Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22
Filtrar
Más filtros












Base de datos
Intervalo de año de publicación
1.
Dev Biol ; 487: 42-56, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35429490

RESUMEN

In mammalian development, oscillatory activation of Notch signaling is required for segmentation clock function during somitogenesis. Notch activity oscillations are synchronized between neighboring cells in the presomitic mesoderm (PSM) and have a period that matches the rate of somite formation. Normal clock function requires cyclic expression of the Lunatic fringe (LFNG) glycosyltransferase, as well as expression of the inhibitory Notch ligand Delta-like 3 (DLL3). How these factors coordinate Notch activation in the clock is not well understood. Recent evidence suggests that LFNG can act in a signal-sending cell to influence Notch activity in the clock, raising the possibility that in this context, glycosylation of Notch pathway proteins by LFNG may affect ligand activity. Here we dissect the genetic interactions of Lfng and Dll3 specifically in the segmentation clock and observe distinctions in the skeletal and clock phenotypes of mutant embryos showing that paradoxically, loss of Dll3 is associated with strong reductions in Notch activity in the caudal PSM. The patterns of Notch activity in the PSM suggest that the loss of Dll3 is epistatic to the loss of Lfng in the segmentation clock, and we present direct evidence for the modification of several DLL1 and DLL3 EGF-repeats by LFNG. We further demonstrate that DLL3 expression in cells co-expressing DLL1 and NOTCH1 can potentiate a cell's signal-sending activity and that this effect is modulated by LFNG, suggesting a mechanism for coordinated regulation of oscillatory Notch activation in the clock by glycosylation and cis-inhibition.


Asunto(s)
Receptores Notch , Somitos , Animales , Regulación del Desarrollo de la Expresión Génica , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Ligandos , Mamíferos/genética , Mesodermo/metabolismo , Receptores Notch/metabolismo , Somitos/metabolismo
2.
Sci Adv ; 7(6)2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33547080

RESUMEN

Calcific aortic valve disease (CAVD) is an increasingly prevalent condition, and endothelial dysfunction is implicated in its etiology. We previously identified nitric oxide (NO) as a calcification inhibitor by its activation of NOTCH1, which is genetically linked to human CAVD. Here, we show NO rescues calcification by an S-nitrosylation-mediated mechanism in porcine aortic valve interstitial cells and single-cell RNA-seq demonstrated NO regulates the NOTCH pathway. An unbiased proteomic approach to identify S-nitrosylated proteins in valve cells found enrichment of the ubiquitin-proteasome pathway and implicated S-nitrosylation of USP9X (ubiquitin specific peptidase 9, X-linked) in NOTCH regulation during calcification. Furthermore, S-nitrosylated USP9X was shown to deubiquitinate and stabilize MIB1 for NOTCH1 activation. Consistent with this, genetic deletion of Usp9x in mice demonstrated CAVD and human calcified aortic valves displayed reduced S-nitrosylation of USP9X. These results demonstrate a previously unidentified mechanism by which S-nitrosylation-dependent regulation of a ubiquitin-associated pathway prevents CAVD.

3.
EMBO Rep ; 20(7): e48247, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31267704

RESUMEN

The Notch signaling pathway is tightly controlled via post-transcriptional regulatory mechanisms that promote or terminate pathway activity. In this issue, Carrieri et al [1] show that phosphorylation of the Notch intracellular domain (NICD) by cyclin-dependent kinases (CDKs) suppresses Notch activity by promoting NICD turnover. These findings link Notch pathway activity to the cell cycle, and the authors propose connections between this regulation and the segmentation clock that times embryonic somitogenesis.


