RESUMEN
Nucleotide-binding leucine-rich repeat (NLR) proteins play a pivotal role in plant immunity by recognizing pathogen effectors1,2. Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity3,4. Unlike most NLRs, the plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation5. The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 (SlNRC2) forms dimers and tetramers and higher-order oligomers at elevated concentrations. Cryo-electron microscopy shows an inactive conformation of SlNRC2 in these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester SlNRC2 from assembling into an active form. Mutations at the dimeric or interdimeric interfaces enhance pathogen-induced cell death and immunity in Nicotiana benthamiana. The cryo-electron microscopy structures unexpectedly show inositol hexakisphosphate (IP6) or pentakisphosphate (IP5) bound to the inner surface of the C-terminal leucine-rich repeat domain of SlNRC2, as confirmed by mass spectrometry. Mutations at the inositol phosphate-binding site impair inositol phosphate binding of SlNRC2 and pathogen-induced SlNRC2-mediated cell death in N. benthamiana. Our study indicates a negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs.
Asunto(s)
Proteínas NLR , Ácido Fítico , Proteínas de Plantas , Multimerización de Proteína , Solanum lycopersicum , Sitios de Unión , Muerte Celular , Microscopía por Crioelectrón , Modelos Moleculares , Mutación , Nicotiana/citología , Nicotiana/inmunología , Nicotiana/metabolismo , Proteínas NLR/antagonistas & inhibidores , Proteínas NLR/química , Proteínas NLR/genética , Proteínas NLR/metabolismo , Proteínas NLR/ultraestructura , Ácido Fítico/metabolismo , Ácido Fítico/química , Inmunidad de la Planta , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/ultraestructura , Unión Proteica , Dominios Proteicos , Solanum lycopersicum/citología , Solanum lycopersicum/genética , Solanum lycopersicum/inmunología , Solanum lycopersicum/metabolismoRESUMEN
In plants, host-pathogen coevolution often manifests in reciprocal, adaptive genetic changes through variations in host nucleotide-binding leucine-rich repeat immune receptors (NLRs) and virulence-promoting pathogen effectors. In grass powdery mildew (PM) fungi, an extreme expansion of a RNase-like effector family, termed RALPH, dominates the effector repertoire, with some members recognized as avirulence (AVR) effectors by cereal NLR receptors. We report the structures of the sequence-unrelated barley PM effectors AVRA6, AVRA7, and allelic AVRA10/AVRA22 variants, which are detected by highly sequence-related barley NLRs MLA6, MLA7, MLA10, and MLA22 and of wheat PM AVRPM2 detected by the unrelated wheat NLR PM2. The AVR effectors adopt a common scaffold, which is shared with the RNase T1/F1 family. We found striking variations in the number, position, and length of individual structural elements between RALPH AVRs, which is associated with a differentiation of RALPH effector subfamilies. We show that all RALPH AVRs tested have lost nuclease and synthetase activities of the RNase T1/F1 family and lack significant binding to RNA, implying that their virulence activities are associated with neo-functionalization events. Structure-guided mutagenesis identified six AVRA6 residues that are sufficient to turn a sequence-diverged member of the same RALPH subfamily into an effector specifically detected by MLA6. Similar structure-guided information for AVRA10 and AVRA22 indicates that MLA receptors detect largely distinct effector surface patches. Thus, coupling of sequence and structural polymorphisms within the RALPH scaffold of PMs facilitated escape from NLR recognition and potential acquisition of diverse virulence functions.
