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The placenta links feto-maternal circulation for exchanges of nutrients, gases, and metabolic wastes between the fetus and mother, being essential for pregnancy process and maintenance. The allantois and mesodermal components of amnion, chorion, and yolk sac are derived from extraembryonic mesoderm (Ex-Mes), however, the mechanisms contributing to distinct components of the placenta and regulation the interactions between allantois and epithelium during chorioallantoic fusion and labyrinth formation remains unclear. Isl1 is expressed in progenitors of the Ex-Mes and allantois the Isl1 mut mouse line is analyzed to investigate contribution of Isl1+ Ex-Mes / allantoic progenitors to cells of the allantois and placenta. This study shows that Isl1 identifies the Ex-Mes progenitors for endothelial and vascular smooth muscle cells, and most of the mesenchymal cells of the placenta and umbilical cord. Deletion of Isl1 causes defects in allantois growth, chorioallantoic fusion, and placenta vessel morphogenesis. RNA-seq and CUT&Tag analyses revealed that Isl1 promotes allantoic endothelial, inhibits mesenchymal cell differentiation, and allantoic signals regulated by Isl1 mediating the inductive interactions between the allantois and chorion critical for chorionic epithelium differentiation, villous formation, and labyrinth angiogenesis. This study above reveals that Isl1 plays roles in regulating multiple genetic and epigenetic pathways of vascular morphogenesis, provides the insight into the mechanisms for placental formation, highlighting the necessity of Isl1 for placenta formation/pregnant maintenance.
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PINCH, an adaptor of focal adhesion complex, plays essential roles in multiple cellular processes and organogenesis. Here, we ablated PINCH1 or both of PINCH1 and PINCH2 in skeletal muscle progenitors using MyoD-Cre. Double ablation of PINCH1 and PINCH2 resulted in early postnatal lethality with reduced size of skeletal muscles and detachment of diaphragm muscles from the body wall. PINCH mutant myofibers failed to undergo multinucleation and exhibited disrupted sarcomere structures. The mutant myoblasts in culture were able to adhere to newly formed myotubes but impeded in cell fusion and subsequent sarcomere genesis and cytoskeleton organization. Consistent with this, expression of integrin ß1 and some cytoskeleton proteins and phosphorylation of ERK and AKT were significantly reduced in PINCH mutants. However, N-cadherin was correctly expressed at cell adhesion sites in PINCH mutant cells, suggesting that PINCH may play a direct role in myoblast fusion. Expression of MRF4, the most highly expressed myogenic factor at late stages of myogenesis, was abolished in PINCH mutants that could contribute to observed phenotypes. In addition, mice with PINCH1 being ablated in myogenic progenitors exhibited only mild centronuclear myopathic changes, suggesting a compensatory role of PINCH2 in myogenic differentiation. Our results revealed a critical role of PINCH proteins in myogenic differentiation.
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Diferenciación Celular , Mioblastos Esqueléticos , Animales , Ratones , Adhesión Celular , Comunicación Celular , Adhesiones Focales/metabolismo , Músculo Esquelético/fisiologíaRESUMEN
In the long-term growth process, alfalfa rhizosphere forms specific microbiome to provide nutrition for its growth and development. However, the effects of different perennial alfalfa cultivars on changes in the rhizosphere soil characteristics and microbiome are not well understood. In this study, 12 perennial alfalfa cultivars were grown continuously for eight years. Rhizosphere samples were tested using Illumina sequencing of the 16S rRNA gene coupled with co-occurrence network analysis to explore the relationship between alfalfa (biomass and crude protein content), soil properties, and the microbial composition and diversity. Redundancy analysis showed SOC and pH had the greatest impact on the composition of the rhizosphere microbial community. Moreover, microbial diversity also contributes to microbial composition. Soil properties (AP, EC, SOC and pH) exhibited a significant positive correlation with soil bacterial communities, which was attributed to the differences between plant cultivars. Partial least squares path modeling (PLS-PM) revealed that microbial biomass and community composition rather than diversity, are the dominant determinants in the rhizosphere soil nitrogen content of perennial alfalfa. Our findings demonstrate that the soil microbial biomass and composition of rhizosphere bacterial communities are strongly affected by cultivar, driving the changes in soil nitrogen content, and variances in the selective capacities of plants.
