RESUMO
Establishing a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss-of-function mouse models showed that FOXO1, a Forkhead family transcription factor, is required for yolk sac (YS) vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that at E8.25, loss of Foxo1 in Tie2-cre expressing cells resulted in increased sprouty 2 (Spry2) and Spry4 expression, reduced arterial gene expression and reduced Kdr (also known as Vegfr2 and Flk1) transcripts without affecting overall endothelial cell identity, survival or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, delta like 4, is significantly reduced in Foxo1 mutant YS without being substantially affected in the embryo proper. We show that FOXO1 binds directly to previously identified Spry2 gene regulatory elements (GREs) and newly identified, evolutionarily conserved Spry4 GREs to repress their expression. Furthermore, overexpression of Spry4 in transient transgenic embryos largely recapitulates the reduced expression of arterial genes seen in conditional Foxo1 mutants. Together, these data reveal a novel role for FOXO1 as a key transcriptional repressor regulating both pre-flow arterial specification and subsequent vessel remodeling within the murine YS.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Remodelação Vascular , Saco Vitelino , Animais , Artérias , Embrião de Mamíferos/metabolismo , Células Endoteliais/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Camundongos , Remodelação Vascular/genética , Saco Vitelino/metabolismoRESUMO
Comprehensive detailed characterization of new mouse models can be challenging due to the individual focus involved in developing these models. Often models are engineered to test a specific hypothesis in a limited number of tissues, stages, and/or other contexts. Whether or not the model produces the desired phenotypes, phenotyping beyond the desired context can be extremely work intensive and these studies are often not undertaken. However, the general information resulting from broader phenotyping can be invaluable to the wider scientific community. The International Mouse Phenotyping Consortium (IMPC) and its subsidiaries, like the Knockout Mouse Project (KOMP), has made great strides in streamlining this process. In particular, the use of microCT has been an invaluable resource in examining internal organ systems throughout fetal/developmental stages. Here, we provide several novel vignettes demonstrating the utility of microCT in uncovering cardiac phenotypes both based on human disease correlations and those that are unpredicted.
Assuntos
Implantação do Embrião , Organogênese , Camundongos , Animais , Humanos , Camundongos Knockout , Microtomografia por Raio-X/métodos , Fenótipo , Imageamento Tridimensional/métodosRESUMO
The SMYD (SET and MYND domain) family of lysine methyltransferases harbor a unique structure in which the methyltransferase (SET) domain is intervened by a zinc finger protein-protein interaction MYND domain. SMYD proteins methylate both histone and non-histone substrates and participate in diverse biological processes including transcriptional regulation, DNA repair, proliferation and apoptosis. Smyd1 is unique among the five family members in that it is specifically expressed in striated muscles. Smyd1 is critical for development of the right ventricle in mice. In zebrafish, Smyd1 is necessary for sarcomerogenesis in fast-twitch muscles. Smyd1 is expressed in the skeletal muscle lineage throughout myogenesis and in mature myofibers, shuttling from nucleus to cytosol during myoblast differentiation. Because of this expression pattern, we hypothesized that Smyd1 plays multiple roles at different stages of myogenesis. To determine the role of Smyd1 in mammalian myogenesis, we conditionally eliminated Smyd1 from the skeletal muscle lineage at the myoblast stage using Myf5(cre). Deletion of Smyd1 impaired myoblast differentiation, resulted in fewer myofibers and decreased expression of muscle-specific genes. Muscular defects were temporally restricted to the second wave of myogenesis. Thus, in addition to the previously described functions for Smyd1 in heart development and skeletal muscle sarcomerogenesis, these results point to a novel role for Smyd1 in myoblast differentiation.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Desenvolvimento Muscular , Proteínas Musculares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/análise , Camundongos , Fibras Musculares Esqueléticas , Proteínas Musculares/análise , Mioblastos/citologia , Fatores de Transcrição/análiseRESUMO
In this work, we report the use of iodine-contrast microCT to perform high-throughput 3D morphological analysis of mouse embryos and neonates between embryonic day 8.5 to postnatal day 3, with high spatial resolution up to 3µm/voxel. We show that mouse embryos at early stages can be imaged either within extra embryonic tissues such as the yolk sac or the decidua without physically disturbing the embryos. This method enables a full, undisturbed analysis of embryo turning, allantois development, vitelline vessels remodeling, yolk sac and early placenta development, which provides increased insights into early embryonic lethality in mutant lines. Moreover, these methods are inexpensive, simple to learn and do not require substantial processing time, making them ideal for high throughput analysis of mouse mutants with embryonic and early postnatal lethality.
