Publications

Fibrosis remains a major unmet medical need. Simplifying principles are needed to better understand fibrosis and to yield new therapeutic approaches. Fibrosis is driven by myofibroblasts that interact with macrophages. A mathematical cell-circuit model predicts two types of fibrosis: hot fibrosis driven by macrophages and myofibroblasts and cold fibrosis driven by myofibroblasts alone. Testing these concepts in cardiac fibrosis resulting from myocardial infarction (MI) and heart failure (HF), we revealed that acute MI leads to cold fibrosis whereas chronic injury (HF) leads to hot fibrosis. MI-driven cold fibrosis is conserved in pigs and humans. We computationally identified a vulnerability of cold fibrosis: the myofibroblast autocrine growth factor loop. Inhibiting this loop by targeting TIMP1 with neutralizing antibodies reduced myofibroblast proliferation and fibrosis post-MI in mice. Our study demonstrates the utility of the concepts of hot and cold fibrosis and the feasibility of a circuit-to-target approach to pinpoint a treatment strategy that reduces fibrosis. A record of this paper's transparent peer review process is included in the supplemental information.

Biofabricating 3D cardiac tissues that mimic the native myocardial tissue is a pivotal challenge in tissue engineering. In this study, we fabricate 3D cardiac tissues with controlled, multidirectional cellular alignment and directed or twisting contractility. We show that multidirectional filamented light can be used to biofabricate high-density (up to 60 × 10⁶ cells mL⁻¹) tissues, with directed uniaxial contractility (3.8x) and improved cell-to-cell connectivity (1.6x gap junction expression). Furthermore, by using multidirectional light projection, we can partially overcome cell-induced light attenuation, and fabricate larger tissues with multidirectional cellular alignment. For example, we fabricate a tri-layered myocardium-like tissue and a bi-layered tissue with torsional contractility. The approach provides a new strategy to rapidly fabricate aligned cardiac tissues relevant to regenerative medicine and biohybrid robotics.

Myocardial injury may ultimately lead to adverse ventricular remodeling and development of heart failure (HF), which is a major cause of morbidity and mortality worldwide. Given the slow pace and substantial costs of developing new therapeutics, drug repurposing is an attractive alternative. Studies of many organs, including the heart, highlight the importance of the immune system in modulating injury and repair outcomes. Glatiramer acetate (GA) is an immunomodulatory drug prescribed for patients with multiple sclerosis. Here, we report that short-term GA treatment improves cardiac function and reduces scar area in a mouse model of acute myocardial infarction and a rat model of ischemic HF. We provide mechanistic evidence indicating that, in addition to its immunomodulatory functions, GA exerts beneficial pleiotropic effects, including cardiomyocyte protection and enhanced angiogenesis. Overall, these findings highlight the potential repurposing of GA as a future therapy for a myriad of heart diseases.

Senescence plays a key role in various physiological and pathological processes. We reported that injury-induced transient senescence correlates with heart regeneration, yet the multi-omics profile and molecular underpinnings of regenerative senescence remain obscure. Using proteomics and single-cell RNA sequencing, here we report the regenerative senescence multi-omic signature in the adult mouse heart and establish its role in neonatal heart regeneration and agrin-mediated cardiac repair in adult mice. We identified early growth response protein 1 (Egr1) as a regulator of regenerative senescence in both models. In the neonatal heart, Egr1 facilitates angiogenesis and cardiomyocyte proliferation. In adult hearts, agrin-induced senescence and repair require Egr1, activated by the integrinFAKERKAkt1 axis in cardiac fibroblasts. We also identified cathepsins as injury-induced senescence-associated secretory phenotype components that promote extracellular matrix degradation and potentially assist in reducing fibrosis. Altogether, we uncovered the molecular signature and functional benefits of regenerative senescence during heart regeneration, with Egr1 orchestrating the process.

Zebrafish have a lifelong cardiac regenerative ability after damage, whereas mammals lose this capacity during early postnatal development. This study investigated whether the declining expression of growth factors during postnatal mammalian development contributes to the decrease of cardiomyocyte regenerative potential. Besides confirming the proliferative ability of neuregulin 1 (NRG1), interleukin (IL)1b, receptor activator of nuclear factor kappa-Β ligand (RANKL), insulin growth factor (IGF)2, and IL6, we identified other potential pro-regenerative factors, with BMP7 exhibiting the most pronounced efficacy. Bmp7 knockdown in neonatal mouse cardiomyocytes and loss-of-function in adult zebrafish during cardiac regeneration reduced cardiomyocyte proliferation, indicating that Bmp7 is crucial in the regenerative stages of mouse and zebrafish hearts. Conversely, bmp7 overexpression in regenerating zebrafish or administration at post-mitotic juvenile and adult mouse stages, in vitro and in vivo following myocardial infarction, enhanced cardiomyocyte cycling. Mechanistically, BMP7 stimulated proliferation through BMPR1A/ACVR1 and ACVR2A/BMPR2 receptors and downstream SMAD5, ERK, and AKT signaling. Overall, BMP7 administration is a promising strategy for heart regeneration.

The tumor microenvironment (TME) is comprised of non-malignant cells that interact with each other and with cancer cells, critically impacting cancer biology. The TME is complex, and understanding it requires simplifying approaches. Here we provide an experimental-mathematical approach to decompose the TME into small circuits of interacting cell types. We find, using female breast cancer single-cell-RNA-sequencing data, a hierarchical network of interactions, with cancer-associated fibroblasts (CAFs) at the top secreting factors primarily to tumor-associated macrophages (TAMs). This network is composed of repeating circuit motifs. We isolate the strongest two-cell circuit motif by culturing fibroblasts and macrophages in-vitro, and analyze their dynamics and transcriptomes. This isolated circuit recapitulates the hierarchy of in-vivo interactions, and enables testing the effect of ligand-receptor interactions on cell dynamics and function, as we demonstrate by identifying a mediator of CAF-TAM interactions - RARRES2, and its receptor CMKLR1. Thus, the complexity of the TME may be simplified by identifying small circuits, facilitating the development of strategies to modulate the TME.

Zebrafish hearts can regenerate by replacing damaged tissue with new cardiomyocytes. Although the steps leading up to the proliferation of surviving cardiomyocytes have been extensively studied, little is known about the mechanisms that control proliferation and redifferentiation to a mature state. We found that the cardiac dyad, a structure that regulates calcium handling and excitation-contraction coupling, played a key role in the redifferentiation process. A component of the cardiac dyad called leucine-rich repeat-containing 10 (Lrrc10) acted as a negative regulator of proliferation, prevented cardiomegaly, and induced redifferentiation. We found that its function was conserved in mammalian cardiomyocytes. This study highlights the importance of the underlying mechanisms required for heart regeneration and their application to the generation of fully functional cardiomyocytes.

Cardiomyocyte proliferation and dedifferentiation have fueled the field of regenerative cardiology in recent years, whereas the reverse process of redifferentiation remains largely unexplored. Redifferentiation is characterized by the restoration of function lost during dedifferentiation. Previously, we showed that ERBB2-mediated heart regeneration has these two distinct phases: transient dedifferentiation and redifferentiation. Here we survey the temporal transcriptomic and proteomic landscape of dedifferentiation-redifferentiation in adult mouse hearts and reveal that well-characterized dedifferentiation features largely return to normal, although elements of residual dedifferentiation remain, even after the contractile function is restored. These hearts appear rejuvenated and show robust resistance to ischemic injury, even 5 months after redifferentiation initiation. Cardiomyocyte redifferentiation is driven by negative feedback signaling and requires LATS1/2 Hippo pathway activity. Our data reveal the importance of cardiomyocyte redifferentiation in functional restoration during regeneration but also protection against future insult, in what could lead to a potential prophylactic treatment against ischemic heart disease for at-risk patients.Shakked et al. highlight the importance of cardiomyocytes' redifferentiation after dedifferentiation and proliferation in cardiac repair and show that redifferentiation depends on negative feedback signaling and LATS1/2 Hippo pathway activity. Cardiomyocyte redifferentiation not only promotes the restoration of cardiac function but also protects against future insult.