Asunto(s)
Proteína Quinasa CDC2 , Quinasas Ciclina-Dependientes , Ciclo Celular , Fosforilación , Transducción de Señal
4.
Elife ; 72018 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-29629872

RESUMEN

Notch signalling maintains stem cell regeneration at the mouse intestinal crypt base and balances the absorptive and secretory lineages in the upper crypt and villus. Here we report the role of Fringe family of glycosyltransferases in modulating Notch activity in the two compartments. At the crypt base, RFNG is enriched in the Paneth cells and increases cell surface expression of DLL1 and DLL4. This promotes Notch activity in the neighbouring Lgr5+ stem cells assisting their self-renewal. Expressed by various secretory cells in the upper crypt and villus, LFNG promotes DLL surface expression and suppresses the secretory lineage . Hence, in the intestinal epithelium, Fringes are present in the ligand-presenting 'sender' secretory cells and promote Notch activity in the neighbouring 'receiver' cells. Fringes thereby provide for targeted modulation of Notch activity and thus the cell fate in the stem cell zone, or the upper crypt and villus.


Asunto(s)
Homeostasis , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Intestinos/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Notch/metabolismo , Células Madre/citología , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al Calcio , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Glucosiltransferasas , Glicosiltransferasas , Péptidos y Proteínas de Señalización Intercelular/genética , Intestinos/citología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas/fisiología , Receptores Acoplados a Proteínas G/fisiología , Receptores Notch/genética , Transducción de Señal , Células Madre/metabolismo
5.
Dev Dyn ; 246(10): 740-748, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28710810

RESUMEN

BACKGROUND: In vertebrate embryos, a "segmentation clock" times somitogenesis. Clock-linked genes, including Lunatic fringe (Lfng), exhibit cyclic expression in the presomitic mesoderm (PSM), with a period matching the rate of somite formation. The clock period varies widely across species, but the mechanisms that underlie this variability are not clear. The half-lives of clock components are proposed to influence the rate of clock oscillations, and are tightly regulated in the PSM. Interactions between Lfng and mir-125a-5p in the embryonic chicken PSM promote Lfng transcript instability, but the conservation of this mechanism in other vertebrates has not been tested. Here, we examine whether this interaction affects clock activity in a mammalian species. RESULTS: Mutation of mir-125 binding sites in the Lfng 3'UTR leads to persistent, nonoscillatory reporter transcript expression in the caudal-most mouse PSM, although dynamic transcript expression recovers in the central PSM. Despite this, expression of endogenous mir-125a-5p is dispensable for mouse somitogenesis. CONCLUSIONS: These results suggest that mir-125a sites in the Lfng 3' untranslated region influence transcript turnover in both mouse and chicken embryos, and support the existence of position-dependent regulatory mechanisms in the PSM. They further suggest the existence of compensatory mechanisms that can rescue the loss of mir-125a-5p in mice. Developmental Dynamics 246:740-748, 2017. © 2017 Wiley Periodicals, Inc.


Asunto(s)
Regiones no Traducidas 3' , Glicosiltransferasas/química , MicroARNs/química , Somitos/citología , Animales , Sitios de Unión , Tipificación del Cuerpo , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Glicosiltransferasas/genética , Mesodermo/metabolismo , Ratones
6.
Elife ; 52016 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-27966429

RESUMEN

The signals that induce the organ of Corti and define its boundaries in the cochlea are poorly understood. We show that two Notch modifiers, Lfng and Mfng, are transiently expressed precisely at the neural boundary of the organ of Corti. Cre-Lox fate mapping shows this region gives rise to inner hair cells and their associated inner phalangeal cells. Mutation of Lfng and Mfng disrupts this boundary, producing unexpected duplications of inner hair cells and inner phalangeal cells. This phenotype is mimicked by other mouse mutants or pharmacological treatments that lower but not abolish Notch signaling. However, strong disruption of Notch signaling causes a very different result, generating many ectopic hair cells at the expense of inner phalangeal cells. Our results show that Notch signaling is finely calibrated in the cochlea to produce precisely tuned levels of signaling that first set the boundary of the organ of Corti and later regulate hair cell development.


