RESUMO
Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.
Assuntos
Células Endoteliais/metabolismo , Glicólise , Neovascularização Fisiológica , Fosfofrutoquinase-2/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Células Endoteliais/citologia , Feminino , Deleção de Genes , Inativação Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfofrutoquinase-2/genética , Pseudópodes/metabolismo , Peixe-ZebraRESUMO
Previous studies have shown that Vasohibin 1 (Vash1) is stimulated by VEGFs in endothelial cells and that its overexpression interferes with angiogenesis in vivo Recently, Vash1 was found to mediate tubulin detyrosination, a post-translational modification that is implicated in many cell functions, such as cell division. Here, we used the zebrafish embryo to investigate the cellular and subcellular mechanisms of Vash1 on endothelial microtubules during formation of the trunk vasculature. We show that microtubules within venous-derived secondary sprouts are strongly and selectively detyrosinated in comparison with other endothelial cells, and that this difference is lost upon vash1 knockdown. Vash1 depletion in zebrafish specifically affected secondary sprouting from the posterior cardinal vein, increasing endothelial cell divisions and cell number in the sprouts. We show that altering secondary sprout numbers and structure upon Vash1 depletion leads to defective lymphatic vessel formation and ectopic lymphatic progenitor specification in the zebrafish trunk.
Assuntos
Proteínas de Ciclo Celular/genética , Desenvolvimento Embrionário/genética , Linfangiogênese/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Sequência Conservada , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Microtúbulos/metabolismo , Modelos BiológicosRESUMO
How developing vascular networks acquire the right balance of arteries, veins and lymphatic vessels to efficiently supply and drain tissues is poorly understood. In zebrafish embryos, the robust and regular 50:50 global balance of intersegmental veins and arteries that form along the trunk prompts the intriguing question of how does the organism keep 'count'? Previous studies have suggested that the ultimate fate of an intersegmental vessel (ISV) is determined by the identity of the approaching secondary sprout emerging from the posterior cardinal vein. Here, we show that the formation of a balanced trunk vasculature involves an early heterogeneity in endothelial cell behaviour and Notch signalling activity in the seemingly identical primary ISVs that is independent of secondary sprouting and flow. We show that Notch signalling mediates the local patterning of ISVs, and an adaptive flow-mediated mechanism subsequently fine-tunes the global balance of arteries and veins along the trunk. We propose that this dual mechanism provides the adaptability required to establish a balanced network of arteries, veins and lymphatic vessels.
Assuntos
Padronização Corporal , Receptores Notch/metabolismo , Peixe-Zebra/embriologia , Animais , Artérias/embriologia , Polaridade Celular , Células Endoteliais/fisiologia , Heterogeneidade Genética , Vasos Linfáticos/embriologia , Fluxo Sanguíneo Regional , Transdução de Sinais , Veias/embriologia , Peixe-Zebra/sangueRESUMO
A 'GGGGCC' repeat expansion in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The exact mechanism resulting in these neurodegenerative diseases remains elusive, but C9 repeat RNA toxicity has been implicated as a gain-of-function mechanism. Our aim was to use a zebrafish model for C9orf72 RNA toxicity to identify modifiers of the ALS-linked phenotype. We discovered that the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (HNRNPK) reverses the toxicity of both sense and antisense repeat RNA, which is dependent on its subcellular localization and RNA recognition, and not on C9orf72 repeat RNA binding. We observed HNRNPK cytoplasmic mislocalization in C9orf72 ALS patient fibroblasts, induced pluripotent stem cell (iPSC)-derived motor neurons and post-mortem motor cortex and spinal cord, in line with a disrupted HNRNPK function in C9orf72 ALS. In C9orf72 ALS/FTD patient tissue, we discovered an increased nuclear translocation, but reduced expression of ribonucleotide reductase regulatory subunit M2 (RRM2), a downstream target of HNRNPK involved in the DNA damage response. Last but not least, we showed that increasing the expression of HNRNPK or RRM2 was sufficient to mitigate DNA damage in our C9orf72 RNA toxicity zebrafish model. Overall, our study strengthens the relevance of RNA toxicity as a pathogenic mechanism in C9orf72 ALS and demonstrates its link with an aberrant DNA damage response, opening novel therapeutic avenues for C9orf72 ALS/FTD.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Doença de Pick , Esclerose Lateral Amiotrófica/patologia , Animais , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Dano ao DNA , Expansão das Repetições de DNA/genética , Demência Frontotemporal/patologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Doença de Pick/genética , RNA/metabolismo , RNA Antissenso , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pbio.1002125.].