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Rare earth elements (REEs) of low concentration are usually beneficial to plant growth, while they are toxic at high concentrations. The effects of treatment with lanthanum (La) (10 and 20 µM), cerium (Ce) (10 and 20 µM), and terbium (Tb) (10 and 20 µM) on seedling growth of alfalfa (Medicago sativa L.), which is one of the most important perennial leguminous forages in the world, were studied. The results showed that all three REE treatments quickened the germination of seeds. The length of shoot under La (20 µM) treatment was significantly shortened (P < 0.05). In addition, treatment with La, Ce, and Tb had a "hormesis effect" on root length. There was a significant decrease in chlorophyll content on treatment with the three REEs, and the degree of decline was in the order of La < Ce < Tb, under the same concentration. In vitro experiments and quantum chemical calculations were further performed to explain why the treatments with REEs reduced the chlorophyll content. In vitro experiments showed that La, Ce, and Tb treatments reduced the absorbance of chlorophyll, and the decrease followed in the order of La > Ce > Tb. Quantum chemical calculations predicted that the decrease in absorption intensity was caused by the reactions between La, Ce, Tb, and chlorophyll, which formed lanthanides-chlorophyll; and there were five types of stable lanthanides-chlorophyll. In conclusion, the decrease in chlorophyll content on treatment with REEs was caused by the change in chlorophyll structure.
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OBJECTIVE: Accumulating evidence has suggested that microRNAs (miRNAs) derived from M2 macrophage-derived exosomes (M2 exosomes) can regulate the progression of hepatocellular carcinoma (HCC). Nevertheless, the effect of miR-27a-3p derived from M2 exosomes on HCC has not been reported. We aim to explore the role of M2 exosomal miR-27a-3p in the cancer stemness of HCC via regulating thioredoxin-interacting protein (TXNIP). METHODS: Exosomes were extracted from transfected M2 macrophages and were then co-cultured with HCC cells. Expression of miR-27a-3p and TXNIP, stemness, proliferation, drug resistance, migration, invasion and in vivo tumorigenicity of HCC cells were determined to assess the role of M2 exosomal miR-27a-3p in HCC. The binding relationship between miR-27a-3p and TXNIP was detected. RESULTS: MiR-27a-3p was upregulated and TXNIP was downregulated in HCC cells, and M2 exosomes further upregulated miR-27a-3p. The upregulated M2 exosomal miR-27a-3p promoted stemness, proliferation, drug resistance, migration, invasion and in vivo tumorigenicity of HCC cells. TXNIP was confirmed as a target gene of miR-27a-3p. CONCLUSION: M2 macrophages-derived exosomal miR-27a-3p promotes cancer stemness of HCC via downregulating TXNIP.
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Carcinoma Hepatocelular/genética , Proteínas Portadoras/genética , Neoplasias Hepáticas/genética , MicroARNs/metabolismo , Células Madre Neoplásicas/patología , Animales , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Regulación hacia Abajo/inmunología , Exosomas/metabolismo , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Regulación hacia Arriba/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Smoothened is a key receptor of the hedgehog pathway, but the roles of Smoothened in cardiac development remain incompletely understood. In this study, we found that the conditional knockout of Smoothened from the mesoderm impaired the development of the venous pole of the heart and resulted in hypoplasia of the atrium/inflow tract (IFT) and a low heart rate. The blockage of Smoothened led to reduced expression of genes critical for sinoatrial node (SAN) development in the IFT. In a cardiac cell culture model, we identified a Gli2-Tbx5-Hcn4 pathway that controls SAN development. In the mutant embryos, the endocardial-to-mesenchymal transition (EndMT) in the atrioventricular cushion failed, and Bmp signalling was downregulated. The addition of Bmp2 rescued the EndMT in mutant explant cultures. Furthermore, we analysed Gli2+ scRNAseq and Tbx5-/- RNAseq data and explored the potential genes downstream of hedgehog signalling in posterior second heart field derivatives. In conclusion, our study reveals that Smoothened-mediated hedgehog signalling controls posterior cardiac progenitor commitment, which suggests that the mutation of Smoothened might be involved in the aetiology of congenital heart diseases related to the cardiac conduction system and heart valves.