Asunto(s)
Glicosiltransferasas/metabolismo , Órgano Espiral/embriología , Proteínas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Glucosiltransferasas , Glicosiltransferasas/genética , Ratones , Mutación , Proteínas/genética
7.
Microsc Microanal ; 22(3): 599-611, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27329311

RESUMEN

The quantity and quality of collagen fibrils in the extracellular matrix (ECM) have a pivotal role in dictating biological processes. Several collagen-binding proteins (CBPs) are known to modulate collagen deposition and fibril diameter. However, limited studies exist on alterations in the fibril ultrastructure by CBPs. In this study, we elucidate how the collagen receptor, discoidin domain receptor 1 (DDR1) regulates the collagen content and ultrastructure in the adventitia of DDR1 knock-out (KO) mice. DDR1 KO mice exhibit increased collagen deposition as observed using Masson's trichrome. Collagen ultrastructure was evaluated in situ using transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. Although the mean fibril diameter was not significantly different, DDR1 KO mice had a higher percentage of fibrils with larger diameter compared with their wild-type littermates. No significant differences were observed in the length of D-periods. In addition, collagen fibrils from DDR1 KO mice exhibited a small, but statistically significant, increase in the depth of the fibril D-periods. Consistent with these observations, a reduction in the depth of D-periods was observed in collagen fibrils reconstituted with recombinant DDR1-Fc. Our results elucidate how DDR1 modulates collagen fibril ultrastructure in vivo, which may have important consequences in the functional role(s) of the underlying ECM.


Asunto(s)
Colágeno/ultraestructura , Receptor con Dominio Discoidina 1/genética , Matriz Extracelular/genética , Animales , Receptor con Dominio Discoidina 1/metabolismo , Ratones , Ratones Noqueados , Unión Proteica
8.
Development ; 143(5): 822-30, 2016 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-26811377

RESUMEN

Vertebrate somitogenesis is regulated by a segmentation clock. Clock-linked genes exhibit cyclic expression, with a periodicity matching the rate of somite production. In mice, lunatic fringe (Lfng) expression oscillates, and LFNG protein contributes to periodic repression of Notch signaling. We hypothesized that rapid LFNG turnover could be regulated by protein processing and secretion. Here, we describe a novel Lfng allele (Lfng(RLFNG)), replacing the N-terminal sequences of LFNG, which allow for protein processing and secretion, with the N-terminus of radical fringe (a Golgi-resident protein). This allele is predicted to prevent protein secretion without altering the activity of LFNG, thus increasing the intracellular half-life of the protein. This allele causes dominant skeletal and somite abnormalities that are distinct from those seen in Lfng loss-of-function embryos. Expression of clock-linked genes is perturbed and mature Hes7 transcripts are stabilized in the presomitic mesoderm of mutant mice, suggesting that both transcriptional and post-transcriptional regulation of clock components are perturbed by RLFNG expression. Contrasting phenotypes in the segmentation clock and somite patterning of mutant mice suggest that LFNG protein may have context-dependent effects on Notch activity.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Glicosiltransferasas/fisiología , Proteínas/genética , Somitos/fisiología , Alelos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Tipificación del Cuerpo/genética , Femenino , Perfilación de la Expresión Génica , Genotipo , Glucosiltransferasas , Glicosiltransferasas/genética , Heterocigoto , Hibridación in Situ , Masculino , Mesodermo/metabolismo , Ratones , Mutación , Fenotipo , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Receptores Notch/metabolismo , Transducción de Señal
9.
Semin Cell Dev Biol ; 49: 68-75, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25483003

RESUMEN

The embryonic vertebrate body axis contains serially repeated elements, somites, which form sequentially by budding from a posterior tissue called the presomitic mesoderm (PSM). Somites are the embryonic precursors of the vertebrae, ribs and other adult structures. Many inherited human diseases are characterized by dysregulated somitogenesis, resulting in skeletal abnormalities that are evident at birth. Several of these conditions, including some cases of autosomal recessive familial spondylocostal dysostosis (SCDO), arise from mutations in the Notch signaling pathway, which has been demonstrated to be a key player in the regulation of somitogenesis. Here, we review the functional roles of the Notch pathway in vertebrate segmentation, focusing on its activities in a clock that times the formation of somites, as well as in the patterning and production of epithelial somites.