The efficacy and safety of gene-therapy strategies for indications like tissue damage hinge on precision; yet, current methods afford little spatial or temporal control of payload delivery. Here, we find that tissue-regeneration enhancer elements (TREEs) isolated from zebrafish can direct targeted, injury-associated gene expression from viral DNA vectors delivered systemically in small and large adult mammalian species. When employed in combination with CRISPR-based epigenome editing tools in mice, zebrafish TREEs stimulated or repressed the expression of endogenous genes after ischemic myocardial infarction. Intravenously delivered recombinant AAV vectors designed with a TREE to direct a constitutively active YAP factor boosted indicators of cardiac regeneration in mice and improved the function of the injured heart. Our findings establish the application of contextual enhancer elements as a potential therapeutic platform for spatiotemporally controlled tissue regeneration in mammals.

Heart regenerative medicine has been gradually evolving from a view of the heart as a nonregenerative organ with terminally differentiated cardiac muscle cells. Understanding the biology of the heart during homeostasis and in response to injuries has led to the realization that cellular communication between all cardiac cell types holds great promise for treatments. Indeed, recent studies highlight new disease-reversion concepts in addition to cardiomyocyte renewal, such as matrix- and vascular-targeted therapies, and immunotherapy with a focus on inflammation and fibrosis. In this review, we will discuss the cross-talk within the cardiac microenvironment and how specific therapies aim to target the hostile cardiac milieu under pathological conditions.

In mammals, the physiological activation of the glucocorticoid receptor (GR) by glucocorticoids (GCs) promotes the maturation of cardiomyocytes during late gestation, but the effect on postnatal cardiac growth and regenerative plasticity is unclear. Here we demonstrate that the GCGR axis restrains cardiomyocyte proliferation during postnatal development. Cardiomyocyte-specific GR ablation in conditional knockout (cKO) mice delayed the postnatal cardiomyocyte cell cycle exit, hypertrophic growth and cytoarchitectural maturation. GR-cKO hearts showed increased expression of genes involved in glucose catabolism and reduced expression of genes promoting fatty acid oxidation and mitochondrial respiration. Accordingly, oxygen consumption in GR-cKO cardiomyocytes was less dependent on fatty acid oxidation, and glycolysis inhibition reverted GR-cKO effects on cardiomyocyte proliferation. GR ablation or transient pharmacological inhibition after myocardial infarction in juvenile and/or adult mice facilitated cardiomyocyte survival, cell cycle re-entry and division, leading to cardiac muscle regeneration along with reduced scar formation. Thus, GR restrains heart regeneration and may represent a therapeutic target.

How do we measure the impact of scientific research? A new study discusses the current publication culture, diverse animal models that are commonly used in cardiovascular studies, the comparison between basic and clinical research paths, and the role of authors and reviewers in bringing these two paths together.

Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes. CaMKII activation results in myotube growth via fusion with mononucleated myoblasts at a fusogenic synapse. Mechanistically, CaMKII interacts with and regulates MYMK and Rac1, and CaMKIIδ/γ knockout mice exhibit smaller regenerated myofibers following injury. In addition, the expression of a dominant negative CaMKII inhibits the formation of large multinucleated myotubes. Finally, we demonstrate the evolutionary conservation of the pathway in chicken myoblasts. We conclude that ERK1/2 represses a signaling cascade leading to CaMKII-mediated fusion of myoblasts to myotubes, providing an attractive target for the cultivated meat industry and regenerative medicine.

Hippo signaling is an evolutionarily conserved pathway that restricts growth and regeneration predominantly by suppressing the activity of the transcriptional coactivator Yap. Using a high-throughput phenotypic screen, we identified a potent and non-toxic activator of Yap. In vitro kinase assays show that the compound acts as an ATP-competitive inhibitor of Lats kinasesthe core enzymes in Hippo signaling. The substance prevents Yap phosphorylation and induces proliferation of supporting cells in the murine inner ear, murine cardiomyocytes, and human Müller glia in retinal organoids. RNA sequencing indicates that the inhibitor reversibly activates the expression of transcriptional Yap targets: upon withdrawal, a subset of supporting-cell progeny exits the cell cycle and upregulates genes characteristic of sensory hair cells. Our results suggest that the pharmacological inhibition of Lats kinases may promote initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear.

During heart development, epicardial cells residing within the outer layer undergo epithelial-mesenchymal transition (EMT) and migrate into the underlying myocardium to support organ growth and morphogenesis. Disruption of epicardial EMT results in embryonic lethality, yet its regulation is poorly understood. Here, we report epicardial EMT within the mesothelial layer of the mouse embryonic heart at ultra-high resolution using scanning electron microscopy combined with immunofluorescence analyses. We identified morphologically active EMT regions that associated with key components of the extracellular matrix, including the basement membrane-associated proteoglycan agrin. Deletion of agrin resulted in impaired EMT and compromised development of the epicardium, accompanied by downregulation of Wilms tumor 1. Agrin enhanced EMT in human embryonic stem cell-derived epicardial-like cells by decreasing β-catenin and promoting pFAK localization at focal adhesions, and promoted the aggregation of dystroglycan within the Golgi apparatus in murine epicardial cells. Loss of agrin resulted in dispersal of dystroglycan in vivo, disrupting basement membrane integrity and impairing EMT. Our results provide new insights into the role of the extracellular matrix in heart development and implicate agrin as a crucial regulator of epicardial EMT.

The discovery of endogenous regenerative potential of the heart in zebrafish and neonatal mice has shifted the cardiac regenerative field towards the utilization of intrinsic regenerative mechanisms in the mammalian heart. The goal of these studies is to understand, and eventually apply, the neonatal regenerative mechanisms into adulthood. To facilitate these studies, the last two decades have seen advancements in the development of injury models in adult mice representative of the diversity of cardiac diseases. Here, we provide an overview for a selection of the most common cardiac ischemic injury models and describe a set of methods used to accurately analyze and quantify cardiac outcomes. Importantly, a comprehensive understanding of cardiac regeneration and repair requires a combination of multiple functional, histological, and molecular analyses.

Cardiomyocyte loss after injury results in adverse remodelling and fibrosis, inevitably leading to heart failure. The ERBB2Neuregulin and HippoYAP signalling pathways are key mediators of heart regeneration, yet the crosstalk between them is unclear. We demonstrate that transient overexpression of activated ERBB2 in cardiomyocytes (OE CMs) promotes cardiac regeneration in a heart failure model. OE CMs present an epithelialmesenchymal transition (EMT)-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration and extracellular matrix turnover. We identified YAP as a critical mediator of ERBB2 signalling. In OE CMs, YAP interacts with nuclear-envelope and cytoskeletal components, reflecting an altered mechanical state elicited by ERBB2. We identified two YAP-activating phosphorylations on S352 and S274 in OE CMs, which peak during metaphase, that are ERK dependent and Hippo independent. Viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Together, we reveal a potent ERBB2-mediated YAP mechanotransduction signalling, involving EMT-like characteristics, resulting in robust heart regeneration.

The muscle community is mourning the loss of David Yaffe, emeritus professor at the Weizmann Institute of Science in Israel, who died at the beginning of July. His pioneering work on muscle cell biology played a major role in founding the modern field of myogenesis.