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Cojinetes Endocárdicos/embriología , Cojinetes Endocárdicos/metabolismo , Proteínas Hedgehog/metabolismo , Organogénesis , Transducción de Señal , Nodo Sinoatrial/embriología , Nodo Sinoatrial/metabolismo , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Biología Computacional/métodos , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos , Receptor Smoothened/genética , Receptor Smoothened/metabolismoRESUMEN
The objective of the current work was to demonstrate the equivalence of Mylan's glatiramer acetate (GA) to that of the reference product Copaxone® (COP) using the four criteria for active pharmaceutical ingredient sameness as established by the US Food and Drug Administration (FDA). The reaction scheme used to produce Mylan's glatiramer acetate (MGA) was compared with that of COP, determined from publicly available literature. Comparative analyses of MGA and COP were performed for physicochemical properties such as amino acid composition and molecular weight distributions. Spectroscopic fingerprints were obtained using circular dichroism spectroscopy. Structural signatures for polymerization and depolymerization including total diethylamine (DEA) content, relative proportions of DEA-adducted amino acids, and N-and C-terminal amino acid sequences were probed with an array of highly sensitive analytical methods. Biological activity of the products was assessed using validated murine Experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. MGA is produced using the same fundamental reaction scheme as COP and was shown to have equivalent physicochemical properties and composition. Analyses of multiple structural signatures demonstrated equivalence of MGA and COP with regard to polymerization, depolymerization, and propagational shift. Examination of the impact on prevention and treatment of EAE demonstrated equivalence of MGA and COP with respect to both activity and toxicity, and thereby provided confirmatory evidence of sameness. A rigorous, multi-pronged comparison of MGA and COP produced using an equivalent fundamental reaction scheme demonstrated equivalent physicochemical properties, structural signatures for polymerization and depolymerization, and biological activity as evidenced by comparable effects in EAE. These studies demonstrate the equivalence of MGA and COP, establishing active ingredient sameness by the US Food and Drug Administration (FDA) criteria for GA, and provide compelling evidence that the FDA-approved generic MGA can be substituted for COP for the treatment of patients with relapsing-remitting MS.
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The rare earth element lanthanum (La) has been proven to be beneficial for plant growth with a low concentration, and abscisic acid (ABA) which is a plant hormone also can regulate plant growth. In the present study, we investigated the germination and seedling growth of switchgrass (Panicum virgatum L.) under La (10 µM), ABA (10 µM) and La + ABA treatments. The results showed that La, ABA and La + ABA treatments could not significantly affect the germination and shoot length as compared to the control (P>0.05). However, La treatment increased the root activity and chlorophyll content, and ABA treatment enhanced root length and root activity (P<0.05). La + ABA treatments demonstrated that La could not significantly alleviate the promotion of ABA in root length, while ABA reversed the increase of chlorophyll content caused by La. The coregulation of La and ABA on chlorophyll content was further explored by in vitro experiments and quantum chemical calculations. In vitro experiments revealed that La, ABA, and La + ABA treatments reduced the absorbance of chlorophyll, and quantum chemical calculations indicated that the reduction of absorbance was caused by the reactions between La, ABA and chlorophyll. In vivo and in vitro experiments, together with quantum chemical calculations, demonstrated that both ABA and La could stimulate the production of chlorophyll, while they also could react with chlorophyll to produce La-monochlorophyll, La-bischlorophyll, and ABA adsorbed chlorophyll, which had lower absorbance. La + ABA treatment significantly decreased the chlorophyll content in vivo. This phenomenon was due to the fact that La and ABA formed LaABA compound, which markedly reduced the concentrations of ABA and La, and the effect of promoting chlorophyll production was overcome by the effect of reducing chlorophyll absorbance.