Asunto(s)
Receptores Notch/fisiología , Transducción de Señal , Somitos/embriología , Animales , Tipificación del Cuerpo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Humanos , Somitos/metabolismo
10.
Cancer Res ; 74(19): 5435-5448, 2014 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-25139440

RESUMEN

High-grade gliomas are characterized by exuberant vascularization, diffuse invasion, and significant chemoresistance, resulting in a recurrent phenotype that makes them impossible to eradicate in the long term. Targeting protumoral signals in the glioma microenvironment could have significant impact against tumor cells and the supporting niche that facilitates their growth. Fibulin-3 is a protein secreted by glioma cells, but absent in normal brain, that promotes tumor invasion and survival. We show here that fibulin-3 is a paracrine activator of Notch signaling in endothelial cells and promotes glioma angiogenesis. Fibulin-3 overexpression increased tumor VEGF levels, microvascular density, and vessel permeability, whereas fibulin-3 knockdown reduced vessel density in xenograft models of glioma. Fibulin-3 localization in human glioblastomas showed dense fiber-like condensations around tumor blood vessels, which were absent in normal brain, suggesting a remarkable association of this protein with tumor endothelium. At the cellular level, fibulin-3 enhanced endothelial cell motility and association to glioma cells, reduced endothelial cell sprouting, and increased formation of endothelial tubules in a VEGF-independent and Notch-dependent manner. Fibulin-3 increased ADAM10/17 activity in endothelial cells by inhibiting the metalloprotease inhibitor TIMP3; this resulted in increased Notch cleavage and increased expression of DLL4 independently of VEGF signaling. Inhibition of ADAM10/17 or knockdown of DLL4 reduced the proangiogenic effects of fibulin-3 in culture. Taken together, these results reveal a novel, proangiogenic role of fibulin-3 in gliomas, highlighting the relevance of this protein as an important molecular target in the tumor microenvironment.


Asunto(s)
Neoplasias Encefálicas/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glioma/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Neoplasias Encefálicas/irrigación sanguínea , Proteínas de Unión al Calcio , Línea Celular Tumoral , Modelos Animales de Enfermedad , Glioma/irrigación sanguínea , Humanos , Neovascularización Patológica , Ratas
11.
Int J Dev Biol ; 58(1): 65-70, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24860997

RESUMEN

Delta-like homologue 1 (Dlk1), an atypical Notch ligand, is known to have roles in growth and development, stem cell maintenance, and cancer. Evidence suggests that Dlk1 expression patterns are more complex than previously appreciated, with multiple isoforms expressed in various tissues in both the embryo and adult. However, the early embryonic expression of Dlk1 has not been well examined. Given that tissue specific Dlk1 knockouts have to date failed to recapitulate phenotypes associated with the conventional Dlk1 loss of function model, a better understanding of early Dlk1 expression is important. To address this question, we have examined Dlk1 expression during the early stages of mouse embryogenesis. Dlk1 expression was first detected at Theiler Stage 14 (TS14), and its expression pattern persisted in specific tissues through TS20. Further, we found that all known Dlk1 splice isoforms were expressed in early embryogenesis, with Dlk1-A and Dlk1-C/C2 isoforms being expressed at the highest levels. The broad co-expression of multiple Dlk1 isoforms corroborates recent work suggesting that Dlk1-mediated signaling may act through multiple DLK1 isoforms to balance differentiation.


Asunto(s)
Empalme Alternativo , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Esbozos de los Miembros/metabolismo , Mesodermo/metabolismo , Animales , Proteínas de Unión al Calcio , Células Cultivadas , Embrión de Mamíferos/citología , Femenino , Hibridación in Situ , Esbozos de los Miembros/embriología , Mesodermo/embriología , Ratones , Morfogénesis/fisiología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
12.
Dev Biol ; 388(2): 159-69, 2014 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-24560643