Assuntos
Desenvolvimento Embrionário , Imageamento Tridimensional/métodos , Camundongos/embriologia , Microtomografia por Raio-X/métodos , Animais , Animais Recém-Nascidos , Meios de Contraste , Decídua/ultraestrutura , Feminino , Genes Letais , Estudos de Associação Genética , Idade Gestacional , Hidrogéis , Iodo , Fenótipo , Coloração e Rotulagem/métodos , Saco Vitelino/ultraestruturaRESUMO
Mesoderm posterior 1 (Mesp1) is well recognized for its role in cardiac development, although it is expressed broadly in mesodermal lineages. We have previously demonstrated important roles for Mesp1 and Ets variant 2 (Etv2) during lineage specification, but their relationship has not been defined. This study reveals that Mesp1 binds to the proximal promoter and transactivates Etv2 gene expression via the CRE motif. We also demonstrate the protein-protein interaction between Mesp1 and cAMP-responsive element binding protein 1 (Creb1) in vitro and in vivo. Utilizing transgenesis, lineage tracing, flow cytometry, and immunostaining technologies, we define the lineage relationship between Mesp1- and Etv2-expressing cell populations. We observe that the majority of Etv2-EYFP(+) cells are derived from Mesp1-Cre(+) cells in both the embryo and yolk sac. Furthermore, we observe that the conditional deletion of Etv2, using a Mesp1-Cre transgenic strategy, results in vascular and hematopoietic defects similar to those observed in the global deletion of Etv2 and that it has embryonic lethality by embryonic day 9.5. In summary, our study supports the hypothesis that Mesp1 is a direct upstream transactivator of Etv2 during embryogenesis and that Creb1 is an important cofactor of Mesp1 in the transcriptional regulation of Etv2 gene expression.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Fatores de Transcrição/genética , Ativação Transcricional , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Western Blotting , Linhagem Celular , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Células NIH 3T3 , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismoRESUMO
Etv2 is an essential transcriptional regulator of hematoendothelial lineages during embryogenesis. Although Etv2 downstream targets have been identified, little is known regarding the upstream transcriptional regulation of Etv2 gene expression. In this study, we established a novel methodology that utilizes the differentiating ES cell and embryoid body system to define the modules and enhancers embedded within the Etv2 promoter. Using this system, we defined an autoactivating role for Etv2 that is mediated by two adjacent Ets motifs in the proximal promoter. In addition, we defined the role of VEGF/Flk1-Calcineurin-NFAT signaling cascade in the transcriptional regulation of Etv2. Furthermore, we defined an Etv2-Flt1-Flk1 cascade that serves as a negative feedback mechanism to regulate Etv2 gene expression. To complement and extend these studies, we demonstrated that the Flt1 null embryonic phenotype was partially rescued in the Etv2 conditional knockout background. In summary, these studies define upstream and downstream networks that serve as a transcriptional rheostat to regulate Etv2 gene expression.
Assuntos
Células da Medula Óssea/citologia , Endotélio/citologia , Expressão Gênica , Fatores de Transcrição/genética , Animais , Calcineurina/metabolismo , Linhagem da Célula , Elementos Facilitadores Genéticos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
All terminally differentiated organs face two challenges, maintaining their cellular identity and restricting organ size. The molecular mechanisms responsible for these decisions are of critical importance to organismal development, and perturbations in their normal balance can lead to disease. A hallmark of heart failure, a condition affecting millions of people worldwide, is hypertrophic growth of cardiomyocytes. The various forms of heart failure in human and animal models share conserved transcriptome remodeling events that lead to expression of genes normally silenced in the healthy adult heart. However, the chromatin remodeling events that maintain cell and organ size are incompletely understood; insights into these mechanisms could provide new targets for heart failure therapy. Using a quantitative proteomics approach to identify muscle-specific chromatin regulators in a mouse model of hypertrophy and heart failure, we identified upregulation of the histone methyltransferase Smyd1 during disease. Inducible loss-of-function studies in vivo demonstrate that Smyd1 is responsible for restricting growth in the adult heart, with its absence leading to cellular hypertrophy, organ remodeling, and fulminate heart failure. Molecular studies reveal Smyd1 to be a muscle-specific regulator of gene expression and indicate that Smyd1 modulates expression of gene isoforms whose expression is associated with cardiac pathology. Importantly, activation of Smyd1 can prevent pathological cell growth. These findings have basic implications for our understanding of cardiac pathologies and open new avenues to the treatment of cardiac hypertrophy and failure by modulating Smyd1.
