaired Muscle Growth, and Severe Myopathy in Zebrafish.

The development and growth of fish skeletal muscles require myoblast fusion to generate multinucleated myofibers. While zebrafish fast-twitch muscle can fuse to generate multinucleated fibers, the slow-twitch muscle fibers remain mononucleated in zebrafish embryos and larvae. The mechanism underlying the fiber-type-specific control of fusion remains elusive. Recent genetic studies using mice identified a long-sought fusion factor named Myomixer. To understand whether Myomixer is involved in the fiber-type specific fusion, we analyzed the transcriptional regulation of myomixer expression and characterized the muscle growth phenotype upon genetic deletion of myomixer in zebrafish. The data revealed that overexpression of Sonic Hedgehog (Shh) drastically inhibited myomixer expression and blocked myoblast fusion, recapitulating the phenotype upon direct genetic deletion of myomixer from zebrafish. The fusion defect in myomixer mutant embryos could be faithfully rescued upon re-expression of zebrafish myomixer gene or its orthologs from shark or human. Interestingly, myomixer mutant fish survived to adult stage though were notably smaller than wildtype siblings. Severe myopathy accompanied by the uncontrolled adipose infiltration was observed in both fast and slow muscle tissues of adult myomixer mutants. Collectively, our data highlight an indispensable role of myomixer gene for cell fusion during both embryonic muscle development and post-larval muscle growth.

LETMD1 is required for mitochondrial structure and thermogenic function of brown adipocytes.

Obesity and metabolic disorders caused by energy surplus pose an increasing concern within the global population. Brown adipose tissue (BAT) dissipates energy through mitochondrial non-shivering thermogenesis, thus representing a powerful agent against obesity. Here we explore the novel role of a mitochondrial outer membrane protein, LETM1-domain containing 1 (LETMD1), in BAT. We generated a knockout (Letmd1KO ) mouse model and analyzed BAT morphology, function and gene expression under various physiological conditions. While the Letmd1KO mice are born normally and have normal morphology and body weight, they lose multilocular brown adipocytes completely and have diminished mitochondrial abundance, DNA copy number, cristae structure, and thermogenic gene expression in the intrascapular BAT, associated with elevated reactive oxidative stress. In consequence, the Letmd1KO mice fail to maintain body temperature in response to acute cold exposure without food and become hypothermic within 4 h. Although the cold-exposed Letmd1KO mice can maintain body temperature in the presence of food, they cannot upregulate expression of uncoupling protein 1 (UCP1) and convert white to beige adipocytes, nor can they respond to adrenergic stimulation. These results demonstrate that LETMD1 is essential for mitochondrial structure and function, and thermogenesis of brown adipocytes.

Feedback regulation of Notch signaling and myogenesis connected by MyoD-Dll1 axis.

Muscle precursor cells known as myoblasts are essential for muscle development and regeneration. Notch signaling is an ancient intercellular communication mechanism that plays prominent roles in controlling the myogenic program of myoblasts. Currently whether and how the myogenic cues feedback to refine Notch activities in these cells are largely unknown. Here, by mouse and human gene gain/loss-of-function studies, we report that MyoD directly turns on the expression of Notch-ligand gene Dll1 which activates Notch pathway to prevent precautious differentiation in neighboring myoblasts, while autonomously inhibits Notch to facilitate a myogenic program in Dll1 expressing cells. Mechanistically, we studied cis-regulatory DNA motifs underlying the MyoD-Dll1-Notch axis in vivo by characterizing myogenesis of a novel E-box deficient mouse model, as well as in human cells through CRISPR-mediated interference. These results uncovered the crucial transcriptional mechanism that mediates the reciprocal controls of Notch and myogenesis.

Generation of mouse conditional knockout alleles in one step using the i-GONAD method.

The Cre/loxP system is a powerful tool for gene function study in vivo. Regulated expression of Cre recombinase mediates precise deletion of genetic elements in a spatially- and temporally-controlled manner. Despite the robustness of this system, it requires a great amount of effort to create a conditional knockout model for each individual gene of interest where two loxP sites must be simultaneously inserted in cis The current undertaking involves labor-intensive embryonic stem (ES) cell-based gene targeting and tedious micromanipulations of mouse embryos. The complexity of this workflow poses formidable technical challenges, thus limiting wider applications of conditional genetics. Here, we report an alternative approach to generate mouse loxP alleles by integrating a unique design of CRISPR donor with the new oviduct electroporation technique i-GONAD. Showing the potential and simplicity of this method, we created floxed alleles for five genes in one attempt with relatively low costs and a minimal equipment setup. In addition to the conditional alleles, constitutive knockout alleles were also obtained as byproducts of these experiments. Therefore, the wider applications of i-GONAD may promote gene function studies using novel murine models.

