RESUMEN
Evolutionary transitions from egg laying (oviparity) to live birth (viviparity) are common across various taxa. Many species also exhibit genetic variation in egg-laying mode or display an intermediate mode with laid eggs containing embryos at various stages of development. Understanding the mechanistic basis and fitness consequences of such variation remains experimentally challenging. Here, we report highly variable intra-uterine egg retention across 316 Caenorhabditis elegans wild strains, some exhibiting strong retention, followed by internal hatching. We identify multiple evolutionary origins of such phenotypic extremes and pinpoint underlying candidate loci. Behavioral analysis and genetic manipulation indicates that this variation arises from genetic differences in the neuromodulatory architecture of the egg-laying circuitry. We provide experimental evidence that while strong egg retention can decrease maternal fitness due to in utero hatching, it may enhance offspring protection and confer a competitive advantage. Therefore, natural variation in C. elegans egg-laying behaviour can alter an apparent trade-off between different fitness components across generations. Our findings highlight underappreciated diversity in C. elegans egg-laying behavior and shed light on its fitness consequences. This behavioral variation offers a promising model to elucidate the molecular changes in a simple neural circuit underlying evolutionary shifts between alternative egg-laying modes in invertebrates.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Oviposición/genética , Oviparidad , Proteínas de Caenorhabditis elegans/genética , Evolución BiológicaRESUMEN
Wild populations of the model organism C. elegans represent a valuable resource, allowing for genetic characterization underlying natural phenotypic variation. Here we provide a simple protocol on how to sample and rapidly identify C. elegans wild isolates. We outline how to find suitable habitats and organic substrates, followed by describing isolation and identification of C. elegans live cultures based on easily recognizable morphological characteristics, molecular barcodes, and mating tests. This protocol uses standard laboratory equipment and requires little prior knowledge of C. elegans biology.
Asunto(s)
Caenorhabditis elegans , Ecosistema , Animales , Caenorhabditis elegans/genética , Reproducción/genéticaRESUMEN
Maintaining reproduction in highly variable, often stressful, environments is an essential challenge for all organisms. Even transient exposure to mild environmental stress may directly damage germ cells or simply tax the physiology of an individual, making it difficult to produce quality gametes. In Caenorhabditis elegans, a large fraction of germ cells acts as nurse cells, supporting developing oocytes before eventually undergoing so-called physiological germ cell apoptosis. Although C. elegans apoptosis has been extensively studied, little is known about how germline apoptosis is influenced by ecologically relevant environmental stress. Moreover, it remains unclear to what extent germline apoptosis contributes to maintaining oocyte quality, and thus offspring viability, in such conditions. Here we show that exposure to diverse environmental stressors, likely occurring in the natural C. elegans habitat (starvation, ethanol, acid, and mild oxidative stress), increases germline apoptosis, consistent with previous reports on stress-induced apoptosis. Using loss-of-function mutant alleles of ced-3 and ced-4, we demonstrate that eliminating the core apoptotic machinery strongly reduces embryonic survival when mothers are exposed to such environmental stressors during early adult life. In contrast, mutations in ced-9 and egl-1 that primarily block apoptosis in the soma but not in the germline, did not exhibit such reduced embryonic survival under environmental stress. Therefore, C. elegans germ cell apoptosis plays an essential role in maintaining offspring fitness in adverse environments. Finally, we show that ced-3 and ced-4 mutants exhibit concomitant decreases in embryo size and changes in embryo shape when mothers are exposed to environmental stress. These observations may indicate inadequate oocyte provisioning due to the absence of germ cell apoptosis. Taken together, our results show that the central genes of the apoptosis pathway play a key role in maintaining gamete quality, and thus offspring fitness, under ecologically relevant environmental conditions.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Caspasas/genética , Proteínas de la Membrana/genética , Oocitos/citología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Represoras/genética , Animales , Apoptosis , Caenorhabditis elegans/efectos de los fármacos , Etanol/toxicidad , Femenino , Ácido Clorhídrico/toxicidad , Masculino , Mutación , Oocitos/efectos de los fármacos , Oocitos/crecimiento & desarrollo , Estrés Oxidativo , Paraquat/toxicidad , Reproducción/efectos de los fármacos , Estrés FisiológicoRESUMEN
Genetic assimilation-the evolutionary process by which an environmentally induced phenotype is made constitutive-represents a fundamental concept in evolutionary biology. Thought to reflect adaptive phenotypic plasticity, matricidal hatching in nematodes is triggered by maternal nutrient deprivation to allow for protection or resource provisioning of offspring. Here, we report natural Caenorhabditis elegans populations harboring genetic variants expressing a derived state of near-constitutive matricidal hatching. These variants exhibit a single amino acid change (V530L) in KCNL-1, a small-conductance calcium-activated potassium channel subunit. This gain-of-function mutation causes matricidal hatching by strongly reducing the sensitivity to environmental stimuli triggering egg-laying. We show that reestablishing the canonical KCNL-1 protein in matricidal isolates is sufficient to restore canonical egg-laying. While highly deleterious in constant food environments, KCNL-1 V530L is maintained under fluctuating resource availability. A single point mutation can therefore underlie the genetic assimilation-by either genetic drift or selection-of an ancestrally plastic trait.