Background: Ischemic heart diseases are leading causes of death and reduced life quality worldwide. Although revascularization strategies significantly reduce mortality after acute myocardial infarction (MI), a large number of patients with MI develop chronic heart failure over time. We previously reported that a fragment of the extracellular matrix protein agrin promotes cardiac regeneration after MI in adult mice. Methods: To test the therapeutic potential of agrin in a preclinical porcine model, we performed ischemia-reperfusion injuries using balloon occlusion for 60 minutes followed by a 3-, 7-, or 28-day reperfusion period. Results: We demonstrated that local (antegrade) delivery of recombinant human agrin to the infarcted pig heart can target the affected regions in an efficient and clinically relevant manner. A single dose of recombinant human agrin improved heart function, infarct size, fibrosis, and adverse remodeling parameters 28 days after MI. Short-term MI experiments along with complementary murine studies revealed myocardial protection, improved angiogenesis, inflammatory suppression, and cell cycle reentry as agrin's mechanisms of action. Conclusions: A single dose of agrin is capable of reducing ischemia-reperfusion injury and improving heart function, demonstrating that agrin could serve as a therapy for patients with acute MI and potentially heart failure.

During cardiac development, cardiomyocytes form complex inner wall structures called trabeculae. Despite significant investigation into this process, the potential role of metabolism has not been addressed. Using single cell resolution imaging in zebrafish, we find that cardiomyocytes seeding the trabecular layer actively change their shape while compact layer cardiomyocytes remain static. We show that Erbb2 signaling, which is required for trabeculation, activates glycolysis to support changes in cardiomyocyte shape and behavior. Pharmacological inhibition of glycolysis impairs cardiac trabeculation, and cardiomyocyte-specific loss- and gain-of-function manipulations of glycolysis decrease and increase trabeculation, respectively. In addition, loss of the glycolytic enzyme pyruvate kinase M2 impairs trabeculation. Experiments with rat neonatal cardiomyocytes in culture further support these observations. Our findings reveal new roles for glycolysis in regulating cardiomyocyte behavior during cardiac wall morphogenesis.

While the heart regenerates poorly in mammals, efficient heart regeneration occurs in zebrafish. Studies in zebrafish have resulted in a model in which preexisting cardiomyocytes dedifferentiate and reinitiate proliferation to replace the lost myocardium. To identify which processes occur in proliferating cardiomyocytes we have used a single-cell RNA-sequencing approach. We uncovered that proliferating border zone cardiomyocytes have very distinct transcriptomes compared to the nonproliferating remote cardiomyocytes and that they resemble embryonic cardiomyocytes. Moreover, these cells have reduced expression of mitochondrial genes and reduced mitochondrial activity, while glycolysis gene expression and glucose uptake are increased, indicative for metabolic reprogramming. Furthermore, we find that the metabolic reprogramming of border zone cardiomyocytes is induced by Nrg1/ErbB2 signaling and is important for their proliferation. This mechanism is conserved in murine hearts in which cardiomyocyte proliferation is induced by activating ErbB2 signaling. Together these results demonstrate that glycolysis regulates cardiomyocyte proliferation during heart regeneration.

The adult mammalian heart regenerates poorly after injury and, as a result, ischemic heart diseases are among the leading causes of death worldwide. The recovery of the injured heart is dependent on orchestrated repair processes including inflammation, fibrosis, cardiomyocyte survival, proliferation, and contraction properties that could be modulated in patients. In this work we designed an automated high-throughput screening system for small molecules that induce cardiomyocyte proliferation in vitro and identified the small molecule Chicago Sky Blue 6B (CSB). Following induced myocardial infarction, CSB treatment reduced scar size and improved heart function of adult mice. Mechanistically, we show that although initially identified using in vitro screening for cardiomyocyte proliferation, in the adult mouse CSB promotes heart repair through (i) inhibition of CaMKII signaling, which improves cardiomyocyte contractility; and (ii) inhibition of neutrophil and macrophage activation, which attenuates the acute inflammatory response, thereby contributing to reduced scarring. In summary, we identified CSB as a potential therapeutic agent that enhances cardiac repair and function by suppressing postinjury detrimental processes, with no evidence for cardiomyocyte renewal.

The robust capacity of skeletal muscle stem cells (SkMSCs, or satellite cells) to regenerate into new muscles in vivo has offered promising therapeutic options for the treatment of degenerative muscle diseases. However, the practical use of SkMSCs to treat muscle diseases is limited, owing to their inability to expand in vitro under defined cultivation conditions without loss of engraftment efficiency. To develop an optimal cultivation condition for SkMSCs, we investigated the behavior of SkMSCs on synthetic maltose-binding protein (MBP)-fibro blast growth factor 2 (FGF2)-immobilized matrix in vitro. We found that the chemically well-defined, xeno-free MBP-FGF2-immobilized matrix effectively supports SkMSC growth without reducing their differentiation potential in vitro. Our data highlights the possible application of the MBP-FGF2 matrix for SkMSC expansion in vitro.

Attaining proper organ size during development and regeneration hinges on the activity of mitogenic factors. Here, we performed a large-scale chemical screen in embryonic zebrafish to identify cardiomyocyte mitogens. Although commonly considered antiproliferative, vitamin D analogs like alfacalcidol had rapid, potent mitogenic effects on embryonic and adult cardiomyocytes in vivo. Moreover, pharmacologic or genetic manipulation of vitamin D signaling controlled proliferation in multiple adult cell types and dictated growth rates in embryonic and juvenile zebrafish. Tissue-specific modulation of vitamin D receptor (VDR) signaling had organ-restricted effects, with cardiac VDR activation causing cardiomegaly. Alfacalcidol enhanced the regenerative response of injured zebrafish hearts, whereas VDR blockade inhibited regeneration. Alfacalcidol activated cardiac expression of genes associated with ErbB2 signaling, while ErbB2 inhibition blunted its effects on cell proliferation. Our findings identify vitamin D as mitogenic for cardiomyocytes and other cell types in zebrafish and indicate a mechanism to regulate organ size and regeneration.

In vertebrate hearts, the ventricular trabecular myocardium develops as a sponge-like network of cardiomyocytes that is critical for contraction and conduction, ventricular septation, papillary muscle formation and wall thickening through the process of compaction(1). Defective trabeculation leads to embryonic lethality(2-4) or non-compaction cardiomyopathy (NCC)(5). There are divergent views on when and how trabeculation is initiated in different species. In zebrafish, trabecular cardiomyocytes extrude from compact myocardium(6), whereas in chicks, chamber wall thickening occurs before overt trabeculation(7). In mice, the onset of trabeculation has not been described, but is proposed to begin at embryonic day 9.0, when cardiomyocytes form radially oriented ribs(2). Endocardium-myocardium communication is essential for trabeculation, and numerous signalling pathways have been identified, including Notch(2,8) and Neuregulin (NRG)(4). Late disruption of the Notch pathway causes NCe. Whereas it has been shown that mutations in the extracellular matrix (ECM) genes Has2 and Vcan prevent the formation of trabeculae in mice(9,10) and the matrix metalloprotease ADAMTS1 promotes trabecular termination(3,) the pathways involved in ECM dynamics and the molecular regulation of trabeculation during its early phases remain unexplored. Here we present a model of trabeculation in mice that integrates dynamic endocardial and myocardial cell behaviours and ECM remodelling, and reveal new epistatic relationships between the involved signalling pathways. NOTCH1 signalling promotes ECM degradation during the formation of endocardial projections that are critical for individualization of trabecular units, whereas NRG1 promotes myocardial ECM synthesis, which is necessary for trabecular rearrangement and growth. These systems interconnect through NRG1 control of Vegfa, but act antagonistically to establish trabecular architecture. These insights enabled the prediction of persistent ECM and cardiomyocyte growth in a mouse NCC model, providing new insights into the pathophysiology of congenital heart disease.

The adult mammalian heart is non-regenerative owing to the post-mitotic nature of cardiomyocytes. The neonatal mouse heart can regenerate, but only during the first week of life. Here we show that changes in the composition of the extracellular matrix during this week can affect cardiomyocyte growth and differentiation in mice. We identify agrin, a component of neonatal extracellular matrix, as required for the full regenerative capacity of neonatal mouse hearts. In vitro, recombinant agrin promotes the division of cardiomyocytes that are derived from mouse and human induced pluripotent stem cells through a mechanism that involves the disassembly of the dystrophin-glycoprotein complex, and Yap- and ERK-mediated signalling. In vivo, a single administration of agrin promotes cardiac regeneration in adult mice after myocardial infarction, although the degree of cardiomyocyte proliferation observed in this model suggests that there are additional therapeutic mechanisms. Together, our results uncover a new inducer of mammalian heart regeneration and highlight fundamental roles of the extracellular matrix in cardiac repair.