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Ascomicetos , Ascomicetos/metabolismo , Grano Comestible/genética , Grano Comestible/metabolismo , Ribonucleasa T1/genética , Ribonucleasa T1/metabolismo , Polimorfismo Genético , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/metabolismoRESUMEN
Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) detect pathogen effectors to trigger immune responses1. Indirect recognition of a pathogen effector by the dicotyledonous Arabidopsis thaliana coiled-coil domain containing NLR (CNL) ZAR1 induces the formation of a large hetero-oligomeric protein complex, termed the ZAR1 resistosome, which functions as a calcium channel required for ZAR1-mediated immunity2-4. Whether the resistosome and channel activities are conserved among plant CNLs remains unknown. Here we report the cryo-electron microscopy structure of the wheat CNL Sr355 in complex with the effector AvrSr356 of the wheat stem rust pathogen. Direct effector binding to the leucine-rich repeats of Sr35 results in the formation of a pentameric Sr35-AvrSr35 complex, which we term the Sr35 resistosome. Wheat Sr35 and Arabidopsis ZAR1 resistosomes bear striking structural similarities, including an arginine cluster in the leucine-rich repeats domain not previously recognized as conserved, which co-occurs and forms intramolecular interactions with the 'EDVID' motif in the coiled-coil domain. Electrophysiological measurements show that the Sr35 resistosome exhibits non-selective cation channel activity. These structural insights allowed us to generate new variants of closely related wheat and barley orphan NLRs that recognize AvrSr35. Our data support the evolutionary conservation of CNL resistosomes in plants and demonstrate proof of principle for structure-based engineering of NLRs for crop improvement.
Asunto(s)
Canales de Calcio , Microscopía por Crioelectrón , Proteínas NLR , Proteínas de Plantas , Receptores Inmunológicos , Triticum , Arabidopsis/inmunología , Arabidopsis/metabolismo , Arginina , Canales de Calcio/química , Canales de Calcio/inmunología , Canales de Calcio/metabolismo , Cationes/metabolismo , Leucina , Proteínas NLR/química , Proteínas NLR/inmunología , Proteínas NLR/metabolismo , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Receptores Inmunológicos/química , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Triticum/inmunología , Triticum/metabolismo , Secuencias de Aminoácidos , Secuencia Conservada , ElectrofisiologíaRESUMEN
Introgressions of chromosomal segments from related species into wheat are important sources of resistance against fungal diseases. The durability and effectiveness of introgressed resistance genes upon agricultural deployment is highly variable-a phenomenon that remains poorly understood, as the corresponding fungal avirulence genes are largely unknown. Until its breakdown, the Pm17 resistance gene introgressed from rye to wheat provided broad resistance against powdery mildew (Blumeria graminis). Here, we used quantitative trait locus (QTL) mapping to identify the corresponding wheat mildew avirulence effector AvrPm17. It is encoded by two paralogous genes that exhibit signatures of reoccurring gene conversion events and are members of a mildew sublineage specific effector cluster. Extensive haplovariant mining in wheat mildew and related sublineages identified several ancient virulent AvrPm17 variants that were present as standing genetic variation in wheat powdery mildew prior to the Pm17 introgression, thereby paving the way for the rapid breakdown of the Pm17 resistance. QTL mapping in mildew identified a second genetic component likely corresponding to an additional resistance gene present on the 1AL.1RS translocation carrying Pm17. This gene remained previously undetected due to suppressed recombination within the introgressed rye chromosomal segment. We conclude that the initial effectiveness of 1AL.1RS was based on simultaneous introgression of two genetically linked resistance genes. Our results demonstrate the relevance of pathogen-based genetic approaches to disentangling complex resistance loci in wheat. We propose that identification and monitoring of avirulence gene diversity in pathogen populations become an integral part of introgression breeding to ensure effective and durable resistance in wheat.
Asunto(s)
Resistencia a la Enfermedad , Introgresión Genética , Enfermedades de las Plantas , Secale , Triticum , Mapeo Cromosómico , Resistencia a la Enfermedad/genética , Fitomejoramiento , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Sitios de Carácter Cuantitativo , Secale/genética , Secale/microbiología , Triticum/genética , Triticum/microbiologíaAsunto(s)
COVID-19 , Vacunas contra la Influenza , Gripe Humana , Humanos , Gripe Humana/epidemiología , SARS-CoV-2 , Estaciones del AñoRESUMEN
BACKGROUND: The ongoing novel coronavirus (COVID-19) global pandemic has resulted in significant levels of morbidity and mortality worldwide, particularly among the elderly and immuno-suppressed groups. Although adequate hand hygiene (HH) behaviour and compliance is widely accepted as being the most effective self-protective measure in preventing the spread of diseases like COVID-19, previous research suggests that normal hand hygiene compliance is poor, but generally improves during a disease pandemic. This research aimed to evaluate the hand hygiene behaviour and compliance of the general public in the initial weeks of the COVID-19 pandemic in Northern Ireland (NI). METHODS: This cross-sectional study involved the use of infrared-imaging cameras to observe the hand hygiene behaviour and compliance of the general public when using one set of male and female public restrooms. RESULTS: The findings of this study indicated that the level of hand hygiene compliance of the general public was poor in the initial weeks, with 82.93% overall not washing their hands adequately. CONCLUSIONS: Inadequate HH behaviour and compliance may have added significantly to the rapid rate of spread of COVID-19 in the initial weeks of the pandemic in NI. Current public health campaigns do not appear, based on this study, to have the desired impact and may need to be reviewed or re-enforced in order to achieve the levels of hand hygiene compliance required to slow the spread of COVID-19 and other communicable diseases in the future.