Human myotube formation is determined by MyoD-Myomixer/Myomaker axis.

Myoblast fusion is essential for formations of myofibers, the basic cellular and functional units of skeletal muscles. Recent genetic studies in mice identified two long-sought membrane proteins, Myomaker and Myomixer, which cooperatively drive myoblast fusion. It is unknown whether and how human muscles, with myofibers of tremendously larger size, use this mechanism to achieve multinucleations. Here, we report an interesting fusion model of human myoblasts where Myomaker is sufficient to induce low-grade fusion, while Myomixer boosts its efficiency to generate giant myotubes. By CRISPR mutagenesis and biochemical assays, we identified MyoD as the key molecular switch of fusion that is required and sufficient to initiate Myomixer and Myomaker expression. Mechanistically, we defined the E-box motifs on promoters of Myomixer and Myomaker by which MyoD induces their expression for multinucleations of human muscle cells. Together, our study uncovered the key molecular apparatus and the transcriptional control mechanism underlying human myoblast fusion.

Fusogenic micropeptide Myomixer is essential for satellite cell fusion and muscle regeneration.

Regeneration of skeletal muscle in response to injury occurs through fusion of a population of stem cells, known as satellite cells, with injured myofibers. Myomixer, a muscle-specific membrane micropeptide, cooperates with the transmembrane protein Myomaker to regulate embryonic myoblast fusion and muscle formation. To investigate the role of Myomixer in muscle regeneration, we used CRISPR/Cas9-mediated genome editing to generate conditional knockout Myomixer alleles in mice. We show that genetic deletion of Myomixer in satellite cells using a tamoxifen-regulated Cre recombinase transgene under control of the Pax7 promoter abolishes satellite cell fusion and prevents muscle regeneration, resulting in severe muscle degeneration after injury. Satellite cells devoid of Myomixer maintain expression of Myomaker, demonstrating that Myomaker alone is insufficient to drive myoblast fusion. These findings, together with prior studies demonstrating the essentiality of Myomaker for muscle regeneration, highlight the obligatory partnership of Myomixer and Myomaker for myofiber formation throughout embryogenesis and adulthood.

Requirement of the fusogenic micropeptide myomixer for muscle formation in zebrafish.

Skeletal muscle formation requires fusion of mononucleated myoblasts to form multinucleated myofibers. The muscle-specific membrane proteins myomaker and myomixer cooperate to drive mammalian myoblast fusion. Whereas myomaker is highly conserved across diverse vertebrate species, myomixer is a micropeptide that shows relatively weak cross-species conservation. To explore the functional conservation of myomixer, we investigated the expression and function of the zebrafish myomixer ortholog. Here we show that myomixer expression during zebrafish embryogenesis coincides with myoblast fusion, and genetic deletion of myomixer using CRISPR/Cas9 mutagenesis abolishes myoblast fusion in vivo. We also identify myomixer orthologs in other species of fish and reptiles, which can cooperate with myomaker and substitute for the fusogenic activity of mammalian myomixer. Sequence comparison of these diverse myomixer orthologs reveals key amino acid residues and a minimal fusogenic peptide motif that is necessary for promoting cell-cell fusion with myomaker. Our findings highlight the evolutionary conservation of the myomaker-myomixer partnership and provide insights into the molecular basis of myoblast fusion.

Control of muscle formation by the fusogenic micropeptide myomixer.

Skeletal muscle formation occurs through fusion of myoblasts to form multinucleated myofibers. From a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) loss-of-function screen for genes required for myoblast fusion and myogenesis, we discovered an 84-amino acid muscle-specific peptide that we call Myomixer. Myomixer expression coincides with myoblast differentiation and is essential for fusion and skeletal muscle formation during embryogenesis. Myomixer localizes to the plasma membrane, where it promotes myoblast fusion and associates with Myomaker, a fusogenic membrane protein. Myomixer together with Myomaker can also induce fibroblast-fibroblast fusion and fibroblast-myoblast fusion. We conclude that the Myomixer-Myomaker pair controls the critical step in myofiber formation during muscle development.

Pten is necessary for the quiescence and maintenance of adult muscle stem cells.