The human heart is continually operating as amuscular pump, contracting, on average, 80 times per minute to propel 8000 liters of blood through body tissues each day.Whereas damaged skeletal muscle has a profound capacity to regenerate, heart muscle, at least in mammals, has poor regenerative potential.This deficiency is attributable to the lack of resident cardiac stem cells, combined with roadblocks that limit adult cardiomyocytes from entering the cell cycle and completing division. Insights for regeneration have recently emerged from studies of animals with an elevated innate capacity for regeneration, the innovation of stem cell and reprogramming technologies, and a clearer understanding of the cardiomyocyte genetic program and key extrinsic signals. Methods to augment heart regeneration now have potential to counteract the high morbidity and mortality of cardiovascular disease.

Regeneration in mammals is restricted to distinct tissues and occurs mainly by expansion and maturation of resident stem cells. During regeneration, even subtle mutations in the proliferating cells may cause a detrimental effect by eliciting abnormal differentiation or malignant transformation. Indeed, cancer in mammals has been shown to arise through deregulation of stem cells maturation, which often leads to a differentiation block and cell transformation. In contrast, lower organisms such as amphibians retain a remarkable regenerative capacity in various organs, which occurs via de- and re-differentiation of mature cells. Interestingly, regenerating amphibian cells are highly resistant to oncogenic transformation. Therapeutic approaches to improve mammalian regeneration mainly include stem-cell transplantations; but, these have proved unsuccessful in non-regenerating organs such as the heart. A recently developed approach is to induce de-differentiation of mature cardiomyocytes using factors that trigger their re-entry into the cell cycle. This novel approach raises numerous questions regarding the balance between transformation and regeneration induced by dedifferentiation of mature mammalian somatic cells. Can this balance be controlled artificially? Do de-differentiated cells acquire the protection mechanisms seen in regenerating cells of lower organisms? Is this model unique to the cardiac tissue, which rarely develops tumors? This review describes regeneration processes in both mammals and lower organisms and, particularly, the ability of regenerating cells to avoid transformation. By comparing the characteristics of mammalian embryonic and somatic cells, we discuss therapeutic strategies of using various cell populations for regeneration. Finally, we describe a novel cardiac regeneration approach and its implications for regenerative medicine.

Novel regenerative therapies may stem from deeper understanding of the mechanisms governing cardiovascular lineage diversification. Using enhancer mapping and live imaging in avian embryos, and genetic lineage tracing in mice, we investigated the spatio-temporal dynamics of cardiovascular progenitor populations. We show that expression of the cardiac transcription factor Nkx2.5 marks a mesodermal population outside of the cardiac crescent in the extraembryonic and lateral plate mesoderm, with characteristics of hemogenic angioblasts. Extra-cardiac Nkx2.5 lineage progenitors migrate into the embryo and contribute to clusters of CD41⁺/CD45⁺and RUNX1⁺cells in the endocardium, the aorta-gonad-mesonephros region of the dorsal aorta and liver. We also demonstrated that ectopic expression of Nkx2.5 in chick embryos activates the hemoangiogenic gene expression program. Taken together, we identified a hemogenic angioblast cell lineage characterized by transient Nkx2.5 expression that contributes to hemogenic endothelium and endocardium, suggesting a novel role for Nkx2.5 in hemoangiogenic lineage specification and diversification.

Organogenesis and regeneration require coordination of cellular proliferation, regulated in part by secreted growth factors and cognate receptors, with tissue nutrient supply provided by expansion and patterning of blood vessels. Here we reveal unexpected combinatorial integration of a growth factor co-receptor with a heterodimeric partner and ligand known to regulate angiogenesis and vascular patterning. We show that ErbB2, which can mediate epidermal growth factor (EGF) and neuregulin signalling in multiple tissues, is unexpectedly expressed by endothelial cells where it partners with neuropilin 1 (Nrp1) to form a functional receptor for the vascular guidance molecule semaphorin 3d (Sema3d). Loss of Sema3d leads to improper patterning of the coronary veins, a phenotype recapitulated by endothelial loss of ErbB2. These findings have implications for possible cardiovascular side-effects of anti-ErbB2 therapies commonly used for cancer, and provide an example of integration at the molecular level of pathways involved in tissue growth and vascular patterning.

Background The developmental origin of the c-kit expressing progenitor cell pool in the adult heart has remained elusive. Recently, it has been discovered that the injured heart is enriched with c-kit⁺ cells, which also express the hematopoietic marker CD45. Methods and results In this study, we characterize the phenotype and transcriptome of the c-kit +/CD45 +/CD11b +/Flk-1 +/Sca-1 ± (B-type) cell population, originating from the left atrial appendage. These cells are defined as cardiac macrophage progenitors. We also demonstrate that the CD45 + progenitor cell population activates heart development, neural crest and pluripotency-associated pathways in vitro, in conjunction with CD45 down-regulation, and acquire a c-kit +/CD45 -/CD11b -/Flk-1 -/Sca-1 + (A-type) phenotype through cell fusion and asymmetric division. This putative spontaneous reprogramming evolves into a highly proliferative, partially myogenic phenotype (C-type). Conclusions Our data suggests that A-type cells and cardiac macrophage precursor cells (B-type) have a common lineage origin, possibly resolving some current conundrums in the field of cardiac regeneration.

Mitochondrial carrier homolog 2 (MTCH2) is a repressor of mitochondrial oxidative phosphorylation (OXPHOS), and its locus is associated with increased BMI in humans. Here, we demonstrate that mice deficient in muscle MTCH2 are protected from diet-induced obesity and hyperinsulinemia and that they demonstrate increased energy expenditure. Deletion of muscle MTCH2 also increases mitochondrial OXPHOS and mass, triggers conversion from glycolytic to oxidative fibers, increases capacity for endurance exercise, and increases heart function. Moreover, metabolic profiling of mice deficient in muscle MTCH2 reveals a preference for carbohydrate utilization and an increase in mitochondria and glycolytic flux in muscles. Thus, MTCH2 is a critical player in muscle biology, modulating metabolism and mitochondria mass as well as impacting whole-body energy homeostasis.Errata: In the originally published version of this article, there was a mistake in Figure S1B: the two fields originally presented were adjacent views of the same gastrocnemius muscle sample. At the time of image acquisition, the images presented in the paper were mislabeled. To correct for this mistake, the authors returned to the original slides (prepared at the original time of collection; prior to publication) and chose two different experiments containing three mice from each genotype (a total of six mice/slides) and acquired images of each. The authors have used these pictures to prepare a corrected version of Figure S1, which has now been corrected online and appears below.

The murine neonatal heart can regenerate after injury through cardiomyocyte (CM) proliferation, although this capacity markedly diminishes after the first week of life. Neuregulin-1 (NRG1) administration has been proposed as a strategy to promote cardiac regeneration. Here, using loss- and gain-of-function genetic tools, we explore the role of the NRG1 co-receptor ERBB2 in cardiac regeneration. NRG1-induced CM proliferation diminished one week after birth owing to a reduction in ERBB2 expression. CM-specific Erbb2 knockout revealed that ERBB2 is required for CM proliferation at embryonic/neonatal stages. Induction of a constitutively active ERBB2 (caERBB2) in neonatal, juvenile and adult CMs resulted in cardiomegaly, characterized by extensive CM hypertrophy, dedifferentiation and proliferation, differentially mediated by ERK, AKT and GSK3 2/2-catenin signalling pathways. Transient induction of caERBB2 following myocardial infarction triggered CM dedifferentiation and proliferation followed by redifferentiation and regeneration. Thus, ERBB2 is both necessary for CM proliferation and sufficient to reactivate postnatal CM proliferative and regenerative potentials.