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COVID-19 , Infección Hospitalaria , Higiene de las Manos , Anciano , Estudios Transversales , Femenino , Adhesión a Directriz , Desinfección de las Manos , Humanos , Masculino , Irlanda del Norte/epidemiología , Pandemias , SARS-CoV-2 , Cuartos de BañoRESUMEN
Every year, thousands of people from the UK travel to other countries for work and leisure. Europe, and particularly Spain, is one of the most popular travel destinations for people from the UK. However, it is known that travel to other countries can enhance the risk of communicable disease transmission from person to person, especially when a new one emerges. Adequate hand hygiene behaviour and compliance is widely accepted as being a simple, effective method in preventing the spread of communicable diseases that may be contracted during travel abroad. There is a well-established body of work investigating hand hygiene practice and compliance in community settings, but no recent studies have examined the hand hygiene practice and compliance of the general population when travelling abroad or in a cross-European context. The findings of this study indicated that most UK members of the general population when travelling abroad have a good level of understanding of the importance of adequate hand hygiene practice and compliance and its role regarding communicable disease prevention and control. As such, self-reported levels of compliance were high. Similar findings were made for Spanish members of the general population. However, while self-reported perceptions of adequacy of hand hygiene performance were relatively high, particularly among UK respondents, this was not supported by responses specifically focused on hand hygiene behaviour. However, differences in self-reported adequacy regarding the importance of handwashing versus hand drying, the number of steps that should be followed and the length of time that should be spent washing and drying hands were found for each group. This suggests that self-reported compliance may reflect intention to practice hand hygiene rather than true compliance. It also suggests that there are gaps in knowledge regarding the adequate method of hand hygiene among the cohort as a whole, and indeed these differences may account be a factor in for the high transmission rates of communicable disease when travelling abroad.
Asunto(s)
Infección Hospitalaria , Higiene de las Manos , Europa (Continente) , Adhesión a Directriz , Mano , Desinfección de las Manos , Humanos , Autoinforme , EspañaRESUMEN
Nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs) in plants can detect avirulence (AVR) effectors of pathogenic microbes. The Mildew locus a (Mla) NLR gene has been shown to confer resistance against diverse fungal pathogens in cereal crops. In barley, Mla has undergone allelic diversification in the host population and confers isolate-specific immunity against the powdery mildew-causing fungal pathogen Blumeria graminis forma specialis hordei (Bgh). We previously isolated the Bgh effectors AVRA1, AVRA7, AVRA9, AVRA13, and allelic AVRA10/AVRA22, which are recognized by matching MLA1, MLA7, MLA9, MLA13, MLA10 and MLA22, respectively. Here, we extend our knowledge of the Bgh effector repertoire by isolating the AVRA6 effector, which belongs to the family of catalytically inactive RNase-Like Proteins expressed in Haustoria (RALPHs). Using structural prediction, we also identified RNase-like folds in AVRA1, AVRA7, AVRA10/AVRA22, and AVRA13, suggesting that allelic MLA recognition specificities could detect structurally related avirulence effectors. To better understand the mechanism underlying the recognition of effectors by MLAs, we deployed chimeric MLA1 and MLA6, as well as chimeric MLA10 and MLA22 receptors in plant co-expression assays, which showed that the recognition specificity for AVRA1 and AVRA6 as well as allelic AVRA10 and AVRA22 is largely determined by the receptors' C-terminal leucine-rich repeats (LRRs). The design of avirulence effector hybrids allowed us to identify four specific AVRA10 and five specific AVRA22 aa residues that are necessary to confer MLA10- and MLA22-specific recognition, respectively. This suggests that the MLA LRR mediates isolate-specific recognition of structurally related AVRA effectors. Thus, functional diversification of multi-allelic MLA receptors may be driven by a common structural effector scaffold, which could be facilitated by proliferation of the RALPH effector family in the pathogen genome.
