RESUMEN
Neurodevelopmental disorder with microcephaly, hypotonia and variable brain anomalies (NMIHBA) is an autosomal recessive neurodevelopmental and neurodegenerative disorder characterized by global developmental delay and severe intellectual disability. Microcephaly, progressive cortical atrophy, cerebellar hypoplasia and delayed myelination are neurological hallmarks in affected individuals. NMIHBA is caused by biallelic variants in PRUNE1 encoding prune exopolyphosphatase 1. We provide in-depth clinical description of two affected siblings harboring compound heterozygous variant alleles, c.383G > A (p.Arg128Gln), c.520G > T (p.Gly174*) in PRUNE1. To gain insights into disease biology, we biochemically characterized missense variants within the conserved N-terminal aspartic acid-histidine-histidine (DHH) motif and provide evidence that they result in the destabilization of protein structure and/or loss of exopolyphosphatase activity. Genetic ablation of Prune1 results in midgestational lethality in mice, associated with perturbations to embryonic growth and vascular development. Our findings suggest that NMIHBA results from hypomorphic variant alleles in humans and underscore the potential key role of PRUNE1 exopolyphoshatase activity in neurodevelopment.
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Ácido Anhídrido Hidrolasas/deficiencia , Discapacidad Intelectual/patología , Microcefalia/patología , Hipotonía Muscular/patología , Mutación , Trastornos del Neurodesarrollo/patología , Monoéster Fosfórico Hidrolasas/genética , Alelos , Animales , Preescolar , Femenino , Humanos , Lactante , Discapacidad Intelectual/etiología , Discapacidad Intelectual/metabolismo , Masculino , Ratones , Microcefalia/etiología , Microcefalia/metabolismo , Hipotonía Muscular/etiología , Hipotonía Muscular/metabolismo , Trastornos del Neurodesarrollo/etiología , Trastornos del Neurodesarrollo/metabolismo , Linaje , FenotipoRESUMEN
Angiogenesis is largely driven by motile endothelial tip-cells capable of invading avascular tissue domains and enabling new vessel formation. Highly responsive to Vascular Endothelial Growth-Factor-A (VEGFA), endothelial tip-cells also suppress angiogenic sprouting in adjacent stalk cells, and thus have been a primary therapeutic focus in addressing neovascular pathologies. Surprisingly, however, there remains a paucity of specific endothelial tip-cell markers. Here, we employ transcriptional profiling and a lacZ reporter allele to identify Kcne3 as an early and selective endothelial tip-cell marker in multiple angiogenic contexts. In development, Kcne3 expression initiates during early phases of angiogenesis (E9) and remains specific to endothelial tip-cells, often adjacent to regions expressing VEGFA. Consistently, Kcne3 activation is highly responsive to exogenous VEGFA but maintains tip-cell specificity throughout normal retinal angiogenesis. We also demonstrate endothelial tip-cell selectivity of Kcne3 in several injury and tumor models. Together, our data show that Kcne3 is a unique marker of sprouting angiogenic tip-cells and offers new opportunities for investigating and targeting this cell type.
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Células Endoteliales/fisiología , Neovascularización Patológica/genética , Neovascularización Fisiológica/genética , Canales de Potasio con Entrada de Voltaje/genética , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Animales Recién Nacidos , Diferenciación Celular/genética , Células Cultivadas , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Embrión de Mamíferos , Células Endoteliales/patología , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfogénesis/genética , Neovascularización Patológica/metabolismo , Embarazo , Retina/metabolismo , Retina/patología , Vasos Retinianos/metabolismo , Vasos Retinianos/patologíaRESUMEN
The Adisintegrin and metalloprotease domain-containing (ADAM) family of proteins is involved in cell adhesion, migration, proteolysis, and signaling. Many ADAMs are required for reproduction; however, the role of Adam6 has remained largely unknown. In the course of humanizing the mouse immunoglobulin heavy chain (IgH) locus, we generated Adam6-deficient mice that demonstrate severe subfertility. We decided to elucidate the role of ADAM6 in fertility and explore the underlying mechanisms. Despite normal sperm development and motility, Adam6-deficient mice display diminished male fertility, have abnormal sperm adhesion, and most importantly cannot transition from uterus to oviduct. To test whether ADAM6 is required for sperm's binding to extracellular matrix (ECM) components, we used a panel of ECM components and showed that unlike normal sperm, Adam6-deficient sperm cannot bind fibronectin, laminin, and tenascin. Reintroduction of Adam6 into these deficient mice repaired sperm interaction with ECM, restored male fertility, and corrected the sperm transport deficit. Together, our data suggest that ADAM6, either alone or in complex with other proteins, aids sperm transport through the female reproductive tract by providing a temporary site of attachment of sperm to ECM components prior to ascent into the oviduct.