It has been more than 30 years since the publication of the new head hypothesis, which proposed that the vertebrate head is an evolutionary novelty resulting from the emergence of neural crest and cranial placodes. Neural crest generates the skull and associated connective tissues, whereas placodes produce sensory organs. However, neither crest nor placodes produce head muscles, which are a crucial component of the complex vertebrate head. We discuss emerging evidence for a surprising link between the evolution of head muscles and chambered hearts-both systems arise from a common pool of mesoderm progenitor cells within the cardiopharyngeal field of vertebrate embryos. We consider the origin of this field in non-vertebrate chordates and its evolution in vertebrates.

The developmental paths that lead to the formation of skeletal muscles in the head are distinct from those operating in the trunk. Craniofacial muscles are associated with head and neck structures. In the embryo, these structures derive from distinct mesoderm populations. Distinct genetic programs regulate different groups of muscles within the head to generate diverse muscle specifications. Developmental and lineage studies in vertebrates and invertebrates demonstrated an overlap in progenitor populations derived from the pharyngeal mesoderm that contribute to certain head muscles and the heart. These studies reveal that the genetic program controlling pharyngeal muscles overlaps with that of the heart. Indeed cardiac and craniofacial birth defects are often linked. Recent studies suggest that early chordates, the last common ancestor of tunicates and vertebrates, had an ancestral pharyngeal mesoderm lineage that later during evolution gave rise to both heart and craniofacial structures. This chapter summarizes studies related to the origins, signaling, genetics, and evolution of the head musculature, highlighting its heterogeneous characteristics in all these aspects.

Cardiomyocyte (CM) maturation in mammals is accompanied by a sharp decline in their proliferative and regenerative potential shortly after birth. In this study, we explored the role of the mechanical properties of the underlying matrix in the regulation of CM maturation. We show that rat and mouse neonatal CMs cultured on rigid surfaces exhibited increased myofibrillar organization, spread morphology, and reduced cell cycle activity. In contrast, compliant elastic matrices induced features of CM dedifferentiation, including disorganized sarcomere network, rounding, and conspicuous cell-cycle re-entry. The rigid matrix facilitated nuclear division (karyokinesis) leading to binucleation, while compliant matrices promoted CM mitotic rounding and cell division (cytokinesis), associated with loss of differentiation markers. Moreover, the compliant matrix potentiated clonal expansion of CMs that involves multiple cell divisions. Thus, the compliant microenvironment facilitates CM dedifferentiation and proliferation via its effect on the organization of the myoskeleton. Our findings may be exploited to design new cardiac regenerative approaches.

The developmental mechanisms that control head muscle formation are distinct from those that operate in the trunk. Head and neck muscles derive from various mesoderm populations in the embryo and are regulated by distinct transcription factors and signaling molecules. Throughout the last decade, developmental, and lineage studies in vertebrates and invertebrates have revealed the peculiar nature of the pharyngeal mesoderm that forms certain head muscles and parts of the heart. Studies in chordates, the ancestors of vertebrates, revealed an evolutionarily conserved cardiopharyngeal field that progressively facilitates the development of both heart and craniofacial structures during vertebrate evolution. This ancient regulatory circuitry preceded and facilitated the emergence of myogenic cell types and hierarchies that exist in vertebrates. This chapter summarizes studies related to the origins, signaling circuits, genetics, and evolution of the head musculature, highlighting its heterogeneous characteristics in all these aspects, with a special focus on the FGF-ERK pathway. Additionally, we address the processes of head muscle regeneration, and the development of stem cell-based therapies for treatment of muscle disorders.

Cardiac and craniofacial developmental programs are intricately linked during early embryogenesis, which is also reflected by a high frequency of birth defects affecting both regions. The molecular nature of the crosstalk between mesoderm and neural crest progenitors and the involvement of endothelial cells within the cardio-craniofacial field are largely unclear. Here we show in the mouse that genetic ablation of vascular endothelial growth factor receptor 2 (Flk1) in the mesoderm results in early embryonic lethality, severe deformation of the cardio-craniofacial field, lack of endothelial cells and a poorly formed vascular system. We provide evidence that endothelial cells are required for migration and survival of cranial neural crest cells and consequently for the deployment of second heart field progenitors into the cardiac outflow tract. Insights into the molecular mechanisms reveal marked reduction in Transforming growth factor beta 1 (Tgfb1) along with changes in the extracellular matrix (ECM) composition. Our collective findings in both mouse and avian models suggest that endothelial cells coordinate cardio-craniofacial morphogenesis, in part via a conserved signaling circuit regulating ECM remodeling by Tgfb1.

The transition between the proliferation and differentiation of progenitor cells is a key step in organogenesis, and alterations in this process can lead to developmental disorders. The extracellular signal-regulated kinase 1/2 (ERK) signaling pathway is one of the most intensively studied signaling mechanisms that regulates both proliferation and differentiation. How a single molecule (e.g. ERK) can regulate two opposing cellular outcomes is still a mystery. Using both chick and mouse models, we shed light on the mechanism responsible for the switch from proliferation to differentiation of head muscle progenitors and implicate ERK subcellular localization. Manipulation of the fibroblast growth factor (FGF)-ERK signaling pathway in chick embryos in vitro and in vivo demonstrated that blockage of this pathway accelerated myogenic differentiation, whereas its activation diminished it. We next examined whether the spatial subcellular localization of ERK could act as a switch between proliferation (nuclear ERK) and differentiation (cytoplasmic ERK) of muscle progenitors. A myristoylated peptide that blocks importin 7-mediated ERK nuclear translocation induced robust myogenic differentiation of muscle progenitor/stem cells in both head and trunk. In the mouse, analysis of Sprouty mutant embryos revealed that increased ERK signaling suppressed both head and trunk myogenesis. Our findings, corroborated by mathematical modeling, suggest that ERK shuttling between the nucleus and the cytoplasm provides a switch-like transition between proliferation and differentiation of muscle progenitors.

Background-Akt1 is a key signaling molecule in multiple cell types, including endothelial cells. Accordingly, Akt1 was proposed as a therapeutic target for ischemic injury in the context of myocardial infarction (MI). The aim of this study was to use multimodal in vivo imaging to investigate the impact of systemic Akt1 deficiency on cardiac function and angiogenesis before and after MI. Methods and Results-In vivo cardiac MRI was performed before and at days 1, 8, 15, and 29 to 30 after MI induction for wild-type, heterozygous, and Akt1-deficient mice. Noninfarcted hearts were imaged using ex vivo stereomicroscopy and microcomputed tomography. Histological examination was performed for noninfarcted hearts and for hearts at days 8 and 29 to 30 after MI. MRI revealed mildly decreased baseline cardiac function in Akt1 null mice, whereas ex vivo stereomicroscopy and microcomputed tomography revealed substantially reduced coronary macrovasculature. After MI, Akt1^-/- mice demonstrated significantly attenuated ventricular remodeling and a smaller decrease in ejection fraction. At 8 days after MI, a larger functional capillary network at the remote and border zone, accompanied by reduced scar extension, preserved cardiac function, and enhanced border zone wall thickening, was observed in Akt1^-/- mice when compared with littermate controls. Conclusions-Using multimodal imaging to probe the role of Akt1 in cardiac function and remodeling after MI, this study revealed reduced adverse remodeling in Akt1-deficient mice after MI. Augmented myocardial angiogenesis coupled with a more functional myocardial capillary network may facilitate revascularization and therefore be responsible for preservation of infarcted myocardium.