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Ácido Abscísico/metabolismo , Clorofila/metabolismo , Lantano/metabolismo , Panicum/crecimiento & desarrollo , Reguladores del Crecimiento de las Plantas/metabolismo , Plantones/crecimiento & desarrollo , Germinación , Panicum/metabolismo , Plantones/metabolismoRESUMEN
OBJECTIVE: To investigate the correlation between the level of glucose in serum and the development of acute pancreatitis (AP). METHODS: Data of 153 AP cases were collected, in which there were 130 patients with mild AP (MAP), 4 with moderate-severe AP (MSAP) and 19 with severe AP (SAP). At the time of admission, following indexes of patients were recorded: glucose, APACHE II score, TNF-α and C-reaction protein (CRP). RESULTS: At the time of admission, the levels of glucose in serum and APACHE II scores in the MSAP and SAP groups were significantly higher than those in the MAP group, but after treatment, the level of glucose in serum was recovered in 95.8% of the patients in the MAP group, while this digit in the SAP group remained to be 68.4%; in the SAP group, the levels of TNF-α and CRP in patients with sustained hypertension were significantly higher than those with non-persistent hypertension; in terms of the length of stay in hospital, the SAP group was shorter than that in the non-treatment group, and the difference had statistical significance (pâ¯<â¯0.05). Moreover, we found that the level of glucose in serum was positively correlated with the APACHE II scores, TNF-α and CRP. CONCLUSION: Glucose level in serum can be used as one of the indicators for evaluating the severity and development of AP in clinical practice.
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BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a severe liver disease, which influences the health of people worldwide. However, the mechanism modulating the pathogenesis of NAFLD remains elusive. It was reported that nuclear enriched abundant transcript 1 (NEAT1) and microRNA-140 (miR-140) could regulate lipogenesis, but whether they could influence NAFLD are still unknown. METHODS: HepG2 cells were treated by free fatty acids (FFA) to establish the model of NAFLD in vitro, and C57 mice were treated by high-fat diet to establish the model of NAFLD in vivo. Cell transfection was applied to regulate the expression of NEAT1 and miR-140. Western blotting and qRT-PCR were applied for measuring expression of protein and mRNA, respectively. HE staining and Oil Red O staining were used for observing liver tissues. RESULTS: NEAT1 and miR-140 are upregulated in hepacytes under the NAFLD conditions. NEAT1 directly binds to miR-140 and acts synergistically with miR-140 to exacerbate the progression of NAFLD. Reciprocally, silence of miR-140 or NEAT1 alleviates the severity of NAFLD. The mechanistical study shows that the axis of NEAT1-miR-140 inactivates AMPK/SREBP-1 signaling during the NAFLD. . CONCLUSION: The NEAT1-miR-140 axis play a crucial role in modulation of NAFLD via inactivation of AMPK/SREBP1 signaling. This study may provide a novel insight for the treatment of NAFLD.