RESUMEN
Adaptive developmental plasticity is a common phenomenon across diverse organisms and allows a single genotype to express multiple phenotypes in response to environmental signals. Developmental plasticity is thus thought to reflect a key adaptation to cope with heterogenous habitats. Adaptive plasticity often relies on highly regulated processes in which organisms sense environmental cues predictive of unfavourable environments. The integration of such cues may involve sophisticated neuro-endocrine signaling pathways to generate subtle or complete developmental shifts. A striking example of adaptive plasticity is found in the nematode C. elegans, which can undergo two different developmental trajectories depending on the environment. In favourable conditions, C. elegans develops through reproductive growth to become an adult in three days at 20 °C. In contrast, in unfavourable conditions (high population density, food scarcity, elevated temperature) larvae can adopt an alternative developmental stage, called dauer. dauer larvae are highly stress-resistant and exhibit specific anatomical, metabolic and behavioural features that allow them to survive and disperse. In C. elegans, the sensation of environmental cues is mediated by amphid ciliated sensory neurons by means of G-coupled protein receptors. In favourable environments, the perception of pro-reproductive cues, such as food and the absence of pro-dauer cues, upregulates insulin and TGF-ß signaling in the nervous system. In unfavourable conditions, pro-dauer cues lead to the downregulation of insulin and TGF-ß signaling. In favourable conditions, TGF-ß and insulin act in parallel to promote synthesis of dafachronic acid (DA) in steroidogenic tissues. Synthetized DA binds to the DAF-12 nuclear receptor throughout the whole body. DA-bound DAF-12 positively regulates genes of reproductive development in all C. elegans tissues. In poor conditions, the inhibition of insulin and TGF-ß signaling prevents DA synthesis, thus the unliganded DAF-12 and co-repressor DIN-1 repress genes of reproductive development and promote dauer formation. Wild C. elegans have often been isolated as dauer larvae suggesting that dauer formation is very common in nature. Natural populations of C. elegans have colonized a great variety of habitats across the planet, which may differ substantially in environmental conditions. Consistent with divergent adaptation to distinct ecological niches, wild isolates of C. elegans and other nematode species isolated from different locations show extensive variation in dauer induction. Quantitative genetic and population-genomic approaches have identified many quantitative trait loci (QTL) associated with differences in dauer induction as well as a few underlying causative molecular variants. In this review, we summarize how C. elegans dauer formation is genetically regulated and how this trait evolves- both within and between species.
TITLE: Génétique et évolution de la plasticité développementale chez le nématode C. elegans : induction environnementale du stade dauer. ABSTRACT: La plasticité phénotypique est un phénomène très courant au cours duquel des phénotypes différents sont exprimés en fonction de facteurs environnementaux. La plasticité, lorsque qu'elle est dite « adaptative ¼, permet aux organismes de faire face à des habitats hétérogènes. Bien que les mécanismes moléculaires régulant la plasticité développementale soient de mieux en mieux compris, nous n'avons encore que peu d'informations sur les bases moléculaires de la variation naturelle et de l'évolution de la plasticité. Le nématode C. elegans présente un exemple emblématique de plasticité adaptative car cette espèce a la capacité d'entrer dans un stade larvaire alternatif appelé « dauer ¼ lorsque les conditions environnementales sont défavorables. Durant ce stade de diapause, les larves peuvent survivre pendant environ trois mois en milieu extrême et reprendre leur développement lorsque les conditions s'améliorent. Nous passons ici en revue les mécanismes moléculaires régulant l'entrée en dauer ainsi que les récents progrès réalisés dans la caractérisation de la variation naturelle et l'évolution de l'induction de ce stade de résistance chez C. elegans comme chez d'autres espèces de nématodes.