RESUMO
Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments.
Assuntos
Vasos Sanguíneos/citologia , Endotélio Vascular/citologia , Animais , Polaridade Celular , Modelos BiológicosRESUMO
To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Bα regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Bα in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Bα in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell-extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Bα controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Bα, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Bα/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell-matrix adhesion.
Assuntos
Endotélio Vascular/fisiologia , Regulação da Expressão Gênica/fisiologia , Histona Desacetilases/metabolismo , Neovascularização Fisiológica/fisiologia , Proteína Fosfatase 2/metabolismo , Grau de Desobstrução Vascular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Adesão Celular/fisiologia , Colágeno , Combinação de Medicamentos , Imunofluorescência , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Processamento de Imagem Assistida por Computador , Laminina , Microscopia Confocal , Proteoglicanas , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA , Grau de Desobstrução Vascular/genética , Peixe-ZebraRESUMO
The correct development of blood vessels is crucial for all aspects of tissue growth and physiology in vertebrates. The formation of an elaborate hierarchically branched network of endothelial tubes, through either angiogenesis or vasculogenesis, relies on a series of coordinated morphogenic events, but how individual endothelial cells adopt specific phenotypes and how they coordinate their behaviour during vascular patterning is unclear. Recent progress in our understanding of blood vessel formation has been driven by advanced imaging techniques and detailed analyses that have used a combination of powerful in vitro, in vivo and in silico model systems. Here, we summarise these models and discuss their advantages and disadvantages. We then review the different stages of blood vessel development, highlighting the cellular mechanisms and molecular players involved at each step and focusing on cell specification and coordination within the network.
Assuntos
Diferenciação Celular/fisiologia , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Modelos Biológicos , Neovascularização Fisiológica/fisiologia , Animais , Humanos , Morfogênese/fisiologiaRESUMO
Glioblastoma (GBM) remains the most malignant primary brain tumor, with a median survival rarely exceeding 2 years. Tumor heterogeneity and an immunosuppressive microenvironment are key factors contributing to the poor response rates of current therapeutic approaches. GBM-associated macrophages (GAMs) often exhibit immunosuppressive features that promote tumor progression. However, their dynamic interactions with GBM tumor cells remain poorly understood. Here, we used patient-derived GBM stem cell cultures and combined single-cell RNA sequencing of GAM-GBM co-cultures and real-time in vivo monitoring of GAM-GBM interactions in orthotopic zebrafish xenograft models to provide insight into the cellular, molecular, and spatial heterogeneity. Our analyses revealed substantial heterogeneity across GBM patients in GBM-induced GAM polarization and the ability to attract and activate GAMs-features that correlated with patient survival. Differential gene expression analysis, immunohistochemistry on original tumor samples, and knock-out experiments in zebrafish subsequently identified LGALS1 as a primary regulator of immunosuppression. Overall, our work highlights that GAM-GBM interactions can be studied in a clinically relevant way using co-cultures and avatar models, while offering new opportunities to identify promising immune-modulating targets.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Animais , Humanos , Glioblastoma/patologia , Peixe-Zebra , Galectina 1/genética , Galectina 1/metabolismo , Galectina 1/uso terapêutico , Linhagem Celular Tumoral , Macrófagos/metabolismo , Neoplasias Encefálicas/patologia , Microambiente Tumoral/genéticaRESUMO
The molecular basis of lymphangiogenesis remains incompletely characterized. Here, we document a novel role for the PDZ domain-containing scaffold protein synectin in lymphangiogenesis using genetic studies in zebrafish and tadpoles. In zebrafish, the thoracic duct arises from parachordal lymphangioblast cells, which in turn derive from secondary lymphangiogenic sprouts from the posterior cardinal vein. Morpholino knockdown of synectin in zebrafish impaired formation of the thoracic duct, due to selective defects in lymphangiogenic but not angiogenic sprouting. Synectin genetically interacted with Vegfr3 and neuropilin-2a in regulating lymphangiogenesis. Silencing of synectin in tadpoles caused lymphatic defects due to an underdevelopment and impaired migration of Prox-1(+) lymphatic endothelial cells. Molecular analysis further revealed that synectin regulated Sox18-induced expression of Prox-1 and vascular endothelial growth factor C-induced migration of lymphatic endothelial cells in vitro. These findings reveal a novel role for synectin in lymphatic development.