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Proteínas Quinasas Activadas por AMP/metabolismo , MicroARNs/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , ARN Largo no Codificante/metabolismo , Transducción de Señal , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Progresión de la Enfermedad , Silenciador del Gen , Células Hep G2 , Humanos , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , ARN Largo no Codificante/genética , Regulación hacia Arriba/genéticaRESUMEN
Generation of widely differing and specialized cell types from a single totipotent zygote involves large-scale transcriptional changes and chromatin reorganization. Pioneer transcription factors play key roles in programming the epigenome and facilitating recruitment of additional regulatory factors during successive cell lineage specification and differentiation steps. Here we show that Isl1 acts as a pioneer factor driving cardiomyocyte lineage commitment by shaping the chromatin landscape of cardiac progenitor cells. Using an Isl1 hypomorphic mouse line which shows congenital heart defects, genome-wide profiling of Isl1 binding together with RNA- and ATAC-sequencing of cardiac progenitor cells and their derivatives, we uncover a regulatory network downstream of Isl1 that orchestrates cardiogenesis. Mechanistically, we show that Isl1 binds to compacted chromatin and works in concert with the Brg1-Baf60c-based SWI/SNF complex to promote permissive cardiac lineage-specific alterations in the chromatin landscape not only of genes with critical functions in cardiac progenitor cells, but also of cardiomyocyte structural genes that are highly expressed when Isl1 itself is no longer present. Thus, the Isl1/Brg1-Baf60c complex plays a crucial role in orchestrating proper cardiogenesis and in establishing epigenetic memory of cardiomyocyte fate commitment.
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Epigénesis Genética/genética , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Miocitos Cardíacos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Células HEK293 , Humanos , Proteínas con Homeodominio LIM/deficiencia , Imagen por Resonancia Magnética , Ratones , Ratones Noqueados , Ratones Transgénicos , Factores de Transcripción/deficienciaRESUMEN
Background: Transcription factor ISL1 plays a critical role in sympathetic neurogenesis. Expression of ISL1 has been associated with neuroblastoma, a pediatric tumor derived from sympatho-adrenal progenitors, however the role of ISL1 in neuroblastoma remains unexplored. Method: Here, we knocked down ISL1 (KD) in SH-SY5Y neuroblastoma cells and performed RNA-seq and ISL1 ChIP-seq analyses. Results: Analyses of these data revealed that ISL1 acts upstream of multiple oncogenic genes and pathways essential for neuroblastoma proliferation and differentiation, including LMO1 and LIN28B. ISL1 promotes expression of a number of cell cycle associated genes, but represses differentiation associated genes including RA receptors and the downstream target genes EPAS1 and CDKN1A. Consequently, Knockdown of ISL1 inhibits neuroblastoma cell proliferation and migration in vitro and impedes tumor growth in vivo, and enhances neuronal differentiation by RA treatment. Furthermore, genome-wide mapping revealed a substantial co-occupancy of binding regions by ISL1 and GATA3, and ISL1 physically interacts with GATA3, and together they synergistically regulate the aforementioned oncogenic pathways. In addition, analyses of the roles of ISL1 and MYCN in MYCN-amplified and MYCN non-amplified neuroblastoma cells revealed an epistatic relationship between ISL1 and MYCN. ISL1 and MYCN function in parallel to regulate common yet distinct oncogenic pathways in neuroblastoma. Conclusion: Our study has demonstrated that ISL1 plays an essential role in neuroblastoma regulatory networks and may serve as a potential therapeutic target in neuroblastoma.
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Carcinogénesis , Factor de Transcripción GATA3/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/fisiopatología , Mapeo de Interacción de Proteínas , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones SCID , Trasplante de Neoplasias , Unión Proteica , Análisis de Secuencia de ARN , Trasplante HeterólogoRESUMEN
Kidney development involves reciprocal and inductive interactions between the ureteric bud (UB) and surrounding metanephric mesenchyme. Signals from renal stromal lineages are essential for differentiation and patterning of renal epithelial and mesenchymal cell types and renal vasculogenesis; however, underlying mechanisms remain not fully understood. Integrin-linked kinase (ILK), a key component of integrin signaling pathway, plays an important role in kidney development. However, the role of ILK in renal stroma remains unknown. Here, we ablated ILK in renal stromal lineages using a platelet-derived growth factor receptor B ( Pdgfrb) -Cre mouse line, and the resulting Ilk mutant mice presented postnatal growth retardation and died within 3 wk of age with severe renal developmental defects. Pdgfrb-Cre;Ilk mutant kidneys exhibited a significant decrease in UB branching and disrupted collecting duct formation. From E16.5 onward, renal interstitium was disorganized, forming medullary interstitial pseudocysts. Pdgfrb-Cre;Ilk mutants exhibited renal vasculature mispatterning and impaired glomerular vascular differentiation. Impaired glial cell-derived neurotrophic factor/Ret and bone morphogenetic protein 7 signaling pathways were observed in Pdgfrb-Cre;Ilk mutant kidneys. Furthermore, phosphoproteomic and Western blot analyses revealed a significant dysregulation of a number of key signaling pathways required for kidney morphogenesis, including PI3K/AKT and MAPK/ERK in Pdgfrb-Cre;Ilk mutants. Our results revealed a critical requirement for ILK in renal-stromal and vascular development, as well as a noncell autonomous role of ILK in UB branching morphogenesis.
