RESUMEN
Arid3b, a member of the conserved ARID family of transcription factors, is essential for mouse embryonic development but its precise roles are poorly understood. Here, we show that Arid3b is expressed in the myocardium of the tubular heart and in second heart field progenitors. Arid3b-deficient embryos show cardiac abnormalities, including a notable shortening of the poles, absence of myocardial differentiation and altered patterning of the atrioventricular canal, which also lacks epithelial-to-mesenchymal transition. Proliferation and death of progenitors as well as early patterning of the heart appear normal. However, DiI labelling of second heart field progenitors revealed a defect in the addition of cells to the heart. RNA microarray analysis uncovered a set of differentially expressed genes in Arid3b-deficient tissues, including Bhlhb2, a regulator of cardiomyocyte differentiation, and Lims2, a gene involved in cell migration. Arid3b is thus required for heart development by regulating the motility and differentiation of heart progenitors. These findings identify Arid3b as a candidate gene involved in the aetiology of human congenital malformations.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Corazón/embriología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Muerte Celular/genética , Muerte Celular/fisiología , Proliferación Celular , Proteínas de Unión al ADN/genética , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inmunoquímica , Hibridación in Situ , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Left ventricular noncompaction (LVNC) causes prominent ventricular trabeculations and reduces cardiac systolic function. The clinical presentation of LVNC ranges from asymptomatic to heart failure. We show that germline mutations in human MIB1 (mindbomb homolog 1), which encodes an E3 ubiquitin ligase that promotes endocytosis of the NOTCH ligands DELTA and JAGGED, cause LVNC in autosomal-dominant pedigrees, with affected individuals showing reduced NOTCH1 activity and reduced expression of target genes. Functional studies in cells and zebrafish embryos and in silico modeling indicate that MIB1 functions as a dimer, which is disrupted by the human mutations. Targeted inactivation of Mib1 in mouse myocardium causes LVNC, a phenotype mimicked by inactivation of myocardial Jagged1 or endocardial Notch1. Myocardial Mib1 mutants show reduced ventricular Notch1 activity, expansion of compact myocardium to proliferative, immature trabeculae and abnormal expression of cardiac development and disease genes. These results implicate NOTCH signaling in LVNC and indicate that MIB1 mutations arrest chamber myocardium development, preventing trabecular maturation and compaction.
Asunto(s)
Cardiomiopatías/etiología , Ventrículos Cardíacos , Mutación , Receptores Notch/fisiología , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/genética , Secuencia de Aminoácidos , Animales , Cardiomiopatías/genética , Femenino , Células HEK293 , Corazón/embriología , Ventrículos Cardíacos/embriología , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Multimerización de Proteína , Ubiquitina-Proteína Ligasas/fisiología , Pez CebraRESUMEN
The epicardium is the outer epithelial covering the heart. This tissue undergoes an epithelial-to-mesenchymal transition (EMT) to generate mesenchymal epicardial-derived cells (EPDCs) that populate the extracellular matrix of the subepicardium and contribute to the development of the coronary vessels and cardiac interstitial cells. Although epicardial EMT plays a crucial role in heart development, the molecular regulation of this process is incompletely understood. Here we examined the possible role of the EMT regulator Snail1 in this process. Snail1 is expressed in the epicardium and EPDCs during mouse cardiac development. To determine the function of Snail1 in epicardial EMT, we deleted Snail1 in the epicardium using Wt1- and Tbx18-Cre drivers. Unexpectedly, epicardial-specific Snail1 mutants are viable and fertile and do not display any obvious morphological or functional cardiac abnormalities. Molecular analysis of these mice reveals that epicardial EMT occurs normally, and epicardial derivatives are established in these mutants. We conclude that Snail1 is not required for the initiation and progression of embryonic epicardial EMT.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Pericardio/embriología , Factores de Transcripción/metabolismo , Animales , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Ratones , Pericardio/patología , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
An often overlooked aspect of digit development is the special nature of the terminal phalanx, a specialized structure with characteristics distinct from other phalanges, for example the presence of ectodermal derivatives such as nails and claws. Here, we describe the unique ossification pattern of distal phalanges and characteristic gene expression in the digit tips of chick and duck embryos. Our results show that the distal phalanx of chick wing digit 1 is a genuine tip with a characteristic ossification pattern and expression of Bambi and Sp8; however, the terminal phalanx of digits 2* and 3 is not a genuine tip, and these are therefore truncated digits. Bambi and Sp8 expression in the chick wing provides a direct molecular assessment of digit identity changes after experimental manipulations of digit primordia. In contrast, digits 1 and 2 of the duck wing both possess true tips. Although chick wing-tip development was not rescued by application of Fgf8, this treatment induced the development of extra phalanges. Grafting experiments show that competence for tip formation, including nails, is latent in the interdigital tissue. Our results deepen understanding of the mechanisms of digit tip formation, highlighting its developmental autonomy and modular nature, with implications for digit reduction or loss during evolution. * Numbering of wing digits is 1, 2, 3 from anterior to posterior.