RESUMEN

The segmental structure of the axial skeleton is formed during somitogenesis. During this process, paired somites bud from the presomitic mesoderm (PSM), in a process regulated by a genetic clock called the segmentation clock. The Notch pathway and the Notch modulator Lunatic fringe (Lfng) play multiple roles during segmentation. Lfng oscillates in the posterior PSM as part of the segmentation clock, but is stably expressed in the anterior PSM during presomite patterning. We previously found that mice lacking overt oscillatory Lfng expression in the posterior PSM (Lfng(∆FCE)) exhibit abnormal anterior development but relatively normal posterior development. This suggests distinct requirements for segmentation clock activity during the formation of the anterior skeleton (primary body formation), compared to the posterior skeleton and tail (secondary body formation). To build on these findings, we created an allelic series that progressively lowers Lfng levels in the PSM. Interestingly, we find that further reduction of Lfng expression levels in the PSM does not increase disruption of anterior development. However tail development is increasingly compromised as Lfng levels are reduced, suggesting that primary body formation is more sensitive to Lfng dosage than is secondary body formation. Further, we find that while low levels of oscillatory Lfng in the posterior PSM are sufficient to support relatively normal posterior development, the period of the segmentation clock is increased when the amplitude of Lfng oscillations is low. These data support the hypothesis that there are differential requirements for oscillatory Lfng during primary and secondary body formation and that posterior development is less sensitive to overall Lfng levels. Further, they suggest that modulation of the Notch signaling by Lfng affects the clock period during development.


Asunto(s)
Desarrollo Óseo/genética , Dosificación de Gen , Glicosiltransferasas/genética , Somitos/crecimiento & desarrollo , Animales , Ratones , Ratones Transgénicos
13.
Dev Cell ; 24(5): 554-61, 2013 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-23484856

RESUMEN

Somites are embryonic precursors of the axial skeleton and skeletal muscles and establish the segmental vertebrate body plan. Somitogenesis is controlled in part by a segmentation clock that requires oscillatory expression of genes including Lunatic fringe (Lfng). Oscillatory genes must be tightly regulated at both the transcriptional and posttranscriptional levels for proper clock function. Here, we demonstrate that microRNA-mediated regulation of Lfng is essential for proper segmentation during chick somitogenesis. We find that mir-125a-5p targets evolutionarily conserved sequences in the Lfng 3' UTR and that preventing interactions between mir-125a-5p and Lfng transcripts in vivo causes abnormal segmentation and perturbs clock activity. This provides strong evidence that microRNAs function in the posttranscriptional regulation of oscillatory genes in the segmentation clock. Further, this demonstrates that the relatively subtle effects of microRNAs on target genes can have broad effects in developmental situations that have critical requirements for tight posttranscriptional regulation.


Asunto(s)
Regiones no Traducidas 3'/genética , Proteínas Aviares/genética , Relojes Biológicos/genética , Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica , Glicosiltransferasas/genética , MicroARNs/genética , Somitos/metabolismo , Animales , Embrión de Pollo , Hibridación in Situ , Mesodermo/citología , Mesodermo/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Somitos/citología
14.
Cancer Res ; 72(15): 3873-85, 2012 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-22665268

RESUMEN

Malignant gliomas are highly invasive and chemoresistant brain tumors with extremely poor prognosis. Targeting of the soluble factors that trigger invasion and resistance, therefore, could have a significant impact against the infiltrative glioma cells that are a major source of recurrence. Fibulin-3 is a matrix protein that is absent in normal brain but upregulated in gliomas and promotes tumor invasion by unknown mechanisms. Here, we show that fibulin-3 is a novel soluble activator of Notch signaling that antagonizes DLL3, an autocrine inhibitor or Notch, and promotes tumor cell survival and invasion in a Notch-dependent manner. Using a strategy for inducible knockdown, we found that controlled downregulation of fibulin-3 reduced Notch signaling and led to increased apoptosis, reduced self-renewal of glioblastoma-initiating cells, and impaired growth and dispersion of intracranial tumors. In addition, fibulin-3 expression correlated with expression levels of Notch-dependent genes and was a marker of Notch activation in patient-derived glioma samples. These findings underscore a major role for the tumor extracellular matrix in regulating glioma invasion and resistance to apoptosis via activation of the key Notch pathway. More importantly, this work describes a noncanonical, soluble activator of Notch in a cancer model and shows how Notch signaling can be reduced by targeting tumor-specific accessible molecules in the tumor microenvironment.