Assuntos
Cardiomegalia/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Insuficiência Cardíaca/genética , Proteínas Musculares/genética , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/genética , Animais , Western Blotting , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/metabolismo , Crescimento Celular , Ecocardiografia , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Células HeLa , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/metabolismo , Humanos , Camundongos , Camundongos Knockout , Miocárdio/patologia , Miócitos Cardíacos/patologia , Proteômica , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Regulação para Cima , Remodelação Ventricular/genéticaRESUMO
Er71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for ER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an Er71-EYFP transgenic mouse model by fusing the 3.9 kb Er71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the EYFP(+) population of cells in Er71 mutant and wild-type littermates. In the absence of ER71, we observed an increase in the number of EYFP-expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-endothelial program, as compared with wild-type littermate controls. We also generated a novel Er71-Cre transgenic mouse model using the same 3.9 kb Er71 promoter. Genetic fate-mapping studies revealed that the ER71-expressing cells give rise to the hematopoietic and endothelial lineages in the wild-type background. In the absence of ER71, these cell populations contributed to alternative mesodermal lineages, including the cardiac lineage. To extend these analyses, we used an inducible embryonic stem/embryoid body system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71 as a critical regulator of mesodermal fate decisions that acts to specify the hematopoietic and endothelial lineages at the expense of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during embryogenesis.
Assuntos
Desenvolvimento Embrionário/fisiologia , Mesoderma/embriologia , Fatores de Transcrição/metabolismo , Animais , Linhagem da Célula , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Feminino , Genes Reporter , Células-Tronco Hematopoéticas/fisiologia , Mesoderma/citologia , Camundongos , Camundongos Transgênicos , Músculo Esquelético/fisiologia , Mutação , Miocárdio/metabolismo , Fatores de Transcrição/genéticaRESUMO
Independent mouse knockouts of Etv2 and Flk1 are embryonic lethal and lack hematopoietic and endothelial lineages. We previously reported that Flk1 activates Etv2 in the initiation of hematopoiesis and vasculogenesis. However, Flk1 and its ligand VEGF are expressed throughout development, from E7.0 to adulthood, whereas Etv2 is expressed only transiently during embryogenesis. These observations suggest a complex regulatory interaction between Flk1 and Etv2. To further examine the Flk1 and Etv2 regulatory interaction, we transduced Etv2 and Flk1 mutant ES cells with viral integrants that inducibly overexpress Flk1 or Etv2. We demonstrated that forced expression of Etv2 rescued the hematopoietic and endothelial potential of differentiating Flk1 and Etv2 mutant cells. We further discovered that forced expression of Flk1 can rescue that of the Flk1, but not Etv2 mutant cells. Therefore, we conclude that the requirement for Flk1 can be bypassed by expressing Etv2, supporting the notion that disruption of Etv2 expression is responsible for the early phenotypes of the Etv2 and Flk1 mutant embryos.