Asunto(s)
Ascomicetos/fisiología , Hordeum/genética , Leucina/química , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Receptores Inmunológicos/genética , Ribonucleasas/química , Alelos , Secuencia de Aminoácidos , Regulación de la Expresión Génica de las Plantas , Variación Genética , Hordeum/inmunología , Hordeum/microbiología , Inmunidad Innata , Leucina/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/metabolismo , Receptores Inmunológicos/metabolismo , Ribonucleasas/metabolismo , Homología de SecuenciaRESUMEN
BACKGROUND: Most research on hand hygiene compliance in community settings indicates that compliance is poor. It is not conclusive as to whether poster interventions are effective at improving compliance. METHODS: An independent, self-designed poster intervention was installed in one set of male and female public restrooms in a university campus in the UK. The hand hygiene practice and compliance of the university population was measured via indirect observation over a 60 day period. RESULTS: During the pre-intervention observation period, 51.09% of the university population practiced basic hand hygiene compliance (washed hands with water, soap and dried afterwards), and 7.88% practiced adequate hand hygiene compliance (washed hands with water and soap for 20 s or more and dried afterwards for 20 s or more). During the post-intervention observation period, 55.39% of the university population were observed practicing basic hand hygiene compliance, and 7.97% practicing adequate hand hygiene compliance. Gender differences revealed that more females practiced basic hand hygiene in the post-intervention observation period (62.81%) than during the pre-intervention period (49.23%) and this was statistically significant (χ2 = 13.49, p = < 0.01). DISCUSSION: The poster intervention had a limited effect on improving the basic and adequate hand hygiene compliance of the general population when using public restrooms. The use of independent, self-designed posters to improve hand hygiene practice and compliance is largely ineffective in the short term and should be used with caution in future intervention strategies.
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Adhesión a Directriz/estadística & datos numéricos , Higiene de las Manos , Promoción de la Salud/métodos , Carteles como Asunto , Cuartos de Baño , Adulto , Femenino , Higiene de las Manos/métodos , Higiene de las Manos/normas , Higiene de las Manos/estadística & datos numéricos , Humanos , Masculino , Evaluación de Resultado en la Atención de Salud , Reino Unido , UniversidadesRESUMEN
INTRODUCTION: Understanding sex differences in willingness to test and testing experience could aid the design of focus interventions to enhance uptake and engagement with care, treatment and support services. This study determined differences in perceived risk of acquiring HIV, willingness to test and HIV testing experience in an urban fishing community. METHODS: A cross-sectional community survey was conducted in 2013 among men and women in two fishing communities (Chorkor and James Town) in Accra. In all, 554 subjects (≥ 18 years) were involved, 264 in Chorkor and 290 in James Town. Data on demographic characteristics, perceived risk for HIV and willingness to test for HIV and testing experience were collected with a structured questionnaire. Descriptive statistics and Chi square test were used for the analysis at 95% significant level, using SPSS version 21. RESULTS: Of 554 subjects, 329 (59.4%) were females, and median age was 32 years. Overall, only 91(40.4%) men and 118(35.9%) women perceived themselves to be at risk of acquiring HIV. A significant proportion of women were willing to test for HIV compared to men (86.3% vs. 80.0%, P = 0.048). Women were more likely to have ever tested for HIV compared to men (42.2% vs. 28.6%, P = 0.001) and more women had tested within 12 months prior to survey than men (49.6% vs. 40.6%, P = 0.230). Of the number who had tested for HIV infection, a higher proportion of men tested voluntarily 42(65.6%), while a higher proportion of women tested as part of healthcare service received 96(69.1%); (P = 0.001; indicating women vs. men). CONCLUSION: Sex differences in risk perception and willingness to test need more focused public education and behaviour change communication strategies to achieve high coverage. Community-based strategies could improve HIV testing among men whilst more access to testing in health settings should be available to women in these communities.