Assuntos
Proteínas de Transporte/metabolismo , Linfangiogênese , Vasos Linfáticos/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/fisiologia , Peixe-Zebra/fisiologia , Animais , Proteínas de Transporte/genética , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Humanos , Larva/genética , Larva/fisiologia , Neovascularização Fisiológica , Neuropilina-2/genética , Ducto Torácico/embriologia , Ducto Torácico/crescimento & desenvolvimento , Fator C de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Vascular endothelial growth factor (VEGF) regulates angiogenesis, but also has important, yet poorly characterized roles in neuronal wiring. Using several genetic and in vitro approaches, we discovered a novel role for VEGF in the control of cerebellar granule cell (GC) migration from the external granule cell layer (EGL) toward the Purkinje cell layer (PCL). GCs express the VEGF receptor Flk1, and are chemoattracted by VEGF, whose levels are higher in the PCL than EGL. Lowering VEGF levels in mice in vivo or ectopic VEGF expression in the EGL ex vivo perturbs GC migration. Using GC-specific Flk1 knock-out mice, we provide for the first time in vivo evidence for a direct chemoattractive effect of VEGF on neurons via Flk1 signaling. Finally, using knock-in mice expressing single VEGF isoforms, we show that pericellular deposition of matrix-bound VEGF isoforms around PC dendrites is necessary for proper GC migration in vivo. These findings identify a previously unknown role for VEGF in neuronal migration.
Assuntos
Movimento Celular/fisiologia , Cerebelo/fisiologia , Neurônios/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Células Cultivadas , Cerebelo/citologia , Ensaio de Imunoadsorção Enzimática , Cones de Crescimento/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Neurônios/citologia , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: To study whether Notch signaling, which regulates cell fate decisions and vessel morphogenesis, controls lymphatic development. METHODS AND RESULTS: In zebrafish embryos, sprouts from the axial vein have lymphangiogenic potential because they give rise to the first lymphatics. Knockdown of delta-like-4 (Dll4) or its receptors Notch-1b or Notch-6 in zebrafish impaired lymphangiogenesis. Dll4/Notch silencing reduced the number of sprouts producing the string of parchordal lymphangioblasts; instead, sprouts connecting to the intersomitic vessels were formed. At a later phase, Notch silencing impaired navigation of lymphatic intersomitic vessels along their arterial templates. CONCLUSIONS: These studies imply critical roles for Notch signaling in the formation and wiring of the lymphatic network.
Assuntos
Linfangiogênese , Sistema Linfático/metabolismo , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Células COS , Movimento Celular , Proliferação de Células , Chlorocebus aethiops , Técnicas de Cocultura , Embrião não Mamífero/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Linfangiogênese/genética , Sistema Linfático/embriologia , Proteínas de Membrana/genética , RNA Mensageiro/metabolismo , Receptores Notch/genética , Ducto Torácico/embriologia , Ducto Torácico/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The importance of the lymphangiogenic factor VEGF-D and its receptor VEGFR-3 in early lymphatic development remains largely unresolved. We therefore investigated their role in Xenopus laevis tadpoles, a small animal model allowing chemicogenetic dissection of developmental lymphangiogenesis. Single morpholino antisense oligo knockdown of xVEGF-D did not affect lymphatic commitment, but transiently impaired lymphatic endothelial cell (LEC) migration. Notably, combined knockdown of xVEGF-D with xVEGF-C at suboptimal morpholino concentrations resulted in more severe migration defects and lymphedema formation than the corresponding single knockdowns. Knockdown of VEGFR-3 or treatment with the VEGFR-3 inhibitor MAZ51 similarly impaired lymph vessel formation and function and caused pronounced edema. VEGFR-3 silencing by morpholino knockdown, MAZ51 treatment, or xVEGF-C/D double knockdown also resulted in dilation and dysfunction of the lymph heart. These findings document a critical role of VEGFR-3 in embryonic lymphatic development and function, and reveal a previously unrecognized modifier role of VEGF-D in the regulation of embryonic lymphangiogenesis in frog embryos.