The search for developmental mechanisms driving vertebrate organogenesis has paved the way toward a deeper understanding of birth defects. During embryogenesis, parts of the heart and craniofacial muscles arise from pharyngeal mesoderm (PM) progenitors. Here, we reveal a hierarchical regulatory network of a set of transcription factors expressed in the PM that initiates heart and craniofacial organogenesis. Genetic perturbation of this network in mice resulted in heart and craniofacial muscle defects, revealing robust cross-regulation between its members. We identified Lhx2 as a previously undescribed player during cardiac and pharyngeal muscle development. Lhx2 and Tcf21 genetically interact with Tbx1, the major determinant in the etiology of DiGeorge/velo-cardio-facial/22q11.2 deletion syndrome. Furthermore, knockout of these genes in the mouse recapitulates specific cardiac features of this syndrome. We suggest that PM-derived cardiogenesis and myogenesis are network properties rather than properties specific to individual PM members. These findings shed new light on the developmental underpinnings of congenital defects.

Vertebrate movement depends on trunk skeletal muscles, which are derived from the segmented paraxial mesoderm known as somites (Christ and Ordahl 1995). During embryogenesis, muscle precursor cells proliferate extensively prior to their differentiation and fusion into muscle fibers containing multiple nuclei. Skeletal muscle was the first tissue in which a determination gene for cell fate, MyoD, was identified in vertebrates (Weintraub et al. 1991). Molecular and technical advances in the last two decades have resulted in a detailed understanding of the embryology of this tissue, and its genetic regulation by key transcription factors, including the paired/homeobox genes Pax3 and Pax7, and the myogenic regulatory genes Myf5, MyoD, Mrf4, and Myogenin (MRFs: myogenic regulatory factors (Kassar-Duchossoy et al. 2004)). These genes are crucial for regulating muscle cell fate, as shown by genetic loss-of-function analyses. Because many transcription factors that regulate the fate of muscle progenitors have been identified, skeletal muscle tissue constitutes an ideal model for the study of organogenesis and regeneration (Tajbakhsh 2005). Questions related to the inductive processes and the molecular events underpinning embryonic myogenesis are currently under intensive study worldwide. Answers to these questions may provide basic insights into developmental biology, as well as to the growing field of regenerative medicine as myogenesis in adult muscle stem cells recapitulates that of the embryo.

A fundamental aspect of skeletal myogenesis involves extensive rounds of cell fusion, in which individualmyoblasts are incorporated into growing muscle fibers. Here we demonstrate that N-WASp, a ubiquitous nucleation-promoting factor of branched microfilament arrays, is an essential contributor to skeletal muscle-cell fusion in developing mouse embryos. Analysis both in vivo and in primary satellite-cell cultures, shows that disruption of N-WASp function does not interfere with the program of skeletal myogenic differentiation, and does not affect myoblast motility, morphogenesis and attachment capacity. N-WASp - deficient myoblasts, however, fail to fuse. Furthermore, our analysis suggests that myoblast fusion requires N-WASp activity in both partners of a fusing myoblast pair. These findings reveal a specific role for N-WASp during mammalian myogenesis. WASp-family elements appear therefore to act as universal mediators of the myogenic cell-cell fusion mechanism underlying formation of functional muscle fibers, in both vertebrate and invertebrate species.

The embryonic heart is composed of two cell layers: the myocardium, which contributes to cardiac muscle tissue, and the endocardium, which covers the inner lumen of the heart. Whereas significant progress has been made toward elucidating the embryonic origins of the myocardium, the origins of the endocardium remain unclear. Here, we have identified an endocardium-forming field medial to the cardiac crescent, in a continuum with the endothelial plexus. In vivo live imaging of quail embryos revealed that endothelial progenitors, like second/anterior heart field progenitors, migrate to, and enter, the heart from the arterial pole. Furthermore, embryonic endothelial cells implanted into the cardiac crescent contribute to the endocardium, but not to the myocardium. In mouse, lineage analysis focusing on endocardial cells revealed an unexpected heterogeneity in the origins of the endocardium. To gain deeper insight into this heterogeneity, we conditionally ablated Flk1 in distinct cardiovascular progenitor populations; FLK1 is required in vivo for formation of the endocardium in the Mesp1 and Tie2 lineages, but not in the Isl1 lineage. Ablation of Flk1 coupled with lineage analysis in the Isl1 lineage revealed that endothelium-derived Isl1-endocardial cells were significantly increased, whereas Isl1(+) endocardial cells were reduced, suggesting that the endocardium is capable of undergoing regulative compensatory growth. Collectively, our findings demonstrate that the second heart field contains distinct myocardial and endocardial progenitor populations. We suggest that the endocardium derives, at least in part, from vascular endothelial cells.

The pharyngeal mesoderm (PM), located in the head region of the developing embryo, recently triggered renewed interest as the major source of cells contributing to broad regions of the heart as well as to the head musculature. What exactly is PM? In this review, we describe the anatomical and molecular characteristics of this mesodermal population and its relationship to the first and second heart fields in chick and mouse embryos. The regulatory network of transcription factors and signalling molecules that regulate PM development is also discussed. In addition, we summarize recent studies into the evolutionary origins of this tissue and its multipotential contributions to both cardiac and pharyngeal muscle progenitors.

Neural crest development involves epithelial-mesenchymal transition (EMT), during which epithelial cells are converted into individual migratory cells. Notably, the same signaling pathways regulate EMT function during both development and tumor metastasis. p53 plays multiple roles in the prevention of tumor development; however, its precise roles during embryogenesis are less clear. We have investigated the role of p53 in early cranial neural crest (CNC) development in chick and mouse embryos. In the mouse, p53 knockout embryos displayed broad craniofacial defects in skeletal, neuronal and muscle tissues. In the chick, p53 is expressed in CNC progenitors and its expression decreases with their delamination from the neural tube. Stabilization of p53 protein using a pharmacological inhibitor of its negative regulator, MDM2, resulted in reduced SNAIL2 (SLUG) and ETS1 expression, fewer migrating CNC cells and in craniofacial defects. By contrast, electroporation of a dominant-negative p53 construct increased PAX7⁺ SOX9⁺ CNC progenitors and EMT/delamination of CNC from the neural tube, although the migration of these cells to the periphery was impaired. Investigating the underlying molecular mechanisms revealed that p53 coordinates CNC cell growth and EMT/delamination processes by affecting cell cycle gene expression and proliferation at discrete developmental stages; disruption of these processes can lead to craniofacial defects.

The anterior heart field (AHF) encompasses a niche in which mesoderm-derived cardiac progenitors maintain their multipotent and undifferentiated nature in response to signals from surrounding tissues. Here, we investigate the signaling mechanism that promotes the shift from proliferating cardiac progenitors to differentiating cardiomyocytes in chick embryos. Genomic and systems biology approaches, as well as perturbations of signaling molecules, in vitro and in vivo, reveal tight crosstalk between the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signaling pathways within the AHF niche: BMP4 promotes myofibrillar gene expression and cardiomyocyte contraction by blocking FGF signaling. Furthermore, inhibition of the FGF-ERK pathway is both sufficient and necessary for these processes, suggesting that FGF signaling blocks premature differentiation of cardiac progenitors in the AHF. We further revealed that BMP4 induced a set of neural crest-related genes, including MSX1. Overexpression of Msx1 was sufficient to repress FGF gene expression and cell proliferation, thereby promoting cardiomyocyte differentiation. Finally, we show that BMP-induced cardiomyocyte differentiation is diminished following cranial neural crest ablation, underscoring the key roles of these cells in the regulation of AHF cell differentiation. Hence, BMP and FGF signaling pathways act via inter- and intra-regulatory loops in multiple tissues, to coordinate the balance between proliferation and differentiation of cardiac progenitors.

In vertebrates, body musculature originates from somites, whereas head muscles originate from the cranial mesoderm. Neck muscles are located in the transition between these regions. We show that the chick occipital lateral plate mesoderm has myogenic capacity and gives rise to large muscles located in the neck and thorax. We present molecular and genetic evidence to show that these muscles not only have a unique origin, but additionally display a distinct temporal development, forming later than any other muscle group described to date. We further report that these muscles, found in the body of the animal, develop like head musculature rather than deploying the programme used by the trunk muscles. Using mouse genetics we reveal that these muscles are formed in trunk muscle mutants but are absent in head muscle mutants. In concordance with this conclusion, their connective tissue is neural crest in origin. Finally, we provide evidence that the mechanism by which these neck muscles develop is conserved in vertebrates.