Asunto(s)
Caenorhabditis elegans , Interacción Gen-Ambiente , Estadios del Ciclo de Vida/genética , Adaptación Fisiológica/genética , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Ambiente , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Larva/crecimiento & desarrollo , Larva/metabolismo , Transducción de Señal/genéticaRESUMEN
The diversity in sperm shape and size represents a powerful paradigm to understand how selection drives the evolutionary diversification of cell morphology. Experimental work on the sperm biology of the male-hermaphrodite nematode Caenorhabditis elegans has elucidated diverse factors important for sperm fertilization success, including the competitive superiority of larger sperm. Yet despite extensive research, the molecular mechanisms regulating C. elegans sperm size and the genetic basis underlying natural variation in sperm size remain unknown. To address these questions, we quantified male sperm size variation of a worldwide panel of 97 genetically distinct C. elegans strains, allowing us to uncover significant genetic variation in male sperm size. Aiming to characterize the molecular genetic basis of C. elegans male sperm size variation using a genome-wide association study, we did not detect any significant quantitative trait loci. We therefore focused on the genetic analysis of pronounced sperm size differences observed between recently diverged laboratory strains (N2 vs. LSJ1/2). Using mutants and quantitative complementation tests, we demonstrate that variation in the gene nurf-1 underlies the evolution of small sperm in the LSJ lineage. Given the previous discovery that this same nurf-1 variation was central for hermaphrodite laboratory adaptation, the evolution of reduced male sperm size in LSJ strains likely reflects a pleiotropic consequence. Together, our results provide a comprehensive quantification of natural variation in C. elegans sperm size and first insights into the genetic determinants of Caenorhabditis sperm size, pointing at an involvement of the NURF chromatin remodeling complex.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Tamaño de la Célula , Proteínas Cromosómicas no Histona/genética , Espermatozoides/citología , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Linaje de la Célula/genética , Ensamble y Desensamble de Cromatina , Trastornos del Desarrollo Sexual/genética , Trastornos del Desarrollo Sexual/patología , Fertilización/genética , Variación Genética/genética , Estudio de Asociación del Genoma Completo , Masculino , Sitios de Carácter Cuantitativo/genética , Espermatozoides/crecimiento & desarrolloRESUMEN
Environmental conditions experienced during animal development are thought to have sustained impact on maturation and adult lifespan. Here we show that in the model organism C. elegans developmental rate and adult lifespan depend on larval population density, and that this effect is mediated by excreted small molecules. By using the time point of first egg laying as a marker for full maturity, we found that wildtype hermaphrodites raised under high density conditions developed significantly faster than animals raised in isolation. Population density-dependent acceleration of development (Pdda) was dramatically enhanced in fatty acid ß-oxidation mutants that are defective in the biosynthesis of ascarosides, small-molecule signals that induce developmental diapause. In contrast, Pdda is abolished by synthetic ascarosides and steroidal ligands of the nuclear hormone receptor DAF-12. We show that neither ascarosides nor any known steroid hormones are required for Pdda and that another chemical signal mediates this phenotype, in part via the nuclear hormone receptor NHR-8. Our results demonstrate that C. elegans development is regulated by a push-pull mechanism, based on two antagonistic chemical signals: chemosensation of ascarosides slows down development, whereas population-density dependent accumulation of a different chemical signal accelerates development. We further show that the effects of high larval population density persist through adulthood, as C. elegans larvae raised at high densities exhibit significantly reduced adult lifespan and respond differently to exogenous chemical signals compared to larvae raised at low densities, independent of density during adulthood. Our results demonstrate how inter-organismal signaling during development regulates reproductive maturation and longevity.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Longevidad/genética , Receptores Citoplasmáticos y Nucleares/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/biosíntesis , Ácidos Grasos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Organismos Hermafroditas/genética , Organismos Hermafroditas/crecimiento & desarrollo , Larva/genética , Larva/crecimiento & desarrollo , Neuropéptidos/metabolismo , Densidad de Población , Receptores Citoplasmáticos y Nucleares/biosíntesis , Transducción de SeñalRESUMEN
Hermaphroditic organisms are key models in sex allocation research, yet the developmental processes by which hermaphrodite sex allocation can evolve remain largely unknown. Here we use experimental evolution of hermaphrodite-male (androdioecious) Caenorhabditis elegans populations to quantify the developmental changes underlying adaptive shifts in hermaphrodite sex allocation. We show that the experimental evolution of increased early-life self-fertility occurred through modification of a suite of developmental traits: increased self-sperm production, accelerated oogenesis and ovulation, and increased embryo retention. The experimental evolution of increased self-sperm production delayed entry into oogenesis-as expected, given the sequentially coupled production of self-spermatogenesis and oogenesis. Surprisingly, however, delayed oogenesis onset did not delay reproductive maturity, nor did it trade-off with gamete or embryo size. Comparing developmental time dynamics of germline and soma indicates that the evolution of increased sperm production did not delay reproductive maturity due to a globally accelerated larval development during the period of self-spermatogenesis. Overall, heterochrony in gametogenesis and soma can explain adaptive shifts in hermaphrodite sex allocation.
