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1.
Development ; 143(20): 3785-3795, 2016 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-27621059

RESUMEN

Vascular endothelial growth factor C (Vegfc) activates its receptor, Flt4, to induce lymphatic development. However, the signals that act downstream of Flt4 in this context in vivo remain unclear. To understand Flt4 signaling better, we generated zebrafish bearing a deletion in the Flt4 cytoplasmic domain that eliminates tyrosines Y1226 and 1227. Embryos bearing this deletion failed to initiate sprouting or differentiation of trunk lymphatic vessels and did not form a thoracic duct. Deletion of Y1226/7 prevented ERK phosphorylation in lymphatic progenitors, and ERK inhibition blocked trunk lymphatic sprouting and differentiation. Conversely, endothelial autonomous ERK activation rescued lymphatic sprouting and differentiation in flt4 mutants. Interestingly, embryos bearing the Y1226/7 deletion formed a functional facial lymphatic network enabling them to develop normally to adulthood. By contrast, flt4 null larvae displayed hypoplastic facial lymphatics and severe lymphedema. Thus, facial lymphatic vessels appear to be the first functional lymphatic network in the zebrafish, whereas the thoracic duct is initially dispensable for lymphatic function. Moreover, distinct signaling pathways downstream of Flt4 govern lymphatic morphogenesis and differentiation in different anatomical locations.


Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Vasos Linfáticos/citología , Vasos Linfáticos/metabolismo , Factor C de Crecimiento Endotelial Vascular/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Genotipo , Hibridación in Situ , Vasos Linfáticos/embriología , Mutación/genética , Fosforilación/genética , Fosforilación/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Factor C de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas de Pez Cebra/genética
2.
Development ; 140(7): 1497-506, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23462469

RESUMEN

Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfc(um18) could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis.


Asunto(s)
Vasos Sanguíneos/embriología , Sistema Linfático/embriología , Factor C de Crecimiento Endotelial Vascular/genética , Factor C de Crecimiento Endotelial Vascular/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiología , Alelos , Animales , Animales Modificados Genéticamente , Comunicación Autocrina/genética , Comunicación Autocrina/fisiología , Vasos Sanguíneos/crecimiento & desarrollo , Movimiento Celular/genética , Movimiento Celular/fisiología , Codón sin Sentido/fisiología , Embrión no Mamífero , Femenino , Sistema Linfático/crecimiento & desarrollo , Ratones , Ratones Noqueados , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , Comunicación Paracrina/genética , Comunicación Paracrina/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Transducción de Señal/genética , Pez Cebra/embriología , Pez Cebra/genética
4.
J Exp Med ; 219(6)2022 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-35510953

RESUMEN

Genetic alterations in RET lead to activation of ERK and AKT signaling and are associated with hereditary and sporadic thyroid cancer and lung cancer. Highly selective RET inhibitors have recently entered clinical use after demonstrating efficacy in treating patients with diverse tumor types harboring RET gene rearrangements or activating mutations. In order to understand resistance mechanisms arising after treatment with RET inhibitors, we performed a comprehensive molecular and genomic analysis of a patient with RET-rearranged thyroid cancer. Using a combination of drug screening and proteomic and biochemical profiling, we identified an adaptive resistance to RET inhibitors that reactivates ERK signaling within hours of drug exposure. We found that activation of FGFR signaling is a mechanism of adaptive resistance to RET inhibitors that activates ERK signaling. Combined inhibition of FGFR and RET prevented the development of adaptive resistance to RET inhibitors, reduced cell viability, and decreased tumor growth in cellular and animal models of CCDC6-RET-rearranged thyroid cancer.


Asunto(s)
Neoplasias Pulmonares , Neoplasias de la Tiroides , Animales , Proteínas del Citoesqueleto/genética , Humanos , Neoplasias Pulmonares/patología , Proteómica , Proteínas Proto-Oncogénicas c-ret/genética , Receptores de Factores de Crecimiento de Fibroblastos , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/genética
5.
Elife ; 62017 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-28350298

RESUMEN

Thyroid cancer is common, yet the sequence of alterations that promote tumor formation are incompletely understood. Here, we describe a novel model of thyroid carcinoma in zebrafish that reveals temporal changes due to BRAFV600E. Through the use of real-time in vivo imaging, we observe disruption in thyroid follicle structure that occurs early in thyroid development. Combinatorial treatment using BRAF and MEK inhibitors reversed the developmental effects induced by BRAFV600E. Adult zebrafish expressing BRAFV600E in thyrocytes developed invasive carcinoma. We identified a gene expression signature from zebrafish thyroid cancer that is predictive of disease-free survival in patients with papillary thyroid cancer. Gene expression studies nominated TWIST2 as a key effector downstream of BRAF. Using CRISPR/Cas9 to genetically inactivate a TWIST2 orthologue, we suppressed the effects of BRAFV600E and restored thyroid morphology and hormone synthesis. These data suggest that expression of TWIST2 plays a role in an early step of BRAFV600E-mediated transformation.


Asunto(s)
Morfogénesis , Proteínas Proto-Oncogénicas B-raf/metabolismo , Glándula Tiroides/embriología , Glándula Tiroides/fisiología , Hormonas Tiroideas/metabolismo , Neoplasias de la Tiroides/patología , Proteína Relacionada con Twist 2/biosíntesis , Animales , Modelos Animales de Enfermedad , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Proteínas Proto-Oncogénicas B-raf/genética , Proteína Relacionada con Twist 2/genética , Pez Cebra
6.
Dev Dyn ; 236(11): 3077-87, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17948311

RESUMEN

The recent establishment of recombination-based cloning systems has greatly facilitated the analysis of gene function by allowing rapid and high-efficiency generation of plasmid constructs. However, the use of such an approach in zebrafish requires the availability of recombination-compatible plasmids that are appropriate for functional studies in zebrafish embryos. In this work, we describe the construction and validation of Gateway compatible vectors based on commonly used zebrafish plasmids. We have generated pCS-based plasmids that allow rapid generation of both N-terminal and C-terminal fusion proteins, and we demonstrate that mRNA synthesized from these plasmids encodes functional native or fusion proteins in injected zebrafish embryos. In parallel, we have established similar Gateway plasmids containing Tol2 cis elements that promote efficient integration into the zebrafish genome and allow expression of native or fusion proteins in a tissue-specific manner in the zebrafish embryo. Finally, we demonstrate the use of this system to rapidly identify tissue-specific cis elements to aid the establishment of blood vessel-specific transgenic constructs. Taken together, this work provides an important platform for the rapid functional analyses of open reading frames in zebrafish embryos.


Asunto(s)
Clonación Molecular/métodos , Técnicas de Transferencia de Gen , Vectores Genéticos , Plásmidos/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Embrión no Mamífero , Sistemas de Lectura Abierta/genética , Recombinación Genética/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
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