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Actitud Frente a la Salud , Infecciones por VIH/diagnóstico , Tamizaje Masivo/estadística & datos numéricos , Aceptación de la Atención de Salud/estadística & datos numéricos , Factores Sexuales , Población Urbana/estadística & datos numéricos , Adolescente , Adulto , Estudios Transversales , Femenino , Ghana , Infecciones por VIH/psicología , Humanos , Masculino , Persona de Mediana Edad , Percepción , Riesgo , Encuestas y Cuestionarios , Adulto JovenRESUMEN
The potential of international academic partnerships to build global capacity is critical in efforts to improve health in poorer countries. Academic collaborations, however, are challenged by distance, communication issues, cultural differences, and historical context. The Collaborative Health Alliance for Reshaping Training, Education, and Research project (funded by the Bill and Melinda Gates Foundation and implemented through academic medicine and public health and governmental institutions in Michigan and Ghana) took a prospective approach to address these issues. The project had four objectives: to create a "charter for collaboration" (CFC), to improve data-driven policy making, to enhance health care provider education, and to increase research capacity. The goal of the CFC was to establish principles to guide the course of the technical work. All participants participated at an initial conference in Elmina, Ghana. Nine months later, the CFC had been revised and adopted. A qualitative investigation of the CFC's effects identified three themes: the CFC's unique value, the influence of the process of creating the CFC on patterns of communication, and the creation of a context for research and collaboration. Creating the CFC established a context in which implementing technical interventions became an opportunity for dialogue and developing a mutually beneficial partnership. To increase the likelihood that research results would be translated into policy reforms, the CFC made explicit the opportunities, potential problems, and institutional barriers to be overcome. The process of creating a CFC and the resulting document define a new standard in academic and governmental partnerships.
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Investigación Biomédica/educación , Creación de Capacidad/organización & administración , Países en Desarrollo , Personal de Salud/educación , Cooperación Internacional , Formulación de Políticas , Investigación Biomédica/organización & administración , Creación de Capacidad/métodos , Comunicación , Ghana , Agencias Gubernamentales , Personal de Salud/organización & administración , Política de Salud , Humanos , Michigan , Desarrollo de Programa , Evaluación de Programas y Proyectos de SaludRESUMEN
BACKGROUND: In this study, we undertook a comparison of the international, national, and local curricula of Ghanaian medical schools in order to identify any gaps. PURPOSE: To identify gaps in the Ghanaian medical school curriculum. METHODS: The Ministry of Health and the two major sites for medical education in Ghana (UGMS, KNUST) participated using the only internationally accepted and validated set of outcome standards for medical education, the Global Minimum Essential Requirements. The competencies were reviewed by two U.S. consultants (DS, SR) and then edited, revised, and validated by individuals who are deeply involved in medical education in Ghana. RESULTS: The KNUST team validated 6 gaps in their curriculum, and the team from UGMS identified 5. The standards were found by the U.S.-based consultants and validated by the Ghanaian team to have 6 gaps, many of which overlapped with those found in the Ghanaian medical school curricula. CONCLUSIONS: This analysis is a first step to determining physician training competency and an inexpensive method for identifying agreed-upon gaps in the current national requirements and local curriculum.
Asunto(s)
Competencia Clínica/estadística & datos numéricos , Curriculum/estadística & datos numéricos , Países en Desarrollo/estadística & datos numéricos , Facultades de Medicina/estadística & datos numéricos , Países en Desarrollo/economía , Escolaridad , Ghana , Humanos , Desarrollo de Programa , Evaluación de Programas y Proyectos de Salud , Facultades de Medicina/economíaRESUMEN
One of the major needs for medical schools and health systems in less affluent countries is system strengthening through the training and development of faculty, doctors, nurses, and other skilled health care workers. Partnering with medical schools in more affluent countries such as the United States is one potential approach for medical schools in underresourced areas, such as Sub-Saharan Africa. Most commonly, these partnerships have focused on research agendas or limited educational exchanges. In this perspective, the authors present an approach to strengthening collaborative relationships between three medical schools in the United States and four in Sub-Saharan Africa. The approach is explicitly focused on achieving partnerships that enable institutions to improve care. It developed from an initiative to fund partnerships or "collaboratives" that address 10 key learning questions determined to be central to focusing efforts on strengthening education systems and, in turn, improving health in Sub-Saharan Africa. The leaders of the schools involved in these partnerships met multiple times across three years to discuss how their collaboratives could address the ten learning questions including what is the best approach and what are the key ingredients for creating effective, multidimensional collaborations between academic institutions in the North and institutions in Sub-Saharan Africa. Collaboratively, they defined a framework of evidence that can be used for evaluating their current initiatives and, potentially, for structuring future partnerships.