Asunto(s)
Caenorhabditis elegans/genética , Evolución Molecular , Organismos Hermafroditas/genética , Maduración Sexual/genética , Adaptación Fisiológica , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Organismos Hermafroditas/crecimiento & desarrollo , Oogénesis , Autofecundación , EspermatogénesisRESUMEN
Sperm cells provide essential, if usually diminutive, ingredients to successful sexual reproduction. Despite this conserved function, sperm competition and coevolution with female traits can drive spectacular morphological change in these cells. Here, we characterize four repeated instances of convergent evolution of sperm gigantism in Caenorhabditis nematodes using phylogenetic comparative methods on 26 species. Species at the extreme end of the 50-fold range of sperm-cell volumes across the genus have sperm capable of comprising up to 5% of egg-cell volume, representing severe attenuation of the magnitude of anisogamy. Furthermore, we uncover significant differences in mean and variance of sperm size among genotypes, between sexes, and within and between individuals of identical genotypes. We demonstrate that the developmental basis of sperm size variation, both within and between species, becomes established during an early stage of sperm development at the formation of primary spermatocytes, while subsequent meiotic divisions contribute little further sperm size variability. These findings provide first insights into the developmental determinants of inter- and intraspecific sperm size differences in Caenorhabditis. We hypothesize that life history and ecological differences among species favored the evolution of alternative sperm competition strategies toward either many smaller sperm or fewer larger sperm.
Asunto(s)
Caenorhabditis/genética , Tamaño de la Célula , Evolución Molecular , Variación Genética , Espermatozoides/citología , Animales , Caenorhabditis/citología , Femenino , MasculinoRESUMEN
Thermal developmental plasticity represents a key organismal adaptation to maintain reproductive capacity in contrasting and fluctuating temperature niches. Although extensively studied, research on thermal plasticity has mainly focused on phenotypic outcomes, such as adult life history, rather than directly measuring plasticity of underlying developmental processes. How thermal plasticity of developmental phenotypes maps into plasticity of resulting final phenotypes, and how such mapping relationships evolve, thus remain poorly understood. Here we address these questions by quantifying thermal plasticity of Caenorhabditis hermaphrodite germline development. We integrate measurements of germline development and fertility at the upper thermal range in isolates of C. briggsae, C. elegans, and C. tropicalis. First, we compare intra- and interspecific variation in thermal germline plasticity with plasticity in reproductive output. Second, we ask whether the developmental errors leading to fertility break-down at upper thermal limits are evolutionarily conserved. We find that temperature variation modulates spermatogenesis, oogenesis and germ cell progenitor pools, yet the thermal sensitivity of these processes varies among isolates and species, consistent with evolutionary variation in upper thermal limits of hermaphrodite fertility. Although defective sperm function is a major contributor to heat-induced fertility break-down, high temperature also significantly perturbs oogenesis, germline integrity, and mitosis-meiosis progression. Remarkably, the occurrence and frequency of specific errors are strongly species- and genotype-dependent, indicative of evolutionary divergence in thermal sensitivity of distinct processes in germline development. Therefore, the Caenorhabditis reproductive system displays complex genotype-by-temperature interactions at the developmental level, which may remain masked when studying thermal plasticity exclusively at the life history level.