Assuntos
Linfangiogênese/fisiologia , Fator D de Crescimento do Endotélio Vascular/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/fisiologia , Animais , Inativação Gênica , Larva/crescimento & desenvolvimento , Linfangiogênese/genética , Vasos Linfáticos/anormalidades , Vasos Linfáticos/embriologia , Oligodesoxirribonucleotídeos Antissenso/genética , Fator D de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator D de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
How vascular tubes build, maintain and adapt continuously perfused lumens to meet local metabolic needs remains poorly understood. Recent studies showed that blood flow itself plays a critical role in the remodelling of vascular networks, and suggested it is also required for the lumenization of new vascular connections. However, it is still unknown how haemodynamic forces contribute to the formation of new vascular lumens during blood vessel morphogenesis. Here we report that blood flow drives lumen expansion during sprouting angiogenesis in vivo by inducing spherical deformations of the apical membrane of endothelial cells, in a process that we have termed inverse blebbing. We show that endothelial cells react to these membrane intrusions by local and transient recruitment and contraction of actomyosin, and that this mechanism is required for single, unidirectional lumen expansion in angiogenic sprouts. Our work identifies inverse membrane blebbing as a cellular response to high external pressure. We show that in the case of blood vessels such membrane dynamics can drive local cell shape changes required for global tissue morphogenesis, shedding light on a pressure-driven mechanism of lumen formation in vertebrates.
Assuntos
Vasos Sanguíneos/embriologia , Morfogênese , Neovascularização Fisiológica , Actomiosina/metabolismo , Animais , Animais Geneticamente Modificados , Vasos Sanguíneos/citologia , Vasos Sanguíneos/metabolismo , Células Endoteliais/metabolismo , Feminino , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Fluxo Sanguíneo Regional , Imagem com Lapso de Tempo , Peixe-ZebraRESUMO
Formation of a regularly branched blood vessel network is crucial in development and physiology. Here we show that the expression of the Notch ligand Dll4 fluctuates in individual endothelial cells within sprouting vessels in the mouse retina in vivo and in correlation with dynamic cell movement in mouse embryonic stem cell-derived sprouting assays. We also find that sprout elongation and branching associates with a highly differential phase pattern of Dll4 between endothelial cells. Stimulation with pathologically high levels of Vegf, or overexpression of Dll4, leads to Notch dependent synchronization of Dll4 fluctuations within clusters, both in vitro and in vivo. Our results demonstrate that the Vegf-Dll4/Notch feedback system normally operates to generate heterogeneity between endothelial cells driving branching, whilst synchronization drives vessel expansion. We propose that this sensitive phase transition in the behaviour of the Vegf-Dll4/Notch feedback loop underlies the morphogen function of Vegfa in vascular patterning.
Assuntos
Neoplasias Encefálicas/genética , Células Endoteliais/metabolismo , Glioblastoma/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Patológica/genética , Receptores Notch/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proteínas de Ligação ao Cálcio , Movimento Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Genes Reporter , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Transplante de Neoplasias , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Fisiológica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Notch/genética , Retina/citologia , Retina/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Sprouting angiogenesis drives blood vessel growth in healthy and diseased tissues. Vegf and Dll4/Notch signalling cooperate in a negative feedback loop that specifies endothelial tip and stalk cells to ensure adequate vessel branching and function. Current concepts posit that endothelial cells default to the tip-cell phenotype when Notch is inactive. Here we identify instead that the stalk-cell phenotype needs to be actively repressed to allow tip-cell formation. We show this is a key endothelial function of neuropilin-1 (Nrp1), which suppresses the stalk-cell phenotype by limiting Smad2/3 activation through Alk1 and Alk5. Notch downregulates Nrp1, thus relieving the inhibition of Alk1 and Alk5, thereby driving stalk-cell behaviour. Conceptually, our work shows that the heterogeneity between neighbouring endothelial cells established by the lateral feedback loop of Dll4/Notch utilizes Nrp1 levels as the pivot, which in turn establishes differential responsiveness to TGF-ß/BMP signalling.
Assuntos
Receptores de Ativinas Tipo I/metabolismo , Endotélio Vascular/crescimento & desenvolvimento , Neuropilina-1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Notch/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Receptores de Activinas Tipo II , Animais , Fator 2 de Diferenciação de Crescimento/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Fenótipo , Receptor do Fator de Crescimento Transformador beta Tipo I , Proteína Smad2/metabolismo , Proteína Smad3/metabolismoRESUMO
The importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP) Xenopus laevis reporter lines expressing green fluorescent protein (GFP) in blood- and lymph vessels driven by the Flk1 (VEGFR-2) promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP) reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP) tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening.