RESUMO
Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system(1) in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis(2,3). Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.
Assuntos
Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/patologia , Engenharia Genética/métodos , Lesões Pré-Cancerosas/imunologia , Lesões Pré-Cancerosas/patologia , Pele/imunologia , Pele/patologia , Animais , Animais Geneticamente Modificados , Comunicação Celular/fisiologia , Transformação Celular Neoplásica/genética , DNA/administração & dosagem , DNA/genética , Imunidade Inata , Larva/fisiologia , Microinjeções , Plasmídeos/administração & dosagem , Plasmídeos/genética , Lesões Pré-Cancerosas/genética , Fenômenos Fisiológicos da Pele/genética , Fenômenos Fisiológicos da Pele/imunologia , Transgenes , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Blood flow plays crucial roles in vascular development, remodeling and homeostasis, but the molecular pathways required for transducing flow signals are not well understood. In zebrafish embryos, arterial expression of activin receptor-like kinase 1 (alk1), which encodes a TGFß family type I receptor, is dependent on blood flow, and loss of alk1 mimics lack of blood flow in terms of dysregulation of a subset of flow-responsive arterial genes and increased arterial endothelial cell number. These data suggest that blood flow activates Alk1 signaling to promote a flow-responsive gene expression program that limits nascent arterial caliber. Here, we demonstrate that restoration of endothelial alk1 expression to flow-deprived arteries fails to rescue Alk1 activity or normalize arterial endothelial cell gene expression or number, implying that blood flow may play an additional role in Alk1 signaling independent of alk1 induction. To this end, we define cardiac-derived Bmp10 as the crucial ligand for endothelial Alk1 in embryonic vascular development, and provide evidence that circulating Bmp10 acts through endothelial Alk1 to limit endothelial cell number in and thereby stabilize the caliber of nascent arteries. Thus, blood flow promotes Alk1 activity by concomitantly inducing alk1 expression and distributing Bmp10, thereby reinforcing this signaling pathway, which functions to limit arterial caliber at the onset of flow. Because mutations in ALK1 cause arteriovenous malformations (AVMs), our findings suggest that an impaired flow response initiates AVM development.
Assuntos
Receptores de Ativinas/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Artérias Carótidas/enzimologia , Embrião não Mamífero/irrigação sanguínea , Endotélio Vascular/enzimologia , Receptores de Ativinas/genética , Animais , Malformações Arteriovenosas/enzimologia , Malformações Arteriovenosas/patologia , Proteínas Morfogenéticas Ósseas/genética , Contagem de Células , Embrião não Mamífero/metabolismo , Endotelina-1/genética , Endotelina-1/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Miocárdio/enzimologia , Miocárdio/patologia , Fosforilação , Transporte Proteico , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Bone morphogenetic proteins (BMPs) are critical players in development and disease, regulating such diverse processes as dorsoventral patterning, palate formation, and ossification. These ligands are classically considered to signal via BMP receptor-specific Smad proteins 1, 5, and 8. To determine the spatiotemporal pattern of Smad1/5/8 activity and thus canonical BMP signaling in the developing zebrafish embryo, we generated a transgenic line expressing EGFP under the control of a BMP-responsive element. EGFP is expressed in many established BMP signaling domains and is responsive to alterations in BMP type I receptor activity and smad1 and smad5 expression. This transgenic Smad1/5/8 reporter line will be useful for determining ligand and receptor requirements for specific domains of BMP activity, as well as for genetic and pharmacological screens aimed at identifying enhancers or suppressors of canonical BMP signaling.