Satellite cells (SCs) are myogenic stem cells required for regeneration of adult skeletal muscles. A proper balance among quiescence, activation and differentiation is essential for long-term maintenance of SCs and their regenerative function. Here we show a function of Pten (phosphatase and tensin homologue) in quiescent SCs. Deletion of Pten in quiescent SCs leads to their spontaneous activation and premature differentiation without proliferation, resulting in depletion of SC pool and regenerative failure. However, prior to depletion, Pten-null activated SCs can transiently proliferate upon injury and regenerate injured muscles, but continually decline during regeneration, suggesting an inability to return to quiescence. Mechanistically, Pten deletion increases Akt phosphorylation, which induces cytoplasmic translocation of FoxO1 and suppression of Notch signalling. Accordingly, constitutive activation of Notch1 prevents SC depletion despite Pten deletion. Our findings delineate a critical function of Pten in maintaining SC quiescence and reveal an interaction between Pten and Notch signalling.

Conditional Loss of Pten in Myogenic Progenitors Leads to Postnatal Skeletal Muscle Hypertrophy but Age-Dependent Exhaustion of Satellite Cells.

Skeletal muscle stem cells (satellite cells [SCs]) are normally maintained in a quiescent (G0) state. Muscle injury not only activates SCs locally, but also alerts SCs in distant uninjured muscles via circulating factors. The resulting GAlert SCs are adapted to regenerative cues and regenerate injured muscles more efficiently, but whether they provide any long-term benefits to SCs is unknown. Here, we report that embryonic myogenic progenitors lacking the phosphatase and tensin homolog (Pten) exhibit enhanced proliferation and differentiation, resulting in muscle hypertrophy but fewer SCs in adult muscles. Interestingly, Pten null SCs are predominantly in the GAlert state, even in the absence of an injury. The GAlert SCs are deficient in self-renewal and subjected to accelerated depletion during regeneration and aging and fail to repair muscle injury in old mice. Our findings demonstrate a key requirement of Pten in G0 entry of SCs and provide functional evidence that prolonged GAlert leads to stem cell depletion and regenerative failure.

Stage-specific effects of Notch activation during skeletal myogenesis.

Skeletal myogenesis involves sequential activation, proliferation, self-renewal/differentiation and fusion of myogenic stem cells (satellite cells). Notch signaling is known to be essential for the maintenance of satellite cells, but its function in late-stage myogenesis, i.e. post-differentiation myocytes and post-fusion myotubes, is unknown. Using stage-specific Cre alleles, we uncovered distinct roles of Notch1 in mononucleated myocytes and multinucleated myotubes. Specifically, constitutive Notch1 activation dedifferentiates myocytes into Pax7 quiescent satellite cells, leading to severe defects in muscle growth and regeneration, and postnatal lethality. By contrast, myotube-specific Notch1 activation improves the regeneration and exercise performance of aged and dystrophic muscles. Mechanistically, Notch1 activation in myotubes upregulates the expression of Notch ligands, which modulate Notch signaling in the adjacent satellite cells to enhance their regenerative capacity. These results highlight context-dependent effects of Notch activation during myogenesis, and demonstrate that Notch1 activity improves myotube's function as a stem cell niche.

Notch activation drives adipocyte dedifferentiation and tumorigenic transformation in mice.

Liposarcomas (LPSs) are the most common soft-tissue cancer. Because of the lack of animal models, the cellular origin and molecular regulation of LPS remain unclear. Here, we report that mice with adipocyte-specific activation of Notch signaling (Ad/N1ICD) develop LPS with complete penetrance. Lineage tracing confirms the adipocyte origin of Ad/N1ICD LPS. The Ad/N1ICD LPS resembles human dedifferentiated LPS in histological appearance, anatomical localization, and gene expression signature. Before transformation, Ad/N1ICD adipocytes undergo dedifferentiation that leads to lipodystrophy and metabolic dysfunction. Although concomitant Pten deletion normalizes the glucose metabolism of Ad/N1ICD mice, it dramatically accelerates the LPS prognosis and malignancy. Transcriptomes and lipidomics analyses indicate that Notch activation suppresses lipid metabolism pathways that supply ligands to Pparγ, the master regulator of adipocyte homeostasis. Accordingly, synthetic Pparγ ligand supplementation induces redifferentiation of Ad/N1ICD adipocytes and tumor cells, and prevents LPS development in Ad/N1ICD mice. Importantly, the Notch target HES1 is abundantly expressed in human LPS, and Notch inhibition suppresses the growth of human dedifferentiated LPS xenografts. Collectively, ectopic Notch activation is sufficient to induce dedifferentiation and tumorigenic transformation of mature adipocytes in mouse.