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Proteínas ADAM/metabolismo , Infertilidad Masculina/genética , Motilidad Espermática/fisiología , Espermatozoides/fisiología , Proteínas ADAM/genética , Animales , Femenino , Masculino , Ratones , Ratones Noqueados , Oviductos , Motilidad Espermática/genéticaRESUMEN
Trafficking of tissue dendritic cells (DCs) via lymph is critical for the generation of cellular immune responses in draining lymph nodes (LNs). In the current study we found that DCs docked to the basolateral surface of lymphatic vessels and transited to the lumen through hyaluronan-mediated interactions with the lymph-specific endothelial receptor LYVE-1, in dynamic transmigratory-cup-like structures. Furthermore, we show that targeted deletion of the gene Lyve1, antibody blockade or depletion of the DC hyaluronan coat not only delayed lymphatic trafficking of dermal DCs but also blunted their capacity to prime CD8+ T cell responses in skin-draining LNs. Our findings uncovered a previously unknown function for LYVE-1 and show that transit through the lymphatic network is initiated by the recognition of leukocyte-derived hyaluronan.
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Células Dendríticas/inmunología , Células Endoteliales/metabolismo , Glicoproteínas/genética , Ácido Hialurónico/metabolismo , Vasos Linfáticos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Movimiento Celular/inmunología , Células Dendríticas/metabolismo , Endotelio Linfático/citología , Endotelio Linfático/metabolismo , Citometría de Flujo , Glicoproteínas/metabolismo , Humanos , Inmunidad Celular/inmunología , Ganglios Linfáticos/inmunología , Proteínas de Transporte de Membrana , Ratones , Ratones Noqueados , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunologíaRESUMEN
Renal vascular development is a coordinated process that requires ordered endothelial cell proliferation, migration, intercellular adhesion, and morphogenesis. In recent decades, studies have defined the pivotal role of endothelial receptor tyrosine kinases (RPTKs) in the development and maintenance of renal vasculature. However, the expression and the role of receptor tyrosine phosphatases (RPTPs) in renal endothelium are poorly understood, though coupled and counterbalancing roles of RPTKs and RPTPs are well defined in other systems. In this study, we evaluated the promoter activity and immunolocalization of two endothelial RPTPs, VE-PTP and PTPµ, in developing and adult renal vasculature using the heterozygous LacZ knock-in mice and specific antibodies. In adult kidneys, both VE-PTP and PTPµ were expressed in the endothelium of arterial, glomerular, and medullary vessels, while their expression was highly limited in peritubular capillaries and venous endothelium. VE-PTP and PTPµ promoter activity was also observed in medullary tubular segments in adult kidneys. In embryonic (E12.5, E13.5, E15.5, E17.5) and postnatal (P0, P3, P7) kidneys, these RPTPs were expressed in ingrowing renal arteries, developing glomerular microvasculature (as early as the S-shaped stage), and medullary vessels. Their expression became more evident as the vasculatures matured. Peritubular capillary expression of VE-PTP was also noted in embryonic and postnatal kidneys. Compared to VE-PTP, PTPµ immunoreactivity was relatively limited in embryonic and neonatal renal vasculature and evident immunoreactivity was observed from the P3 stage. These findings indicate 1) VE-PTP and PTPµ are expressed in endothelium of arterial, glomerular, and medullary renal vasculature, 2) their expression increases as renal vascular development proceeds, suggesting that these RPTPs play a role in maturation and maintenance of these vasculatures, and 3) peritubular capillary VE-PTP expression is down-regulated in adult kidneys, suggesting a role of VE-PTP in the development of peritubular capillaries.