Asunto(s)
Caenorhabditis/fisiología , Fertilidad , Oogénesis , Espermatogénesis , Animales , Caenorhabditis/embriología , Caenorhabditis/crecimiento & desarrollo , Células Germinativas/crecimiento & desarrollo , Organismos Hermafroditas/crecimiento & desarrollo , Organismos Hermafroditas/fisiología , TemperaturaRESUMEN
Theory and empirical study produce clear links between mating system evolution and inbreeding depression. The connections between mating systems and outbreeding depression, whereby fitness is reduced in crosses of less related individuals, however, are less well defined. Here we investigate inbreeding and outbreeding depression in self-fertile androdioecious nematodes, focusing on Caenorhabditis sp. 11. We quantify nucleotide polymorphism for nine nuclear loci for strains throughout its tropical range, and find some evidence of genetic differentiation despite the lowest sequence diversity observed in this genus. Controlled crosses between strains from geographically separated regions show strong outbreeding depression, with reproductive output of F1s reduced by 36% on average. Outbreeding depression is therefore common in self-fertilizing Caenorhabditis species, each of which evolved androdioecious selfing hermaphroditism independently, but appears strongest in C. sp. 11. Moreover, the poor mating efficiency of androdioecious males extends to C. sp. 11. We propose that self-fertilization is a key driver of outbreeding depression, but that it need not evolve as a direct result of local adaptation per se. Our verbal model of this process highlights the need for formal theory, and C. sp. 11 provides a convenient system for testing the genetic mechanisms that cause outbreeding depression, negative epistasis, and incipient speciation.
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Caenorhabditis/fisiología , Organismos Hermafroditas , Polimorfismo Genético , Autofecundación , Animales , Evolución Biológica , Caenorhabditis/genética , Endogamia , Masculino , Datos de Secuencia Molecular , Reproducción , Análisis de Secuencia de ADNRESUMEN
DUSP6/MKP-3 is a cytoplasmic dual-specificity phosphatase specific for the MAP kinases ERK1/2. Previous data have shown that the MEK/ERK axis exerts a retro-control on its own signaling through transcriptional and post-translational regulation of DUSP6. We first confirm the key role of MEK/ERK in maintaining the levels of dusp6 mRNA, while PI3K/mTOR, p38 MAPK, and JNK signaling pathways had no significant effects. We further show that regulation of dusp6 mRNA stability plays a critical role in ERK-dependent regulation of dusp6 expression. Luciferase reporter constructs indicated that MEK/ERK signaling increased the half-life of dusp6 mRNA in a 3'untranslated region (3'UTR)-dependent manner. In addition, hypoxia, a hallmark of tumor growth, was found to increase both endogenous levels of dusp6 mRNA and the stability of the luciferase reporter constructs containing its 3'UTR, in a HIF-1-dependent manner. Nevertheless, a basal ERK activity was required for the response to hypoxia. Finally, Tristetraprolin (TTP), a member of the TIS11 CCCH zinc finger protein family, and PUM2, an homolog of drosophila pumilio, two proteins regulating mRNA stability reduced the levels of endogenous dusp6 mRNA and the activity of the dusp6/3'UTR luciferase reporter constructs. This study shows that post-transcriptional regulation is a key process in the control of DUSP6 expression.
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Fosfatasa 6 de Especificidad Dual/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/fisiología , Quinasas Quinasa Quinasa PAM/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , Línea Celular , Fosfatasa 6 de Especificidad Dual/genética , Humanos , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Estabilidad Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/fisiología , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMEN
Embryonic stem (ES) cells are pluripotent cells derived from blastocyst-stage embryos. They are characterized by their infinite self-renewal capacity and their ability to differentiate into many cell types in vitro as well as in vivo. The present protocol describes culture conditions that allow efficient differentiation of mouse ES cells toward the endothelial lineage, both in two-dimensional cultures and in three-dimensional, multicellular embryoid bodies. We also provide a protocol for establishing recombinant ES cell clones, giving the example of cells expressing green fluorescent protein and an antibiotic resistance gene under the control of an endothelial-specific promoter. These transgenes allow the visualization and the genetic selection of endothelial cells from a heterogeneous population of differentiating ES cells. Potential applications for these differentiation models are given, including the study of the endothelial cell lineage and its derivatives, the testing of molecules involved in angiogenesis, and the potential use of selected endothelial cells in vivo.