Asunto(s)
Tipificación del Cuerpo/genética , Extremidades/embriología , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/genética , Alas de Animales/embriología , Animales , Embrión de Pollo , Patos , Osteogénesis/genéticaRESUMEN
OBJECTIVE: Calcific aortic valve disease is similar to atherosclerosis in that both diseases result from chronic inflammation and endothelial dysfunction. Heterozygous NOTCH1 mutations have been associated to calcific aortic disease and a bicuspid aortic valve. We investigated whether mice with genetic inactivation of the Notch signaling pathway are prone to develop valve disease when exposed to a predisposing diet. METHODS AND RESULTS: Using Doppler echocardiography, histology, immunohistochemistry, quantitative gene expression analysis, and cell culture assays, we examined the effect of a hypercholesterolemic diet supplemented with vitamin D on mice heterozygous for null mutations in the Notch1 receptor or the effector transcription factor gene RBPJk. After 16 weeks on the hyperlipidemic diet, calcific aortic disease was detected in heterozygous RBPJk mice. Analysis of valve leaflets revealed macrophage infiltration, enhanced collagen deposition, proosteogenic protein expression, and calcification. Heterozygous null Notch1 mice displayed milder histopathologic changes and did not develop any significant hemodynamic disturbance. Valvular disease correlated with reduced expression of the Notch target gene Hey1 in valves of RBPJk heterozygous mice fed the hyperlipidemic diet. Consistent with the in vivo data, Notch signaling inhibition in porcine valve interstitial cells led to downregulation of HEY1 transcription, activation of osteogenic markers, and increased calcified nodule formation. CONCLUSIONS: We show that Notch signaling disruption via RBPJk heterozygous inactivation results in aortic valve disease. Notch1 heterozygous mice do not show functional impairment, suggesting that additional Notch receptors may be involved in aortic valve homeostasis and disease. Our data establish a genetic mouse model of calcific aortic valve disease and may help to identify a patient population with reduced valvular NOTCH signaling at risk for developing this disease.
Asunto(s)
Válvula Aórtica/metabolismo , Calcinosis/etiología , Haploinsuficiencia , Enfermedades de las Válvulas Cardíacas/etiología , Hipercolesterolemia/complicaciones , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/deficiencia , Receptor Notch1/deficiencia , Transducción de Señal , Análisis de Varianza , Animales , Válvula Aórtica/patología , Válvula Aórtica/fisiopatología , Calcinosis/genética , Calcinosis/metabolismo , Calcinosis/patología , Calcinosis/fisiopatología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Colesterol en la Dieta , Modelos Animales de Enfermedad , Ecocardiografía Doppler , Fibrosis , Regulación de la Expresión Génica , Genotipo , Enfermedades de las Válvulas Cardíacas/genética , Enfermedades de las Válvulas Cardíacas/metabolismo , Enfermedades de las Válvulas Cardíacas/patología , Enfermedades de las Válvulas Cardíacas/fisiopatología , Hemodinámica , Heterocigoto , Hipercolesterolemia/etiología , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patología , Hipercolesterolemia/fisiopatología , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Inmunohistoquímica , Ratones , Ratones Noqueados , Osteogénesis/genética , Fenotipo , Receptor Notch1/genética , Volumen Sistólico , Porcinos , Función Ventricular , Vitamina DRESUMEN
The apical ectodermal ridge (AER) is a specialized epithelium located at the distal edge of the limb bud that directs outgrowth along the proximodistal axis. Although the molecular basis for its function is well known, the cellular mechanisms that lead to its maturation are not fully understood. Here, we show that Arid3b, a member of the ARID family of transcriptional regulators, is expressed in the AER in mouse and chick embryos, and that interference with its activity leads to aberrant AER development, in which normal structure is not achieved. This happens without alterations in cell numbers or gene expression in main signalling pathways. Cells that are defective in Arid3b show an abnormal distribution of the actin cytoskeleton and decreased motility in vitro. Moreover, movements of pre-AER cells and their contribution to the AER were defective in vivo in embryos with reduced Arid3b function. Our results show that Arid3b is involved in the regulation of cell motility and rearrangements that lead to AER maturation.