Lkb1 controls brown adipose tissue growth and thermogenesis by regulating the intracellular localization of CRTC3.

Brown adipose tissue (BAT) dissipates energy through Ucp1-mediated uncoupled respiration and its activation may represent a therapeutic strategy to combat obesity. Here we show that Lkb1 controls BAT expansion and UCP1 expression in mice. We generate adipocyte-specific Lkb1 knockout mice and show that, compared with wild-type littermates, these mice exhibit elevated UCP1 expression in BAT and subcutaneous white adipose tissue, have increased BAT mass and higher energy expenditure. Consequently, KO mice have improved glucose tolerance and insulin sensitivity, and are more resistant to high-fat diet (HFD)-induced obesity. Deletion of Lkb1 results in a cytoplasm to nuclear translocation of CRTC3 in brown adipocytes, where it recruits C/EBPß to enhance Ucp1 transcription. In parallel, the absence of Lkb1 also suppresses AMPK activity, leading to activation of the mTOR signalling pathway and subsequent BAT expansion. These data suggest that inhibition of Lkb1 or its downstream signalling in adipocytes could be a novel strategy to increase energy expenditure in the context of obesity, diabetes and other metabolic diseases.

Notch signaling as a novel regulator of metabolism.

Evolutionarily unprepared for modern high-calorie diets and sedentary lifestyles, humans are now unprecedentedly susceptible to metabolic disorders such as obesity, type 2 diabetes (T2D), nonalcoholic fatty liver, and cardiovascular disease. These metabolic conditions are intertwined, together known as metabolic syndrome, and compromise human life quality as well as lives. Notch signaling, a fundamental signal transduction pathway critical for cell-cell communication and development, has recently been recognized as a key player in metabolism. This review summarizes the emerging roles of Notch signaling in regulating the metabolism of various cell and tissue types, with emphasis on the underlying molecular mechanisms and the potential of targeting this signal axis to treat metabolic diseases.

Lkb1 deletion promotes ectopic lipid accumulation in muscle progenitor cells and mature muscles.

Excessive intramyocellular triglycerides (muscle lipids) are associated with reduced contractile function, insulin resistance, and Type 2 diabetes, but what governs lipid accumulation in muscle is unclear. Here we report a role of Lkb1 in regulating lipid metabolism in muscle stem cells and their descendent mature muscles. We used Myod(Cre) and Lkb1(flox/flox) mice to specifically delete Lkb1 in myogenic cells including stem and differentiated cells, and examined the lipid accumulation and gene expression of myoblasts cultured from muscle stem cells (satellite cells). Genetic deletion of Lkb1 in myogenic progenitors led to elevated expression of lipogenic genes and ectopic lipid accumulation in proliferating myoblasts. Interestingly, the Lkb1-deficient myoblasts differentiated into adipocyte-like cells upon adipogenic induction. However, these adipocyte-like cells maintained myogenic gene expression with reduced ability to form myotubes efficiently. Activation of AMPK by AICAR prevented ectopic lipid formation in the Lkb1-null myoblasts. Notably, Lkb1-deficient muscles accumulated excessive lipids in vivo in response to high-fat diet feeding. These results demonstrate that Lkb1 acts through AMPK to limit lipid deposition in muscle stem cells and their derivative mature muscles, and point to the possibility of controlling muscle lipid content using AMPK activating drugs.

Plk1 phosphorylation of PTEN causes a tumor-promoting metabolic state.

One outcome of activation of the phosphatidylinositol 3-kinase (PI3K) pathway is increased aerobic glycolysis, but the upstream signaling events that regulate the PI3K pathway, and thus the Warburg effect, are elusive. Increasing evidence suggests that Plk1, a cell cycle regulator, is also involved in cellular events in addition to mitosis. To test whether Plk1 contributes to activation of the PI3K pathway, and thus aerobic glycolysis, we examined potential targets of Plk1 and identified PTEN as a Plk1 substrate. We hypothesize that Plk1 phosphorylation of PTEN leads to its inactivation, activation of the PI3K pathway, and the Warburg effect. Our data show that overexpression of Plk1 leads to activation of the PI3K pathway and enhanced aerobic glycolysis. In contrast, inhibition of Plk1 causes markedly reduced glucose metabolism in mice. Mechanistically, we show that Plk1 phosphorylation of PTEN and Nedd4-1, an E3 ubiquitin ligase of PTEN, results in PTEN inactivation. Finally, we show that Plk1 phosphorylation of PTEN promotes tumorigenesis in both its phosphatase-dependent and -independent pathways, revealing potentially new drug targets to arrest tumor cell growth.