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Riñón/enzimología , Enfermedades Renales Poliquísticas/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Células del Estroma/enzimología , Animales , Proteína Morfogenética Ósea 7/genética , Proteína Morfogenética Ósea 7/metabolismo , Diferenciación Celular , Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Predisposición Genética a la Enfermedad , Edad Gestacional , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Integrasas/genética , Integrasas/metabolismo , Riñón/anomalías , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Morfogénesis , Fenotipo , Fosfatidilinositol 3-Quinasa/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas c-ret/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de SeñalRESUMEN
Malformations of the sympathetic nervous system have been associated with cardiovascular instability, gastrointestinal dysfunction, and neuroblastoma. A better understanding of the factors regulating sympathetic nervous system development is critical to the development of potential therapies. Here, we have uncovered a temporal requirement for the LIM homeodomain transcription factor ISL1 during sympathetic nervous system development by the analysis of two mutant mouse lines: an Isl1 hypomorphic line and mice with Isl1 ablated in neural crest lineages. During early development, ISL1 is required for sympathetic neuronal fate determination, differentiation, and repression of glial differentiation, although it is dispensable for initial noradrenergic differentiation. ISL1 also plays an essential role in sympathetic neuron proliferation by controlling cell cycle gene expression. During later development, ISL1 is required for axon growth and sympathetic neuron diversification by maintaining noradrenergic differentiation, but repressing cholinergic differentiation. RNA-seq analyses of sympathetic ganglia from Isl1 mutant and control embryos, together with ISL1 ChIP-seq analysis on sympathetic ganglia, demonstrated that ISL1 regulates directly or indirectly several distinct signaling pathways that orchestrate sympathetic neurogenesis. A number of genes implicated in neuroblastoma pathogenesis are direct downstream targets of ISL1. Our study revealed a temporal requirement for ISL1 in multiple aspects of sympathetic neuron development, and suggested Isl1 as a candidate gene for neuroblastoma.
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Neuronas Adrenérgicas/metabolismo , Neuronas Colinérgicas/metabolismo , Ganglios Simpáticos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas con Homeodominio LIM/genética , Neuroblastoma/genética , Factores de Transcripción/genética , Neuronas Adrenérgicas/citología , Animales , Secuencia de Bases , Ciclo Celular/genética , Diferenciación Celular , Linaje de la Célula/genética , Proliferación Celular , Neuronas Colinérgicas/citología , Embrión de Mamíferos , Ganglios Simpáticos/citología , Humanos , Proteínas con Homeodominio LIM/metabolismo , Ratones , Ratones Transgénicos , Cresta Neural/citología , Cresta Neural/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neurogénesis/genética , Cultivo Primario de Células , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
Pericytes are widely believed to function as mesenchymal stem cells (MSCs), multipotent tissue-resident progenitors with great potential for regenerative medicine. Cultured pericytes isolated from distinct tissues can differentiate into multiple cell types in vitro or following transplantation in vivo. However, the cell fate plasticity of endogenous pericytes in vivo remains unclear. Here, we show that the transcription factor Tbx18 selectively marks pericytes and vascular smooth muscle cells in multiple organs of adult mouse. Fluorescence-activated cell sorting (FACS)-purified Tbx18-expressing cells behaved as MSCs in vitro. However, lineage-tracing experiments using an inducible Tbx18-CreERT2 line revealed that pericytes and vascular smooth muscle cells maintained their identity in aging and diverse pathological settings and did not significantly contribute to other cell lineages. These results challenge the current view of endogenous pericytes as multipotent tissue-resident progenitors and suggest that the plasticity observed in vitro or following transplantation in vivo arises from artificial cell manipulations ex vivo.