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Endotelio Vascular/metabolismo , Riñón/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Ratones , Fosforilación/genética , Regiones Promotoras Genéticas/genética , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismoRESUMEN
BACKGROUND: The Eph family of receptor tyrosine kinases plays important roles in neural development. Previous studies have implicated Eph receptors and their ligands, the ephrins, in neuronal migration, axon bundling and guidance to specific targets, dendritic spine formation and neural plasticity. However, specific contributions of EphA5 and EphA6 receptors to the regulation of neuronal cell morphology have not been well studied. RESULTS: Here we show that deletion of EphA5 and EphA6 results in abnormal Golgi staining patterns of cells in the brain, and abnormal spine morphology. CONCLUSION: These observations suggest novel functions of these Eph receptors in the regulation of neuronal and spine structure in brain development and function.
RESUMEN
Secreted frizzled-related protein 4 (SFRP4) is an extracellular regulator of the wingless-type mouse mammary tumor virus integration site family (WNT) pathway. SFRP4 has been implicated in adipocyte dysfunction, obesity, insulin resistance, and impaired insulin secretion in patients with type 2 diabetes. However, the exact role of SFRP4 in regulating whole-body metabolism and glucose homeostasis is unknown. We show here that male Sfrp4(-/-) mice have increased spine length and gain more weight when fed a high-fat diet. The body composition and body mass per spine length of diet-induced obese Sfrp4(-/-) mice is similar to wild-type littermates, suggesting that the increase in body weight can be accounted for by their longer body size. The diet-induced obese Sfrp4(-/-) mice have reduced energy expenditure, food intake, and bone mineral density. Sfrp4(-/-) mice have normal glucose and insulin tolerance and ß-cell mass. Diet-induced obese Sfrp4(-/-) and control mice show similar impairments of glucose tolerance and a 5-fold compensatory expansion of their ß-cell mass. In summary, our data suggest that loss of SFRP4 alters body length and bone mineral density as well as energy expenditure and food intake. However, SFRP4 does not control glucose homeostasis and ß-cell mass in mice.
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Tamaño Corporal/genética , Densidad Ósea/genética , Dieta Alta en Grasa , Ingestión de Alimentos/genética , Metabolismo Energético/genética , Células Secretoras de Insulina/metabolismo , Obesidad , Proteínas Proto-Oncogénicas/genética , Animales , Glucemia/metabolismo , Composición Corporal/genética , Conducta Alimentaria , Técnicas de Sustitución del Gen , Prueba de Tolerancia a la Glucosa , Células HEK293 , Homeostasis/genética , Humanos , Insulina/metabolismo , Masculino , Ratones , Ratones Noqueados , Vía de Señalización Wnt , Microtomografía por Rayos XRESUMEN
G protein-coupled receptor 17 (GPR17) was recently reported to be a Foxo1 target in agouti-related peptide (AGRP) neurons. Intracerebroventricular injection of GPR17 agonists induced food intake, whereas administration of an antagonist to the receptor reduced feeding. These data lead to the conclusion that pharmacological modulation of GPR17 has therapeutic potential to treat obesity. Here we report that mice deficient in Gpr17 (Gpr17(-/-)) have similar food intake and body weight compared with their wild-type littermates. Gpr17(-/-) mice have normal hypothalamic Agrp mRNA expression, AGRP plasma levels, and metabolic rate. GPR17 deficiency in mice did not affect glucose homeostasis or prevent fat-induced insulin resistance. These data do not support a role for GPR17 in the control of food intake, body weight, or glycemic control.