Assuntos
Corpos Embrioides/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Linhagem Celular , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese , Células-Tronco Hematopoéticas/citologia , Masculino , Camundongos , Camundongos Transgênicos , Fatores de Transcrição/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
During embryogenesis, the endothelial and the hematopoietic lineages first appear during gastrulation in the blood island of the yolk sac. We have previously reported that an Ets variant gene 2 (Etv2/ER71) mutant embryo lacks hematopoietic and endothelial lineages; however, the precise roles of Etv2 in yolk sac development remains unclear. In this study, we define the role of Etv2 in yolk sac blood island development using the Etv2 mutant and a novel Etv2-EYFP reporter transgenic line. Both the hematopoietic and the endothelial lineages are absent in the Etv2 mutant yolk sac. In the Etv2-EYFP transgenic mouse, the EYFP reporter is activated in the nascent mesoderm, expressed in the endothelial and blood progenitors, and in the Tie2(+), c-kit(+), and CD41(+) hematopoietic population. The hematopoietic activity in the E7.75 yolk sac was exclusively localized to the Etv2-EYFP(+) population. In the Etv2 mutant yolk sac, Tie2(+) cells are present but do not express hematopoietic or endothelial markers. In addition, these cells do not form hematopoietic colonies, indicating an essential role of Etv2 in the specification of the hematopoietic lineage. Forced overexpression of Etv2 during embryoid body differentiation induces the hematopoietic and the endothelial lineages, and transcriptional profiling in this context identifies Lmo2 as a downstream target. Using electrophoretic mobility shift assay, chromatin immunoprecipitation, transcriptional assays, and mutagenesis, we demonstrate that Etv2 binds to the Lmo2 enhancer and transactivates its expression. Collectively, our studies demonstrate that Etv2 is expressed during and required for yolk sac hematoendothelial development, and that Lmo2 is one of the downstream targets of Etv2.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Endoteliais/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas com Domínio LIM/metabolismo , Fatores de Transcrição/metabolismo , Saco Vitelino/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Imuno-Histoquímica , Proteínas com Domínio LIM/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fatores de Transcrição/genética , Transfecção , Saco Vitelino/citologiaRESUMO
Tissue clearing for whole organ cell profiling has revolutionized biology and imaging for exploration of organs in three-dimensional space without compromising tissue architecture. But complicated, laborious procedures, or expensive equipment, as well as the use of hazardous, organic solvents prevent the widespread adoption of these methods. Here, we report a simple and rapid tissue clearing method, EZ Clear, that can clear whole adult mouse organs in 48 hr in just three simple steps. Samples stay at room temperature and remain hydrated throughout the clearing process, preserving endogenous and synthetic fluorescence, without altering sample size. After wholemount clearing and imaging, samples processed with EZ Clear can be subjected to downstream applications, such as tissue embedding and cryosectioning followed by standard histology or immunofluorescent staining without loss of fluorescence signal from endogenous or synthetic reporters. Furthermore, we demonstrate that wholemount adult mouse brains processed with EZ Clear can be successfully immunolabeled for fluorescent imaging while still retaining signal from endogenous fluorescent reporters. Overall, the simplicity, speed, and flexibility of EZ Clear make it easy to adapt and implement in diverse imaging modalities in biomedical research.
Assuntos
Corantes , Imageamento Tridimensional , Animais , Encéfalo/diagnóstico por imagem , Imageamento Tridimensional/métodos , Camundongos , Solventes , Coloração e RotulagemRESUMO
Iodine-contrast micro-computed tomography (microCT) 3D imaging provides a non-destructive and high-throughput platform for studying mouse embryo and neonate development. Here we provide protocols on preparing mouse embryos and neonates between embryonic day 8.5 (E8.5) to postnatal day 4 (P4) for iodine-contrast microCT imaging. With the implementation of the STABILITY method to create a polymer-tissue hybrid structure, we have demonstrated that not only is soft tissue shrinkage minimized but also the minimum required time for soft tissue staining with iodine is decreased, especially for E18.5 to P4 samples. In addition, we also provide a protocol on using commercially available X-CLARITYTM hydrogel solution to create the similar polymer-tissue hybrid structure on delicate early post-implantation stage (E8.5 to E14.5) embryos. With its simple sample staining and mounting processes, this protocol is easy to adopt and implement for most of the commercially available, stand-alone microCT systems in order to study mouse development between early post-implantation to early postnatal stages. © 2019 by John Wiley & Sons, Inc.
Assuntos
Meios de Contraste/uso terapêutico , Compostos de Iodo/uso terapêutico , Camundongos , Microtomografia por Raio-X/métodos , Animais , Animais Recém-Nascidos , Embrião de Mamíferos , Microtomografia por Raio-X/instrumentaçãoRESUMO
SET and MYND Domain 1 (SMYD1) is a cardiac and skeletal muscle-specific, histone methyl transferase that is critical for both embryonic and adult heart development and function in both mice and men. We report here that skeletal muscle-specific, myogenin (myoG)-Cre-mediated conditional knockout (CKO) of Smyd1 results in perinatal death. As early as embryonic day 12.5, Smyd1 CKOs exhibit multiple skeletal muscle defects in proliferation, morphology, and gene expression. However, all myotonic descendants are not afflicted equally. Trunk muscles are virtually ablated with excessive accumulation of brown adipose tissue (BAT), forelimb muscles are disorganized and improperly differentiated, but other muscles, such as the masseter, are normal. While expression of major myogenic regulators went unscathed, adaptive and innate immune transcription factors critical for BAT development/physiology were downregulated. Whereas classical mitochondrial BAT accumulation went unscathed following loss of SMYD1, key transcription factors, including PRDM16, UCP-1, and CIDE-a that control skeletal muscle vs. adipose fate, were downregulated. Finally, in rare adults that survive perinatal lethality, SMYD1 controls specification of some, but not all, skeletal muscle fiber-types.