The cells of the second heart field (SHF) contribute to the outflow tract and right ventricle, as well as to parts of the left ventricle and atria. Isl1, a member of the LIM-homeodomain transcription factor family, is expressed early in this cardiac progenitor population and functions near the top of a transcriptional pathway essential for heart development. Isl1 is required for the survival and migration of SHF-derived cells into the early developing heart at the inflow and outflow poles. Despite this important role for Isl1 in early heart formation, the transcriptional regulation of Isl1 has remained largely undefined. Therefore, to identify transcription factors that regulate Isl1 expression in vivo, we screened the conserved noncoding sequences from the mouse Isl1 locus for enhancer activity in transgenic mouse embryos. Here, we report the identification of an enhancer from the mouse Isl1 gene that is sufficient to direct expression to the SHF and its derivatives. The Isl1 SHF enhancer contains three consensus Forkhead transcription factor binding sites that are efficiently and specifically bound by Forkhead transcription factors. Importantly, the activity of the enhancer is dependent on these three Forkhead binding sites in transgenic mouse embryos. Thus, these studies demonstrate that Isl1 is a direct transcriptional target of Forkhead transcription factors in the SHF and establish a transcriptional pathway upstream of Isl1 in the SHF.

Keywords: Developmental Biology

Adult skeletal muscle possesses a remarkable regenerative capacity, due to the presence of satellite cells, adult muscle stem cells. We used fate-mapping techniques in avian and mouse models to show that trunk (Pax3⁺) and cranial (MesP1⁺) skeletal muscle and satellite cells derive from separate genetic lineages. Similar lineage heterogeneity is seen within the head musculature and satellite cells, due to their shared, heterogenic embryonic origins. Lineage tracing experiments with Isl1Cre mice demonstrated the robust contribution of Isl1⁺ cells to distinct jaw muscle-derived satellite cells. Transplantation of myofiber-associated, Isl1-derived satellite cells into damaged limb muscle contributed to muscle regeneration. In vitro experiments demonstrated the cardiogenic nature of cranial- but not trunk-derived satellite cells. Finally, overexpression of Isl1 in the branchiomeric muscles of chick embryos inhibited skeletal muscle differentiation in vitro and in vivo, suggesting that this gene plays a role in the specification of cardiovascular and skeletal muscle stem cell progenitors.

Head muscle development has been studied less intensively than myogenesis in the trunk, although this situation is gradually changing, as embryological and genetic insights accumulate. This review focuses on novel studies of the origins, composition and evolution of distinct craniofacial muscles. Cellular and molecular parallels are drawn between cardiac and branchiomeric muscle developmental programs, both of which utilize multiple lineages with distinct developmental histories, and argue for the tissues' common evolutionary origin. In addition, there is increasing evidence that the specification of skeletal muscles in the head appears to be distinct from that operating in the trunk: considerable variation among the different craniofacial muscle groups is seen, in a manner resembling myogenic specification in lower organisms.

Objective: While both play a role in the transcriptional response of hypoxic endothelial cells (ECs), hypoxia- inducible factor-1 alpha (HIF-1 alpha) and HIF-2 alpha differ in their transactivation sites, pointing at potentially different target genes. We studied the discrete and common effects of HIF-1 alpha and HIF-2 alpha on the cytokine expression and vasculogenic properties of ECs. Methods and Results: H5V and bovine aortic ECs were transfected to express HIF-1 alpha, HIF-2 alpha or both. Overexpression of HIF-1 alpha or HIF-2 alpha and, to a greater extent, cotransfection of HIF-1 alpha and HIF-2 alpha resulted in EC activation, as revealed by analysis of the adhesion capacities and adhesion molecule surface expression of ECs. From the paracrine aspect, conditioned medium from HIF-expressing ECs was found to promote the migration and tube formation capacity of wild-type ECs, mostly following HIF-1 alpha and HIF-2 alpha co-expression. Antibody arrays revealed altered expression of multiple cytokines, pointing at consistent additive effects of HIF-1 alpha and HIF-2 alpha on angiogenic protein expression. Finally, HIF-1 alpha and HIF-2 alpha additively promoted vessel formation in vivo, as demonstrated by a Matrigel angiogenesis assay. Conclusion: Our results further clarify the functional roles of HIF-1 alpha and HIF-2 alpha in ECs and for the first time demonstrate a common contribution of HIF-1 alpha and HIF-2 alpha to vasculogenesis. Copyright (C) 2009 S. Karger AG, Basel

Secreted Frizzled-related proteins (sFRPs) are signalling molecules well-known as antagonists of the Wnt pathway, but recent studies indicate that they may have additional functions unrelated to Wnt. A new study demonstrates that mammalian sFRP2 can act as an enhancer of collagen processing in vitro and in vivo, augmenting myocardial injury-driven fibrosis. These findings underscore the biological versatility of sFRP family members.

Background: The tumor suppressor p53 is an important regulator that control various cellular networks, including cell differentiation. Interestingly, some studies suggest that p53 facilitates cell differentiation, whereas others claim that it suppresses differentiation. Therefore, it is critical to evaluate whether this inconsistency represents an authentic differential p53 activity manifested in the various differentiation programs. Methodology/Principal Findings: To clarify this important issue, we conducted a comparative study of several mesenchymal differentiation programs. The effects of p53 knockdown or enhanced activity were analyzed in mouse and human mesenchymal cells, representing various stages of several differentiation programs. We found that p53 down-regulated the expression of master differentiation-inducing transcription factors, thereby inhibiting osteogenic, adipogenic and smooth muscle differentiation of multiple mesenchymall cell types. In contrast, p53 is essential for skeletal muscle differentiation and osteogenic re-programming of skeletal muscle committed cells. Conclusions: These comparative studies suggest that, depending on the specific cell type and the specific differentiation program p53 may exert a positive or a negative effect, and thus can be referred as a "guardian of differentiation" at large.

Bone marrow stromal cells (BMSCs) contain progenitors capable of participating in postnatal angiogenesis. Hypoxia-inducible factors (HIFs) mediate endothelial activation by driving the expression of multiple angiogenic factors. We explored the potential of HIF-1α and HIF-2α modification in BMSCs, as a tool to improve cell-based angiogenic therapy. BMSCs were retrovirally transduced to express stable forms of HIF-1α and HIF-2α. HIF-1α and, to a greater extent, HIF-2α overexpression promoted differentiation of BMSCs to the endothelial lineage, evident by CD31 and Tie-2 expression and improved adhesive properties. Whereas chemotaxis toward stromal-derived factor 1 was higher in both HIF-α-expressing BMSCs, enhanced migration toward vascular endothelial growth factor was found only following overexpression of HIF-2α, supported by a robust expression of its receptor, Flk-1. HIF-α expression was associated with upregulation of angiogenic proteins and improved tube formation. Cytokine arrays of endothelial cells stimulated by medium collected from HIF-α-expressing BMSCs revealed further angiogenic activation and improved adhesive capacity. Eventually, delivery of HIF-2α-transduced BMSCs induced a more robust angiogenic response, compared with shamtransduced or HIF-1α-transduced BMSCs in the corneal micropocket angiogenesis model. Our results support the use of HIF-α genes, particularly HIF-2α, to augment the efficacy of future cell-based therapy.