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Ingestión de Alimentos/genética , Glucosa/metabolismo , Proteínas del Tejido Nervioso/genética , Receptores Acoplados a Proteínas G/genética , Proteína Relacionada con Agouti/metabolismo , Análisis de Varianza , Animales , Secuencia de Bases , Composición Corporal/efectos de los fármacos , Metabolismo Energético/genética , Metabolismo Energético/fisiología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Neuronas/metabolismo , Análisis de Secuencia de ARN , Factores de Tiempo , Microtomografía por Rayos XRESUMEN
Glaucoma is a leading cause of blindness, afflicting more than 60 million people worldwide. Increased intraocular pressure (IOP) due to impaired aqueous humor drainage is a major risk factor for the development of glaucoma. Here, we demonstrated that genetic disruption of the angiopoietin/TIE2 (ANGPT/TIE2) signaling pathway results in high IOP, buphthalmos, and classic features of glaucoma, including retinal ganglion degeneration and vision loss. Eyes from mice with induced deletion of Angpt1 and Angpt2 (A1A2Flox(WB) mice) lacked drainage pathways in the corneal limbus, including Schlemm's canal and lymphatic capillaries, which share expression of the PROX1, VEGFR3, and FOXC family of transcription factors. VEGFR3 and FOXCs have been linked to lymphatic disorders in patients, and FOXC1 has been linked to glaucoma. In contrast to blood endothelium, in which ANGPT2 is an antagonist of ANGPT1, we have shown that both ligands cooperate to regulate TIE2 in the lymphatic network of the eye. While A1A2Flox(WB) mice developed high IOP and glaucoma, expression of ANGPT1 or ANGPT2 alone was sufficient for ocular drainage. Furthermore, we demonstrated that loss of FOXC2 from lymphatics results in TIE2 downregulation, suggesting a mechanism for ocular defects in patients with FOXC mutations. These data reveal a pathogenetic and molecular basis for glaucoma and demonstrate the importance of angiopoietin ligand cooperation in the lymphatic endothelium.
Asunto(s)
Angiopoyetina 1/genética , Glaucoma/patología , Hipertensión Ocular/patología , Receptor TIE-2/genética , Angiopoyetina 2/genética , Animales , Humor Acuoso , Separación Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/metabolismo , Presión Intraocular , Ligandos , Sistema Linfático/patología , Ratones , Ratones Noqueados , Mutación , Malla Trabecular/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1(-/-) mice lack any obvious limb or skeletal defects, Sost(-/-) mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost(-/-); Sostdc1(-/-) mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost(-/-) and Sost(-/-); Sostdc1(-/-) mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling.
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Proteínas Morfogenéticas Óseas/metabolismo , Ectodermo/embriología , Extremidades/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Glicoproteínas/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vía de Señalización Wnt/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Morfogenéticas Óseas/genética , Biología Computacional , Ectodermo/metabolismo , Evolución Molecular , Glicoproteínas/genética , Proteínas Hedgehog/metabolismo , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Noqueados , Análisis por Micromatrices , Factor de Transcripción SOX9/metabolismo , Proteína Gli3 con Dedos de ZincRESUMEN
Optical projection tomography (OPT) is an imaging modality that has, in the last decade, answered numerous biological questions owing to its ability to view gene expression in 3 dimensions (3D) at high resolution for samples up to several cm(3). This has increased demand for a cabinet OPT system, especially for mouse embryo phenotyping, for which OPT was primarily designed for. The Medical Research Council (MRC) Technology group (UK) released a commercial OPT system, constructed by Skyscan, called the Bioptonics OPT 3001 scanner that was installed in a limited number of locations. The Bioptonics system has been discontinued and currently there is no commercial OPT system available. Therefore, a few research institutions have built their own OPT system, choosing parts and a design specific to their biological applications. Some of these custom built OPT systems are preferred over the commercial Bioptonics system, as they provide improved performance based on stable translation and rotation stages and up to date CCD cameras coupled with objective lenses of high numerical aperture, increasing the resolution of the images. Here, we present a detailed description of a custom built OPT system that is robust and easy to build and install. Included is a hardware parts list, instructions for assembly, a description of the acquisition software and a free download site, and methods for calibration. The described OPT system can acquire a full 3D data set in 10 minutes at 6.7 micron isotropic resolution. The presented guide will hopefully increase adoption of OPT throughout the research community, for the OPT system described can be implemented by personnel with minimal expertise in optics or engineering who have access to a machine shop.
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Tomografía Óptica/instrumentación , Algoritmos , Animales , Encéfalo/embriología , Diseño de Equipo , Ratones , Fenómenos Ópticos , Programas InformáticosRESUMEN
Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene's transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon-intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN's artificial intron opens up engineering modalities for the generation of multifunctional alleles.