RESUMO
Developmental, stem cell and cancer biologists are interested in the molecular definition of cellular differentiation. Although single-cell RNA sequencing represents a transformational advance for global gene analyses, novel obstacles have emerged, including the computational management of dropout events, the reconstruction of biological pathways and the isolation of target cell populations. We develop an algorithm named dpath that applies the concept of metagene entropy and allows the ranking of cells based on their differentiation potential. We also develop self-organizing map (SOM) and random walk with restart (RWR) algorithms to separate the progenitors from the differentiated cells and reconstruct the lineage hierarchies in an unbiased manner. We test these algorithms using single cells from Etv2-EYFP transgenic mouse embryos and reveal specific molecular pathways that direct differentiation programmes involving the haemato-endothelial lineages. This software program quantitatively assesses the progenitor and committed states in single-cell RNA-seq data sets in a non-biased manner.
Assuntos
Linhagem da Célula/genética , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Embrionárias Murinas/citologia , Análise de Célula Única , Fatores de Transcrição/metabolismo , Algoritmos , Animais , Agregação Celular , Separação Celular , Análise por Conglomerados , Corpos Embrioides/citologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Software , Transcriptoma/genéticaRESUMO
The Smyd1 gene encodes a lysine methyltransferase specifically expressed in striated muscle. Because Smyd1-null mouse embryos die from heart malformation prior to formation of skeletal muscle, we developed a Smyd1 conditional-knockout allele to determine the consequence of SMYD1 loss in mammalian skeletal muscle. Ablation of SMYD1 specifically in skeletal myocytes after myofiber differentiation using Myf6(cre) produced a non-degenerative myopathy. Mutant mice exhibited weakness, myofiber hypotrophy, prevalence of oxidative myofibers, reduction in triad numbers, regional myofibrillar disorganization/breakdown and a high percentage of myofibers with centralized nuclei. Notably, we found broad upregulation of muscle development genes in the absence of regenerating or degenerating myofibers. These data suggest that the afflicted fibers are in a continual state of repair in an attempt to restore damaged myofibrils. Disease severity was greater for males than females. Despite equivalent expression in all fiber types, loss of SMYD1 primarily affected fast-twitch muscle, illustrating fiber-type-specific functions for SMYD1. This work illustrates a crucial role for SMYD1 in skeletal muscle physiology and myofibril integrity.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Musculares/metabolismo , Atrofia Muscular/enzimologia , Miofibrilas/enzimologia , Miofibrilas/patologia , Fatores de Transcrição/metabolismo , Animais , Feminino , Masculino , Camundongos Knockout , Desenvolvimento Muscular/genética , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Rápida/ultraestrutura , Força Muscular , Atrofia Muscular/patologia , Miofibrilas/ultraestrutura , Tamanho do Órgão , Oxirredução , Regeneração , Sarcolema/metabolismo , Regulação para Cima/genéticaRESUMO
MicroRNAs (miRNAs) are known to regulate critical developmental stages during embryogenesis. Here, we defined an Etv2-miR-130a cascade that regulates mesodermal specification and determination. Ablation of Dicer in the Etv2-expressing precursors resulted in altered mesodermal lineages and embryonic lethality. We identified miR-130a as a direct target of Etv2 and demonstrated its role in the segregation of bipotent hemato-endothelial progenitors toward the endothelial lineage. Gain-of-function experiments demonstrated that miR-130a promoted the endothelial program at the expense of the cardiac program without impacting the hematopoietic lineages. In contrast, CRISPR/Cas9-mediated knockout of miR-130a demonstrated a reduction of the endothelial program without affecting hematopoiesis. Mechanistically, miR-130a directly suppressed Pdgfra expression and promoted the endothelial program by blocking Pdgfra signaling. Inhibition or activation of Pdgfra signaling phenocopied the miR-130a overexpression and knockout phenotypes, respectively. In summary, we report the function of a miRNA that specifically promotes the divergence of the hemato-endothelial progenitor to the endothelial lineage.