The depth of a cell of a multicellular organism is the number of cell divisions it underwent since the zygote, and knowing this basic cell property would help address fundamental problems in several areas of biology. At present, the depths of the vast majority of human and mouse cell types are unknown. Here, we show a method for estimating the depth of a cell by analyzing somatic mutations in its microsatellites, and provide to our knowledge for the first time reliable depth estimates for several cells types in mice. According to our estimates, the average depth of oocytes is 29, consistent with previous estimates. The average depth of B cells ranges from 34 to 79, linearly related to the mouse age, suggesting a rate of one cell division per day. In contrast, various types of adult stem cells underwent on average fewer cell divisions, supporting the notion that adult stem cells are relatively quiescent. Our method for depth estimation opens a window for revealing tissue turnover rates in animals, including humans, which has important implications for our knowledge of the body under physiological and pathological conditions.

The cell lineage tree of a multicellular organism represents its history of cell divisions from the very first cell, the zygote. A new method for high-resolution reconstruction of parts of such cell lineage trees was recently developed based on phylogenetic analysis of somatic mutations accumulated during normal development of an organism. In this study we apply this method in mice to reconstruct the lineage trees of distinct cell types. We address for the first time basic questions in developmental biology of higher organisms, namely what is the correlation between the lineage relation among cells and their (1) function, (2) physical proximity and (3) anatomical proximity. We analyzed B-cells, kidney-, mesenchymal- and hematopoietic-stem cells, as well as satellite cells, which are adult skeletal muscle stem cells isolated from their niche on the muscle fibers (myofibers) from various skeletal muscles. Our results demonstrate that all analyzed cell types are intermingled in the lineage tree, indicating that none of these cell types are single exclusive clones. We also show a significant correlation between the physical proximity of satellite cells within muscles and their lineage. Furthermore, we show that satellite cells obtained from a single myofiber are significantly clustered in the lineage tree, reflecting their common developmental origin. Lineage analysis based on somatic mutations enables performing high resolution reconstruction of lineage trees in mice and humans, which can provide fundamental insights to many aspects of their development and tissue maintenance.

During embryogenesis, paraxial mesoderm cells contribute skeletal muscle progenitors, whereas cardiac progenitors originate in the lateral splanchnic mesoderm (SpM). Here we focus on a subset of the SpM that contributes to the anterior or secondary heart field (AHF/SHF), and lies adjacent to the cranial paraxial mesoderm (CPM), the precursors for the head musculature. Molecular analyses in chick embryos delineated the boundaries between the CPM, undifferentiated SpM progenitors of the AHF/ SHF, and differentiating cardiac cells. We then revealed the regionalization of branchial arch mesoderm: CPM cells contribute to the proximal region of the myogenic core, which gives rise to the mandibular adductor muscle. SpM cells contribute to the myogenic cells in the distal region of the branchial arch that later form the intermandibular muscle. Gene expression analyses of these branchiomeric muscles in chick uncovered a distinct molecular signature for both CPM- and SpM-derived muscles. Islet1 (Isl1) is expressed in the SpM/AHF and branchial arch in both chick and mouse embryos. Lineage studies using Isl1-Cre mice revealed the significant contribution of Isl1⁺ cells to ventral/distal branchiomeric (stylohyoid, mylohyoid and digastric) and laryngeal muscles. By contrast, the Isl1 lineage contributes to mastication muscles (masseter, pterygoid and temporalis) to a lesser extent, with virtually no contribution to intrinsic and extrinsic tongue muscles or extraocular muscles. In addition, in vivo activation of the Wnt/β-catenin pathway in chick embryos resulted in marked inhibition of Isl1, whereas inhibition of this pathway increased Isl1 expression. Our findings demonstrate, for the first time, the contribution of Isl1⁺ SpM cells to a subset of branchiomeric skeletal muscles.

Heart formation requires the coordinated recruitment of multiple cardiac progenitor cell populations derived from both the first and second heart fields. In this study, we have ablated the Bmp receptor 1a and the Wnt effector β-catenin in the developing heart of mice by using MesP1-cre, which acts in early mesoderm progenitors that contribute to both first and second heart fields. Remarkably, the entire cardiac crescent and later the primitive ventricle were absent in MesP1-cre; BmpR1a^lox/lox mutants. Although myocardial progenitor markers such as Nkx2-5 and Isl1 and the differentiation marker MLC2a were detected in the small, remaining cardiac field in these mutants, the first heart field markers, eHand and Tbx-5, were not expressed. We conclude from these results that Bmp receptor signaling is crucial for the specification of the first heart field. In MesP1-cre; β-catenin ^lox/lox mutants, cardiac crescent formation, as well as first heart field markers, were not affected, although cardiac looping and right ventricle formation were blocked. Expression of Isl1 and Bmp4 in second heart field progenitors was strongly reduced. In contrast, in a gain-of-function mutation of β-catenin using MesP1-cre, we revealed an expansion of Isl1 and Bmp4 expressing cells, although the heart tube was not formed. We conclude from these results that Wnt/β-catenin signaling regulates second heart-field development, and that a precise amount and/or timing of Wnt/β-catenin signaling is required for proper heart tube formation and cardiac looping.

In the vertebrate head, mesoderm cells fuse together to form a myofiber, which is attached to specific cranial neural crest (CNC-derived skeletal elements in a highly coordinated manner. Although it has long been recognized that CNC plays a role in the formation of the head musculature, the precise molecular underpinnings of this process remain elusive. In the present study we explored the nature of the crosstalk between CNC and mesoderm cells during head muscle development, employing three models for genetic perturbations of CNC development in mice, as well as experimental ablation of CNC in chick embryos. We demonstrate that although early myogenesis is CNC-independent, the migration, patterning and differentiation of muscle precursors are regulated by CNC. In the absence of CNC cells, accumulated myoblasts are kept in a proliferative state, presumably because of an increase of Fgf8 in adjacent tissues, which leads to abnormalities in both differentiation and subsequent myofiber organization in the head. These results have uncovered a surprising degree of complexity and multiple distinct roles for CNC in the patterning and differentiation of muscles during craniofacial development. We suggest that CNC cells control craniofacial development by regulating positional interactions with mesoderm-derived muscle progenitors that together shape the cranial musculoskeletal architecture in vertebrate embryos.

Recent findings in mouse and zebrafish embryos, as well as in embryonic stem cells, emphasize the critical importance of the Wnt/β-catenin pathway in the regulation of cardiogenesis, and highlight the exquisite timing and specific cellular responses by which this signaling pathway exerts its influence. These studies clearly demonstrate that the Wnt/β-catenin pathway plays distinct, even opposing, roles during various stages of cardiac development.

During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm (i.e. the lateral plate mesoderm and paraxial mesoderm, respectively). In the present study, we have employed both in vitro and in vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and skeletal muscles. Using fate-mapping studies, gene expression analyses, and manipulation of signaling pathways in the chick embryo, we demonstrate that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell populations within the cardiac outflow tract. We further show that Bmp signaling affects the specification of mesoderm cells in the head: application of Bmp4, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm and blocks the differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo. A deeper understanding of mesodermal lineage specification in the vertebrate head is expected to provide insights into the normal, as well as pathological, aspects of heart and craniofacial development.

Recent studies have postulated that distinct regulatory cascades control myogenic differentiation in the head and the trunk. However, although the tissues and signaling molecules that induce skeletal myogenesis in the trunk have been identified, the source of the signals that trigger skeletal muscle formation in the head remain obscure. Here we show that although myogenesis in the trunk paraxial mesoderm is induced by Wnt signals from the dorsal neural tube, myogenesis in the cranial paraxial mesoderm is blocked by these same signals. In addition, BMP family members that are expressed in both the dorsal neural tube and surface ectoderm are also potent inhibitors of myogenesis in the cranial paraxial mesoderm. We provide evidence suggesting that skeletal myogenesis in the head is induced by the BMP inhibitors, Noggin and Gremlin, and the Wnt inhibitor, Frzb. These molecules are secreted by both cranial neural crest cells and by other tissues surrounding the cranial muscle anlagen. Our findings demonstrate that head muscle formation is locally repressed by Wnt and BMP signals and induced by antagonists of these signaling pathways secreted by adjacent tissues.