Asunto(s)
Movimiento Celular/genética , Proteínas de Unión al ADN/fisiología , Ectodermo/embriología , Extremidades/embriología , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Embrión de Pollo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ectodermo/metabolismo , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Morfogénesis/genética , Morfogénesis/fisiologíaRESUMEN
RATIONALE: The proepicardium is a transient structure comprising epicardial progenitor cells located at the posterior limit of the embryonic cardiac inflow. A network of signals regulates proepicardial cell fate and defines myocardial and nonmyocardial domains at the venous pole of the heart. During cardiac development, epicardial-derived cells also contribute to coronary vessel morphogenesis. OBJECTIVE: To study Notch function during proepicardium development and coronary vessel formation in the mouse. METHODS AND RESULTS: Using in situ hybridization, RT-PCR, and immunohistochemistry, we find that Notch pathway elements are differentially activated throughout the proepicardial-epicardial-coronary transition. Analysis of RBPJk-targeted embryos indicates that Notch ablation causes ectopic procardiogenic signaling in the proepicardium that in turn promotes myocardial differentiation in adjacent mesodermal progenitors, resulting in a premature muscularization of the sinus venosus horns. Epicardium-specific Notch1 ablation using a Wt1-Cre driver line disrupts coronary artery differentiation, reduces myocardium wall thickness and myocyte proliferation, and reduces Raldh2 expression. Ectopic Notch1 activation disrupts epicardium development and causes thinning of ventricular walls. CONCLUSIONS: Epicardial Notch modulates cell differentiation in the proepicardium and adjacent pericardial mesoderm. Notch1 is later required for arterial endothelium commitment and differentiation and for vessel wall maturation during coronary vessel development and myocardium growth.
Asunto(s)
Circulación Sanguínea/fisiología , Vasos Coronarios/embriología , Morfogénesis/fisiología , Pericardio/embriología , Receptores Notch/fisiología , Transducción de Señal/fisiología , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/fisiología , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/fisiología , Diferenciación Celular/fisiología , Proliferación Celular , Vasos Coronarios/citología , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/fisiología , Ratones , Ratones Endogámicos , Ratones Transgénicos , Modelos Animales , Mutación , Pericardio/citología , Receptor Notch1/genética , Receptor Notch1/fisiología , Receptores Notch/genéticaRESUMEN
Digit formation is the last step in the skeletal patterning of developing limbs. This process involves important aspects such as determination of chondrogenic versus interdigital areas; growth of digital rays with periodic segmentation to form joints and thus phalanges, and finally tip formation. Traditionally it was believed that the properties of digital rays were fixed at earlier stages, but recently a surprising plasticity of digit primordia at the time of condensation has been demonstrated. This implies the presence of local interactions that are able to modulate the particular programs that make a given digit, but we don't fully understand how they operate. An involvement of signaling from the interdigital spaces and from the apical ectodermal ridge has been proposed. Another interesting question is the formation of the last limb structure, digit tips, which may involve a specific molecular and cellular program. Indeed, the expression of several developmentally important genes is restricted to digit tips at late stages of limb development. Understanding the molecular and cellular interactions that lead to digit morphogenesis has important implications not only in the context of embryonic development (for example, how early cues received by cells are translated into anatomy or what are the mechanisms that control the cease of activity of signaling regions) but also in terms of limb diversification during evolution.
Asunto(s)
Extremidades/embriología , Animales , Embrión de Pollo , HumanosRESUMEN
Mammalian DNA polymerase mu (Polmu), preferentially expressed in secondary lymphoid organs, is shown here to be up-regulated in germinal centers after immunization. Alternative splicing appears to be part of Polmu regulation during an immune response. We generated Polmu-deficient mice that are viable and show no anatomical malformation or serious alteration in lymphoid populations, with the exception of an underrepresentation of the B cell compartment. Young and aged homozygous Polmu(-/-) mice generated similar immune responses after immunization with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken gammaglobulin (CGG), compared with their wild-type littermates. Nonetheless, the kinetics of development of the centroblast population showed significant differences. Hypermutation analysis of the rearranged heavy chain intron region in centroblasts isolated from NP-CGG-immunized Polmu(-/-) mice showed a similar quantitative and qualitative somatic mutation spectrum, but a lower representation of heavily mutated clones. These results suggest that although it is not a critical partner, Polmu modulates the in vivo somatic hypermutation process.