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Células Madre Mesenquimatosas/citología , Especificidad de Órganos , Pericitos/citología , Adipocitos/citología , Envejecimiento/genética , Linaje de la Célula , Cicatriz/patología , Fibroblastos/citología , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Integrasas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Desarrollo de Músculos , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Neuronas/citología , Pericitos/metabolismo , Fenotipo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismoRESUMEN
The sinoatrial node (SAN) is the dominant pacemaker of the heart. Abnormalities in SAN formation and function can cause sinus arrhythmia, including sick sinus syndrome and sudden death. A better understanding of genes and signaling pathways that regulate SAN development and function is essential to develop more effective treatment to sinus arrhythmia, including biological pacemakers. In this review, we briefly summarize the key processes of SAN morphogenesis during development, and focus on the transcriptional network that drives SAN development.
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Cardiopatías/terapia , Marcapaso Artificial , Arritmia Sinusal/etiología , Arritmia Sinusal/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas con Homeodominio LIM/metabolismo , Marcapaso Artificial/efectos adversos , Nodo Sinoatrial/metabolismo , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The sinoatrial node (SAN) maintains a rhythmic heartbeat; therefore, a better understanding of factors that drive SAN development and function is crucial to generation of potential therapies, such as biological pacemakers, for sinus arrhythmias. Here, we determined that the LIM homeodomain transcription factor ISL1 plays a key role in survival, proliferation, and function of pacemaker cells throughout development. Analysis of several Isl1 mutant mouse lines, including animals harboring an SAN-specific Isl1 deletion, revealed that ISL1 within SAN is a requirement for early embryonic viability. RNA-sequencing (RNA-seq) analyses of FACS-purified cells from ISL1-deficient SANs revealed that a number of genes critical for SAN function, including those encoding transcription factors and ion channels, were downstream of ISL1. Chromatin immunoprecipitation assays performed with anti-ISL1 antibodies and chromatin extracts from FACS-purified SAN cells demonstrated that ISL1 directly binds genomic regions within several genes required for normal pacemaker function, including subunits of the L-type calcium channel, Ank2, and Tbx3. Other genes implicated in abnormal heart rhythm in humans were also direct ISL1 targets. Together, our results demonstrate that ISL1 regulates approximately one-third of SAN-specific genes, indicate that a combination of ISL1 and other SAN transcription factors could be utilized to generate pacemaker cells, and suggest ISL1 mutations may underlie sick sinus syndrome.