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Alelos , Silenciador del Gen , Ingeniería Genética/métodos , Mutagénesis Insercional/métodos , Inversión de Secuencia/genética , ADN Nucleotidiltransferasas/metabolismoRESUMEN
The vasculature of the CNS is structurally and functionally distinct from that of other organ systems and is particularly prone to developmental abnormalities and hemorrhage. Although other embryonic tissues undergo primary vascularization, the developing nervous system is unique in that it is secondarily vascularized by sprouting angiogenesis from a surrounding perineural plexus. This sprouting angiogenesis requires the TGF-ß and Wnt pathways because ablation of these pathways results in aberrant sprouting and hemorrhage. We have genetically deleted Gpr124, a member of the large family of long N-terminal group B G protein-coupled receptors, few members of which have identified ligands or well-defined biologic functions in mammals. We show that, in the developing CNS, Gpr124 is specifically expressed in the vasculature and is absolutely required for proper angiogenic sprouting into the developing neural tube. Embryos lacking Gpr124 exhibit vascular defects characterized by delayed vascular penetration, formation of pathological glomeruloid tufts within the CNS, and hemorrhage. In addition, they display defects in palate and lung development, two processes in which TGF-ß and/or Wnt pathways also play important roles. We also show that TGF-ß stimulates Gpr124 expression, and ablation of Gpr124 results in perturbed TGF-ß pathway activation, suggesting roles for Gpr124 in modulating TGF-ß signaling. These results represent a unique function attributed to a long N-terminal group B-type G protein-coupled receptor in a mammalian system.
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Sistema Nervioso Central/irrigación sanguínea , Sistema Nervioso Central/embriología , Neovascularización Fisiológica/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Embrión de Mamíferos , Ingeniería Genética , Técnicas Histológicas , Inmunohistoquímica , Hibridación in Situ , Pulmón/embriología , Pulmón/metabolismo , Ratones , Análisis por Micromatrices , Hueso Paladar/embriología , Hueso Paladar/metabolismo , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Wnt/metabolismoRESUMEN
Obsessive-compulsive disorder (OCD) is a common psychiatric disorder defined by the presence of obsessive thoughts and repetitive compulsive actions, and it often encompasses anxiety and depressive symptoms. Recently, the corticostriatal circuitry has been implicated in the pathogenesis of OCD. However, the etiology, pathophysiology and molecular basis of OCD remain unknown. Several studies indicate that the pathogenesis of OCD has a genetic component. Here we demonstrate that loss of a neuron-specific transmembrane protein, SLIT and NTRK-like protein-5 (Slitrk5), leads to OCD-like behaviors in mice, which manifests as excessive self-grooming and increased anxiety-like behaviors, and is alleviated by the selective serotonin reuptake inhibitor fluoxetine. Slitrk5(-/-) mice show selective overactivation of the orbitofrontal cortex, abnormalities in striatal anatomy and cell morphology and alterations in glutamate receptor composition, which contribute to deficient corticostriatal neurotransmission. Thus, our studies identify Slitrk5 as an essential molecule at corticostriatal synapses and provide a new mouse model of OCD-like behaviors.
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Conducta Animal , Proteínas de la Membrana/deficiencia , Neostriado/fisiopatología , Proteínas del Tejido Nervioso/deficiencia , Trastorno Obsesivo Compulsivo/diagnóstico , Animales , Conducta Compulsiva/genética , Aseo Animal , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Sinapsis , Transmisión SinápticaRESUMEN
Inhibiting angiogenesis has become an effective approach for treating cancer and other diseases. However, our understanding of signaling pathways in tumor angiogenesis has been limited by the embryonic lethality of many gene knockouts. To overcome this limitation, we used the plasticity of embryonic stem (ES) cells to develop a unique approach to study tumor angiogenesis. Murine ES cells can be readily manipulated genetically; in addition, ES cells implanted subcutaneously in mice develop into tumors that contain a variety of cell types (teratomas). We show that ES cells differentiate into bona fide endothelial cells within the teratoma, and that these ES-derived endothelial cells form part of the functional tumor vasculature. Using this powerful and flexible system, the Angiopoietin/Tie2 system is shown to have a key role in the regulation of tumor vessel size. Endothelial differentiation in the ES teratoma model allows gene-targeting methods to be used in the study of tumor angiogenesis.