Asunto(s)
Neoplasias Encefálicas/patología , Proliferación Celular , Resistencia a Antineoplásicos/genética , Proteínas de la Matriz Extracelular/fisiología , Glioma/patología , Receptor Notch1/fisiología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/genética , Glioma/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Desnudos , Comunicación Paracrina/genética , Comunicación Paracrina/fisiología , ARN Interferente Pequeño/farmacología , Ratas , Receptor Notch1/genética , Receptor Notch1/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
15.
Biochim Biophys Acta ; 1812(1): 121-9, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20951801

RESUMEN

Notch signaling is essential for proper cardiac development. We recently identified missense variants in the NOTCH1 receptor in patients with diverse left ventricular outflow tract (LVOT) malformations (NOTCH1(G661S) and NOTCH1(A683T)) that reduce ligand-induced Notch signaling. Here, we examine the molecular mechanisms that contribute to reduced signaling and perturbed development. We find that NOTCH1(A683T) exhibits reduced S1 cleavage due to impaired trafficking through the endoplasmic reticulum (ER). This observation is consistent with improper localization of the variant receptor to the ER and decreased presentation at the cell surface. In contrast, the nearby mutation NOTCH1(G661S) exhibits reduced cell-surface presentation in the absence of overt folding or trafficking defects. To examine the implications of these variants in disease pathogenesis, we investigated their effect on epithelial-to-mesenchymal transition (EMT), a critical process for development of the outflow tract. We find that these LVOT-associated NOTCH1 alleles can contribute to defective EMT in endothelial cell lines through impaired induction of Snail and Hes family members. These data represent the first description of a molecular mechanism underlying NOTCH1 mutations in individuals with LVOT malformations, and have important implications regarding the functional contribution of these alleles to a complex set of developmental defects.


Asunto(s)
Músculo Liso , Mutación Missense , Receptor Notch1/genética , Transducción de Señal/genética , Actinas/metabolismo , Animales , Western Blotting , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Membrana Celular/metabolismo , Técnicas de Cocultivo , Retículo Endoplásmico/metabolismo , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Técnica del Anticuerpo Fluorescente , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Músculo Liso/química , Células 3T3 NIH , Ratas , Receptor Notch1/metabolismo , Proteínas Serrate-Jagged , Transducción de Señal/fisiología , Transfección , Obstrucción del Flujo Ventricular Externo/genética , Obstrucción del Flujo Ventricular Externo/fisiopatología
16.
Dev Dyn ; 238(7): 1803-12, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19479951

RESUMEN

Tight regulation of Notch pathway signaling is important in many aspects of embryonic development. Notch signaling can be modulated by expression of fringe genes, encoding glycosyltransferases that modify EGF repeats in the Notch receptor. Although Lunatic fringe (Lfng) has been shown to play important roles in vertebrate segmentation, comparatively little is known regarding the developmental functions of the other vertebrate fringe genes, Radical fringe (Rfng) and Manic fringe (Mfng). Here we report that Mfng expression is not required for embryonic development. Further, we find that despite significant overlap in expression patterns, we detect no obvious synergistic defects in mice in the absence of two, or all three, fringe genes during development of the axial skeleton, limbs, hindbrain, and cranial nerves.


Asunto(s)
Tipificación del Cuerpo/genética , Huesos/embriología , Desarrollo Embrionario/genética , Extremidades/embriología , Proteínas/fisiología , Rombencéfalo/embriología , Animales , Embrión de Mamíferos , Fertilidad/genética , Fertilidad/fisiología , Viabilidad Fetal/genética , Viabilidad Fetal/fisiología , Eliminación de Gen , Glucosiltransferasas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Familia de Multigenes/genética , Familia de Multigenes/fisiología , Proteínas/genética
17.
Hepatology ; 48(6): 1989-97, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19026002