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Educación Médica/métodos , Relaciones Interinstitucionales , Internacionalidad , África del Sur del Sahara , Conducta Cooperativa , Países en Desarrollo , Humanos , Cooperación Internacional , Relaciones Interprofesionales , Facultades de Medicina , Estados UnidosRESUMEN
Formation of the pulmonary vasculature has been described as occurring by outgrowth of existing vessels (angiogenesis), de novo formation of new vessels (vasculogenesis), or a combination of both processes. Uncertainty about the contribution of angiogenesis and vasculogenesis to pulmonary vascular formation is partly due to methodologic approaches. Evidence in favor of angiogenesis stems from studies that used vascular-filling methods. Such methods identify only directly continuous lumina. Evidence for vasculogenesis has been provided by the use of molecular markers of blood vessel endothelium. Use of both methods has not been combined in the same species, however. We hypothesized, based on published evidence from quail and mouse, that chick pulmonary vascular formation occurs by vasculogenesis. To test that hypothesis, we used vascular filling, serial section, and immunohistochemical methods to analyze the developing lungs of chick embryos from Hamburger and Hamilton stages 20 to 43. Vascular filling suggested that the lumen of the pulmonary arteries sprouted from the sixth pharyngeal arch arteries. However, serial sections and immunohistochemical localization of fetal liver kinase-1 protein, the receptor for vascular endothelial growth factor, showed that the pulmonary arterial tree formed from endothelial cell precursors and coalescence of isolated blood vessels in the mediastinal splanchnic mesenchyme centrally to the developing lung tissue distally. Pulmonary veins grew from the left atrium to the developing lungs. Pulmonary blood vessel formation occurred continuously throughout the embryonic period studied. Our results show that vasculogenesis is the main process by which the pulmonary vasculature forms in the developing chick embryo.
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Pulmón/embriología , Arteria Pulmonar/embriología , Circulación Pulmonar/fisiología , Venas Pulmonares/embriología , Animales , Embrión de Pollo , Inmunohistoquímica , Pulmón/irrigación sanguínea , Neovascularización Fisiológica/fisiologíaRESUMEN
We report the first ubiquitous green fluorescent protein expression in chicks using a lentiviral vector approach, with eGFP under the control of the phosphoglycerol kinase promoter. Several demonstrations of germline transmission in chicks have been reported previously, using markers that produce tissue-specific, but not ubiquitous, expression. Using embryos sired by a heterozygous male, we demonstrate germline transmission in the embryonic tissue that expresses eGFP uniformly, and that can be used in tissue transplants and processed by in situ hybridization and immunocytochemistry. Transgenic tissue is identifiable by both fluorescence microscopy and immunolabeling, resulting in a permanent marker identifying transgenic cells following processing of the tissue. Stable integration of the transgene has allowed breeding of homozygous males and females that will be used to produce transgenic embryos in 100% of eggs laid upon reaching sexual maturity. These results demonstrate that a transgenic approach in the chick model system is viable and useful even though a relatively long generation time is required. The transgenic chick model will benefit studies on embryonic development, as well as providing the pharmaceutical industry with an economical bioreactor.