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Células Madre Embrionarias/enzimología , Células Madre Embrionarias/patología , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/enzimología , Neovascularización Patológica , Proteínas Tirosina Quinasas Receptoras/fisiología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/fisiología , Angiopoyetinas/antagonistas & inhibidores , Animales , Diferenciación Celular , Línea Celular , Modelos Animales de Enfermedad , Células Endoteliales/enzimología , Células Endoteliales/patología , Ratones , Ratones SCID , Neoplasias Experimentales/etiología , Receptor TIE-2 , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/deficiencia , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Teratoma/irrigación sanguínea , Teratoma/enzimología , Teratoma/etiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Blood vessel formation is controlled by the balance between pro- and antiangiogenic pathways. Although much is known about the factors that drive sprouting of neovessels, the factors that stabilize and pattern neovessels are undefined. The expression of angiomodulin (AGM), a vascular endothelial growth factor (VEGF)-A binding protein, was increased in the vasculature of several human tumors as compared to normal tissue, raising the hypothesis that AGM may modulate VEGF-A-dependent vascular patterning. To elucidate the expression pattern of AGM, we developed an AGM knockin reporter mouse (AGM(lacZ/+)), with which we demonstrate that AGM is predominantly expressed in the vasculature of developing embryos and adult organs. During physiological and pathological angiogenesis, AGM is upregulated in the angiogenic vasculature. Using the zebrafish model, we found that AGM is restricted to developing vasculature by 17 to 22 hours postfertilization. Blockade of AGM activity with morpholino oligomers results in prominent angiogenesis defects in vascular sprouting and remodeling. Concurrent knockdown of both AGM and VEGF-A results in synergistic angiogenesis defects. When VEGF-A is overexpressed, the compensatory induction of the VEGF-A receptor, VEGFR2/flk-1, is blocked by the simultaneous injection of AGM morpholino oligomers. These results demonstrate that the vascular-specific marker AGM modulates vascular remodeling in part by temporizing the proangiogenic effects of VEGF-A.
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Proteínas de Neoplasias/metabolismo , Neoplasias/irrigación sanguínea , Neovascularización Patológica/metabolismo , Neovascularización Fisiológica , Neovascularización Retiniana/metabolismo , Piel/irrigación sanguínea , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genotipo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfolinas/metabolismo , Proteínas de Neoplasias/genética , Neovascularización Patológica/genética , Neovascularización Patológica/fisiopatología , Neovascularización Fisiológica/genética , Oligonucleótidos Antisentido/metabolismo , Fenotipo , Regiones Promotoras Genéticas , Neovascularización Retiniana/genética , Neovascularización Retiniana/fisiopatología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas , Pez Cebra/embriología , Proteínas de Pez Cebra/genéticaRESUMEN
The EphA5 receptor tyrosine kinase plays key roles in axon guidance during development. However, the presence of EphA5 protein in the nervous system has not been fully characterized. To examine EphA5 localization better, mutant mice, in which the EphA5 cytoplasmic domain was replaced with beta-galactosidase, were analyzed for both temporal and regional changes in the distribution of EphA5 protein in the developing and adult nervous system. During embryonic development, high levels of EphA5 protein were found in the retina, olfactory bulb, cerebral neocortex, hippocampus, pretectum, tectum, cranial nerve nuclei, and spinal cord. Variations in intensity were observed as development proceeded. Staining of pretectal nuclei, tectal nuclei, and other areas of the mesencephalon became more diffuse after maturity, whereas the cerebral neocortex gained more robust intensity. In the adult, receptor protein continued to be detected in many areas including the olfactory nuclei, neocortex, piriform cortex, induseum griseum, hippocampus, thalamus, amygdala, hypothalamus, and septum. In addition, EphA5 protein was found in the claustrum, stria terminalis, barrel cortex, and striatal patches, and along discrete axon tracts within the corpus callosum of the adult. We conclude that EphA5 function is not limited to the developing mouse brain and may play a role in synaptic plasticity in the adult.