RESUMEN

UNLABELLED: Alagille syndrome (AGS) is a heterogeneous developmental disorder associated with bile duct paucity and various organ anomalies. The syndrome is caused by mutations in JAG1, which encodes a ligand in the Notch signaling pathway, in the majority of cases and mutations in the NOTCH2 receptor gene in less than 1% of patients. Although a wide array of JAG1 mutations have been identified in the AGS population, these mutational variants have not accounted for the wide phenotypic variability observed in patients with this syndrome. The Fringe genes encode glycosyltransferases, which modify Notch and alter ligand-receptor affinity. In this study, we analyzed double heterozygous mouse models to examine the Fringe genes as potential modifiers of the Notch-mediated hepatic phenotype observed in AGS. We generated mice that were haploinsufficient for both Jag1 and one of three paralogous Fringe genes: Lunatic (Lfng), Radical (Rfng), and Manic (Mfng). Adult Jag1(+/-)Lfng(+/-) and Jag1(+/-)Rfng(+/-) mouse livers exhibited widespread bile duct proliferation beginning at 5 weeks of age and persisting up to 1 year. The Jag1(+/-)Mfng(+/-) livers showed a subtle, yet significant increase in bile duct numbers and bile duct to portal tract ratios. These abnormalities were not observed in the newborn period. Despite the portal tract expansion by bile ducts, fibrosis was not increased and epithelial to mesenchymal transition was not shown in the affected portal tracts. CONCLUSION: Mice heterozygous for mutations in Jag1 and the Fringe genes display striking bile duct proliferation, which is not apparent at birth. These findings suggest that the Fringe genes may regulate postnatal bile duct growth and remodeling, and serve as candidate modifiers of the hepatic phenotype in AGS.


Asunto(s)
Síndrome de Alagille/patología , Conductos Biliares/patología , Proteínas de Unión al Calcio/genética , Glicosiltransferasas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Hígado/patología , Proteínas de la Membrana/genética , Proteínas/genética , Síndrome de Alagille/genética , Síndrome de Alagille/metabolismo , Animales , Conductos Biliares/anomalías , Conductos Biliares/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proliferación Celular , Modelos Animales de Enfermedad , Glucosiltransferasas , Glicosiltransferasas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Hígado/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Fenotipo , Proteínas/metabolismo , Proteínas Serrate-Jagged , Transducción de Señal/fisiología
18.
Biochim Biophys Acta ; 1783(12): 2384-90, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18706457

RESUMEN

During vertebrate segmentation, oscillatory activation of Notch signaling is important in the clock that regulates the timing of somitogenesis. In mice, the cyclic activation of NOTCH1 requires the periodic expression of Lunatic fringe (Lfng). For LFNG to play a role in the segmentation clock, its cyclic transcription must be coupled with post-translational mechanisms that confer a short protein half-life. LFNG protein is cleaved and released into the extracellular space, and here we examine the hypothesis that this secretion contributes to a short LFNG intracellular half-life, facilitating rapid oscillations within the segmentation clock. We localize N-terminal protein sequences that control the secretory behavior of fringe proteins and find that LFNG processing is promoted by specific proprotein convertases including furin and SPC6. Mutations that alter LFNG processing increase its intracellular half-life without preventing its secretion. These mutations do not affect the specificity of LFNG function in the Notch pathway, thus regulation of protein half-life affects the duration of LFNG activity without altering its function. Finally, the embryonic expression pattern of Spc6 suggests a role in terminating LFNG activity during somite patterning. These results have important implications for the mechanisms that contribute to the tight control of Notch signaling during vertebrate segmentation.