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Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Células Endoteliales/fisiología , Células Madre/fisiología , Animales , Trasplante de Células , Células Cultivadas , Embrión de Mamíferos/citología , Células Endoteliales/citología , Matriz Extracelular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Ratones Desnudos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/citologíaRESUMEN
Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.
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Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Serina/metabolismo , Animales , Línea Celular , Fosfatasa 6 de Especificidad Dual , Estabilidad de Enzimas , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Tirosina Fosfatasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The partial sequence of the increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom revealed a strong homology to vascular endothelial growth factor (VEGF)-A. We now report its complete amino acid sequence determined by Edman degradation and its biological effects on mouse and human vascular endothelial cells. ICPP is a homodimeric protein linked by cysteine disulfide bonds of 25115 Da revealed by mass spectrometry. Each monomer is composed of 110 amino acids including eight cysteine residues and a pyroglutamic acid at the N-terminal extremity. ICPP shares 52% sequence identity with human VEGF but lacks the heparin binding domain and Asn glycosylation site. Besides its strong capillary permeability activity, ICPP was found to be a potent in vitro angiogenic factor when added to mouse embryonic stem cells or human umbilical vein endothelial cells. ICPP was found to be as potent as human VEGF165 in activating p42/p44 MAPK, in reinitiation of DNA synthesis in human umbilical vein endothelial cells, and in promoting in vitro angiogenesis of mouse embryonic stem cells. All these biological actions, including capillary permeability in mice, were fully inhibited by 1 microm of a new specific VEGF receptor tyrosine kinase inhibitor (ZM317450) from AstraZeneca that belongs to the anilinocinnoline family of compounds. Indeed, up to a 30 times higher concentration of inhibitor did not affect platelet-derived growth factor, epidermal growth factor, FGF-2, insulin, alpha-thrombin, or fetal calf serum-induced p42/p44 MAPK and reinitiation of DNA synthesis. Therefore, we conclude that this venom-derived ICPP exerts its biological action (permeability and angiogenesis) through activation of VEGF receptor signaling (VEGF-R2 and possibly VEGF-R1).
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Permeabilidad Capilar/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Proteínas/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Transducción de Señal , Venenos de Víboras/química , Secuencia de Aminoácidos , Animales , Línea Celular , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Datos de Secuencia Molecular , Peso Molecular , Proteínas/antagonistas & inhibidores , Proteínas/farmacología , Receptores de Factores de Crecimiento Endotelial Vascular , Homología de Secuencia de Aminoácido , ViperidaeRESUMEN
Large scale purification of endothelial cells is of great interest as it could improve tissue transplantation, reperfusion of ischemic tissues and treatment of pathologies in which an endothelial cell dysfunction exists. In this study, we describe a novel genetic approach that selects for endothelial cells from differentiating embryonic stem (ES) cells. Our strategy is based on the establishment of ES-cell clones that carry an integrated puromycin resistance gene under the control of a vascular endothelium-specific promoter, tie-1. Using EGFP as a reporter gene, we first confirmed the endothelial specificity of the tie-1 promoter in the embryoid body model and in cells differentiated in 2D cultures. Subsequently, tie-1-EGFP ES cells were used as recipients for the tie-1-driven puror transgene. The resulting stable clones were expanded and differentiated for seven days in the presence of VEGF before puromycin selection. As expected, puromycin-resistant cells were positive for EGFP and also expressed several endothelial markers, including CD31, CD34, VEGFR-1, VEGFR-2, Tie-1, VE-cadherin and ICAM-2. Release from the puromycin selection resulted in the appearance of alpha-smooth muscle actin-positive cells. Such cells became more numerous when the population was cultured on laminin-1 or in the presence of TGF-beta1, two known inducers of smooth muscle cell differentiation. The hypothesis that endothelial cells or their progenitors may differentiate towards a smooth muscle cell phenotype was further supported by the presence of cells expressing both CD31 and alpha-smooth muscle actin markers. Finally, we show that purified endothelial cells can incorporate into the neovasculature of transplanted tumors in nude mice. Taken together, these results suggest that application of endothelial lineage selection to differentiating ES cells may become a useful approach for future pro-angiogenic and endothelial cell replacement therapies.