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Proliferación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas con Homeodominio LIM/metabolismo , Contracción Miocárdica/fisiología , Nodo Sinoatrial/embriología , Factores de Transcripción/metabolismo , Animales , Ancirinas/genética , Ancirinas/metabolismo , Supervivencia Celular , Cromatina/genética , Cromatina/metabolismo , Eliminación de Gen , Proteínas con Homeodominio LIM/genética , Ratones , Ratones Transgénicos , Unión Proteica , Síndrome del Seno Enfermo/embriología , Síndrome del Seno Enfermo/genética , Síndrome del Seno Enfermo/patología , Nodo Sinoatrial/citología , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genéticaRESUMEN
The voltage-gated Na(+) channel Nav 1.5 is essential for action potential (AP) formation and electrophysiological homoeostasis in the heart. The ubiquitin-proteasome system (UPS) is a major degradative system for intracellular proteins including ion channels. The ubiquitin protein ligase E3 component N-recognin (UBR) family is a part of the UPS; however, their roles in regulating cardiac Nav 1.5 channels remain elusive. Here, we found that all of the UBR members were expressed in cardiomyocytes. Individual knockdown of UBR3 or UBR6, but not of other UBR members, significantly increased Nav 1.5 protein levels in neonatal rat ventricular myocytes, and this effect was verified in HEK293T cells expressing Nav 1.5 channels. The UBR3/6-dependent regulation of Nav 1.5 channels was not transcriptionally mediated, and pharmacological inhibition of protein biosynthesis failed to counteract the increase in Nav 1.5 protein caused by UBR3/6 reduction, suggesting a degradative modulation of UBR3/6 on Nav 1.5. Furthermore, the effects of UBR3/6 knockdown on Nav 1.5 proteins were abolished under the inhibition of proteasome activity, and UBR3/6 knockdown reduced Nav 1.5 ubiquitylation. The double UBR3-UBR6 knockdown resulted in comparable increases in Nav 1.5 proteins to that observed for single knockdown of either UBR3 or UBR6. Electrophysiological recordings showed that UBR3/6 reduction-mediated increase in Nav 1.5 protein enhanced the opening of Nav 1.5 channels and thereby the amplitude of the AP. Thus, our findings indicate that UBR3/6 regulate cardiomyocyte Nav 1.5 channel protein levels via the ubiquitin-proteasome pathway. It is likely that UBR3/6 have the potential to be a therapeutic target for cardiac arrhythmias.
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Miocardio/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Proteínas de Neoplasias/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Potenciales de Acción , Animales , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Miocitos Cardíacos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , ARN Interferente Pequeño/metabolismo , Ratas Sprague-DawleyRESUMEN
Specialized myocytes of the cardiac conduction system (CCS) are essential to coordinate sequential contraction of cardiac atria and ventricles. Anomalies of the CCS can result in lethal cardiac arrhythmias, including sick sinus syndrome and atrial or ventricular fibrillation. To develop future therapies and regenerative medicine aimed at cardiac arrhythmias, it is important to understand formation and function of distinct components of the CCS. Essential to this understanding is the development of CCS-specific markers. In this review, we briefly summarize available mouse models of CCS markers and focus on those involving the hyperpolarization cation-selective nucleotide-gated cation channel, HCN4, which selectively marks all components of the specialized CCS in adult heart. Recent studies have revealed, however, that HCN4 expression during development is highly dynamic in cardiac precursors. These studies have offered insights into the contributions of the first and second heart field to myocyte and conduction system lineages and suggested the timing of allocation of specific conduction system precursors during development. Altogether, they have highlighted the utility of HCN4 as a cell surface marker for distinct components of the CCS at distinct stages of development, which can be utilized to facilitate purification and characterization of CCS precursors in mouse and human model systems and pave the way for regenerative therapies.
Asunto(s)
Biomarcadores/metabolismo , Sistema de Conducción Cardíaco/fisiología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Proteínas Musculares/metabolismo , Canales de Potasio/metabolismo , Animales , Linaje de la Célula , Sistema de Conducción Cardíaco/citología , RatonesRESUMEN
Tetralogy of Fallot (TOF) is a complex congenital heart defect and the microRNAs regulation in TOF development is largely unknown. Herein, we explored the role of miRNAs in TOF. Among 75 dysregulated miRNAs identified from human heart tissues, miRNA-940 was the most down-regulated one. Interestingly, miRNA-940 was most highly expressed in normal human right ventricular out-flow tract comparing to other heart chambers. As TOF is caused by altered proliferation, migration and/or differentiation of the progenitor cells of the secondary heart field, we isolated Sca-1(+) human cardiomyocyte progenitor cells (hCMPC) for miRNA-940 function analysis. miRNA-940 reduction significantly promoted hCMPCs proliferation and inhibited hCMPCs migration. We found that JARID2 is an endogenous target regulated by miRNA-940. Functional analyses showed that JARID2 also affected hCMPCs proliferation and migration. Thus, decreased miRNA-940 affects the proliferation and migration of the progenitor cells of the secondary heart field by targeting JARID2 and potentially leads to TOF development.