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Encéfalo/metabolismo , Receptor EphA5/metabolismo , Médula Espinal/metabolismo , Animales , Western Blotting , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Galactósidos , Hibridación in Situ , Indoles , Ratones , Ratones Transgénicos , Receptor EphA5/genética , Retina/embriología , Retina/crecimiento & desarrollo , Retina/metabolismo , Médula Espinal/embriología , Médula Espinal/crecimiento & desarrollo , beta-Galactosidasa/genéticaRESUMEN
Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.
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Linfocitos B/inmunología , Células Dendríticas/inmunología , Eliminación de Gen , Receptor Notch1/genética , Linfocitos T/inmunología , Timo/citología , Animales , Linaje de la Célula , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Receptor Notch1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Expression of the hyaluronan receptor LYVE-1 is one of few available criteria used to discriminate lymphatic vessels from blood vessels. Until now, endothelial LYVE-1 expression was reported to be restricted to lymphatic vessels and to lymph node, liver, and spleen sinuses. Here, we provide the first evidence that LYVE-1 is expressed on blood vessels of the yolk sac during mouse embryogenesis. LYVE-1 is ubiquitously expressed in the yolk sac capillary plexus at E9.5, then becomes progressively down-regulated on arterial endothelium during vascular remodelling. LYVE-1 is also expressed on intra-embryonic arterial and venous endothelium at early embryonic stages and on endothelial cells of the lung and endocardium throughout embryogenesis. These findings have important implications for the use of LYVE-1 as a specific marker of the lymphatic vasculature during embryogenesis and neo-lymphangiogenesis. Our data are also the first demonstration, to our knowledge, that the mouse yolk sac is devoid of lymphatic vessels.
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Vasos Sanguíneos/metabolismo , Glicoproteínas/biosíntesis , Vasos Linfáticos/metabolismo , Animales , Vasos Sanguíneos/embriología , Endocardio/citología , Endocardio/embriología , Endocardio/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Inmunohistoquímica , Pulmón/citología , Pulmón/embriología , Pulmón/metabolismo , Vasos Linfáticos/embriología , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos C57BL , Microscopía ConfocalRESUMEN
Colon cancer stem cells are believed to originate from a rare population of putative CD133+ intestinal stem cells. Recent publications suggest that a small subset of colon cancer cells expresses CD133, and that only these CD133+ cancer cells are capable of tumor initiation. However, the precise contribution of CD133+ tumor-initiating cells in mediating colon cancer metastasis remains unknown. Therefore, to temporally and spatially track the expression of CD133 in adult mice and during tumorigenesis, we generated a knockin lacZ reporter mouse (CD133lacZ/+), in which the expression of lacZ is driven by the endogenous CD133 promoters. Using this model and immunostaining, we discovered that CD133 expression in colon is not restricted to stem cells; on the contrary, CD133 is ubiquitously expressed on differentiated colonic epithelium in both adult mice and humans. Using Il10-/-CD133lacZ mice, in which chronic inflammation in colon leads to adenocarcinomas, we demonstrated that CD133 is expressed on a full gamut of colonic tumor cells, which express epithelial cell adhesion molecule (EpCAM). Similarly, CD133 is widely expressed by human primary colon cancer epithelial cells, whereas the CD133- population is composed mostly of stromal and inflammatory cells. Conversely, CD133 expression does not identify the entire population of epithelial and tumor-initiating cells in human metastatic colon cancer. Indeed, both CD133+ and CD133- metastatic tumor subpopulations formed colonospheres in in vitro cultures and were capable of long-term tumorigenesis in a NOD/SCID serial xenotransplantation model. Moreover, metastatic CD133- cells form more aggressive tumors and express typical phenotypic markers of cancer-initiating cells, including CD44 (CD44+CD24-), whereas the CD133+ fraction is composed of CD44lowCD24+ cells. Collectively, our data suggest that CD133 expression is not restricted to intestinal stem or cancer-initiating cells, and during the metastatic transition, CD133+ tumor cells might give rise to the more aggressive CD133(- )subset, which is also capable of tumor initiation in NOD/SCID mice.