Asunto(s)
Furina/metabolismo , Glicosiltransferasas/fisiología , Proproteína Convertasa 5/metabolismo , Procesamiento Proteico-Postraduccional , Somitos/fisiología , Fosfatasa Alcalina/metabolismo , Animales , Western Blotting , Tipificación del Cuerpo , Proteínas de Unión al Calcio/metabolismo , Cicloheximida/farmacología , Técnica del Anticuerpo Fluorescente , Furina/genética , Semivida , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Mutación/genética , Proproteína Convertasa 5/genética , Receptor Notch1/metabolismo , Proteínas Serrate-Jagged
19.
Hum Mol Genet ; 17(18): 2886-93, 2008 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-18593716

RESUMEN

Congenital aortic valve stenosis (AVS), coarctation of the aorta (COA) and hypoplastic left heart syndrome (HLHS) are congenital cardiovascular malformations that all involve the left ventricular outflow tract (LVOT). They are presumably caused by a similar developmental mechanism involving the developing endothelium. The exact etiology for most LVOT malformations is unknown, but a strong genetic component has been established. We demonstrate here that mutations in the gene NOTCH1, coding for a receptor in a developmentally important signaling pathway, are found across the spectrum of LVOT defects. We identify two specific mutations that reduce ligand (JAGGED1) induced NOTCH1 signaling. One of these mutations perturbs the S1 cleavage of the receptor in the Golgi. These findings suggest that the levels of NOTCH1 signaling are tightly regulated during cardiovascular development, and that relatively minor alterations may promote LVOT defects. These results also establish for the first time that AVS, COA and HLHS can share a common pathogenetic mechanism at the molecular level, explaining observations of these defects co-occurring within families.


Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Ventrículos Cardíacos/anomalías , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Mutación Missense , Receptor Notch1/genética , Transducción de Señal , Obstrucción del Flujo Ventricular Externo/genética , Obstrucción del Flujo Ventricular Externo/fisiopatología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Ventrículos Cardíacos/fisiopatología , Humanos , Proteína Jagged-1 , Ligandos , Masculino , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Receptor Notch1/química , Receptor Notch1/metabolismo , Alineación de Secuencia , Proteínas Serrate-Jagged , Obstrucción del Flujo Ventricular Externo/congénito , Población Blanca/genética
20.
Development ; 135(5): 899-908, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18234727

RESUMEN

The Notch pathway plays multiple roles during vertebrate somitogenesis, functioning in the segmentation clock and during rostral/caudal (R/C) somite patterning. Lunatic fringe (Lfng) encodes a glycosyltransferase that modulates Notch signaling, and its expression patterns suggest roles in both of these processes. To dissect the roles played by Lfng during somitogenesis, a novel allele was established that lacks cyclic Lfng expression within the segmentation clock, but that maintains expression during R/C somite patterning (Lfng(DeltaFCE1)). In the absence of oscillatory Lfng expression, Notch activation is ubiquitous in the PSM of Lfng(DeltaFCE1) embryos. Lfng(DeltaFCE1) mice exhibit severe segmentation phenotypes in the thoracic and lumbar skeleton. However, the sacral and tail vertebrae are only minimally affected in Lfng(DeltaFCE1) mice, suggesting that oscillatory Lfng expression and cyclic Notch activation are important in the segmentation of the thoracic and lumbar axial skeleton (primary body formation), but are largely dispensable for the development of sacral and tail vertebrae (secondary body formation). Furthermore, we find that the loss of cyclic Lfng has distinct effects on the expression of other clock genes during these two stages of development. Finally, we find that Lfng(DeltaFCE1) embryos undergo relatively normal R/C somite patterning, confirming that Lfng roles in the segmentation clock are distinct from its functions in somite patterning. These results suggest that the segmentation clock may employ varied regulatory mechanisms during distinct stages of anterior/posterior axis development, and uncover previously unappreciated connections between the segmentation clock, and the processes of primary and secondary body formation.


Asunto(s)
Tipificación del Cuerpo/fisiología , Desarrollo Óseo , Huesos/embriología , Glicosiltransferasas/genética , Animales , Animales Recién Nacidos , Huesos/anomalías , ADN/genética , Regulación del Desarrollo de la Expresión Génica , Genotipo , Glicosiltransferasas/deficiencia , Hibridación in Situ , Ratones , Oscilometría , Receptores Notch/genética , Receptores Notch/fisiología , Eliminación de Secuencia , Columna Vertebral/anomalías , Saco Vitelino/fisiología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...