Asunto(s)
Animales Modificados Genéticamente , Vectores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Lentivirus/genética , Animales , Embrión de Pollo , Pollos , Desarrollo Embrionario , Femenino , Marcadores Genéticos , Proteínas Fluorescentes Verdes/genética , Heterocigoto , Homocigoto , Inmunohistoquímica , Hibridación in Situ , Masculino , Microscopía Fluorescente , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Distribución Tisular , TransgenesRESUMEN
A new, rapid method is described for combining in situ hybridization and immunocytochemistry to define cell populations and to map three-dimensional movements of groups of labeled cells within developing chick embryos. The method allows fluorescently labeled cells to be followed in living embryos and subsequently detected as a permanent reaction product for detailed three-dimensional analysis by immunocytochemistry in histological serial sections. Cell identity can be ascertained using a specific riboprobe and in situ hybridization. With this approach, the movements of two groups of cells can be mapped simultaneously (using two different fluorescent trackers and, subsequently, two different chromogens for immunocytochemistry) to analyze relative movements within an embryo, and when combined with in situ hybridization with a specific riboprobe for cell identity, allows fate mapping studies to be conducted using molecular criteria, rather than solely at morphological/positional criteria. The improved method enables the investigator to extract substantially more information from individual embryos, maximizing the results obtained from labor-intensive fate mapping studies.
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Embrión de Pollo/citología , Embrión de Pollo/metabolismo , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Animales , Trasplante de Células , Embrión de Pollo/embriología , Técnicas de Cultivo de Embriones , Colorantes Fluorescentes/análisis , Microinyecciones , Microscopía Fluorescente , ParafinaRESUMEN
Gastrulation is characterized by the extensive movements of cells. Fate mapping is used to follow such cell movements as they occur over time, and prospective fate maps have been constructed for several stages of the model organisms used in modern studies in developmental biology. In chick embryos, detailed fate maps have been constructed for both prospective mesodermal and ectodermal cells. However, the origin and displacement of the prospective endodermal cells during crucial periods in gastrulation remain unclear. This study had three aims. First, we determined the primitive-streak origin of the endoderm using supravital fluorescent markers, and followed the movement of the prospective endodermal cells as they dispersed to generate the definitive endodermal layer. We show that between stages 3a/b and 4, the intraembryonic definitive endoderm receives contributions mainly from the rostral half of the primitive streak, and that endodermal movements parallel those of ingressing adjacent mesodermal subdivisions. Second, the question of the epiblast origin of the endodermal layer was addressed by precisely labeling epiblast cells in a region known to give rise to prospective somitic cells, and following their movement as they underwent ingression through the primitive streak. We show that the epiblast clearly contributes prospective endodermal cells to the primitive streak, and subsequently to definitive endoderm of the area pellucida. Finally, the relationship between the hypoblast and the definitive endoderm was defined by following labeled rostral primitive-streak cells over a short period of time as they contributed to the definitive endoderm, and combining this with in situ hybridization with a riboprobe for Crescent, a marker of the hypoblast. We show that as the definitive endodermal layer is laid down, there is cell-cell intercalation at its interface with the displaced hypoblast cells. These data were used to construct detailed prospective fate maps of the endoderm in the chick embryo, delineating the origins and migrations of endodermal cells in various rostrocaudal levels of the primitive streak during key periods in early development.
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Endodermo/fisiología , Gástrula/fisiología , Animales , Carbocianinas/metabolismo , Embrión de Pollo , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Succinimidas/metabolismoRESUMEN
Patterning of the ventral head has been attributed to various cell populations, including endoderm, mesoderm, and neural crest. Here, we provide evidence that head and heart development may be influenced by a ventral midline endodermal cell population. We show that the ventral midline endoderm of the foregut is generated directly from the extreme rostral portion of Hensen's node, the avian equivalent of the Spemann organizer. The endodermal cells extend caudally in the ventral midline from the prechordal plate during development of the foregut pocket. Thus, the prechordal plate appears as a mesendodermal pivot between the notochord and the ventral foregut midline. The elongating ventral midline endoderm delimits the right and left sides of the ventral foregut endoderm. Cells derived from the midline endoderm are incorporated into the endocardium and myocardium during closure of the foregut pocket and fusion of the bilateral heart primordia. Bilateral ablation of the endoderm flanking the midline at the level of the anterior intestinal portal leads to randomization of heart looping, suggesting that this endoderm is partitioned into right and left domains by the midline endoderm, thus performing a function similar to that of the notochord in maintaining left-right asymmetry. Because of its derivation from the dorsal organizer, its extent from the forebrain through the midline of the developing face and pharynx, and its participation in formation of a single midline heart tube, we propose that the ventral midline endoderm is ideally situated to function as a ventral organizer of the head and heart.