Assuntos
Linhagem da Célula , Mesoderma/citologia , MicroRNAs/genética , Fatores de Transcrição/genética , Animais , Células Cultivadas , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Hematopoese , Mesoderma/metabolismo , Camundongos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Smyd1/Bop is an evolutionary conserved histone methyltransferase previously shown by conventional knockout to be critical for embryonic heart development. To further explore the mechanism(s) in a cell autonomous context, we conditionally ablated Smyd1 in the first and second heart fields of mice using a knock-in (KI) Nkx2.5-cre driver. Robust deletion of floxed-Smyd1 in cardiomyocytes and the outflow tract (OFT) resulted in embryonic lethality at E9.5, truncation of the OFT and right ventricle, and additional defects consistent with impaired expansion and proliferation of the second heart field (SHF). Using a transgenic (Tg) Nkx2.5-cre driver previously shown to not delete in the SHF and OFT, early embryonic lethality was bypassed and both ventricular chambers were formed; however, reduced cardiomyocyte proliferation and other heart defects resulted in later embryonic death at E11.5-12.5. Proliferative impairment prior to both early and mid-gestational lethality was accompanied by dysregulation of transcripts critical for endoplasmic reticulum (ER) stress. Mid-gestational death was also associated with impairment of oxidative stress defense-a phenotype highly similar to the previously characterized knockout of the Smyd1-interacting transcription factor, skNAC. We describe a potential feedback mechanism in which the stress response factor Tribbles3/TRB3, when directly methylated by Smyd1, acts as a co-repressor of Smyd1-mediated transcription. Our findings suggest that Smyd1 is required for maintaining cardiomyocyte proliferation at minimally two different embryonic heart developmental stages, and its loss leads to linked stress responses that signal ensuing lethality.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático , Coração/crescimento & desenvolvimento , Proteínas Musculares/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Estresse Oxidativo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Chlorocebus aethiops , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Humanos , Metilação , Camundongos , Dados de Sequência Molecular , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcrição Gênica , Regulação para CimaRESUMO
Previous reports regarding the genetic hierarchy between Ets related protein 71 (Er71/Etv2) and Flk1 is unclear. In the present study, we pursued a genetic approach to define the molecular cascade between Etv2 and Flk1. Using a transgenic Etv2-EYFP reporter mouse, we examined the expression pattern of Etv2 relative to Flk1 in the early conceptus. Etv2-EYFP was expressed in subset of Flk1 positive cells during primitive streak stages, suggesting that Flk1 is upstream of Etv2 during gastrulation. Analysis of reporter gene expression in Flk1 and Etv2 mutant mice further supports the hypothesis that Flk1 is necessary for Etv2 expression. The frequency of cells expressing Flk1 in Etv2 mutants is only modestly altered (21% decrease), whereas expression of the Etv2-EYFP transgenic reporter was severely reduced in the Flk1 null background. We further demonstrate using transcriptional assays that, in the presence of Flk1, the Etv2 promoter is activated by VEGF, the Flk1 ligand. Pharmacological inhibition studies demonstrate that VEGF mediated activation is dependent on p38 MAPK, which activates Creb. We identify the VEGF response element in the Etv2 promoter and demonstrate that Creb binds to this motif by EMSA and ChIP assays. In summary, we provide new evidence that VEGF activates Etv2 by signaling through Flk1, which activates Creb through the p38 MAPK signaling cascade.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Gastrulação/genética , Fatores de Transcrição/genética , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Animais , Sequência de Bases , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Ligação Proteica , Elementos de Resposta , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ativação Transcricional , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The vertebrate heart is assembled during embryogenesis in a modular manner from different populations of precursor cells. The right ventricular chamber and outflow tract are derived primarily from a population of progenitors known as the anterior heart field. These regions of the heart are severely hypoplastic in mutant mice lacking the myocyte enhancer factor 2C (MEF2C) and BOP transcription factors, suggesting that these cardiogenic regulatory factors may act in a common pathway for development of the anterior heart field and its derivatives. We show that Bop expression in the developing heart depends on the direct binding of MEF2C to a MEF2-response element in the Bop promoter that is necessary and sufficient to recapitulate endogenous Bop expression in the anterior heart field and its cardiac derivatives during mouse development. The Bop promoter also directs transcription in the skeletal muscle lineage, but only cardiac expression is dependent on MEF2. These findings identify Bop as an essential downstream effector gene of MEF2C in the developing heart, and reveal a transcriptional cascade involved in development of the anterior heart field and its derivatives.